Growth factor TGF-β induces intestinal epithelial cell (IEC-6) differentiation: miR-146b as a regulatory component in the negative feedback loop

TGF-β is a potent pleiotropic factor that promotes small intestinal cell differentiation. The role of microRNAs in the TGF-β induction of intestinal epithelial phenotype is largely unknown. We hypothesized that microRNAs are functionally involved in TGF-β-induced intestinal cell growth. In this study, TGF-β caused a morphological change of IEC-6 cells and stimulated expression of the epithelial cell markers alkaline phosphatase, villin, and aminopeptidase N. By global microRNA profiling during TGF-β-induced intestinal crypt cell (IEC-6) differentiation, we identified 19 differentially expressed microRNAs. We showed by real-time Q-PCR that miR-146b expression increased rapidly after TGF-β treatment; sequence analysis and in vitro assays revealed that miR-146b targets SIAH2, an E3 ubiquitin ligase, with decreased protein expression upon IEC-6 cell differentiation. Transfection of miR-146b inhibitor before TGF-β treatment blocked the down-regulation of SIAH2 in response to TGF-β. Moreover, SIAH2 over-expression during TGF-β treatment caused a significant decrease in Smad7 protein expression in IEC-6 cells. Furthermore, activation of the ERK1/2 pathway is active in the up-regulation of miR-146b by TGF-β. These findings suggest a novel mechanism whereby TGF-β signaling during IEC-6 cell differentiation may be modulated in part by microRNAs, and we propose a key role for miR-146b in the homeostasis of growth factor TGF-β signaling through a negative feedback regulation involving down-regulation of SIAH2 repressed Smad7 activities.     

Sevoflurane protects cardiomyocytes against hypoxia/reperfusion injury via LINC01133/miR-30a-5p axis

Previous studies failed to elucidate the detailed mechanisms of anesthetic preconditioning as a protective approach against ischemic/reperfusion (I/R) injury in cells. The present study mainly centered on discovering the mechanisms of Sevoflurane (Sev) in preventing cardiomyocytes against I/R injury. Human cardiomyocyte AC16 cell line was used to simulate I/R injury based on a hypoxia/reperfusion (H/R) model. After Sev treatment, cell viability and apoptosis were detected by MTT assay and flow cytometry, respectively. Lactate dehydrogenase (LDH) content was measured using an LDH Detection Kit. Relative mRNA and protein expressions of LINC01133, miR-30a-5p and apoptosis-related proteins were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. Target gene of miR-30a-5p and their potential binding sites were predicted using Starbase and confirmed by dual-luciferase reporter assay. Cell behaviors were assessed again after miR-30a-5p and LINC01133 transfection. Sev could improve cell viability, reduce LDH leakage, and down-regulate the expressions of apoptosis-related proteins (Bax, cleaved caspase-3 and cleaved caspase-9) and LINC01133 as well as up-regulate miR-30a-5p and Bcl-2 expressions in H/R cells. MiR-30a-5p was the target of LINC01133, and up-regulating miR-30a-5p enhanced the effects of Sev in H/R cells, with a suppression on H/R-induced activation of the p53 signaling pathway. However, up-regulating LINC01133 reversed the enhancing effects of miR-30a-5p on Sev pretreatment in H/R cells. Sev could protect cardiomyocytes against H/R injury through the miR-30a-5p/LINC01133 axis, which may provide a possible therapeutic method for curing cardiovascular I/R injury.     

MiR-3691-5p is upregulated in docosahexaenoic acid-treated vascular endothelial cell and targets serpin family E member 1

Endothelium, the inner cellular lining of blood vessels, has an important role in the regulation of physiological processes and its dysfunction may initiate cardiovascular complications. Previous investigations have revealed that dietary docosahexaenoic acid (DHA) is related to a lower possibility of cardiovascular disease and mortality. Until now, the molecular mechanisms in the biological activities of DHA remain largely unknown. MicroRNAs (miRNAs) play a vital role in regulating gene expression. Thus, we aimed to investigate whether DHA improves the dysfunction via regulating miRNAs. To understand the protective effects of DHA through modulating miR-3691-5p and its target genes for palmitic acid (PAL) induced apoptosis in endothelial cells. The present study demonstrated that DHA upregulated miR-3691-5p expression, and downregulated the expression of its target gene serpin family E member 1 (SERPINE1). MiR-mimics and inhibitors modulation results indicated that miR-3691-5p regulates endothelial apoptosis through activating antiapoptotic response which controlled by the STAT3 signaling pathway. In conclusion, we have shown that PAL-induced apoptosis could be decreased by DHA treatment through miR-3691-5/SERPINE1 pathways.     

miRNA expression profiling uncovers a role of miR-302b-3p in regulating skin fibroblasts senescence

Numbers of emerging evidence suggest that variable microRNA (miRNA) expression facilitates the aging process. In this study, we distinguished aberrant miRNA expression in aged skin and explored the biological functions and potential mechanism of upregulated miR-302b-3p. At first, miRNA microarray analysis was examined to explore miRNA expression profiling in the skin of aging mice model by D -galactose (d -gal) injection. We identified 29 aberrant miRNAs in aged mice skin. Next, KEGG enrichment analysis was conducted with DIANA-miPath v3.0, which was revealed that enrichment pathways involved in such processes as extracellular matrix-receptor interaction, MAPK signaling pathway, and mammalian target of rapamycin (mTOR) signaling pathway. The target genes of deregulated miRNAs were predicted from four bioinformatic algorithms (miRDB, Targetscan, miRwalk, and Tarbase). The interaction network of miRNAs and their targets were visualized using Cytoscape software. As a result, we found that some hub genes (including JNK2, AKT1/2/3, PAK7, TRPS1, BCL2L11, and IKZF2) were targeted by 12 potential miRNAs (including miR-302b-3p, miR-291a-5p, miR-139-3p, miR-467c-3p, miR-186-3p, etc.). Subsequently, we identified five upregulated miRNA via quantitative polymerase chain reaction and all of them were confirmed increased significantly in aged skin tissues compared with young control tissues. Among them, high expression of miR-302b-3p was verified in both aged skin tissues and senescence fibroblasts. Furthermore, miR-302b-3p mimic accelerated skin fibroblast senescence and suppressed the longevity-associated gene Sirtuin 1(Sirt1) expression, whereas miR-302b-3p inhibitor could delay skin fibroblast senescence and contribute Sirt1 expression. In addition, we demonstrated that c-Jun N-terminal kinase 2(JNK2) is a direct target of miR-302b-3p by a luciferase reporter assay. An inverse correlation was verified in fibroblasts between miR-302b-3p and JNK2. Most importantly, siRNA JNK2 confirmed that low expression of JNK2 could accelerate fibroblasts senescence. In conclusion, our results indicated that overexpressed miR-302b-3p plays an important biological role in accelerating skin aging process via directly targeting JNK2 gene.     

Regulatory effects of lncRNA ATB targeting miR-200c on proliferation and apoptosis of colorectal cancer cells

This investigation was intended to elucidate whether long noncoding RNA (lncRNA)-activated by transforming growth factor-β (ATB) interacting with miR-200c could mediate colorectal cancer (CRC) progression, offering potential strategies for diagnosing and treating CRC. Here totally 315 patients with CRC were recruited, and their CRC tissues and adjacent normal tissues were gathered. Concurrently, four colon cancer cell lines (ie, SW620, Lovo, HCT116, and SW480) and the human colon mucosal epithelial cell line (NCM460) were also purchased. Moreover, si-ATB, si-NC, miR-200c mimic, miR-200c inhibitor, and miR-NC were prepared for transfection into the CRC cells, and their effects on CRC cell lines were evaluated based on the conduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, and flow cytometry assay. Eventually, the Luciferase reporter gene assay was carried out to judge if there existed a targeted relationship between ATB and miR-200c. The results of Cox regression analyses suggested that overexpressed lncRNA ATB, underexpressed miR-200c, poor tumor differentiation, lymph-vascular invasion, and perineural invasion were symbolic of shortened survival of the patients with CRC (all P < .05). Besides, transfection of pcDNA3.1-ATB and miR-200c inhibitor could boost the viability and proliferation of Lovo and SW620 cell lines (all P < .05). Meanwhile, the expressions of p53 and p21 were also reduced under treatments of pcDNA3.1-ATB and miR-200c inhibitor (P < .05). In addition, CDK2 seemed to reverse the contribution of miR-200c to intensifying viability and proliferation of Lovo and SW420 cell lines (P < .05). Furthermore, ATB might downregulate miR-200c expression by targeting it (P < .05), and CDK2 was subjected to dual regulation of both ATB and miR-200c (P < .05). In conclusion, the lncRNA ATB/miR-200c/CDK2 signaling was responsible for intensified proliferation and prohibited apoptosis of CRC cells, which might provide effective approaches for diagnosing and treating CRC.     

Tanshinone IIA inhibits the adipogenesis and inflammatory response in ox-LDL-challenged human monocyte-derived macrophages via regulating miR-130b/WNT5A

Atherosclerosis is a kind of chronic cardiovascular disease, characterized by oxidized low-density lipoprotein (ox-LDL) accumulation in macrophage. Tanshinone IIA (Tan), a lipophilic pharmacologically activate compound from Salvia miltiorrhiza Bunge, has been indicated to exert cardioprotective roles. Nevertheless, the biological role of Tan and regulatory mechanism in atherosclerosis are not fully established. In present study, atherosclerosis model was established in THP-1-derived macrophages by treatment of ox-LDL. The adipogenesis was measured by Nile red staining. The expressions of inflammatory factors, microRNA-130b (miR-130b) and WNT5A were measured by quantitative real-time polymerase chain reaction or Western blot. The target association between miR-130b and WNT5A was explored via luciferase activity and RNA immunoprecipitation assay. The results showed that exposure of Tan inhibited ox-LDL-induced adipogenesis and expressions of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha in THP-1-derived macrophages. miR-130b expression was decreased in THP-1-derived macrophages treated by ox-LDL and its overexpression attenuated adipogenesis as well as inflammatory response. miR-130b knockdown reversed the regulatory effect of Tan on adipogenesis and inflammatory response in THP-1-derived macrophages stimulated by ox-LDL. In addition, WNT5A acted as a functional target of miR-130b and inhibited by Tan and miR-130b. As a conclusion, Tan decreased the adipogenesis and inflammatory response by mediating miR-130b and WNT5A, providing a novel theoretical foundation for treatment of atherosclerosis.     

LncRNA TP73-AS1 is a novel regulator in cervical cancer via miR-329-3p/ARF1 axis

Cervical cancer holds one of the highest morbidity and mortality in various types of cancers. It even leads to the most number of cancer-related deaths of women. A lot of research has indicated that the anomalous expression of long noncoding RNAs (lncRNAs) would induce carcinogenesis and is associated with poor prognosis of patients with cancer. However, the function and mechanism of many lncRNAs still call for further research. Tumor Protein P73 Antisense RNA 1 (TP73-AS1) is no exception. LncRNA TP73-AS1 has been found to promote cancer progressions in various cancers. It is upregulated in cervical cancer cells. The proliferation and migration ability of cervical cancer cells can also be boosted by TP73-AS1 in return. Meanwhile, miRNA-329-3p is downregulated in cervical cancer cells and could bind with both TP73-AS1 and ADP Ribosylation Factor 1 (ARF1). TP73-AS1 inhibited miR-329-3p expression while miR-329-3p inhibited ARF1 expression. More importantly, TP73-AS1 can positively regulate ARF1 expression. Based on all these experiments, TP73-AS1 regulates ARF1 expression by competitively binding with miR-329-3p, thus regulating cervical cancer progression. Further rescue assays confirmed TP73-AS1 regulates cervical cell proliferation and migration via miR-329-3p/ARF1. TP73-AS1 might serve as a novel regulator in cervical cancer.