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MicroRNAs (miRNAs) are small regulatory RNAs that participate in various biological processes by silencing target genes. In Arabidopsis, microRNA163 (miR163) was found to be involved in seed germination, root development, and biotic resistance. However, the regulatory roles of miR163 remain unclear. In the current study, the mir163 mutant was investigated to comprehensively understand and characterize its functions in Arabidopsis. RNA-sequencing and Gene Ontology enrichment analyses revealed that miR163 might be involved in "response to stimulus" and "metabolic process". Interestingly, "response to stress", including heat, cold, and oxidative stress, was enriched under the subcategory of "response to stimulus". We observed that miR163 and PXMT were repressed and induced under heat stress, respectively. Furthermore, the study detected significant differences in seed germination rate, hypocotyl length, and survival rate, indicating a variation in the thermotolerance between WT and mir163 mutant. The results revealed that the mir163 mutant had a lesser degree of germination inhibition by heat treatment than WT. In addition, the mir163 mutant showed a better survival rate and longer hypocotyl length under heat treatment than the WT. The metabolomes of WT and mir163 mutant were further analyzed. The contents of benzene derivatives and flavonoids were affected by miR163, which could enhance plants' defense abilities. In conclusion, miR163/targets regulated the expression of stress-responsive genes and the accumulation of defense-related metabolites to alter stress tolerance., (© 2023 Scandinavian Plant Physiology Society.)

Field-grown rice (Oryza sativa L.), when exposed to various environmental stresses, produces high amounts of reactive oxygen species, such as H 2 O 2 . MicroRNAs (miRNAs) play crucial roles in plant stress responses. This study characterized the functions of H 2 O 2 -regulated miRNAs in rice. Small RNA deep sequencing revealed that miR156 levels decreased following H 2 O 2 treatment. Searches of the rice transcriptome and degradome databases indicated that OsSPL2 and OsTIFY11b are miR156-target genes. Interactions between miR156 and OsSPL2 and OsTIFY11b were confirmed using transient expression assays through agroinfiltration. In addition, the levels of OsSPL2 and OsTIFY11b transcripts were lower in transgenic rice plants overexpressing miR156 than in wild-type plants. The OsSPL2-GFP and OsTIFY11b-GFP proteins were localized to the nucleus. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated interactions between OsSPL2 and OsTIFY11b. Furthermore, OsTIFY11b interacted with OsMYC2 to regulate the expression of OsRBBI3-3, which encodes a proteinase inhibitor. The results suggested that H 2 O 2 accumulation in rice suppresses the expression of miR156, and induces the expression of its target genes, OsSPL2 and OsTIFY11b, whose proteins interact in the nucleus to regulate the expression of OsRBBI3-3, which is involved in plant defense., (© 2023 John Wiley & Sons Ltd.)

Myostatin (MSTN) is a growth and differentiation factor that regulates proliferation and differentiation of myoblasts, which in turn controls skeletal muscle growth. It may regulate myoblast differentiation by influencing miRNA expression, and the present study aimed to clarify its precise mechanism of action. Here, we found that MSTN -/- pigs showed an overgrowth of skeletal muscle and upregulated miR-455-3p level. Intervention of MSTN expression using siMSTN in C2C12 myoblasts also showed that siMSTN significantly increased the expression of miR-455-3p. It was found that miR-455-3p directly targeted the 3'-untranslated region of Smad2 by dual-luciferase assay. qRT-PCR, Western blotting, and immunofluorescence analyses indicated that miR-455-3p overexpression or Smad2 silencing in C2C12 myoblasts significantly promoted myoblast differentiation. Furthermore, siMSTN significantly increased the expression of GATA3 . The levels of miR-455-3p were considerably reduced in C2C12 myoblasts following GATA3 knockdown. Consistently, GATA3 knockdown also reduced the enhanced miR-455-3p expression caused by siMSTN. Finally, we illustrated that GATA3 has a role in myoblast differentiation regulation. Taken together, we identified the expression profiles of miRNAs in MSTN -/- pigs and found that miR-455-3p positively regulates myoblast differentiation. In addition, we revealed that MSTN acts through the GATA3/miR-455-3p/Smad2 cascade to regulate muscle development.

Loss of muscle mass can lead to diseases such as sarcopenia, diabetes, and obesity, which can worsen the quality of life and increase the incidence of disease. Therefore, understanding the mechanism underlying skeletal muscle differentiation is vital to prevent muscle diseases. We previously found that microRNA-320 (miR-320) is highly expressed in the lean muscle-type pigs, but its regulatory role in myogenesis remains unclear. The bioinformatics prediction indicated that miR-320 could bind to the 3 'untranslated region of growth factor receptor-bound protein-2 (Grb2). We hypothesized that miR-320 targets Grb2 to regulate myoblasts differentiation. To verify this, we transfected miR-320 mimic and inhibitor into C2C12 myoblasts to assess the role of miR-320 during myoblasts differentiation. We used real-time qPCR, luciferase reporter assays, and western blotting to confirm that miR-320 directly targets Grb2 to promote myoblasts differentiation. Moreover, by using a dexamethasone-induced atrophic model of myotubes, we discovered that miR-320 promotes the repair of damaged myotubes. Our findings expand understanding of miRNAs and genes related to regulating skeletal muscle differentiation, and provide insight into underlying therapeutic strategies for muscle diseases., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Originally characterized as an oncoprotein overexpressed in many forms of cancer that participates in numerous cellular pathways, DEK has since been well described regarding the regulation of epigenetic markers and transcription factors in neurons. However, its role in neuropathic allodynia processes remain elusive and intriguingly complex. Here, we show that DEK, which is induced in spinal dorsal horn neurons after spinal nerve ligation (SNL), is regulated by miR-489-3p. Moreover, SNL-induced decrease in miR-489-3p expression increased the expression of DEK, which recruited TET1 to the promoter fragments of the Bdnf, Grm5, and Stat3 genes, thereby enhancing their transcription in the dorsal horn. Remarkably, these effects were also induced by intrathecally administering naïve animals with miR-489-3p inhibitor, which could be inhibited by knockdown of TET1 siRNA or DEK siRNA. Conversely, delivery of intrathecal miR-489-3p-mimic into SNL rats attenuated allodynia behavior and reversed protein expression coupled to the promoter segments in the dorsal horn. Thus, a spinal miR-489-3p/DEK/TET1 transcriptional axis may contribute to neuropathic allodynia. These results may provide a new target for treating neuropathic allodynia., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background/aim: Sarcomatoid renal cell carcinoma (sRCC) is an aggressive subtype of RCC. In the current study, we investigated whether the POLR2A and RUNX2 genes are involved in RCC-related signaling pathway and associated with miR-378., Materials and Methods: Thirteen formalin-fixed and paraffin-embedded RCC samples were collected. Three software programs were used to predict the potential gene regulation in RCC by miR-378 and immunohistochemistry analysis was applied to confirm the expression of targeted proteins in FFPE samples. MicroRNA-378 transfection, analysis of mRNA and protein expression via Western Blotting and cell apoptosis analysis via flow cytometry for POLR2A and RUNX2 were further studied in four renal cell carcinoma cell lines., Results: Both the mRNA and protein expression levels of POLR2A and RUNX2 in sRCC cell lines and were significantly higher than those in other subtypes of RCC, and similar results were obtained in clinical samples (p<0.01). Second, overexpression of miR-378 significantly suppressed the expression of POLR2A and RUNX2 in sRCC cells (p<0.01) and enhanced apoptosis (p<0.05)., Conclusion: miR-378 significantly inhibits the expression of POLR2A and RUNX2 in sRCC and further promotes apoptosis of sRCC cells. We speculated that the apoptosis mechanism in sRCC occurs via regulation of the ERK2 and PI3K/AKT pathways, which might distinguish it from other subtypes of RCC., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide. Emerging evidence has revealed the vital functions of microRNAs (miRNAs) in cancer malignant progressions. miR-375 has been verified to serve as an antioncogene in tumorigenesis and a potential therapeutic target in various types of cancer. In this study, we aimed to determine the role of miR-375 in the regulation of chemoresistance and metastasis of HCC. Differentially expressed miR-375 and NCAPG2 were externally validated using expression data from The Cancer Genome Atlas (TCGA) database. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression levels of miR-375 in HCC tissues and cell lines. miR-375 mimics and NCAPG2-overexpression were transfected into HepG2 and Huh7 cells to establish miR-375 overexpression models. Cell Counting Kit-8, Transwell, and flow cytometry experiments were conducted to monitor cell proliferation, migration, and apoptosis. The targeting relationship between miR-375 and non-SMC condensin II complex subunit G 2 (NCAPG2) was determined by qRT-PCR, western blot, and luciferase reporter gene assay. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using Gene Set Enrichment Analysis (GSEA). The pathway enrichment analysis was used to predict the potential pathways for further study. miR-375 was significantly downregulated in HCC tissues and cells compared to adjacent tissue and normal hepatocyte cell line respectively while NCAPG2 was upregulated. The targeting relationship was verified by luciferase reporting assay, and miR-375 could target the 3'UTR of NCAPG2 mRNA and effectively suppress NCAPG2 protein expression. Replenishing of miR-375 significantly repressed HCC cell proliferation and migration, and induced cell apoptosis. Overexpression of NCAPG2 recovered those biological abilities in miR-375 overexpressed cells. Collective data suggested that miR-375 served as a tumor suppressor via regulating NCAPG2. Replenishing of miR-375 or knockout of NCAPG2 could be therapeutically exploited for HCC.

The screening and diagnosis of cancer are hallmarks of medicine in the aging population. Recently, microRNAs have shown potential for use as biomarkers, which could advance the field of diagnostics. The presence of miRNA-141 in the serum has been well described in several malignancies. However, the invasive approach used for sampling represents the major limitation for its practical application and, hence, its notable absence as a method for screening the general population. In light of this, we aimed to develop a high-sensitivity microRNA (miR) biosensor for application in the diagnosis of all miR-141-associated cancers, such as colorectal cancer (CRC) and breast cancer (BC). The novelty lies in our dual-probe design, which is reliant on the hybridization of the fluorescein isothiocyanate (FITC) targeting probe onto an existing sample of urinary miR-141 in the first step, followed by complementary binding with a biotinylated probe that has been coated on a modified screen-printed carbon electrode (SPCE). The hybridization of the probe and sensor produces signals via the catalytic reduction of H 2 O 2 at HRP-modified SPCEs in the presence of H 2 O, which was measured by either cyclic voltammetry or chronoamperometry (CA) currents. In our study, the detection and expression of miR-141 in a cohort of colorectal cancer ( n = 6) and breast cancer ( n = 4) samples showed that its levels were significantly higher than in a healthy cohort ( n = 9) ( p < 0.004). Moreover, our miR sensor demonstrated high stability, reliability, and sensitivity ( p < 0.0001). This work hopefully provides new information for the detection and monitoring of de novo and existing cancers.

Background: Long noncoding RNAs (lncRNA) are important in the growth and metastasis of colon cancer. The objective of this study was to describe the potential role of lncRNA NEAT1 in the progression of colon cancer., Methods: Quantitative real-time polymerase chain reaction was used for detecting NEAT1, miR-185-5p, and IGF2 in colon cancer cells and tissues. The potential diagnostic value of NEAT1 in colon cancer was analyzed with the receiver operating characteristic curve. Kaplan-Meier method was applied for evaluating the association between NEAT1 expression and the overall survival of osteosarcoma patients, whereas Transwell assay was introduced to examine the potential invasion and migration of colon cancer cells. In addition, the binding of NEAT1/IGF2 to miR-185-5p was confirmed by RNA pull-down and RNA-binding protein immunoprecipitation assays and dual-luciferase reporter gene assay. Finally, rescue experiments were conducted to confirm the role of NEAT1/miR-185-5p/IGF2 axis in colon cancer., Results: Colon cancer patients with low NEAT1 expression presented with longer overall survival than those with high expression. The migration and invasion of colon cancer cells were considerably promoted by overexpressed NEAT1. Both NEAT1 and IGF2 bound to miR-185-5p., Conclusion: NEAT1 upregulate IGF2 expression through absorbing miR-185-5p to enhances the migration and invasion of colon cancer cells., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Plant microRNAs (miRNAs) are non-coding RNAs, which are composed of 20-24 nucleotides. MiRNAs play important roles in plant growth and responses to biotic and abiotic stresses. Wounding is one of the most serious stresses for plants; however, the regulation of miRNAs in plants upon wounding is not well studied. In this study, miR2111, a wound-repressed miRNA, identified previously in sweet potato (Ipomoea batatas cv Tainung 57) by small RNA deep sequencing was chosen for further analysis. Based on sweet potato transcriptome database, F-box/kelch repeat protein (IbFBK), a target gene of miR2111, was identified. IbFBK is a wound-inducible gene, and the miR2111-induced cleavage site in IbFBK mRNA is between the 10th and 11th nucleotides of miR2111. IbFBK is a component of the E3 ligase SCF (SKP1-Cullin-F-box) complex participating in protein ubiquitination and degradation. The results of yeast two-hybrid and bimolecular fluorescence complementation assays demonstrate that IbFBK was conjugated with IbSKP1 through the F-box domain in IbFBK N-terminus to form SCF complex, and interacted with IbCNR8 through the kelch-repeat domain in IbFBK C-terminus. The interaction of IbFBK and IbCNR8 may lead to the ubiquitination and degradation of IbCNR8. In conclusion, the suppression of miR2111 resulted in the increase of IbFBK, and may regulate protein degradation of IbCNR8 in sweet potato responding to wounding., 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 © 2019. Published by Elsevier B.V.)

In recent years, a growing body of evidence has provided support for the important role of microRNAs (miRNAs) in the progression of human cancers. A recent study showed that a novel miRNA miR-3650 expression was significantly decreased in hepatocellular carcinoma (HCC). However, the precise role of miR-3650 in HCC have remained poorly understood. In this study, we found that miR-3650 expression was frequently decreased in HCC tissues. Low expression of miR-3650 is positively associated with tumor metastasis and poor survival of HCC patients. Forced expression of miR-3650 significantly inhibited the migration and epithelial-mesenchymal transition (EMT) of HCC cells. Through bioinformatic analysis and luciferase assays, we confirmed that neurofascin (NFASC) is a directly target mRNA of miR-3650. Rescue experiment demonstrated that NAFSC overexpression could partially counteracted the inhibitory effect of miR-3650 in HCC metastasis and EMT. In conclusion, our findings are the first time to demonstrate that reduced expression of miR-3650 in HCC was correlated with tumor metastasis and poor survival. MiR-3650 repressed HCC migration and EMT by directly targeting NFASC. Our findings suggested that miR-3650 may serve as a potential prognostic marker and promising application in HCC therapy.

The regulatory role of long noncoding RNA (lncRNA) growth arrest-specific transcript 5 ( GAS5 ) in both cancerous and noncancerous cells have been widely reported. This study aimed to evaluate the role of lncRNA GAS5 in heart failure caused by myocardial infarction. We reported that silence of lncRNA GAS5 attenuated hypoxia-triggered cell death, as cell viability was increased and apoptosis rate was decreased. This phenomenon was coupled with the down-regulated expression of p53, Bax and cleaved caspase-3, as well as the up-regulated expression of CyclinD1, CDK4 and Bcl-2. At the meantime, the expression of four heart failure-related miRNAs was altered when lncRNA GAS5 was silenced ( miR-21 and miR - 142 - 5p were up-regulated; miR-30b and miR-93 were down-regulated). RNA immunoprecipitation assay results showed that lncRNA GAS5 worked as a molecular sponge for miR - 142 - 5p . More interestingly, the protective actions of lncRNA GAS5 silence on hypoxia-stimulated cells were attenuated by miR - 142 - 5p suppression. Besides, TP53INP1 was a target gene for miR - 142 - 5p . Silence of lncRNA GAS5 promoted the activation of PI3K/AKT and MEK/ERK signaling pathways in a miR - 142 - 5p -dependent manner. Collectively, this study demonstrated that silence of lncRNA GAS5 protected H9c2 cells against hypoxia-induced injury possibly via sponging miR - 142 - 5p , functionally releasing TP53INP1 mRNA transcripts that are normally targeted by miR - 142 - 5p .

MiRNAs play diverse roles in plant development and defense responses by binding to their mRNA targets based on sequence complementarity. Here, we investigated a wound-related miR408 and its target genes in sweet potato (Ipomoea batatas) by small RNA deep sequencing and transcriptome analysis. The expression patterns of miR408 and the miR408 precursor were significantly repressed by wounding and jasmonate (JA). In contrast, expression of the putative target genes IbKCS (3-ketoacyl-CoA synthase 4), IbPCL (plantacyanin), and IbGAUT (galacturonosyltransferase 7-like) of miR408 was increased following wounding, whereas only IbKCS was increased after JA treatment. Target cleavage site mapping and Agrobacterium-mediated transient assay demonstrated that IbKCS, IbPCL, and IbGAUT were the targets of miR408. The expression of miR408 target genes was repressed in transgenic sweet potatoes overexpressing miR408. These data indicated a relationship between miR408 and its target genes. Notably, miR408-overexpressing plants showed a semi-dwarf phenotype and attenuated resistance to insect feeding, while transgenic plants overexpressing IbKCS exhibited more insect resistance than plants overexpressing only the empty vector. Collectively, sweet potato reduces the abundance of miR408 upon wounding to elevate the expression of IbKCS, IbPCL, and IbGAUT. The expression of IbKCS enhances the defense system against herbivore wounding.

Accumulating evidence has indicated that aberrantly expressed microRNAs (miRs) are extensively involved in cancer development and progression. MiR-639 has been reported to act as tumor promoter in various types of cancer. However, the biological function and underlying molecular mechanism of miR-639 in thyroid carcinoma (TC) have not been intensively investigated. Herein the present study aimed to investigate the functional role of miR-639 in TC. We found that miR-639 expression was upregulated in TC cells and clinical tissues. Overexpression of miR-639 promoted TC cell proliferation and cell cycle, with increased expression of CyclinE and c-myc, whereas miR-639-in reverses the function. Using prediction software and luciferase reporter assay, we found that CDKN1A was a target of miR-639. CDKN1A small interfering RNA (siRNA) abrogated the role of miR-639-in on cell proliferation of TC. In summary, our data demonstrated that miR-639 upregulation was associated with development of TC, miR-639 promoted cell proliferation and cell cycle by targeting CDKN1A in TC., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

MicroRNAs (miRNAs) have been shown to function as either oncogenes or tumor suppressors by negatively regulating target genes involved in tumor initiation and progression. In this study, we demonstrated that down-regulation of miR-33a-3p in human primary hepatocellular cancer (HCC) specimens was significantly associated with metastases and poor survival. Over-expression of miR-33a-3p in HepG2 cells remarkably suppressed not only cell growth, migration and invasion, but also tumor growth and metastases in the chick embryo chorioallantoic membrane (CAM) assay, and down-regulated Pre-B-Cell Leukemia Homeobox 3 (PBX3) expression. Conversely, inhibition of miR-33a-3p in Bel-7402 cells resulted in increased of cell growth, spreading and invasion. Furthermore, rescue experiments by over-expression PBX3 completely eliminated the inhibitory effects of miR-33a-3p on tumor growth and metastasis, both in vitro and in vivo. The luciferase assay showed that 3'-untranslated regions (3'-UTRs) of PBX3 were inhibited significantly by miR-33a-3p, while mutations in the miR-33a-3p pairing residues rescued the luciferase expression. Taken together, our findings suggest that miR-33a-3p suppressed the malignant phenotype while also inhibiting PBX3 expression in hepatocellular cancer, implying that miR-33a-3p may be a promising biomarkers and therapy target for HCC intervention., Competing Interests: CONFLICTS OF INTERESTS The authors declare no potential conflicts of interest in the study.

The aim of the present study was to assess the effects of sprouty homolog 2 (SPRY2) gene regulation by miR-21 on the occurrence, development and tumor metastasis in multiple myeloma (MM). The miR-21 expression lentiviral vector (LV)-anti-miR-21 and a liposome transfection method were used to screen MM cell lines with stable silent SPRY2. Real-time quantitative polymerase chain reaction (PCR) and western blot analyses were used to detect SPRY2 expression and miR-21 protein expression levels. An MTT assay was used to assess cell proliferation. Flow cytometry was used for analysis of cell cycle. A scratch test/wound healing assay was used to detect the cell migration ability. A Transwell assay was used to detect the cell invasion ability. Real-time quantitative PCR and western blot analysis showed that in the MM cell lines with high endogenous miR-21 expression (RPMI8226 and KM3), SPRY2 expression was significantly lower. Conversely, in the U266 cell line with low endogenous miR-21 expression, SPRY2 expression was significantly higher, and the gray values of miR-21 and SPRY2 protein in the respective cell lines showed statistically significant differences (P<0.01). Following transfection of U266 cells, the expression of miR-21 in the U266/LV-anti-miR21 lentiviral multiplicity of infection (MOI) 20 group and -MOI 40 group decreased significantly compared with that in the untransfected U266 group (P<0.05). SPRY2 protein expression in U266 cells transfected with miR-21 mimics was significantly reduced compared with that in the non-transfected (untreated) group and the negative control-transfected group (P<0.01). An MTT assay showed that compared with the non-transfected and negative control groups, the cell growth rate as well as the proliferation rate were significantly decreased in the transfection group 48, 72 and 96 h after transfection (P<0.01). Flow cytometric analysis showed that 48 and 72 h after transfection of U266 cells with miR-21 mimics, the apoptotic rates were (24.7 ± 1.97 and 38.6 ± 1.56%) in the U266 group, (27.3 ± 1.72 and 37.3 ± 1.59%) in the siRNA group and (12.7 ± 1.27 and 22.1 ± 1.63%) in the U266/miR-21 group. Compared with the two control groups, the apoptotic rate in the U266/miR-21 group was significantly decreased and the G0/G1 phase cell population was significantly reduced (P<0.05). Scratch experiments showed that the cell migration ability was significantly reduced in the transfection group 24 and 48 h after transfection (P<0.05). A Transwell invasion assay confirmed that the number of U266 cells which migrated through a Matrigel-covered polyphosphate membrane significantly decreased in the transfection group 24 and 48 h after transfection. The cell-penetrating ability was also significantly decreased (P<0.05). In conclusion, the downregulation of SPRY2 gene expression mediated by miR-21 promotes the proliferation and invasion of MM cells in vitro, suggesting that miR-21 may be a novel potential molecular therapeutic target in the treatment of MM.

The aim of the present study was to investigate the expression level of microRNA 21 (miR‑21) in the peripheral blood of patients with multiple myeloma (MM) and to investigate the correlation between miR‑21 and sprouty homolog 2 (SPRY2) gene expression levels in MM. A total of 30 patients with MM, 15 with monoclonal gammopathy of undetermined significance (MGUS) and 20 normal control (NC) outpatients were selected for the detection of miR‑21 and SPRY2 expression using reverse transcription-quantitative polymerase chain reaction. In addition, western blot analysis was performed to detect the expression of miR‑21 and SPRY2 in MM cell lines. The expression of miR‑21 in U‑266 cells following lipofectamine transfection of fluorescence‑labeled miR‑21 mimic/inhibitor was observed using a fluorescence microscope and the expression level of SPRY2 in the miR‑21 mimic/inhibitor‑transfected U‑266 cells was detected using western blot analysis. The miR‑21 expression level in the circulating serum of the MM patient group was significantly higher (P<0.01) than that of the MGUS and NC groups. The MM cell lines with high endogenous miR‑21 expression exhibited an expression level of SPRY2 that was significantly lower than that in the MM cells with low endogenous miR‑21 expression. The transfection efficiency of fluorescence‑labeled miR‑21 mimic/inhibitor was >90%. Compared with the miR‑21 expression level in untreated U‑266 cells (0.82±0.13), the expression level of miR‑21 was increased by 120.2‑fold in miR‑21 mimic‑transfected cells (98.6±14.2; P<0.001) and was decreased by 61.9% in the miR‑21 inhibitor‑transfected cells (0.37±0.06; P<0.05). The grayscale value of protein bands demonstrated that SPRY2 protein expression significantly decreased in miR‑21 mimic‑transfected U‑266 cells compared with that in the inhibitor‑transfected, siRNA‑transfected and untreated cells (P<0.01). miR‑21 may represent a negative regulator involved in the downregulation of SPRY2 in MM. miR‑21 is closely associated with the pathogenesis, progression and prognosis of MM and may thus be used as an indicator of poor MM prognosis.

Decreased GLUT4 expression and impaired GLUT4 cell membrane translocation are involved in type 2 diabetes mellitus (T2DM) pathogenesis so the factors impacting GLUT4 expression may be associated with T2DM. In this study, we identified four miRNAs: miR-31, miR-93, miR-146a, and miR-199a which suppress GLUT4 expression in HEK293T cells. Subsequently, we determined expression of these four miRNAs in plasma samples of T2DM patients, T2DM susceptible individuals, and healthy controls and found miR-199a was overexpressed in patients' plasma compared with healthy control. Because the miR-199a binding site in GLUT4 3'UTR is highly conserved among vertebrates, we detected the glucose uptake in rat L6 myoblast cells through gain- and loss-of-function of miR-199a. We found that miR-199a can repress glucose uptake in L6 cells, which was rescued by GLUT4 overexpression. These results indicate that T2DM patients may have a high level miR-199a that reduce GLUT4 expression and contribute to the insulin resistance. Hence, miR-199a may be a novel biomarker for risk estimation and classification in T2DM patients.

Objective: To investigate the regulative effect of microRNA-338-3p on colorectal carcinoma cell invasion and migration., Methods: The microRNA-338-3p expression pattern of colorectal carcinoma tissues and cell lines was detected by real-time reverse transcriptase polymerase chain reaction. The protein level of smoothened was detected by western blot analysis. Furthermore, colorectal carcinoma cells were pretreated with or without anti-smoothened-small interfering ribonucleic acid prior to the addition of pre-microRNA-338-3p or anti-microRNA-338-3p. The status of colorectal carcinoma cell invasion and that of migration were detected by transwell assay and wound healing assay, respectively., Results: The expression of microRNA-338-3p was significantly down-regulated in colorectal carcinoma tissues in comparison with those in the adjacent non-tumorous tissues, and the value was negatively related to advanced tumor, node, metastasis stage and local invasion. The expression of microRNA-338-3p in colorectal carcinoma cells transfected with pre-microRNA-338-3p p was significantly increased. Furthermore, over-expression of microRNA-338-3p inhibited the expression of smoothened protein in colorectal carcinoma cells, which showed obviously suppressed invasion and migration ability. The expression of microRNA-338-3p in colorectal carcinoma cells transfected with anti-microRNA-338-3p was significantly decreased. Moreover, the down-regulated expression of microRNA-338-3p caused the up-regulated expression of smoothened protein in colorectal carcinoma cells, which showed significantly enhanced invasion and migration ability. However, anti-smoothened-small interfering ribonucleic acid largely, but not completely, reversed the effects induced by blockage of microRNA-338-3p, suggesting that the regulative effect of microRNA-338-3p on colorectal carcinoma cell invasion and migration was indeed mediated by smoothened. Additionally, smoothened was identified as a direct target of microRNA-338-3p by luciferase assay., Conclusions: MicroRNA-338-3p could inhibit colorectal carcinoma cell invasion and migration by inhibiting smoothened expression.

Aim: To investigate the regulative effect of miRNA (miR)-221 on colorectal carcinoma (CRC) cell radiosensitivity and the underlying mechanisms., Methods: A human CRC-derived cell line was cultured conventionally and exposed to different doses of X-rays (0, 2, 4, 6 and 8 Gy). The total RNA and protein of the cells were extracted 24 h after irradiation, and the alteration of miR-221 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene mRNA expression was detected by real-time reverse transcriptase polymerase chain reaction (PCR). The protein alteration of PTEN in the cells was detected by Western blotting. Caco2 cells were pretreated with or without anti-PTEN-siRNA prior to the addition of pre-miR-221 or anti-miR-221 using Lipofectamine 2000. Colony formation assay and flow cytometry analysis were used to measure the surviving cell fraction and the sensitizing enhancement ratio after irradiation. Additionally, PTEN 3'-untranslated region fragment was PCR amplified and inserted into a luciferase reporter plasmid. The luciferase reporter plasmid construct was then transfected into CRC cells together with pre-miR-221 or anti-miR-221, and the luciferase activity in the transfected cells was detected., Results: The X-ray radiation dose had a significant effect on the expression of miR-221 and PTEN protein in human Caco2 cells in a dose-dependent manner. The miR-221 expression level improved gradually with the increase in irradiation dose, while the PTEN protein expression level reduced gradually. miR-221 expression was significantly reduced in the anti-miR-221 group compared with the pre-miR-221 and negative control groups (P < 0.01). Anti-miR-221 upregulated expression of PTEN protein and enhanced the radiosensitivity of Caco2 cells (P < 0.01). Moreover, the inhibitory effect was dramatically abolished by pretreatment with anti-PTEN-siRNA, suggesting that the enhancement of radiosensitivity was indeed mediated by PTEN. A significant increase of luciferase activity was detected in CRC cells that were cotransfected with the luciferase reporter plasmid construct and anti-miR-221 (P < 0.01)., Conclusion: Anti-miR-221 can enhance the radiosensitivity of CRC cells by upregulating PTEN.

Aim: To construct a lentivirus-based inhibitor with specific secondary structure that could exert long-term suppression on microRNA-338-3p (miR-338-3p), thus elucidating its molecular function in colorectal carcinoma cells., Methods: The miR-338-3p inhibitor sequence was synthesized and inserted into pLV-THM plasmid. HEK-293T cells were co-transfected with the lentiviral vectors pLV-THM-miR-338-3p-inhibitor, psPAX2, and pMD2.G. The supernatant containing the lentivirus particles was harvested to determine the viral titer, and then used to infect colorectal carcinoma-derived SW-620 cells. eGFP(+) cells were sorted using flow cytometry. The expression of miR-338-3p in SW-620 cells was determined with real-time RT-PCR, and the expression of the smoothened (SMO) protein was detected using Western blot analysis. The migration ability of the transfected SW-620 cells was assessed with transwell assay., Results: Restriction endonuclease analysis and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p-inhibitor was successfully constructed. The expression of miR-338-3p in SW-620 cells was significantly decreased by infection with the lentivirus pLV-THM-miR-338-3p-inhibitor. Moreover, the down-regulated expression of miR-338-3p caused up-regulated expression of the SMO protein in SW-620 cells, which showed significantly enhanced migration in transwell assay., Conclusion: The construction of the lentiviral vector pLV-THM-miR-338-3p-inhibitor with specific secondary structure provides a basis for further studies the molecular function of miR-338-3p in colorectal carcinoma. miR-338-3p may suppress SMO gene expression and thereby inhibit colorectal carcinoma migration.

Aim: To investigate the regulative effect of miRNA-338-3p (miR-338-3p) on cell growth in colorectal carcinoma (CRC)., Methods: The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed. The recombinant viral vector encoding the pre-miR-338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus. The supernatant containing the lentivirus particles was harvested to determine the viral titer, and this supernatant was then used to transduce CRC-derived cell line, SW-620. Flow cytometry was utilized for sorting the green fluorescent protein (GFP)⁺ cells to establish the SW-620 cell line stably expressing pre-miR-338-3p or miR-338-3p-inhibitor. Moreover, the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction, and Western blotting was used to detect the expression of the smoothened (SMO, the possible target of miR-338-3p) protein in SW-620 cells. Furthermore, the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry, respectively., Results: Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully. GFP was expressed after the SW-620 cells were transduced by the lentivirus. Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased (relative expression 3.91 ± 0.51 vs 2.36 ± 0.44, P < 0.01). Furthermore, overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells, which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate (CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%, P < 0.01]. Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased (relative expression 0.92 ± 0.29 vs 2.36 ± 0.44, P < 0.01). Moreover, the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells, which showed significantly enhanced proliferation ability (CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%, P < 0.01). However, anti-SMO-siRNA largely, but not completely, reversed the effects induced by blockage of miR-338-3p, suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO., Conclusion: miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.

MicroRNAs (miRNAs) are small noncoding RNAs which post-transcriptionally regulate gene expression by directing mRNA cleavage or translational inhibition. miRNAs play multiple roles in the growth, development and stress responses in plants. However, little is known of the wounding-responsive miRNAs and their regulation. Here, we investigated the expression patterns of microR828 (miR828) on wounding in sweet potato (Ipomoea batatas cv Tainung 57). The expression of miR828 was only detected in leaves, and was induced by wounding rather than by ethylene, hydrogen peroxide (H2O2), methyl jasmonate or nitric oxide (NO). Moreover, cyclic guanosine monophosphate (cGMP) was necessary for miR828 accumulation in leaves on wounding. Two miR828 target candidates, named IbMYB and IbTLD, were obtained by cDNA cloning, and their mRNA cleavage caused by miR828 was confirmed by cleavage site mapping, agro-infiltration and transgenics studies. The reduction in IbMYB and IbTLD expression coincided with the induction of miR828, demonstrating that IbMYB and IbTLD might be miR828 targets. Furthermore, transgenic sweet potato overexpressing miR828 precursor affected lignin and H2O2 contents. These results showed that cGMP could regulate wounding-responsive miR828, which repressed the expression of IbMYB and IbTLD. Subsequently, lignin and H2O2 were accumulated to participate in defense mechanisms., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Objective: To investigate the regulatory effect of microRNA-221 (MIR221) on CDKN1C/p57 expression in colon carcinoma cells in vitro., Methods: Caco2 cells were treated with or without anti-p57-siRNA prior to the addition of pre-MIR221 or anti-MIR221. The MIR221 expression pattern was detected by real-time RT-PCR, and the mRNA and protein levels of CDKN1C/p57 expression were detected using semi-quantitative RT-PCR and Western blotting. Caco2 cell proliferation following the treatment was detected with MTT assay. CDKN1C/p57 3'-UTR fragment was amplified by PCR from the genome DNA of human colon and inserted into a luciferase reporter plasmid. The luciferase reporter plasmid construct was then transfected into Caco2 cells along with pre-MIR221 or anti-MIR221, and the luciferase activity in the transfected cells was detected., Results: MIR221-specific inhibitor significantly up-regulated CDKN1C/p57 protein expression in Caco2 cells (P<0.01). Anti-MIR221 could markedly inhibit Caco2 cell proliferation, and the inhibitory effect was obviously abolished by pretreatment with anti-p57-siRNA, suggesting that the inhibition was mediated by CDKN1C/p57 (P<0.01). A significant increase of luciferase activity was detected in Caco2 cells co-transfected with the luciferase reporter plasmid construct and anti-MIR221 (P<0.01)., Conclusions: MIR221 can interact with the target site on the 3'-UTR of CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing to promote colon carcinoma cell proliferation, suggesting the value of MIR221 as a potential target for treatment of colon carcinoma.

Objective: To investigate the expression of microRNA-221 (miR-221) and CDKN1C/P57 in colorectal carcinoma (CRC) and adjacent non-cancerous tissues. The effect of miR-221-specific inhibitor on cell proliferation and apoptosis in CRC cells was also assessed., Methods: The expression of miR-221 was detected by real-time RT-PCR. CDKN1C/P57 mRNA and corresponding protein expression pattern were detected by semi-quantitative RT-PCR and Western-blot. The specific 2'-methoxy-modified RNA oligonucleotide of miR-221(miRNA inhibitor,anti-miR-221) was designed, synthesized and transfected into Caco2 cell by liposome. Finally, the status of CRC cell proliferation and apoptosis were detected by MTT assay and flow cytometry., Results: The expression of miR-221 was significantly up-regulated in CRC tissues as compared to the adjacent non-cancerous tissues(2.041±1.401 vs. 0.806±0.341, P<0.01). There was no significant difference in CDKN1C/P57 mRNA expression between CRC and non-cancerous tissues, whereas CDKN1C/P57 protein markedly decreased in CRC (3.019±1.708 vs. 0.972±0.316, P<0.01). miR-221-specific inhibitor significantly enhanced CDKN1C/P57 protein expression, inhibited proliferation of CRC cells and induced apoptosis of CRC cells(P<0.01)., Conclusions: miR-221 inhibits CDKN1C/P57 expression by post-transcriptional gene silencing to promote CRC development and progression. miR-221-specific inhibitor potentially inhibits the growth of CRC cells. Therefore, it may be a new target for the biologic therapy for CRC.

Objective: To investigate miRNA-221 expression in human colorectal carcinoma (CRC) cells and the effects of miR-221-specific inhibitor on the proliferation and apoptosis of CRC cells., Methods: Four human CRC cell lines (HT-29, Lovo, SW-480, and CaCO2) were examined for miRNA-221 expression using real-time Q-PCR. The specific 2,-methoxy-modified RNA oligonucleotides of miR-221 (anti-miR-221) were synthesized and transfected into Caco2 cells via liposome, and the changes in the expression of miR-221 in the cells were detected by real-time Q-PCR. The proliferation and apoptosis of the transfected CRC cells were detected using MTT assay and flow cytometry., Results: The 4 human CRC cells showed significantly upregulated expression of miR-221 compare with HUVECs (P<0.01). The miR-221-specific inhibitor, anti-miR-221, significantly inhibited the expression of miR-221 in Caco2 cells and suppressed the cell proliferation, causing also obvious cell apoptosis (P<0.01)., Conclusion: The miR-221-specific inhibitor shows potent inhibitory effect on the growth of CRC cells, suggesting its value as a potential anti-tumor candidate for treatment of CRC.

Aim: To investigate the regulatory effect of microRNA-221 (miR-221) on CDKN1C/p57 expression in colorectal carcinoma (CRC)., Methods: Thirty four CRC and adjacent non-tumorous tissue samples were collected individually. Total RNA and protein were isolatedand from these samples and four human CRC-derived cell lines (including HT-29, Lovo, SW-480 and Caco2). MiR-221 expression was examined using real-time RT-PCR. CRC cells were treated with or without anti-p57-siRNA prior to the addition of pre-miR-221 or anti-miR-221. The mRNA and protein levels of CDKN1C/p57 were examined using semi-quantitative RT-PCR and Western blot, respectively. CRC cell proliferation and apoptosis were assessed using MTT assay and flow cytometry, respectively. The CDKN1C/p57 3'-UTR fragment was amplified using PCR from the genomic DNA of human colon cells and inserted into a luciferase reporter construct. The reporter construct was then transfected into CRC cells together with pre-miR-221 or anti-miR-221, and the luciferase activity in the transfected cells was examined., Results: MiR-221 expression was significantly up-regulated in 90% of CRC samples compared to that in the adjacent non-tumorous tissue, and the expression level was positively correlated to an advanced TNM stage and local invasion. There was no significant difference in CDKN1C/p57 mRNA expression between CRC and corresponding non-tumorous tissues, whereas CDKN1C/p57 protein expression was markedly decreased in the CRC samples. A significant inverse correlation between miR-221 and CDKN1C/p57 expression was found in CRC cells. Moreover, a miR-221-specific inhibitor significantly increased CDKN1C/p57 protein expression in CRC cells. Anti-miR-221 markedly inhibited CRC cell proliferation and induced apoptosis. This inhibitory effect was abolished by pretreatment with anti-p57-siRNA, suggesting that the inhibition was mediated by CDKN1C/p57. A significant increase of the luciferase activity was observed in CRC cells co-transfected with the luciferase reporter construct and anti-miR-221., Conclusion: MiR-221 binds to the target site in the 3'-UTR of the CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing to promote CRC occurrence and progress, therefore serving as a potential therapeutic target for the prevention and treatment of CRC.