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Background: Diabetic nephropathy-related osteoporosis (DNOP) is the most common comorbid bone metabolic disorder associated with diabetes mellitus (DM). The Liuwei Dihuang Pill (LWD) is a traditional Chinese herbal medicine widely used to treat diabetic complications, including diabetic nephropathy (DN). This study aimed to identify the biomarkers of the mechanisms of DNOP in LWD with systems biology approaches., Methods: Herein, we performed an integrated analysis of the GSE51674 and GSE63446 datasets from the GEO database via weighted gene co-expression network and network pharmacology (WGCNA) analysis. In addition, a network pharmacology approach, including bioactive compounds, was used with oral bioavailability (OB) and drug-likeness (DL) evaluation. Next, target prediction, functional enrichment analysis, network analysis, and virtual docking were used to investigate the mechanisms of LWD in DNOP., Results: WGCNA successfully identified 63 DNOP-related miRNAs. Among them, miR-574 was significantly upregulated in DN and OP samples. A total of 117 targets of 22 components associated with LWD in DNOP were obtained. The cellular response to nitrogen compounds, the AGERAGE signaling pathway in diabetic complications, and the MAPK signaling pathway were related to the main targets. Network analysis showed that kaempferol and quercetin were the most significant components. MAPK1 was identified as a potential target of miR-574 and the hub genes in the protein-protein interaction (PPI) network. The docking models demonstrated that kaempferol and quercetin had a strong binding affinity for Asp 167 of MAPK1., Conclusion: This study demonstrated that miR-574 may play important roles in DNOP, and the therapeutic effects of kaempferol and quercetin on LWD in DNOP might be mediated by miR-574 by targeting MAPK1. Our results provide new perspectives for further studies on the anti-DNOP mechanism of LWD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The mechanism related to ovarian follicular is complex, which has not been fully elucidated. Abundant reports have confirmed that the ovarian function development is closely related to sympathetic innervation. As one of the major neurotransmitters, norepinephrine (NE) is considered an effective regulator of ovarian functions like granulosa cell (GC) apoptosis. However, the mechanism between NE and GC apoptosis in rat is still unclear. In our study, GCs were isolated and cultured in vitro with NE treatment. The apoptosis of GCs was facilitated by NE. Wilms tumor 1 (WT1) was found to be significantly downregulated in GCs after NE treatment, and overexpression of WT1 repressed apoptosis in rat GCs induced by NE. rno-miR-128-3p was found to be significantly enhanced by NE in GCs, and inhibition of rno-miR-128-3p repressed apoptosis in rat GCs induced by NE. Mechanistically, rno-miR-128-3p interacted with WT1 and repressed its expression. In summary, inhibition of rno-miR-128-3p may enhance WT1 expression, and then repress NE-induced apoptosis in rat GCs. Our research may provide a new insight for the improvement of ovarian follicular development., (© 2021. The Author(s).)

Prolactin (PRL) is a pituitary hormone that regulates multiple physiological processes. However, the mechanisms of PRL synthesis have not been fully elucidated. The aims of the present study were to study the functions and the related mechanisms of miR-375 regulating PRL synthesis. We initially found that miR-375 mainly expressed in the lactotrophs of mouse pituitary gland. To identify the function of miR-375 in the pituitary gland, the miR-375 knockout mice were generated by using Crispr/Cas9 technique. The results showed that miR-375 knockout resulted in the decline of pituitary PRL mRNA and protein levels by 75.7 and 60.4%, respectively, and the serum PRL level reduced about 46.1%, but had no significant effect on FSH, LH and TSH. Further, we identified that Estrogen receptor 1 (alpha) (Esr1) was a downstream molecule of miR-375. The real-time PCR and Western blot results showed that ESR1 mRNA and protein levels markedly decreased by 40.9 and 42.9% in the miR-375 knockout mouse pituitary, and these were subsequently confirmed by the in vitro study using transfections of miR-375 mimics and inhibitors in pituitary lactotroph GH4 cells. Further, Rasd1 was predicted by bioinformatic tools and proved to be the direct target of miR-375 in lactotrophs using the dual-luciferase reporter assay. Rasd1-siRNA transfection results revealed the negative effect of Rasd1 in regulating ESR1. Collectively, the results presented here demonstrate that miR-375 positively modulates PRL synthesis through Rasd1 and Esr1, which are crucial for understanding the regulating mechanisms of pituitary hormone synthesis.

Background: The complications caused by diabetes mellitus (DM) are the focus of clinical treatment. However, little is known about diabetic enteropathy (DE) and its potential underlying mechanism., Methods: Intestinal epithelial cells (IECs) and intestinal epithelial stem cells (IESCs) were harvested from BKS.Cg-Dock7 m +/+Lepr db /JNju (DM) mice, and the expression of R-Spondin 3 (Rspo3) was detected by RT-qPCR, Western blotting, immunohistochemistry, and immunofluorescence. The role of Rspo3 in the abnormal differentiation of IECs during DM was confirmed by knockdown experiments. Through miRNA expression profiling, bioinformatics analysis, and RT-qPCR, we further analyzed the differentiation-related miRNAs in the IECs from mice with DM., Results: Abnormal differentiation of IECs was observed in the mice with DM. The expression of Rspo3 was upregulated in the IECs from the mice with DM. This phenomenon was associated with Rspo3 overexpression. Additionally, Rspo3 is a major determinant of Lgr5+ stem cell identity in the diabetic state. Microarray analysis, bioinformatics analysis, and luciferase reporter assays revealed that microRNA (miR)-380-5p directly targeted Rspo3. Moreover, miR-380-5p upregulation was observed to attenuate the abnormal differentiation of IECs by regulating Rspo3 expression., Conclusions: Together, our results provide definitive evidence of the essential role of Rspo3 in the differentiation of IECs in DM.

Breast cancer is the second most prevalent cancer in women worldwide. Long non‑coding RNAs (lncRNAs) have been identified as important regulators of tumorigenesis and tumor metastasis. lncRNA FGD5‑AS1 has been previously reported as a carcinogenic gene, however its role in breast cancer has yet to be investigated. The present study aimed to understand the function of lncRNA FGD5‑AS1 in breast cancer and examine the underlying molecular mechanisms. Sample tissues for downstream gene expression profiling were collected from patients with breast cancer (n=23). The effect of FGD5‑AS1 overexpression on cell viability, invasion and migration has been studied in breast cancer cells (MDA‑MB‑231). Changes in glycolysis were monitored by comparing glucose consumption, lactate production and ATP levels. Using StarBase and TargetScan databases a putative interaction between FGD5‑AS1, miR‑195‑5p and SNF1‑like kinase 2 (NUAK2) was predicted in silico . Expression levels of FGD5‑AS1, has‑miR‑195‑5p and NUAK2 were validated by reverse transcription‑quantitative PCR and interactions were validated using dual‑luciferase reporter assays and RNA pull‑down. High expression of lncRNA FGD5‑AS1 was detected in breast cancer tissue samples and disease model cell lines. Silencing of FGD5‑AS1 led to decreased cell proliferation, migration and invasion. It was identified that at a molecular level FGD5‑AS1 serves as a sponge of miR‑195‑5p and alters the expression of its downstream target gene NUAK2. In breast cancer lncRNA FGD5‑AS1 serve a key role in glycolysis and tumor progression via the miR‑195‑5p/NUAK2 axis. The findings of the present study indicated FGD5‑AS1 as a candidate target for intervention in patients with breast cancer.

Background: Circular RNA (circRNA) is highly expressed in the brain tissue, but its molecular mechanism in cerebral ischemia-reperfusion remains unclear. Here, we explored the role and underlying mechanisms of circRNA antisense non-coding RNA in the INK4 locus (circ_ANRIL) in oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell injury., Results: The expression of circ_ANRIL in OGD/R-induced human brain microvascular endothelial cells (HBMECs) was significantly up-regulated, while that of miR-622 was significantly down-regulated. Overexpression of circ_ANRIL significantly inhibited the proliferation of OGD/R-induced HBMECs and aggravated OGD/R-induced cell apoptosis. Moreover, circ_ANRIL overexpression further increased the secretion of interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 in OGD/R-treated HBMECs. The results of bioinformatics analysis and luciferase reporter assay indicated that circ_ANRIL served as an miR-622 sponge to negatively regulate the expression of miR-622 in OGD/R-treated HBMECs. Additionally, circ_ANRIL silencing exerted anti-apoptotic and anti-inflammatory effects by positively regulating the expression of miR-622. Furthermore, inhibition of OGD/R-induced activation of the nuclear factor (NF)-κB pathway by circ_ANRIL silencing was significantly reversed by treatment with miR-622 inhibitor., Conclusions: Knockdown of circ_ANRIL improved OGD/R-induced cell damage, apoptosis, and inflammatory responses by inhibiting the NF-κB pathway through sponging miR-622.

MicroRNAs are essential post-transcriptional regulators and may act as the noninvasive biomarkers for disease diagnosis. Sensitive and multiplexed detection of microRNAs may facilitate the accurate and early clinical diagnosis, but the available methods are usually compromised by using organic dyes as the signal probes, laborious chemical and enzymatic manipulations, and the complicated reaction schemes. Here we reported a siRNA-directed self-assembled quantum dot (QD) biosensor for facile and simultaneous detection of multiple microRNAs. In this biosensor, the binding of microRNA targets with corresponding QD nanoprobes leads to the formation of siRNA duplexes, which not only induces the spectrally resolved coding of microRNAs, but also facilitates the assembly of QDs:magnetic nanoparticle bioconjugates for the isolation and enrichment of target microRNAs. The disassembled QDs can be sensitively detected by single-molecule detection, enabling quantitatively sensing of microRNAs at the single-particle level. This biosensor employs only QDs as the signal reporters, which can simultaneously detect multiple microRNAs from the same sample and achieves femtomolar sensitivity and single-base mismatch selectivity without the involvement of any target labeling and amplification steps. Moreover, it can be successfully applied for simultaneous detection of circulating microRNAs in clinical serum samples, holding great potential in non-invasive early diagnosis and biomedical researches., 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 B.V. All rights reserved.)

Background: Intervertebral disc degeneration (IVDD) is a well-known cause of lower back pain, which is induced by multiple factors including increased apoptosis and decreased survival of nucleus pulposus cells. In this study, we evaluate the effect and potential mechanism of miR-660 on the nucleus pulposus cells apoptosis induced by TNF-α., Methods: First, we collected tissue of nucleus pulposus from IVDD and healthy controls. General characteristic of the IVDD and healthy control was also collected. And, we also collected nucleus pulposus cells that stimulated by TNF-α or control. miRNA microarray was performed to identify the differentially expressed miRNAs. Apoptosis rate and miR-660 relative expression was measured after stimulated with different concentration of TNF-α to identify the optimal concentration of TNF-α. Second, we successfully constructed antigomiR-660 to block the miR-660 expression in nucleus pulposus cells and then stimulated with TNF-α (100 ng/ml, 12 h). The apoptosis rates and relative protein expression were then measured again. The target association between miR-660 and SAA1 was confirmed by dual-luciferase reporter., Results: There was no significant difference between the age (IVDD: 39 ± 10 years, healthy controls: 36 ± 7 years), BMI and sex between IVDD and healthy controls. Microarray analysis found that miR-660 was significantly up-regulated in IVDD and TNF-α treated groups, which was further identified by PCR. We found that the rate of apoptosis and miR-660 expression increased with TNF-α concentration increased. Finally, TNF-a with 100 ng/ml was used for further experiment. Compared with TNF-α group, TNF-α + antigomiR-660 could significantly down-regulated the apoptosis rate and relative protein (c-Caspase3 and c-Caspase7). Dual-luciferase reporter revealed that miR-660 could directly binding to the SAA1 at 80-87 sites. Compared with TNF-α alone group, TNF-α + antigomiR-660 significantly up-regulated the SAA1 expression (P < 0.05)., Conclusion: These results indicated that knockdown of miR-660 protected the nucleus pulposus from apoptosis that induced TNF-α via up-regulation of SAA1. Further studies should focus on the role of miR-660 in protecting IVDD in vivo.

Objective: Chemoresistance has been a major problem in cancer chemotherapy. The present study aimed to investigate the effect of Rosmarinic acid (RA) on chemoresistance to 5-Fu and its molecular mechanism in gastric carcinoma., Methods: CCK8 cell proliferation and apoptosis assay were used to evaluate the effect of RA on chemoresistance to 5-Fu in GC cells. RNA microarray was used to identify miRNA involved. Expression level of miRNA in GC cells was determined by RT-PCR. Down- or up-regulating of miRNA in the GC cells was performed by transfection of RNA interference or expression vectors in the GC cells. Double luciferase reporter assay was used to verify miRNA target genes. Expression of P-glycoprotein and Bax was analyzed with Western blot., Results: RA treated SGC7901/5-Fu cells showed significant increased chemosensitivity to 5-Fu. The IC50 of 5-Fu was significantly reduced in RA treated SGC7901/5-Fu cells (70.43 ± 1.06 μg/mL) compared to untreated SGC7901/5-Fu cells (208.6 ± 1.09 μg/mL) (P < 0.05). Apoptosis rate was significantly increased in RA+5-Fu treated SGC7901/5-Fu cells compared to 5-FU treatment alone (P < 0.01). Two miRNAs, namely miR-642a-3p and miR-6785-5p, were identified to be involved in the chemo-sensitizing effect of RA in the SGC7901/5-Fu cells. RA treated SGC7901/5-Fu cells showed reduced expression levels of miR-642a-3p and miR-6785-5p compared to untreated SGC7901/5-Fu cells (P < 0.05). Down- or up-regulation of miR-6785-5p increased or reduced chemosensitivity of gastric carcinoma cells to 5-Fu, respectively. RA treated SGC7901/5-Fu and the SGC7901/5-Fu-Si cells showed significantly increased FOXO4 expression (P < 0.01). Double luciferase reporter assay confirmed miR-6785-5p directly targets FOXO4 to regulate its expression. RA significantly reduced P-gp expression and increased Bax expression in SGC7901/5-Fu and the SGC7901/5-Fu-Si cells (P < 0.05)., Conclusion: RA enhances chemosensitivity of resistant gastric carcinoma SGC7901 cells to 5-Fu by downregulating miR-6785-5p and miR-642a-3p and increasing FOXO4 expression. These study suggest the potential for RA as a multidrug resistance-reversing agent in GC., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Introduction: miRNAs dysregulate in spinal cord injury (SCI) and have been demonstrated to play a crucial role in neurite outgrowth. However, the underlying mechanism remains elusive. In this study, we constructed a mouse model of SCI, extracted RNA from injured spinal cord tissue for the use of microarray assay. miR-181d-5p which is one of the most significantly expressed miRNAs in miRNA-mRNA network, abundantly expressed in center system and highly conserved across different spices, was chosen for our further study., Aims: To demonstrate whether miR-181d-5p can promote neurite outgrowth in PC12 cells via PI3K/Akt signaling pathway, we performed function analysis of miR-181d-5p with LV-miR-181d-5p and LV-sh-GFP to infect PC12 cells., Results: Through microarray assay analysis, we totally found 262 significantly expressed miRNAs and 2973 target genes in SCI and observed that their expression dynamically changed postinjury. Here, we provided enough evidences that the overexpression of miR181d-5p significantly decreased the expression of PTEN, upregulated p-Akt expression, increased neurite outgrowth-related proteins (GAP-43 and NF-200) and synaptic vesicle-related proteins (Synapsin and PSD95), and then promoted neurite outgrowth in PC12 cells. Furthermore, we confirmed that miR-181d-5p could directly target to the 3'-UTR of PTEN mRNA through dual-luciferase report assay., Conclusions: Our study supports that aberrant expression of miRNAs is involved in the pathogenesis of SCI, miR-181d-5p plays an important role in neurite growth in PC12 cells via PI3K/Akt signaling pathway and may be a candidate target for the treatment of SCI in the future., (© 2017 John Wiley & Sons Ltd.)

Osteogenic differentiation of mesenchymal stem cells (MSCs) is a complex process, which is regulated by various factors including microRNAs. Our preliminary data showed that the expression of endogenous miR-20a was increased during the course of osteogenic differentiation. Simultaneously, the expression of osteoblast markers and regulators BMP2, BMP4, Runx2, Osx, OCN and OPN was also elevated whereas adipocyte markers PPARγ and osteoblast antagonist, Bambi and Crim1, were downregulated, thereby suggesting that miR-20a plays an important role in regulating osteoblast differentiation. To validate this hypothesis, we tested its effects on osteogenic differentiation by introducing miR-20a mimics and lentiviral-miR20a-expression vectors into hMSCs. We showed that miR-20a promoted osteogenic differentiation by the upregulation of BMP/Runx2 signaling. We performed bioinformatics analysis and predicted that PPARγ, Bambi and Crim1 would be potential targets of miR-20a. PPARγ is a negative regulator of BMP/Runx2 signaling whereas Bambi or Crim1 are antagonists of the BMP pathway. Furthermore, we confirmed that all these molecules were indeed the targets of miR-20a by luciferase reporter, quantitative RT-PCR and western blot assays. Similarly to miR-20a overexpression, the osteogenesis was enhanced by the silence of PPARγ, Bambi or Crim1 by specific siRNAs. Taken together, for the first time, we demonstrated that miR-20a promoted the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARγ, Bambi and Crim1, the negative regulators of BMP signaling.

MicroRNAs (miRNAs) are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition. Computer-based approaches for miRNA gene identification are being considered as indispensable in miRNAs research. Similarly, experimental approaches for detection of miRNAs are crucial to the testing and validating of computational algorithms. The detection of miRNAs in tissues or cells can supply valuable information for investigating the biological function of these molecules. Selective and highly sensitive detection methods will pave the way for extended understanding of miRNA function within organisms. In this review, we summarize the various computational methods for identification of miRNAs as well as the methodologies that have been developed to detection miRNAs.

MicroRNAs (miRNAs) are endogenous single-stranded RNAs of 18-22 nt in length, which can regulate the complementary mRNAs at the post-transcriptional level by cleavage or repression of translation of the target mRNAs. Studies have shown that the majority of animal miRNAs are transcribed from independent transcription units, and someare transcribed together with their host genes. However, the nature of the primary transcript of intergenic miRNAs remains unknown. Silkworm (Bombyx mori) miRNAs are representative of those of the Lepidoptera insects and many of them are conserved in Caenorhabditis elegans and other animal species. To date, little is known about the transcriptional regulation of silkworm miRNA genes. We performed the genomic analysis on the silkworm miRNA transcripts around the promoter region including the transcription start site (TSS) and the TATA-box, and on the organization of the miRNA cluster. In 73 pre-miRNAs from the silkworm 131 promoters were detected via a bioinformatics approach. Among them the portion of non-conserved promoters is greater than that of the conserved ones. The genomic organization of pre-miRNAs of the silkworm was globally analyzed and it was determined that 11 of them we reorganized into five clusters. Sequence alignment showed that paralogs existed for some of the miRNAs in the cluster. These results may increase the understanding of the specific sequences upstream of the pre-miRNAs and of the functional implications of miRNA clusters in the silkworm.

MicroRNAs (miRNAs) are a recently discovered family of endogenous, noncoding RNA molecules approximately 22 nt in length. miRNAs modulate gene expression post-transcriptionally by binding to complementary sequences in the coding or 3' untranslated region of target messenger RNAs (mRNAs). It is now clear that the biogenesis and function of miRNAs are related to the molecular mechanisms of various clinical diseases, and that they can potentially regulate every aspect of cellular activity, including differentiation and development, metabolism, proliferation, apoptotic cell death, viral infection and tumorgenesis. Here, we review recent advances in miRNA research, and discuss the diverse roles of miRNAs in disease.

microRNAs (miRNAs) are 20-24 nucleotide (nt) RNAs that regulate eukaryotic gene expression post-transcriptionally by the degradation or translational inhibition of their target messenger RNAs (mRNAs). To identify miRNA target genes will help a lot by understanding their biological functions. Sophisticated computational approaches for miRNA target prediction, and effective biological techniques for validating these targets now play a central role in elucidating their functions. Owing to the imperfect complementarity of animal miRNAs with their targets, it is difficult to judge the accuracy of the prediction. Complexity of regulation by miRNA-mediated targets at protein and mRNAs levels has made it more challenging to identify the targets. To date, only a few miRNAs targets are confirmed. In this article, we review the methods of miRNA target prediction and the experimental validation for their corresponding mRNA targets in animals.

The small regulatory non-coding RNA molecules, known as microRNAs (miRNAs), have been recognized as potential regulator of gene expression and modulate the gene function at the post-transcriptional level. It is now clear that miRNA biogenesis and function are related to the molecular mechanisms of various clinical diseases, which can potentially regulate every aspect of cellular activity, including differentiation and development, metabolism, proliferation, apoptotic cell death viral infection and tumorgenesis. Here, we review recent work and provide insight into the diverse roles of miRNAs.