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Infertility affects approximately 10-15% of couples worldwide who are attempting to conceive, with male infertility accounting for 50% of infertility cases. Male infertility is related to various factors such as hormone imbalance, urogenital diseases, environmental factors, and genetic factors. Owing to its relationship with genetic factors, male infertility cannot be diagnosed through routine examination in most cases, and is clinically called 'idiopathic male infertility.' Recent studies have provided evidence that microRNAs (miRNAs) are expressed in a cell-or stage-specific manner during spermatogenesis. This review focuses on the role of miRNAs in male infertility and spermatogenesis. Data were collected from published studies that investigated the effects of miRNAs on spermatogenesis, sperm quality and quantity, fertilization, embryo development, and assisted reproductive technology (ART) outcomes. Based on the findings of these studies, we summarize the targets of miRNAs and the resulting functional effects that occur due to changes in miRNA expression at various stages of spermatogenesis, including undifferentiated and differentiating spermatogonia, spermatocytes, spermatids, and Sertoli cells (SCs). In addition, we discuss potential markers for diagnosing male infertility and predicting the varicocele grade, surgical outcomes, ART outcomes, and sperm retrieval rates in patients with non-obstructive azoospermia (NOA)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shi, Yu, Guo, Sui, Tian, Ni, Chen, Jiang, Jiang, Lu and Lin.)

Neuroblastoma (NB) is a challenging pediatric extracranial solid tumor characterized by a poor prognosis and resistance to chemotherapy. Identifying targets to enhance chemotherapy sensitivity in NB is of utmost importance. Increasing evidence implicates long noncoding RNAs (lncRNAs) play important roles in cancer, but their functional roles remain largely unexplored. Here, we analyzed our RNA sequencing data and identified the upregulated lncRNA ZNF674-AS1 in chemotherapy non-responsive NB patients. Elevated ZNF674-AS1 expression is associated with poor prognosis and high-risk NB. Importantly, targeting ZNF674-AS1 expression in NB cells suppressed tumor growth in vivo. Further functional studies have revealed that ZNF674-AS1 constrains cisplatin sensitivity by suppressing pyroptosis and promoting cell proliferation. Moreover, ZNF674-AS1 primarily relies on CA9 to fulfill its functions on cisplatin resistance. High CA9 levels were associated with high-risk NB and predicted poor patient outcomes. Mechanistically, ZNF674-AS1 directly interacted with the RNA binding protein IGF2BP3 to enhance the stability of CA9 mRNA by binding with CA9 transcript, leading to elevated CA9 expression. As a novel regulator of CA9, IGF2BP3 positively upregulated CA9 expression. Together, these results expand our understanding of the cancer-associated function of lncRNAs, highlighting the ZNF674-AS1/IGF2BP3/CA9 axis as a constituting regulatory mode in NB tumor growth and cisplatin resistance. These insights reveal the pivotal role of ZNF674-AS1 inhibition in recovering cisplatin sensitivity, thus providing potential therapeutic targets for NB treatment., (© 2024. The Author(s).)

Background: Hyperlipidemia is a major risk factor for vascular endothelial injury and atherosclerosis leading to cardiovascular diseases. Early diagnosis of vascular endothelial injury is important for the prevention and prognosis of cardiovascular diseases. This study aimed to investigate sensitive circulating microRNA (miRNA) as a potential diagnostic biomarker of vascular endothelial injury in a hyperlipidemic rat model., Methods: The miRNA expression profile was detected by miRNA microarray. The hyperlipidemic rat model was established by intraperitoneal injection of vitamin D 3 combined with a high-fat diet. Plasma miRNA levels were measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)., Results: No significant difference was found in the types of highly expressed miRNAs between human umbilical artery endothelial cells (HUAEC) and human umbilical vein endothelial cells (HUVEC). A total of 10 highly expressed miRNAs in endothelial cells were selected as candidate miRNAs, including miR-21, miR-126, let-7a, miR-23a, miR-221, miR-125b, miR-26a, miR-29a, miR-16, and miR-100. The plasma levels of let-7a, miR-126, miR-21, and miR-26a were significantly elevated in hyperlipidemic rats at 30 and 50 days after modeling, while the plasma level of miR-29a was significantly decreased. No significant change was found in the plasma levels of miR-125b, miR-23a, miR-221, miR-100, and miR-16. Interestingly, a significant reduction in plasma miR-29 level was detected as early as 20 days after modeling, which was earlier than for soluble intercellular adhesion molecule‑1 (sICAM-1)., Conclusion: The plasma levels of endothelial cell-enriched miRNAs were correlated with vascular endothelial injury induced by hyperlipidemia. miR-29a might serve as a potential early diagnostic biomarker of endothelial injury-related diseases., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

The majority of molecularly targeted therapies in clinical use target disease-related proteins, but only a small fraction (∼1.5%) of human genome is protein-coding region. Considering that ∼70% of human genome is transcribed to noncoding RNAs, targeting noncoding RNAs rather than protein-coding RNAs can significantly expand the proportion of human genome that can be manipulated. H19 long noncoding RNA (lncRNA) is aberrantly expressed in a variety of cancer types and actively contributes to multiple steps of tumorigenesis. Therefore, we selected H19 as a representative target and designed synthetic anti-H19 construct for the self-assembly and delivery of anti-H19 small RNA (sRNA) to prevent colorectal cancer development and metastasis based on the natural ability of the host liver to package sRNA-encapsulating small extracellular vesicles (sEVs) and the endogenous circulating sEVs to transfer sRNA. As anticipated, the synthetic anti-H19 construct successfully generated anti-H19 sRNA-encapsulating sEVs and exhibited high silencing efficiency on H19 lncRNA in an ex vivo model. In orthotopic and lung metastasis mouse models of colorectal cancer, the anti-H19 construct exhibited significantly superior therapeutic efficacy over 5-fluorouracil (5-Fu) in preventing primary tumor growth and lung metastasis. Particularly, the anti-H19 sRNA-encapsulating sEVs were generated in a nontoxic, nonimmunogenic and biocompatible manner. In summary, this study demonstrates that the in vivo self-assembled anti-H19 sRNA can serve as a new therapeutic agent for colorectal cancer., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The present study focuses on the role of the long noncoding RNA (lncRNA) NEAT1 in regulating autophagy during the ischemia‒reperfusion (I/R) injury process and its possible regulatory mechanism based on the results of laboratory experiments. Neuro-2a (N2a) cells and BV-2 microglial cells were cultured separately, and oxygen-glucose deprivation/reoxygenation (OGD/R) was induced in vitro to mimic cerebral I/R injury. The expression of lncRNA NEAT1 was measured after reoxygenation for different durations, and the results showed that NEAT1 expression was significantly different after OGD/R for 12 h; thus, cell models of NEAT1 overexpression and knockdown were constructed. Knockdown of NEAT1 effectively relieved reperfusion injury. In an N2a and BV-2 cell coculture system, knockdown of NEAT1 reduced autophagic flow in neuronal cells after reperfusion. To clarify the mechanism of NEAT1 after neuronal I/R injury, label-free quantitative proteomics (LFQ) was used to identify the differentially expressed proteins (DEPs) in NEAT1 knockdown neurons after OGD/R for 12 h. Additionally, Gene Ontology (GO) enrichment, protein‒protein interaction (PPI) network and parallel-reaction monitoring (PRM) quantitative analyses were carried out; the results showed that the expression levels of the autophagy-related proteins Gaa, Glb1, Prkaa1, Kif23, Sec24a and Vps25 were significantly reduced and that these proteins interact. In summary, this study shows that NEAT1 can regulate the interactions between autophagy-related proteins after neuronal I/R injury, reducing the level of autophagy and relieving neuronal reperfusion injury., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

MicroRNAs (miRNAs) play a vital role in the nerve regulation of honey bees ( Apis mellifera ). This study aims to investigate the differences in expression of miRNAs in a honey bee's brain for olfactory learning tasks and to explore their potential role in a honey bee's olfactory learning and memory. In this study, 12 day old honey bees with strong and weak olfactory performances were utilized to investigate the influence of miRNAs on olfactory learning behavior. The honey bee brains were dissected, and a small RNA-seq technique was used for high-throughput sequencing. The data analysis of the miRNA sequences revealed that 14 differentially expressed miRNAs (DEmiRNAs) between the two groups, strong (S) and weak (W), for olfactory performance in honey bees were identified, which included seven up-regulated and seven down-regulated. The qPCR verification results of the 14 miRNAs showed that four miRNAs (miR-184-3p, miR-276-3p, miR-87-3p, and miR-124-3p) were significantly associated with olfactory learning and memory. The target genes of these DEmiRNAs were subjected to the GO database annotation and KEGG pathway enrichment analyses. The functional annotation and pathway analysis showed that the neuroactive ligand-receptor interaction pathway, oxidative phosphorylation, biosynthesis of amino acids, pentose phosphate pathway, carbon metabolism, and terpenoid backbone biosynthesis may be a great important pathway related to olfactory learning and memory in honey bees. Our findings together further explained the relationship between olfactory performance and the brain function of honey bees at the molecular level and provides a basis for further study on miRNAs related to olfactory learning and memory in honey bees.

Wilms tumor is a common abdominal malignant tumor in children. However, the molecular mechanism of Wilms tumor is unclear. GSE66405 and GSE197047 were obtained from the Gene Expression Omnibus database. To identify differentially expressed genes (DEGs) in Wilms tumor, the R package "limma" was used. Weighted gene co-expression network analysis was performed to identify the significant module. The list of DEGs was input into the Search Tool for the Retrieval of Interacting Genes database to construct a protein-protein interaction network for predicting core genes. Gene Ontology analysis and the Kyoto Encyclopedia of Genes and Genomes analysis are computational methods for assessing gene function and biological pathways. The genome was analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes and developed by gene set enrichment analysis. Comparative Toxicogenomics Database analysis was performed to find the diseases most related to the core genes. TargetScan was used to screen for miRNAs that regulate hub genes. A total of 925 DEGs were identified. The differently expressed genes were mainly enriched in the metabolic pathway, AMPK signaling pathway, ErbB signaling pathway, mRNA detection pathway, and folded protein binding. A total of 16 core genes (HNRNPK, PABPC1, HNRNPD, NCL, YBX1, EIF4G1, KHDRBS1, HNRNPAB, HSPA4, EEF2, HSP90AA1, EEF1A1, A TP5A1, SDHA, CCT4, CCT5) were obtained. chaperonin containing TCP-1 subunit 4 (CCT4) was downregulated in tumor tissue samples, which may have reverse regulatory significance for Wilms tumor. CCT4, HSP90AA1, NCL, PABPC1, and YBX1 were found to be associated with kidney disease, acute kidney injury, edema, tumor metastasis, transitional cell carcinoma, necrosis, and inflammation. The research found that the related miRNA of the CCT4 gene was hsamiR-7-5p. CCT4 might play an essential role in the occurrence and development of Wilms tumor, and they may participate in the occurrence and development of Wilms tumor through the ERBB signal pathway. CCT4 may be a promising biomarker of Wilms tumor., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Osteosarcoma (OS) is a bone tumour affecting adolescents. Dysregulation of Barx homeobox 1 (BARX1) expression is involved in various cancers, but its function and mechanism in the process of OS are undefined. This study revealed that BARX1 expression is higher in OS tissue than in adjacent normal tissue. Downregulation of BARX1 in OS cells significantly suppressed their proliferation and migration, whereas enforced expression of exogenous BARX1 exerted the opposite effects on OS cells. Subsequently, heat shock 70-kDa protein 6 (HSPA6) expression was clearly increased after BARX1 overexpression in OS cells, as confirmed by RNA sequencing. The dual-luciferase reporter assay confirmed that HSPA6 expression is directly regulated by BARX1. The in vitro assay indicated that silencing HSPA6 expression attenuated OS proliferation and migration induced by BARX1. A dual immunofluorescence labelling assay provided further evidence that BARX1 was overexpressed and associated with HSPA6 overexpression in OS tumour tissue. In conclusion, BARX1 promotes OS cell proliferation and migration by inducing the expression of HSPA6, which plays an oncogenic role in OS. BARX1 and HSPA6 can potentially act as novel therapeutic targets for OS., (© 2023. The Author(s).)

Recently, long noncoding RNAs (lncRNAs) and the Warburg effect have been reported to play important roles in the radio-sensitivity of tumor cells. Survival correlates with pathologic responses to chemoradiotherapy and improving responses to radiation may translate into improved survival. This study aims to examine the effects and mechanisms of lncRNA DGCR5 and the Warburg effect on ESCC cell radiosensitivity. Levels of DGCR5, miR-195 and hexokinase 2 (HK2) expression in ESCC tissues and cells were determined and their clinical significance was analyzed. TE-1 and KYSE150 cells received a 6 Gy dose of X-ray radiation and their survival, proliferation and apoptosis were evaluated using colony formation assays, CCK-8 assays, and flow cytometry, respectively. Lactic acid production and glucose consumption were also examined in both cell types. Finally, the expression of apoptotic proteins was assessed using Western blotting. Analysis revealed that DGCR5 and HK2 were overexpressed in ESCC, while miR-195 was under expressed. Moreover, it was demonstrated that down-regulation of DGCR5 inhibited cell proliferation and promoted apoptosis, resulting in increased radiosensitivity by inhibition of the Warburg Effect. Conversely, overexpression of DGCR5 exhibited an opposite phenomenon in vitro. When investigating the mechanism, we identified that miR-195 was predicted to be a direct downstream target of DGCR5. Meanwhile, HK2 was predicted to be a direct downstream target of miR-195. Dual-luciferase reporter assays verified the direct interaction between these molecules. Finally, in vivo experiments were utilized to validate that knockdown of DGCR5 suppressed the Warburg effect via targeting of the miR-195/HK2 axis to increase the radiosensitivity of ESCC. Our study reveals that down-regulation of DGCR5 resulted in inhibition of the Warburg effect through interaction with the miR-195/HK2 axis increasing ESCC cell apoptosis after irradiation, thus enhancing cell radiosensitivity., (©2023 by Radiation Research Society. All rights of reproduction in any form reserved.)

Methyltransferase-like protein 3 (METTL3) mediated N6-Methyladenosine (m 6 A) modification has been implicated in many physiological and pathological processes. However, its function and mechanism in kidney aging are not entirely clear. Here, we investigated changes in m 6 A levels of aging kidneys and the role of METTL3 in senescent renal tubular epithelial cells and its potential mechanisms. First, we used the naturally aged mouse model and the D-galactose (D-gal)-induced aged mouse model. Dot blot and m 6 A RNA methylation quantification showed significantly decreased m 6 A levels in both models. In addition, we observed that METTL3 was down-regulated in D-gal-induced senescent human renal tubular epithelial cell line (HK-2). METTL3 reduction was associated with senescence-related phenotypes of HK-2 cells. We also found that miR-181a-5p attenuated HK-2 senescence by targeting the NF-κB pathway. Moreover, METTL3 was able to promote the maturation of miR-181a-5p and then inhibited the expression of NF-κB and IL-1α. Taken together, we demonstrate that the METTL3/miR-181a-5p/NF-κB axis counteracts HK-2 senescence. Our results suggest that METTL3 may be a novel biomarker and a potential therapy target for kidney aging., Competing Interests: Conflict of interest The authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is characterized by microglia activation that leads to neuroinflammation. Heat shock transcription factor 1 (HSF1) is known to exert neuroprotective effects on neurodegenerative diseases. This study sought to analyse the role and mechanism of HSF1 in PD-induced neuroinflammation. The PD mouse models were established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Animal behaviour capacities and neuronal damage were assessed via behavioural tests, tyrosine hydroxylase (TH) staining, and immunofluorescence. Levels of HSF1, miR-214-3p, nuclear factor of activated T cells 2 (NFATc2), and neuroinflammatory factors were detected via RT-qPCR, Western blotting, and ELISA.Binding relationships between HSF1 and miR-214-3p, miR-214-3p, and NFATc2 were tested via dual-luciferase or chromatin immunoprecipitation assays. Functional rescue experiments were designed to confirm the roles of miR-214-3p and NFATc2. HSF1 expression in brain tissues was downregulated upon MPTP treatment. HSF1 overexpression reduced motor deficits and loss of dopaminergic neurons, increased TH-positive neurons, and repressed neuroinflammation and micro-glia activation. Mechanically, HSF1 bound to the miR-214-3p promoter to increase its expression and inhibited NFATc2 transcription. miR-214-3p downregulation or NFATc2 overexpression reversed the inhibition of HSF1 overexpression on neuroinflammation and microglia activation. Overall, our findings unveiled the therapeutic role of HSF1 in PD-induced neuroinflammation and microglia activation via regulating miR-214-3p and NFATc2.

Background and Purpose: MicroRNAs (miRNAs) are highly expressed in the central nervous system and play important roles in ischaemic stroke pathogenesis. However, the role of miRNAs in cerebral ischaemia-reperfusion injury remains unclear. Here, we investigated the role of miR-140-3p in regulating oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury in vitro to identify a new biomarker for research on ischaemic stroke., Methods: The differential expression of miR-140-3p and Tyro3 in OGD/R-exposed N2a cells was verified by qRT-PCR. N2a cells were transfected with miR-140-3p mimic, miR-140-3p inhibitor, Tyro3 or siTyro3, and qRT-PCR, Western blotting, the Cell counting kit-8 (CCK-8) assay, Hoechst 33342/PI staining and flow cytometry analyses were performed to measure miRNA, mRNA and protein expression; cell viability; and apoptosis., Results: OGD/R-exposed N2a cells exhibited increased miR-140-3p expression, decreased viability, reduced Bcl-2 protein expression and increased Bax and Caspase-3 protein expression and apoptosis; the miR-140-3p mimic markedly amplified these changes, exacerbating OGD/R-induced injury to N2a cells, while the miR-140-3p inhibitor reversed these changes and alleviated OGD/R-induced injury. OGD/R-exposed N2a cells expressed less Tyro3, and Tyro3 overexpression increased cell viability and Bcl-2 protein expression, reduced Bax and Caspase-3 protein expression, and alleviated OGD/R-induced injury. However, silencing Tyro3 reversed these changes and exacerbated OGD/R-induced injury. MiR-140-3p directly bound the Tyro3 mRNA 3'UTR. Rescue experiments indicated that the miR-140-3p mimic-induced changes in cell viability and protein expression were alleviated by Tyro3 overexpression and that the miR-140-3p inhibitor-induced changes in cell viability and protein expression were alleviated by silencing Tyro3. Tyro3 overexpression increased cell viability and PI3K and p-Akt protein expression, but these effects were weakened by the addition of LY294002., Conclusions: MiR-140-3p directly targets Tyro3 to regulate cell viability and apoptosis of OGD/R-exposed N2a cells through the PI3K/Akt pathway, suggesting that miR-140-3p is a novel biomarker and therapeutic target for ischaemic stroke., Competing Interests: Competing Interest The authors have no relevant financial or nonfinancial interests to disclose., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cetuximab (CET) resistance in colorectal cancer (CRC) is responsible to poor prognosis to some extent. M2 macrophage polarization is closely correlated with drug resistance to cancers. Therefore, this study aims to investigate whether the mechanism of HCG18 on CET resistance to CRC involving in M2 macrophage polarization., Methods: Clinic samples and SW620 cells with/without M0 macrophage co-culture served as experimental subjects. CET treatment was performed to induce SW620 cell resistant to CET. qRT-PCR and western blot were employed to evaluate the mRNA and protein expression of genes. The capabilities of cell viability, proliferation, migration and invasion were examined using CCK-8, clone formation assay and transwell. ELISA was employed to examine the protein concentrations of IL-10 and TGF-β1. StarBase and luciferase activity assay were conducted to consolidate the interactions among HCG18, miR-365a-3p and FOXO1., Results: In clinical samples and CRC cells, the abundance of HCG18 was enhanced whereas miR-365a-3p was reduced. Besides, HCG18 expression in CET-resistant tumor tissues was higher than that in CET-sensitive tumor tissues and the trend of miR-365a-3p was opposite to that of HCG18. HCG18 knockdown attenuated macrophage-induced CET resistance in SW620 cells and suppressed M2 polarization of THP-1 cells. Mechanistically, HCG18 interacted with miR-365a-3p and miR-365a-3p targeted FOXO1. MiR-365a-3p inhibitor abolished HCG18 knockdown-mediated inhibition of CET resistance, while FOXO1 knockdown compromised the influences of miR-365a-3p inhibitor. FOXO1 could positively regulate CSF-1 expression to promote M2 macrophage polarization and macrophage-induced CET resistance., Conclusion: Our results revealed that HCG18 promoted M2 macrophage polarization to facilitate CET resistance to CRC cells through modulating miR-365a-3p/FOXO1/CSF-1 axis., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Background: Intestinal microbiota plays an important role in maintaining the microecological balance of the gastrointestinal tract in various animals. Disturbances in the intestinal microbiota may lead to the proliferation of potentially pathogenic bacteria that become the dominant species, leading to intestinal immune disorders, intestinal inflammation, and other intestinal diseases. Numerous studies have been confirmed that high-altitude exposure affects the normal function of the intestine and the composition of the intestinal microbiota. However, it is still necessary to reveal the changes in intestinal microbiota in high-altitude exposure environments, and clarify the relationship between the proliferation of potentially pathogenic bacteria and intestinal injury in this environment. In addition, explored probiotics that may have preventive effects against intestinal diseases., Methods and Results: C57BL/6 mice were randomly divided into three groups, a high-altitude group (HA), control group (C), and high-altitude probiotic group (HAP). The HA and HAP groups were subjected to hypoxia modeling for 14 days in a low-pressure oxygen chamber with daily gavage of 0.2 mL of normal saline (HA) and Lactobacillus johnsonii YH1136 bacterial fluid (HAP), while the control group was fed normally. L. johnsonii YH1136 was isolated from feces of a healthy Tibetan girl in Baingoin county, the Nagqu region of the Tibet Autonomous Region, at an altitude of 5000 meters. Our observations revealed that gavage of YH1136 was effective in improving the damage to the intestinal barrier caused by high-altitude exposure to hypoxic environments and helped to reduce the likelihood of pathogenic bacteria infection through the intestinal barrier. It also positively regulates the intestinal microbiota to the extent of Lactobacillus being the dominant microbiome and reducing the number of pathogenic bacteria. By analyzing the expression profile of ileal microRNAs and correlation analysis with intestinal microbiota, we found that Staphylococcus and Corynebacterium1 cooperated with miR-196a-1-3p and miR-3060-3p, respectively, to play a regulatory role in the process of high-altitude hypoxia-induced intestinal injury., Conclusion: These findings revealed the beneficial effect of L. johnsonii YH1136 in preventing potential endogenous pathogenic bacteria-induced intestinal dysfunction in high-altitude environments. The mechanism may be related to the regulation of intestinal injury from the perspective of the gut microbiota as well as miRNAs., Competing Interests: HW was employed by the Guangzhou Beneco Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wan, Zhang, Jia, Qin, Sun, Xin, Zeng, Jing, Fang, Pan, Zeng, Bai, Wang, Ma and Ni.)

Background: CRNDE is known to be an important predictive factor of prognosis in many tumors; however, its role in cisplatin resistance is still unknown in ovarian cancer. The aim of the current research was to investigate the association between CRNDE and cisplatin resistance., Materials and Methods: QRT-PCR and in situ hybridization assay were employed to detect the expression of CRNDE in ovarian cancer cells and tissues; CCK8 assay, AnnexinV-FITC apoptosis assay and Trans-well assay, to determine the cell proliferation, apoptosis and invasion; and RNA-pull down assay, mass spectrometry analysis, gene microarray to search the targeted gene of CRNDE and SRSF1. Association of CRNDE with SRSF1 was determined in ovarian cancer cells and nude mice., Results: It was found that CRNDE and SRSF1 expression were higher in the cisplatin resistant ovarian cancer cells than their control cells. High expression of CRNDE and SRSF1 led to cisplatin resistance. While inhibition of CRNDE or SRSF1 sensitized ovarian cancer to cisplatin in vitro and in vivo. Moreover, as indicated in RIP assay, SRSF1 was potentially the targeted gene of CRNDE, and CRNDE promoting SRSF1 expression to induce cisplatin resistance; as indicated in gene microassay, there was significantly positive correlation between SRSF1 and TIA1, and SRSF1 promoting TIA1 expression., Conclusion: In conclusion, CRNDE induced cisplatin resistance in ovarian cancer through SRSF1/TIA1 signaling pathway; thus, CRNDE inhibitor or SRSF1 inhibitor combined with cisplatin might act as a novel promising approach to ovarian cancer., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

MicroRNAs (miRNAs) are regulatory RNA molecules that bind to target messenger RNAs (mRNAs) and affect the stability or translational efficiency of the bound mRNAs. Single or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian cells. These reporter systems, however, are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase (usually Renilla) used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect cells. In this study, we replaced the SV40 promoter in the psiCHECK-2 reporter vector, which is widely used with mammalian cell lines, with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors, designated psiCHECK-2-TK and psiCHECK-2-AC5.1, respectively. Only psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target (Renilla)/reference (firefly) luciferase activity ratios in mammalian (HeLa and HEK293) and insect (Sf9, S2, Helicoverpa zea fat body and ovary) cell lines, while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell line. Moreover, psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target, the 3'-untranslated region of heat shock protein 90, in both mammalian and H. zea cell lines, but psiCHECK-2 failed to do so in H. zea cell lines. Furthermore, psiCHECK-2-TK with the target sequence, HzMasc (H. zea Masculinizer), accurately differentiated between H. zea cell lines with or without the negative regulation factor (miRNA or piRNA) of HzMasc. These data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells., (© 2021 Institute of Zoology, Chinese Academy of Sciences.)

MicroRNA-1269 (miR-1296) promotes esophageal cancer. However, its role in other cancers, such as glioblastoma (GBM) is unclear. We predicted that miR-1269 might interact with long non-coding RNA (lncRNA) SLC16A1 Antisense RNA 1 (SLC16A1-AS1), a critical player in GBM. We then studied the interaction between SLC16A1-AS1 and miR-1269 in GBM. In this study, paired GBM and non-tumor tissues were used to analyze the expression of SLC16A1-AS1 and premature and mature miR-1269. The interaction of SLC16A1-AS1 with premature miR-1269 was analyzed with RNA pull-down assay and dual-luciferase reporter assay. Cellular fractionation assay was applied to determine the subcellular location of SLC16A1-AS1. Overexpression assays were applied to determine the role of SLC16A1-AS1 in miR-1269 maturation. BrdU, Transwell and cell apoptosis assays were performed to analyze the role of SLC16A1-AS1 and miR-1269 in GBM cell proliferation, migration, and invasion. Interestingly, we observed the upregulation of premature miR-1269 and downregulation of mature miR-1269 in GBM. SLC16A1-AS1 was also overexpressed in GBM. The direct interaction of SLC16A1-AS1 with premature miR-1269 was observed. SLC16A1-AS1 suppressed miR-1269 maturation and promoted cell proliferation, migration, and invasion, and inhibited cell apoptosis, while miR-1269 displayed the opposite trend. SLC16A1-AS1 partly reversed the effects of miR-1269 on GBM cell proliferation, movement and apoptosis. Moreover, SLC16A1-AS1 overexpression increased the level of ki-67, CDK4 and Bcl-2 in LN-229 and LN-18 cells. However, miR-1269 could partly reverse the effect of SLC16A-AS1 on protein levels. Overall, miR-1269 is downregulated in GBM and its maturation is regulated by SLC16A1-AS1.

Surgery for colorectal cancer (CRC) can cause damage to the intestinal mucosal barrier and lead to bacterial invasion. This study mainly analyzed whether propofol (PPF) could protect the intestinal mucosal barrier damage caused by CRC surgery, and explored its molecular mechanism. A mouse CRC model was constructed using azomethane and dextran sulfate sodium. During anesthesia, continuous intravenous injection of PPF was used for intervention. The influences of PPF on intestinal mucosal permeability and bacterial invasion were detected. The levels of microRNA (miR)-155, Toll-like receptor 4 (TLR4)/NF-κB in the intestinal mucosa, and the location of miR-155 were detected by fluorescence in situ hybridization (FISH). Mouse macrophages were used to analyze the regulation of miR-155 on the secretion of inflammatory cytokines through the TLR4/NF-κB pathway. PPF treatment promoted the expression of tight junction protein in the intestinal mucosa, protected the intestinal barrier, inhibited the translocation of intestinal bacteria, and increased the level of the beneficial bacterium Lactobacillus on the mucosal surface. In addition, PPF treatment could inhibit the expression of miR-155, TLR4/NF-KB, and reverse inflammatory response. miR-155 was expressed in macrophages of intestinal mucosa tissue. Overexpression of miR-155 promoted the nuclear translocation of NF-κB and the expression of inflammatory cytokines in macrophages. The use of VIPER to inhibit TLR4 reversed the pro-inflammatory effects of miR-155. PPF might inhibit the activation of the NF-κB pathway by downregulating miR-155 expression, thereby reducing the secretion of inflammatory cytokines. This might be the mechanism by which PPF protected the intestinal barrier of CRC surgical model mice., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Subclinical necrotic enteritis (SNE) is one of the serious threats to the poultry industry. Probiotics have been proven to exert beneficial effects in controlling SNE. However, their exact mechanisms have not been fully elucidated. Moreover, few studies have focused on their impact on microRNAs (miRNAs). Therefore, the present study aimed to explore the miRNA expression profiles in the ileum of broiler chickens during probiotic supplementation for controlling SNE. A total of 180 newly hatched male broilers were randomly allocated into three groups, including a negative control group, an SNE infection group, and a Bacillus licheniformis H2 pretreatment group. Illumina high-throughput sequencing was conducted to identify the miRNA expression of the three groups. Results showed that 628 miRNAs, including 582 known miRNAs and 46 novel miRNAs, were detected in the miRNA libraries. The target genes of 57 significantly differentially expressed miRNAs were predicted and annotated. Moreover, they were found to be partly enriched in pathways related to immunity and inflammation such as tumor necrosis factor receptor binding, immune response-regulating signaling pathway, Toll-like receptor 2 signaling pathway, interleukin-15 production, activation of NF-κB-inducing kinase activity, and MAP kinase tyrosine/serine/threonine phosphatase activity. Some of the target genes of 57 miRNAs were related to the MAPK signaling pathway. Furthermore, the expression of several miRNAs, which may be involved in the MAPK signaling pathway, was significantly affected by SNE induction and showed no significant difference in the presence of H2. All these findings provide comprehensive miRNA expression profiles of three different treatment groups. They further suggest that H2 could exert beneficial effects in controlling SNE through immune and inflammatory response associated with altered miRNA expression, such as the MAPK signaling pathway.

Long-term exposure to arsenic, a widely distributed environmental toxicant, may result in damage to various organs, including the liver. Mice exposed chronically to arsenite developed hepatic damage, inflammation, and fibrosis, as well as increased levels of microRNA-21 (miR-21) and hypoxia-inducible factor (HIF)-1α. The levels of miR-21 and HIF-1α were also enhanced in primary hepatocytes and L-02 cells exposed to arsenite. The culture media from these cells induced the activation of hepatic stellate cells (HSCs), as demonstrated by up-regulation of the protein levels of α-smooth muscle actin (α-SMA) and collagen1A2 (COL1A2) and by increased activity in gel contractility assays. For L-02 cells, knockdown of miR-21 blocked the arsenite-induced up-regulation of HIF-1α and vascular endothelial growth factor (VEGF), which prevented the activation of LX-2 cells induced by medium from arsenite-exposed L-02 cells. However, these effects were reversed by down-regulation of von Hippel Lindau protein (pVHL). In arsenite-treated L-02 cells, miR-21 knockdown elevated the levels of ubiquitination and accelerated the degradation of HIF-1α via pVHL. In the livers of miR-21 -/- mice exposed chronically to arsenite, there were less hepatic damage, lower fibrosis, lower levels of HIF-1α and VEGF, and higher levels of pVHL than for wild-type mice. In summary, we propose that miR-21, acting via the HIF-1α/VEGF signaling pathway, is involved in arsenite-induced hepatic fibrosis through mediating aberrant cross-talk of hepatocytes and HSCs. The findings provide evidence relating to the pathogenesis of hepatic fibrosis induced by exposure to arsenic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Key Message: This study is the first to demonstrate that GA 4 -induced dormancy release is associated with the NF-Y complex, which interacts with gibberellin inhibitor RGL2 in Japanese apricot. Seasonal dormancy is not only vital for the survival in cold winter but also affects flowering of temperate fruit trees and the dormancy release depends on the accumulation of the cold temperatures (Chilling requirement-CR). To understand the mechanism of dormancy release in deciduous fruit crops, we compared miRNA sequencing data during the transition stage from paradormancy to dormancy release in the Japanese apricot and found that the miR169 family showed significant differentially up-regulated expression during dormancy induction and was down-regulated during the dormancy release periods. The 5' RACE assay and RT-qPCR validated its target gene NUCLEAR FACTOR-Y subunit A (NF-YA), which exhibited the opposite expression pattern. Further study showed that exogenous GA 4 could inhibit the expression of the gibberellic acid (GA) signal transduction suppressor PmRGL2 (RGA-LIKE 2) and promote the expression of NF-Y. Moreover, the interaction between the NF-Y family and GA inhibitor PmRGL2 was verified by the yeast-two-hybrid (Y2H) system and a bimolecular fluorescence complementarity (BiFC) experiment. These results suggest that synergistic regulation of the NF-Y and PmRGL2 complex leads to the activation of dormancy release induced by GA 4 . These findings will help to elucidate the functional and regulatory roles of miR169 and NF-Y complex in seasonal bud dormancy induced by GA in Japanese apricot and provide new insights for the discovery of dormancy release mechanisms in woody plants.

Increasing evidence demonstrates that Lin28B plays critical roles in numerous biological processes including cell proliferation and stemness maintenance. However, the molecular mechanisms underlying Lin28B nuclear translocation remain poorly understood. Here, we found for the first time that KRAS promoted Lin28B nuclear translocation through PKCβ, which directly bound to and phosphorylated Lin28B at S243. Firstly, we observed that Lin28B was upregulated in pancreatic cancer, contributing to cellular migration and proliferation. Furthermore, nuclear Lin28B upregulated TET3 messenger RNA and protein levels by blocking the production of mature let-7i. Subsequently, increased TET3 expression could also promote the expression of Lin28B, thereby forming a Lin28B/let-7i/TET3 feedback loop. Our results suggest that the KRAS/Lin28B axis drives the let-7i/TET3 pathway to maintain the stemness of pancreatic cancer cells. These findings illuminate the distinct mechanism of Lin28B nuclear translocation and its important roles in KRAS-driven pancreatic cancer, and have important implications for development of novel therapeutic strategies for this cancer., (© 2020 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The promoter methylation mode of microribonucleic acid (miRNA) plays a crucial role in the process of hepatocellular carcinoma (HCC). Therefore, the primary purpose of this study was to screen and verify the miRNA methylation sites associated with the overall survival (OS) and clinical characteristics of HCC patients. Methylation-related data were from the Cancer Genome Atlas (TCGA). R software was utilized to screen the methylation sites. The least absolute shrinkage and selection operator algorithm was utilized to develop the miRNA promoter methylation models. Then, methylation-specific polymerase chain reaction was performed with 146 HCC tissues to verify the accuracy of the vascular infiltration-related model. Additionally, we verified the functions of vascular infiltration-related miRNA by utilizing cells transfected with miR-199a-3p mimic. The model for predicting OS of HCC patients contained eight methylation sites. The Kaplan-Meier analysis suggested that the model could divide HCC patients into high- and low-risk groups (P < .0001). COX regression analysis suggested that the model (P < .001; 95% CI, 1.264-2.709) and T category (P < .001; 95% CI, 1.472-3.119) were independent risk factors for affecting OS of HCC patients. The model for predicting vascular infiltration, pathological grade, and clinical stage contained 7, 10, and 9 methylation sites respectively, with their area under the receiver operating characteristic curve (AUC) values 0.667, 0.745, and 0.725, respectively. The functional analysis suggested that miRNA methylation is involved in various biological processes such as WNT, MAPK, and mTOR signaling pathways. The accuracy of the vascular infiltration-related model was consistent with our previous bioinformatics assay. And upregulation of miR-199a-3p decreased migration and invasion abilities. The screened miRNA promoter methylation sites can be served as biomarkers for judging OS, vascular infiltration, pathology grade, and clinical stage. It can also provide new targets for improving the treatment and prognosis of HCC patients., (© 2020 Wiley Periodicals, Inc.)

Purpose: Cervical cancer is one of the most fatal diseases among women in under-developed countries. To improve cervical cancer treatment, discovery of new targets is needed. In this study, we investigated the expression of NUP210, miR-22, and Fas in cervical cancer tissues and their functions in cell cycle regulation., Materials and Methods: We detected and compared the expression levels of NUP210, miR-22, and Fas in cervical cancer tissues with paired normal tissues using immunohistochemistry, Western blot, and real-time quantitative polymerase chain reaction. NUP210 was knocked down in HeLa cells via lentivirus, followed by cell cycle and proliferation analysis. Using a luciferase reporter assay, we explored the link between miR-22 and NUP210. We overexpressed miR-22 in HeLa cells and analyzed cell cycle and proliferation function. We then overexpressed miR-22 in NUP210 knockdown cells to explore the connection between Fas and miR-22-NUP210 signaling., Results: We found that NUP210 was overexpressed in cervical cancer patients. Knocking down NUP210 restored cell apoptosis and proliferation. We confirmed miR-22 as a regulator of NUP210 and verified that miR-22 was inhibited in cervical cancer development. We also found that restoring miR-22 expression could induce cell apoptosis. Finally, we found that miR-22-regulated expression of NUP210 could alter Fas expression and, in turn, elicit cell cycle arrest and proliferation., Conclusion: miR-22 in cervical cancer is downregulated, resulting in NUP210 overexpression and inhibition of Fas-induced cell apoptosis., Competing Interests: The authors have no potential conflicts of interest to disclose., (© Copyright: Yonsei University College of Medicine 2020.)

Radiation-induced pulmonary fibrosis (RIPF) is a life-threatening complication of thoracic radiotherapy, which contributes to continued deterioration in pulmonary function. Sphingosine-1 phosphate receptor 3 (S1PR3) has been identified as a crucial molecule in fibrosis. Accumulating evidence indicated that the inhibition of the S1PRs ameliorates fibrogenesis. Thus, this study aims to explore whether S1PR3 participates in RIPF and elucidates the molecular mechanisms underlying S1PR3-modulated epithelial-mesenchymal transition (EMT) in transforming growth factor-β1-induced pulmonary epithelia. A recombinant adeno-associated viral-mediated S1PR3 (AAV-S1PR3) gene therapy analyzed the effect of S1PR3 gene deficiency on the altered histology structure and molecular mechanisms in the lung of mice with whole-lung irradiation. Compared with the AAV-negative control mice, AAV-mediated S1PR3 knockdown in the lung of mice attenuated pulmonary fibrosis induced by the radiation, as indicated by the alleviation of collagen accumulation, lessened histopathological alterations, and the suppression of inflammatory cells infiltration. S1PR3 deficiency reversed the RIPF concomitantly with abrogated EMT-related protein (α-smooth muscle actin). Consistently, S1PR3-deficient pulmonary epithelia inhibited the EMT process changes and fibrosis formation. Furthermore, S1PR3 was designated as one of the target genes for microRNA-495-3p (miR-495-3p). The inhibition of miR-495-3p promoted the expression of S1PR3 in pulmonary epithelia, whereas the overexpression of miR-495-3p inhibited the S1PR3/SMAD2/3 pathway and suppressed the EMT process. Collectively, miR-495-3p might be a negative regulator of the EMT process in fibrosis formation by inhibiting the targeted S1PR3 gene. These results established a link between the S1PR3 gene, the EMT process, and the fibrosis, suggesting the pharmacological blockage of S1PR3 as a potential therapeutic strategy for RIPF., (© 2019 Wiley Periodicals, Inc.)

Objective: The long non-coding RNA zinc finger E-box-binding homeobox 1 antisense 1 (ZEB1-AS1) acts as an oncogenic regulator in many human tumours. In the present study, we identify the role and potential molecular biological mechanisms of ZEB1-AS1 in colon adenocarcinoma (COAD)., Methods: QRT-PCR was used to detect the expression of ZEB1-AS1, miR-455-3p and p21-activated kinases 2 (PAK2) in COAD tissues. CCK8 assay, EdU assay, transwell assay and scratch wound assay were used to explore the biological function of ZEB1-AS1 in COAD cells. Bioinformatics, luciferase reporter assays and an RNA pull-down assay were used to demonstrate the mechanism of ZEB1-AS1. We further explore the role of ZEB1-AS1 in vivo though xenograft tumour assay., Results: We found that ZEB1-AS1 expression was significantly up-regulated in COAD tissues, and high ZEB1-AS1 level was correlated with the poor prognosis of COAD patients. MiR-455-3p plays an anti-cancer role in COAD by targeting PAK2. We confirmed that ZEB1-AS1 promotes PAK2 expression by sponging miR-455-3p, thus facilitating COAD cell growth and metastasis., Conclusions: To sum up, this result illustrates the novel molecular mechanism of ZEB1-AS1 in COAD and provides a new target for the diagnosis and treatment of COAD patients., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Osteoarthritis (OA), a type of joint diseases, could result in breakdown of joint cartilage and underlying bone. Accumulating evidences suggested that long non-coding RNAs play important roles in OA progression. However, the underlying mechanism of H19 in OA is still not fully explored. The expression levels of H19 and miR-106a-5p in OA samples from patients or cultured chondrocytes were examined by quantitative real time polymerase chain reaction. Cell proliferation and apoptosis were analysed by MTT assay and flow cytometry, respectively. Western blotting was employed to detect the expression levels of PCNA, CyclinD1, Caspase 3 and Cleaved Caspase 3. StarBase database, luciferase assay and RNA immunoprecipitation were introduced to confirm the relationship between H19 and miR-106a-5p. The correlation of H19 and miR-106a-5p was analysed by Spearman rank analysis. H19 expression was upregulated, while miR-106a-5p level was downregulated in OA samples and IL-1b-treated chondrocytes. H19 overexpression inhibited the proliferation and induced apoptosis in IL-1b-treated chondrocytes, while H19 knockdown induced the opposite effect. Luciferase and RIP assay demonstrated that miR-106a-5p was a direct target of H19. miR-106a-5p overexpression led to proliferation promotion and apoptosis inhibition in chondrocytes treated by IL-1β and it reversed the effect of H19 addition. We conclude that H19 could regulate proliferation and apoptosis of chondrocytes treated by IL-1β in OA via sponging miR-106a-5p.

Objective: Increasing evidence demonstrated that long non-coding RNA (lncRNA) played a vital role in human tumorigenesis and progression, including colorectal cancer (CRC). However, the underlying mechanisms are still largely unknown. The aim of this study was to investigate the function of lncRNA LINC00324 on the development of CRC and explore the possible mechanisms., Materials and Methods: The expression level of LINC00324 and miR-214-3p was measured by quantitative real time polymerase chain reaction (qRT-PCR) in CRC cells. The effects on cell proliferation, migration and invasion were assessed by MTT and transwell assays, respectively. In addition, the protein levels of cyclin D1, p21, p27 and three MMPs were detected by Western blot analysis. The target of LINC00324 was predicted by online software and confirmed by luciferase reporter assay., Results: We first detected the expression of LIN00324 was increased while miR-214-3p was decreased in CRC cells. Knockdown of LIN00324 suppressed proliferation, migration and invasion in SW620 and HCT15 cells. Moreover, overexpression of miR-214-3p also inhibited CRC cell proliferation, migration and invasion. Then, we identified miR-214-3p as directly target of LINC00324 and the expression of miR-214-3p was downregulated by LINC00324. In addition, inhibiting miR-214-3p reversed the effects of LINC00324 on CRC cell proliferation, migration and invasion., Conclusions: Our results proved that LINC00324 regulated CRC cell proliferation, migration and invasion by sponging miR-214-3p, suggesting that it might be a potential therapeutic target for CRC therapy.

Nuclear receptor interacting protein (NRIP1), also known as RIP140, is a transcriptional co-regulator required for the maintenance of energy homeostasis and ovulation. Although several studies have identified roles for NRIP1 in various cell processes, the biological functions of NRIP1 in esophageal squamous cell carcinoma (ESCC) remain unknown. Herein, we demonstrate that NRIP1 inhibits the migration and invasion of ESCC cells. Further mechanistic studies revealed that NRIP1 is directly targeted by miR-548-3p and miR-576-5p. We then identified that miR-548-3p and miR-576-5p regulate the migration and invasion of ESCC cells by inhibiting NRIP1 expression. Interestingly, the expression of miR-548-3p and miR-576-5p in ESCC cell lines and ESCC tissues is up-regulated and NRIP1 is down-regulated relative to controls. A statistically significant inverse association was found between the expression levels of miR-548-3p/miR-576-5p and NRIP1. These combined results reveal novel functions for miR-548-3p, miR-576-5p, and NRIP1 in regulating ESCC cell migration and invasion which are important functions for the metastatic process in esophageal cancer.

Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer. MicroRNAs have been increasingly implicated in NSCLC and may serve as novel therapeutic targets to combat cancer. Here we investigated the functional implication of miR-188 in NSCLC. We first analyzed miR-188 expression in both NSCLC clinical samples and cancer cell lines. Next we investigated its role in A549 and H2126 cells with cell proliferation, migration, and apoptosis assays. To extend the in vitro study, we employed both xenograft model and LSL- K-ras G12D lung cancer model to examine the role of miR-188 in tumorigenesis. Last we tested MAP3K3 as miR-188 target in NSCLC model. MiR-188 expression was significantly downregulated at the NSCLC tumor sites and lung cancer cells. In vitro transfection of miR-188 reduced cell proliferation and migration potential and promoted cell apoptosis. In xenograft model, miR-188 inhibited tumor growth derived from cancer cells. Intranasal miR-188 administration reduced tumor formation in NSCLC animal model. MAP3K3 was validated as direct target of miR-188. Knocking down MAP3K3 in mice also inhibited tumorigenesis in LSL- K-ras G12D model. Our results demonstrate that miR-188 and its downstream target MAP3K3 could be a potential therapeutic target for NSCLC.

Purpose: lncRNA H19 has been considered as an oncogenic lncRNA in many human tumours. In the present study, we identify the role and molecular mechanism of lncRNA H19 in melanoma., Method: QRT-PCR was used to detect the expression of lncRNA H19 and E2F3 was detected in melanoma tissues. Cell counting kit-8 (CCK8), representative metabolites analysis was used to explore the biological function of lncRNA H19, miR-106a-5p and E2F3 in melanoma cells. Bioinformatics, luciferase reporter assays, MS2-RIP and RNA pull-down assay was used to demonstrate the molecular mechanism of lncRNA H19 in melanoma. We further test the function of lncRNA H19 in vivo though Xenograft tumour assay., Results: We found that lncRNA H19 was increased in melanoma tissue, and lncRNA H19 was correlated with poor prognosis of melanoma patients. miR-106a-5p acts as a tumour suppressor in melanoma by targeting E2F3. E2F3 affects the melanoma cell glucose metabolism and growth. We also demonstrated that lncRNA H19 may function as the sponge of miR-106a-5p to up-regulate E2F3 expression, and consequently promote the glucose metabolism and growth of melanoma., Conclusions: This result elucidates a new mechanism for lncRNA H19 in melanoma development and provides a survival indicator and potential therapeutic target for melanoma patients.

The microRNAs (miRNAs) comprise a broad class of non-coding small endogenous RNAs that are associated with many biological processes through the regulation of target genes, such as leaf morphogenesis and polarity, biotic and abiotic stress responses, and root and flower development. We identified a miRNA that affects flower development, miR319a, in Prunus mume. The Pm-miR319a target, Pm-TCP4, was validated by 5'RACE. The higher expression of Pm-TCP4 in imperfect flowers showed that Pm-TCP4 might promote pistil abortion. Further experiments showed that Pm-miR319a negatively regulates the expression of Pm-TCP4 mRNAs and affected pistil development. Sixteen downstream genes of Pm-TCP4 related to flower development were predicted. Previous studies have shown that they have an impact on the development of pistils. In this study it was established that Pm-miR319a indirectly regulates the development of pistils by regulating its target gene Pm-TCP4.

Background/aims: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and has the third highest mortality rate among all cancers. MicroRNAs are a class of endogenous, single-stranded short noncoding RNAs. The purpose of this study was to study the role of microRNA-873 in HCC., Methods: The expression of miRNA-873 and tumor suppressor in lung cancer 1 (TSLC1) in HCC tissues and cell lines was detected by real-time quantitative RT-PCR (RT-qPCR) or western blot. A CCK-8 assay was used to examine cell proliferation; flow cytometry was used to assess the cell cycle; the Transwell migration assay was used to test for metastasis. Luciferase assays were performed to assess whether TSLC1 was a novel target of miRNA-873., Results: We showed that miRNA-873 was upregulated in HCC tissues and cell lines compared with the normal control. Knockdown of miRNA-873 inhibited the growth and metastasis of HepG2 and accelerated G1 phase arrest, while overexpression of miRNA-873 had the opposite effect. The dual-luciferase reporter assays revealed that TSLC1 was a novel target of miRNA-873. Further study showed that TSLC1 was decreased in HCC tissues and cell lines. There was a negative correlation between the expression levels of TSLC1 and miRNA-873. The effect of miRNA-873 overexpression was neutralized by TSLC1. We also found that miRNA-873 activated the PI3K/AKT/mTOR signaling pathway and promoted HCC., Conclusions: Our data demonstrated that miRNA-873 promoted HCC progression by targeting TSLC1 and provided a new target for the therapy of HCC., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide. The regeneration capacity of the adult mammalian heart is very limited, so that the lost cells are replaced by fibrotic scar. This is followed by remodeling of the surrounding myocardium, which includes cardiac hypertrophy and fibrosis, and makes the ventricular wall thicken and stiffen. This adverse cardiac remodeling leads to impaired cardiac function and eventually leads to heart failure. Extensive studies have revealed that microRNAs (miRNAs) play an essential role in cardiovascular diseases. microRNA-22 (miR-22) is one of the most abundant miRNA in the heart. Many studies have demonstrated that miR-22 plays critical roles in MI and subsequent cardiac remodeling. In this review, we summarized the recent research progresses, including the regulatory effects of miR-22 in oxidative stress, cardiac apoptosis, autophagy, hypertrophy, fibrosis and regeneration.

MDR1 is highly expressed in MDR A2780DX5 ovarian cancer cells, MDR SGC7901R gastric cancer cells and recurrent tumours. It pumps cytoplasmic agents out of cells, leading to decreased drug accumulation in cells and making cancer cells susceptible to multidrug resistance. Here, we identified that miR-495 was predicted to target ABCB1, which encodes protein MDR1. To reduce the drug efflux and reverse MDR in cancer cells, we overexpressed a miR-495 mimic in SGC7901R and A2780DX cells and in transplanted MDR ovarian tumours in vivo. The results indicated that the expression of MDR1 in the above cells or tumours was suppressed and that subsequently the drug accumulation in the MDR cells was decreased, cell death was increased, and tumour growth was inhibited after treatment with taxol-doxorubicin, demonstrating increased drug sensitivity. This study suggests that pre-treatment with miR-495 before chemotherapy could improve the curative effect on MDR1-based MDR cancer., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Ovarian cancer is the leading cause of death among gynecologic diseases in Western countries. We have previously identified a miR-200-E-cadherin axis that plays an important role in ovarian inclusion cyst formation and tumor invasion. The purpose of this study was to determine if the miR-200 pathway is involved in the early stages of ovarian cancer pathogenesis by studying the expression levels of the pathway components in a panel of clinical ovarian tissues, and fallopian tube tissues harboring serous tubal intraepithelial carcinomas (STICs), a suggested precursor lesion for high-grade serous tumors., Methods: RNA prepared from ovarian and fallopian tube epithelial and stromal fibroblasts was subjected to quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) to determine the expression of miR-200 families, target and effector genes and analyzed for clinical association. The effects of exogenous miR-200 on marker expression in normal cells were determined by qRT-PCR and fluorescence imaging after transfection of miR-200 precursors., Results: Ovarian epithelial tumor cells showed concurrent up-regulation of miR-200, down-regulation of the four target genes (ZEB1, ZEB2, TGFβ1 and TGFβ2), and up-regulation of effector genes that were negatively regulated by the target genes. STIC tumor cells showed a similar trend of expression patterns, although the effects did not reach significance because of small sample sizes. Transfection of synthetic miR-200 precursors into normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cells confirmed reduced expression of the target genes and elevated levels of the effector genes CDH1, CRB3 and EpCAM in both normal OSE and FTE cells. However, only FTE cells had a specific induction of CA125 after miR-200 precursor transfection., Conclusions: The activation of the miR-200 pathway may be an early event that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian carcinomas (HGSOC) usually express high levels of CA125, the induction of CA125 expression in FTE cells by miR-200 precursor transfection is consistent with the notion that HGSOC has an origin in the distal fallopian tube.

Non-small cell lung cancer is one of the most common cancers and the leading cause of cancer death worldwide. Genetic variants in regulatory regions of some miRNAs might be involved in non-small cell lung cancer susceptibility and survival. rs12220909 (G/C) genetic polymorphism in miR-4293 has been shown to be associated with decreased risk of esophageal squamous cell carcinoma. However, the influence of rs12220909 genetic variation on non-small cell lung cancer susceptibility has not been reported. In order to evaluate the potential association between miR-4293 rs12220909 and non-small cell lung cancer risk in a Chinese population, we performed a case-control study among 998 non-small cell lung cancer cases and 1471 controls. The data shows that miR-4293 rs12220909 was significantly associated with decreased susceptibility to non-small cell lung cancer (GC vs.GG: OR = 0.681, 95%CI = 0.555-0.835, P = 2.19E-4; GG vs. GC+CC: OR = 0.687, 95%CI = 0.564-0.837, P = 1.95E-4), which indicates that rs12220909 in miR-4293 may play a significant role in the development of non-small cell lung cancer.

Increased production of soluble fms-like tyrosine kinase-1 (sFlt-1) from placenta is one of the major contributors to the development of preeclampsia. Our previous study has shown that hydrogen sulfide (H 2 S) inhibits sFlt-1 release in placenta. In the present study, we sought to investigate whether endogenous H 2 S affects sFlt-1 production and elucidate which H 2 S-producing enzyme is responsible for its effect in placenta. It was found that, besides cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), 3-mercaptopyruvatesulfurtransferase (3-MST) was identified in human placenta and mainly localized in syncytiotrophoblasts. There was no significant difference in expression level of 3-MST among preeclamptic and normal placentas. Treatment of cultured syncytiotrophoblasts with NaHS and l-cysteine suppressed sFlt-1 mRNA expression and caused a decrease in sFlt-1 protein content in culture media of the cells. Transfection of syncytiotrophoblasts with CBS siRNA and CSE siRNA reversed the above effects of l-cysteine. Furthermore, NaHS and l-cysteine treatment decreased the half-life of sFlt-1 mRNA and increased the expression of miR-133b targeting sFlt-1. MiR-133b expression was downregulated in preeclamptic placentas and correlated with the level of CBS and CSE. These results indicate that H 2 S is an important regulatory factor in sFlt-1 production in placenta. Reduced H 2 S generation in placenta contributes to development of preeclampsia by enhancing sFlt-1 production., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

CD4(+)CD25(+)FoxP3(+) thymic-derived regulatory T cells (tTregs) are indispensable for maintaining immune system equilibrium. Adoptive transfer of tTregs is an effective means of suppressing graft-versus-host disease (GVHD) in murine models and in early human clinical trials. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an ubiquitin-conjugating enzyme that mediates nuclear factor κB (NF-κB) activation, plays an essential role in modulating regulatory T cell survival and function. MicroRNAs (miRNAs) are noncoding RNAs, which mediate RNA silencing and posttranscriptional gene repression. By performing comprehensive TaqMan Low Density Array miRNA assays, we identified 10 miRNAs differentially regulated in human tTreg compared with control T cells. One candidate, miR-146b, is preferentially and highly expressed in human naive tTregs compared with naive CD4 T cells. miRNA prediction software revealed that TRAF6 was the one of the top 10 scored mRNAs involved tTreg function with the highest probability as a potential miR-146b target. Antagomir-mediated knockdown of miRNA-146b, but not another miRNA-146 family member (miRNA-146a), enhanced TRAF6 expression. TRAF6, in turn, increases NF-κB activation, which is essential for tTreg function as well as Foxp3 protein and antiapoptotic gene expression, and downregulates proapoptotic gene expression. miR-146b knockdown increased the nuclear localization and expression of genes regulated by NF-κB, which was associated with enhanced tTreg survival, proliferation, and suppressive function measured in vitro and in vivo. TRAF6 inhibition had the opposite effects. We conclude that an miR-146b-TRAF6-NF-κB-FoxP3 signaling pathway restrains regulatory T cell survival, proliferation, and suppressor function. In vitro exposure of human tTregs to miR-146b antagomirs can be exploited to improve the clinical efficacy of human adoptive tTreg transfer in a GVHD setting., (© 2016 by The American Society of Hematology.)

Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, and its expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 in vitro and in vivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mitochondrial oxidative damage is critically involved in cardiac ischemia reperfusion (I/R) injury. MicroRNA-22 (miR-22) has been predicted to potentially target sirtuin-1 (Sirt1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), both of which are known to provide protection against mitochondrial oxidative injury. The present study aims to investigate whether miR-22 is involved in the regulation of cardiac I/R injury by regulation of mitochondrial function. We found that miR-22 level was significantly increased in rat hearts subjected to I/R injury, as compared with the sham group. Intra-myocardial injection of 20 ug miR-22 inhibitor reduced I/R injury as evidenced by significant decreases in cardiac infarct size, serum lactate dehydrogenase (LDH) and creatine kinase (CK) levels and the number of apoptotic cardiomyocytes. H9c2 cardiomyocytes exposed to hypoxia/reoxygenation (H/R) insult exhibited an increase in miR-22 expression, which was blocked by reactive oxygen species (ROS) scavenger and p53 inhibitor. In addition, miR-22 inhibitor attenuated, whereas miR-22 mimic aggravated H/R-induced injury in H9c2 cardiomyocytes. MiR-22 inhibitor per se had no significant effect on cardiac mitochondrial function. Mitochondria from rat receiving miR-22 inhibitor 48h before ischemia were found to have a significantly less mitochondrial superoxide production and greater mitochondrial membrane potential and ATP production as compared with rat receiving miR control. In H9c2 cardiomyocyte, it was found that miR-22 mimic aggravated, whilst miR-22 inhibitor significantly attenuated H/R-induced mitochondrial damage. By using real time PCR, western blot and dual-luciferase reporter gene analyses, we identified Sirt1 and PGC1α as miR-22 targets in cardiomyocytes. It was found that silencing of Sirt1 abolished the protective effect of miR-22 inhibitor against H/R-induced mitochondrial dysfunction and cell injury in cardiomyocytes. Taken together, our findings reveal a novel molecular mechanism for cardiac mitochondrial dysfunction during myocardial I/R injury at the miRNA level and demonstrate the therapeutic potential of miR-22 inhibition for acute myocardial I/R injury by maintaining cardiac mitochondrial function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are increasingly recognized as oncogenes or tumor suppressors in laryngeal squamous cell carcinoma (LSCC). In this study, we analyzed the roles of miR-365a-3p, miR-143-5p, and miR-494-3p in LSCC using Annexin V/propidium iodide double staining and flow cyto-metry, along with a Transwell migration and invasion assay. The results showed that miR-365a-3p inhibitor significantly facilitated cell apoptosis and suppressed cell cycle progression, migration, and invasion in Hep-2 cells. However, miR-143-5p and miR-494-3p had no such influences. We then investigated the role of miR-365a-3p in LSCC in vivo and found that miR-365a-3p inhibitor suppressed LSCC xenograft tumor growth and metastasis in xenograft mouse models. Moreover, miR-365a-3p inhibitor significantly decreased the expression of p-AKT (Ser473), which indicated that miR-365a-3p can mediate PI3K/AKT signaling pathway transduction via p-AKT (Ser473) in LSCC. The data suggest that miR-365a-3p may act as an oncomiR and may promote growth and metastasis in LSCC via the PI3K/AKT signaling pathway, and thus miR‑365a-3p may be a potential therapeutic target for treatment of LSCC.

Unlabelled: Hydrogen sulfide (H2S) has been implicated to angiogenesis in various tissues. We sought to investigate the role of hydrogen sulfide (H2S) in regulating production of vascular endothelial growth factor (VEGF) proteins, the key factors of angiogenesis and vasculogenesis, in placenta., Methods: Placental tissues were obtained from pregnant women with preeclampsia and healthy pregnant women who underwent elective cesarean section. Explants and trophoblasts were isolated from healthy placentas and treated with H2S donor and precursor. Western blotting was used to determine the levels of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). The levels of VEGF mRNA, miR miR-200c,-20a and -20b were determined by quantitative real time PCR., Results: NaHS and l-cysteine increased VEGF but not placenta growth factor (PlGF) production in cultured explants and trophoblasts. Transfection of CBS and CSE siRNA reversed the stimulatory effect of l-cysteine on VEGF production in placental cells. H2S prolonged the half-life of VEGF mRNA and decreased the expression of miR-200c,-20a and -20b in placental cells. MiR-200c mimic and inhibitor affected VEGF mRNA and protein level, whereas miR-20a or -20b mimic and inhibitor affect VEGF protein release but not mRNA expression. The expression level of miR-200c,-20a and -20b as well as the level of CBS, CSE and VEGF were downregulated in preeclamptic placentas., Conclusion: H2S produced via CSE and CBS plays a critical role in VEGF production in human placenta. Reduced expression of CSE and CBS may contribute to the abnormal production of angiogenic factors in preeclamptic placenta., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hydrogen sulfide, generated in the myocardium predominantly via cystathionine-γ-lyase (CSE), is cardioprotective. Our previous study has shown that estrogens enhance CSE expression in myocardium of female rats. The present study aims to explore the mechanisms by which estrogens regulate CSE expression, in particular to clarify the role of estrogen receptor subtypes and the transcriptional factor responsible for the estrogenic effects. We found that either the CSE inhibitor or the CSE small interfering RNA attenuated the protective effect of 17β-estradiol (E2) against H2O2- and hypoxia/reoxygenation-induced injury in primary cultured neonatal cardiomyocytes. E2 stimulates CSE expression via estrogen receptor (ER)-α both in cultured cardiomyocytes in vitro and in the myocardium of female mice in vivo. A specificity protein-1 (Sp-1) consensus site was identified in the rat CSE promoter and was found to mediate the E2-induced CSE expression. E2 increases ERα and Sp-1 and inhibits microRNA (miR)-22 expression in myocardium of ovariectomized rats. In primary cardiomyocytes, E2 stimulates Sp-1 expression through the ERα-mediated down-regulation of miR-22. It was confirmed that both ERα and Sp-1 were targeted by miR-22. In the myocardium of ovariectomized rats, the level of miR-22 inversely correlated to CSE, ERα, Sp-1, and antioxidant biomarkers and positively correlated to oxidative biomarkers. In summary, this study demonstrates that estrogens stimulate Sp-1 through the ERα-mediated down-regulation of miR-22 in cardiomyocytes, leading to the up-regulation of CSE, which in turn results in an increase of antioxidative defense. Interaction of ERα, miR-22, and Sp-1 may play a critical role in the control of oxidative stress status in the myocardium of female rats.

MicroRNAs (miRNAs) have been implicated in DNA repair pathways through transcriptional responses to DNA damaging agents or through predicted miRNA regulation of DNA repair genes. We hypothesized that additional DNA damage regulating miRNAs could be identified by screening a library of 810 miRNA mimetics for the ability to alter cellular sensitivity to ionizing radiation (IR). A prostate cancer Metridia luciferase cell model was applied to examine the effects of individual miRNAs on IR sensitivity. A large percentage of miRNA mimetics were found to increase cellular sensitivity to IR, while a smaller percentage were protective. Two of the most potent IR sensitizing miRNAs, miR-890 and miR-744-3p, significantly delayed IR induced DNA damage repair. Both miRNAs inhibited the expression of multiple components of DNA damage response and DNA repair. miR-890 directly targeted MAD2L2, as well as WEE1 and XPC, where miR-744-3p directly targeted RAD23B. Knock-down of individual miR-890 targets by siRNA was not sufficient to ablate miR-890 radiosensitization, signifying that miR-890 functions by regulating multiple DNA repair genes. Intratumoral delivery of miR-890 mimetics prior to IR therapy significantly enhanced IR therapeutic efficacy. These results reveal novel miRNA regulation of DNA repair and identify miR-890 as a potent IR sensitizing agent., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The previous studies in this lab discovered that microRNA-885-3p (miR-885-3p) was regulated by a sulfated polysaccharide that bound to bone morphogenetic protein receptor, type IA (BMPR1A) to inhibit angiogenesis. However, its specific role and its mechanism of action in tumor cells have not been elucidated. We show that miR-885-3p markedly suppresses angiogenesis in vitro and in vivo. MiR-885-3p inhibits Smad1/5/8 phosphorylation and downregulates DNA-binding protein inhibitor ID-1 (Id1), a proangiogenic factor, by targeting BMPR1A, leading to impaired angiogenesis. Overexpression or silencing of BMPR1A affects angiogenesis in a Smad/Id1-dependent manner. We further show that miR-885-3p impairs the growth of HT-29 colon cancer cell xenografts in nude mice by suppressing angiogenesis through disruption of BMPR1A and Smad/Id1 signaling. These results support a novel role for miR-885-3p in tumor angiogenesis by targeting BMPR1A, which regulates a proangiogenic factor, and provide new evidence that targeting miRNAs might be an effective therapeutic strategy for improving colon cancer treatment.

The protective effect of hydrogen sulfide (H2S) against myocardial ischemia/reperfusion (IR) injury via anti-apoptotic signaling is well established, but the underlying mechanism remains unclear. Recently, miRNAs have been identified as important mediators of myocardial injury by regulating apoptosis-related genes. It was found in our previous preliminary study that microRNA-1 (miR-1) expression underwent a significant change in IR group compared to H2S preconditioned group, indicating that miR-1 possessed myocyte-specific properties. In the present study, we intended to see whether miR-1 participated in H2S protection of cardiomyocytes against IR-induced apoptosis by regulating apoptosis-related genes. Cardiomyocytes of neonatal rats were subjected to hypoxia/reoxygenation (HR) injury with or without H2S preconditioning, while the myocardium of adult SD rats was subjected to IR with or without H2S preconditioning. It was found that HR injury increased apoptosis of cardiac myocytes, up-regulated the expression of miR-1, and down-regulated the expression of Bcl-2. H2S preconditioning attenuated cardiomyocyte apoptosis and LDH release, as well as enhanced cell viability following HR injury. MiR-1 was up-regulated by HR and down-regulated by H2S preconditioning. In contrast, Bcl-2 was down-regulated by HR and up-regulated by H2S preconditioning. In addition, Bcl-2 protein was down-regulated by the miR-1 mimic in a dose-dependent manner. H2S also attenuated IR-induced cardiomyocyte apoptosis in vivo. MiR-1 regulated H2S protection of cardiomyocytes against IR-induced apoptosis by stimulating Bcl-2. These results implicate miR-1 as an important regulator of H2S on the IR myocardium.

Breast cancer (BC) is one of the most common cancers in women, and it can often metastasize to the bone. The mechanism of BC bone metastasis remains unclear and requires in-depth investigation. In a previous study, we found the expression of matrix metalloproteinase 2 (MMP2) to be significantly more pronounced at metastatic bone sites than at orthotopic sites. MicroRNA expression profiling showed miR-106b to be markedly downregulated during BC bone metastasis. However, the specific manner in which MMP2 and miR-106b are involved in the BC bone metastasis is still unclear. In the present study, we found MMP2 expression in orthotopic tumor tissue to be related to the risk of bone metastasis in BC patients. MiR-106b levels in orthotopic tumor tissue showed a negative correlation with MMP2 expression and breast cancer bone metastasis. MMP2 was shown to be a direct target of miR-106b. Both gain- and loss-of-function studies showed that MMP2 could promote the migration and invasion of BC cells and that miR-106b could suppress both. The blockage of MMP2 by RNA interference mimicked the anti-migration and anti-invasion effects of miR-106b, and introduction of MMP2 antagonized the function of miR-106b. MMP2 was also found to regulate the ERK signaling cascade and so adjust the bone microenvironment to favor osteoclastogenesis and bone metastasis. These results suggest that MMP2 upregulation plays an important role in BC bone metastasis through ERK pathways, and miR-106b directly regulates MMP2 expression. The miR-106b/MMP2/ERK pathway may be a promising therapeutic target for inhibiting BC bone metastasis., (© 2013 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)