Differential expression of basal microRNAs’ patterns in human dental pulp stem cells

MicroRNAs (miRNAs) are small non‐coding RNAs that regulate translation of mRNA into protein and play a crucial role for almost all biological activities. However, the identification of miRNAs from mesenchymal stem cells (MSCs), especially from dental pulp, is poorly understood. In this study, dental pulp stem cells (DPSCs) were characterized in terms of their proliferation and differentiation capacity. Furthermore, 104 known mature miRNAs were profiled by using real‐time PCR. Notably, we observed 19 up‐regulated miRNAs and 29 significantly down‐regulated miRNAs in DPSCs in comparison with bone marrow MSCs (BM‐MSCs). The 19 up‐regulated miRNAs were subjected to ingenuity analysis, which were composed into 25 functional networks. We have chosen top 2 functional networks, which comprised 10 miRNA (hsa‐miR‐516a‐3p, hsa‐miR‐125b‐1‐3p, hsa‐miR‐221‐5p, hsa‐miR‐7, hsa‐miR‐584‐5p, hsa‐miR‐190a, hsa‐miR‐106a‐5p, hsa‐mir‐376a‐5p, hsa‐mir‐377‐5p and hsa‐let‐7f‐2‐3p). Prediction of target mRNAs and associated biological pathways regulated by each of this miRNA was carried out. We paid special attention to hsa‐miR‐516a‐3p and hsa‐miR‐7‐5p as these miRNAs were highly expressed upon validation with qRT‐PCR analysis. We further proceeded with loss‐of‐function analysis with these miRNAs and we observed that hsa‐miR‐516a‐3p knockdown induced a significant increase in the expression of WNT5A. Likewise, the knockdown of hsa‐miR‐7‐5p increased the expression of EGFR. Nevertheless, further validation revealed the role of WNT5A as an indirect target of hsa‐miR‐516a‐3p. These results provide new insights into the dynamic role of miRNA expression in DPSCs. In conclusion, using miRNA signatures in human as a prediction tool will enable us to elucidate the biological processes occurring in DPSCs.     

Rotavirus‐induced miR‐142‐5p elicits proviral milieu by targeting non‐canonical transforming growth factor beta signalling and apoptosis in cells

MicroRNA (miRNA) expression is significantly influenced by viral infection, because of either host antiviral defences or proviral factors resulting in the modulation of viral propagation. This study was undertaken to identify and analyse the significance of cellular miRNAs during rotavirus (SA11 or KU) infection. Sixteen differentially regulated miRNAs were identified during rotavirus infection of which hsa‐miR‐142‐5p was up‐regulated and validated by quantitative polymerase chain reaction. Exogenous expression of miR‐142‐5p inhibitor resulted in a significant reduction of viral titer indicating proviral role of miR‐142‐5p. Functional studies of hsa‐miR‐142‐5p identified its role in transforming growth factor beta (TGFβ) signalling as TGFβ receptor 2 and SMAD3 were degraded during both hsa‐miR‐142‐5p overexpression and rotavirus infection. TGFβ is induced during rotavirus infection, which may promote apoptosis by activation of non‐canonical pathways in HT29 cells. However, up‐regulated miR‐142‐5p resulted in the inhibition of TGFβ‐induced apoptosis suggesting its anti‐apoptotic function. Rotavirus NSP5 was identified as a regulator of miR‐142‐5p expression. Concurrently, NSP5‐HT29 cells showed inhibition of TGFβ‐induced apoptosis and epithelial to mesenchymal transition by blocking non‐canonical pathways. Overall, the study identified proviral function of hsa‐miR‐142‐5p during rotavirus infection. In addition, modulation of TGFβ‐induced non‐canonical signalling in microsatellite stable colon cancer cells can be exploited for cancer therapeutics.     

Effect of the interaction between MiR‐200b‐3p and DNMT3A on cartilage cells of osteoarthritis patients

The aim of this research is to explore the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of osteoarthritis (OA) cartilage cells. Quantitative RT‐PCR was performed to analyse the expression of miR‐200b‐3p, DNMT3A, MMP1, MMP3, MMP9, MMP13 and COL II in normal and OA cartilage tissues. The dual‐luciferase reporter assay and Western blot assay were conducted to confirm the targeting relationship between miR‐200b‐3p and DNMT3A. We also constructed eukaryotic expression vector to overexpress miR‐200b‐3p and DNMT3A. We detected the expression level of MMPs and COL II in stable transfected cartilage cells using RT‐PCR and Western blot. Cell proliferation and apoptosis were evaluated using the MTS, pellet culture and Hoechst 33342 staining method. Finally, we explored the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of OA cartilage cells. The results of RT‐PCR indicated that both miR‐200b‐3p and COL II were down‐regulated in OA cartilage tissues, while the expression of DNMT3A and MMPs was up‐regulated in OA cartilage tissues. The expressions of DNMT3A, MMPs and COL II detected by Western blot showed the same trend of the results of RT‐PCR. The dual‐luciferase reporter assay and Western blot assay confirmed the targeting relationship between miR‐200b‐3p and DNMT3A. In overexpressed miR‐200b‐3p cartilage cells, DNMT3A and MMPs were significantly down‐regulated, COL II was significantly up‐regulated, cell viability was enhanced and apoptosis rate was decreased (P < 0.05). In overexpressed DNM3T cartilage cells, MMPs were significantly up‐regulated, COL II was significantly down‐regulated, cell viability was weakened and apoptosis rate was increased (P < 0.05). MiR‐200b‐3p inhibited the secretion of MMPs, promoted the synthesis of COL II and enhanced the growth and proliferation of OA cartilage cells through inhibiting the expression of DNMT3A.     

Identification of cell and disease specific microRNAs in glomerular pathologies

MicroRNAs (miRs) are small non‐coding RNAs that regulate gene expression in physiological processes as well as in diseases. Currently miRs are already used to find novel mechanisms involved in diseases and in the future, they might serve as diagnostic markers. To identify miRs that play a role in glomerular diseases urinary miR‐screenings are a frequently used tool. However, miRs that are detected in the urine might simply be filtered from the blood stream and could have been produced anywhere in the body, so they might be completely unrelated to the diseases. We performed a combined miR‐screening in pooled urine samples from patients with different glomerular diseases as well as in cultured human podocytes, human mesangial cells, human glomerular endothelial cells and human tubular cells. The miR‐screening in renal cells was done in untreated conditions and after stimulation with TGF‐β. A merge of the detected regulated miRs led us to identify disease‐specific, cell type‐specific and cell stress‐induced miRs. Most miRs were down‐regulated following the stimulation with TGF‐β in all cell types. Up‐regulation of miRs after TGF‐β was cell type‐specific for most miRs. Furthermore, urinary miRs from patients with different glomerular diseases could be assigned to the different renal cell types. Most miRs were specifically regulated in one disease. Only miR‐155 was up‐regulated in all disease urines compared to control and therefore seems to be rather unspecific. In conclusion, a combined urinary and cell miR‐screening can improve the interpretation of screening results. These data are useful to identify novel miRs potentially involved in glomerular diseases.     

MicroRNA‐203 mimics age‐related aortic smooth muscle dysfunction of cytoskeletal pathways

Increased aortic stiffness is a biomarker for subsequent adverse cardiovascular events. We have previously reported that vascular smooth muscle Src‐dependent cytoskeletal remodelling, which contributes to aortic plasticity, is impaired with ageing. Here, we use a multi‐scale approach to determine the molecular mechanisms behind defective Src‐dependent signalling in an aged C57BL/6 male mouse model. Increased aortic stiffness, as measured in vivo by pulse wave velocity, was found to have a comparable time course to that in humans. Bioinformatic analyses predicted several miRs to regulate Src‐dependent cytoskeletal remodelling. qRT‐PCR was used to determine the relative levels of predicted miRs in aortas and, notably, the expression of miR‐203 increased almost twofold in aged aorta. Increased miR‐203 expression was associated with a decrease in both mRNA and protein expression of Src, caveolin‐1 and paxillin in aged aorta. Probing with phospho‐specific antibodies confirmed that overexpression of miR‐203 significantly attenuated Src and extracellular signal regulated kinase (ERK) signalling, which we have previously found to regulate vascular smooth muscle stiffness. In addition, transfection of miR‐203 into aortic tissue from young mice increased phenylephrine‐induced aortic stiffness ex vivo, mimicking the aged phenotype. Upstream of miR‐203, we found that DNA methyltransferases (DNMT) 1, 3a, and 3b are also significantly decreased in the aged mouse aorta and that DNMT inhibition significantly increases miR‐203 expression. Thus, the age‐induced increase in miR‐203 may be caused by epigenetic promoter hypomethylation in the aorta. These findings indicate that miR‐203 promotes a re‐programming of Src/ERK signalling pathways in vascular smooth muscle, impairing the regulation of stiffness in aged aorta.     

Regulation of miR319 during cold stress in sugarcane

MicroRNAs (miRNAs) are part of a novel mechanism of gene regulation that is active in plants under abiotic stress conditions. In the present study, 12 miRNAs were analysed to identify miRNAs differentially expressed in sugarcane subjected to cold stress (4 °C). The expression of miRNAs assayed by stem–loop RT‐PCR showed that miR319 is up‐regulated in sugarcane plantlets exposed to 4 °C for 24 h. The induction of miR319 expression during cold stress was observed in both roots and shoots. Sugarcane miR319 was also regulated by treatment with abscisic acid. Putative targets of this miRNA were identified and their expression levels were decreased in sugarcane plantlets exposed to cold. The cleavage sites of two targets were mapped using a 5′ RACE PCR assay confirming the regulation of these genes by miR319. When sugarcane cultivars contrasting in cold tolerance were subjected to 4 °C, we observed up‐regulation of miR319 and down‐regulation of the targets in both varieties; however, the changes in expression were delayed in the cold‐tolerant cultivar. These results suggest that differences in timing and levels of the expression of miR319 and its targets could be tested as markers for selection of cold‐tolerant sugarcane cultivars.     

MicroRNA‐503 promotes angiotensin II‐induced cardiac fibrosis by targeting Apelin‐13

Cardiac fibrosis is a major cause of heart failure. MicroRNAs (miRs) are important epigenetic regulators of cardiac function and cardiovascular diseases, including cardiac fibrosis. This study aimed to explore the role of miR‐503 and its mechanisms in regulating cardiac fibrosis. miR‐503 was found up‐regulated in the mouse LV tissues subjected to transverse aortic constriction (TAC) and in neonatal cardiac fibroblasts (CFs) cultured with Angiotension II. The role of miR‐503 in regulating CF cell proliferation and/or collagen production in mice neonatal CFs were determined using an MTT assay and RT‐PCR respectively. Forced expression of miR‐503 increased the cellular proliferation and collagen production in mice neonatal CFs. The effects were abrogated by cotransfection with AMO‐503 (a specific inhibitor of miR‐503). Injection of antagomiR‐503 elevated cardiac function and inhibited the expression of connective tissue growth factor (CTGF) and transforming growth factor (TGF)‐β in the TAC mice. Additional analysis revealed that Apelin‐13 is a direct target of miR‐503, as the overexpression of miR‐503 decreased the protein and mRNA expression levels of Apelin‐13. In the CFs with pre‐treatment of AngII, we transfected AMO‐503 into the cells treated with siRNA‐APLN. siRNA‐APLN abolished the effects of AMO‐503 on the production of collagen I and III and the expression of TGF‐β and CTGF. Furthermore, pre‐treatment of CFs with Apelin‐13 (1–100 nmol/l) inhibited angiotensin II‐mediated collagen production and activation of CTGF and TGF‐β. So we conclude that miR‐503 promotes cardiac fibrosis via miR‐503‐Apelin‐13‐TGF‐β‐CTGF‐collagen production pathway. Thus, miR‐503 is a promising therapeutic target for reducing cardiac fibrosis.     

miRNA‐182/Deptor/mTOR axis regulates autophagy to reduce intestinal ischaemia/reperfusion injury

It had been reported miR‐182 was down‐regulated after intestinal ischaemia/reperfusion (I/R) damage. However, its role and potential mechanisms are still unknown. This study was aimed to elucidate the function of miR‐182 in intestinal I/R injury and the underlying mechanisms. The model of intestinal injury was constructed in wild‐type and Deptor knockout (KO) mice. Haematoxylin‐eosin staining, Chiu's score and diamine oxidase were utilized to detect intestinal damage. RT‐qPCR assay was used to detected miR‐182 expression. Electronic microscopy was used to detect autophagosome. Western blot was applied to detect the expression of Deptor, S6/pS6, LC3‐II/LC3‐I and p62. Dual‐luciferase reporter assay was used to verify the relationship between miR‐182 and Deptor. The results showed miR‐182 was down‐regulated following intestinal I/R. Up‐regulation of miR‐182 reduced intestinal damage, autophagy, Deptor expression and enhanced mTOR activity following intestinal I/R. Moreover, suppression of autophagy reduced intestinal damage and inhibition of mTOR by rapamycin aggravated intestinal damage following intestinal I/R. Besides, damage of intestine was reduced and mTOR activity was enhanced in Deptor KO mice. In addition, Deptor was the target gene of miR‐182 and was indispensable for the protection of miR‐182 on intestine under I/R condition. Together, our research implicated up‐regulation of miR‐182 inhibited autophagy to alleviate intestinal I/R injury via mTOR by targeting Deptor.     

Derepression of c‐Fos caused by MicroRNA‐139 down‐regulation contributes to the metastasis of human hepatocellular carcinoma

This study investigates whether the anti‐metastasis effect of microRNA‐139 (miR‐139) on hepatocellular carcinoma (HCC) is mediated through regulating c‐fos expression. The expression levels of miR‐139 and c‐fos in human HCC cell sublines with high (MHCC97H) and low (MHCC97L) spontaneous metastatic potentials were quantified using QPCR or Western blot. miR‐139 mimics was transfected into MHCC97H cells to overexpress miR‐139, and miR‐139 inhibitor was transfected into MHCC97L cells to down‐express miR‐139. The effect of overexpression or down‐expression of miR‐139 on c‐fos expression of MHCC97H and MHCC97L cells was evaluated using QPCR and Western blot. The 3′ untranslated region segments of FOS containing the miR‐139 binding sites were amplified by PCR, and the luciferase activity in the transfected cells was assayed. In comparison with the expression level of miR‐139 in MHCC97L cells, the expression level in MHCC97H cells was significantly decreased, whereas c‐Fos was significantly up‐regulated in MHCC97H. The overexpression of miR‐139 significantly inhibited the expression of c‐fos in MHCC97H cells, and the down‐expression of miR‐139 significantly promoted the expression of c‐fos in MHCC97L cells. miR‐139 suppressed the luciferase activity of the pGL‐FOS by approximately 40% compared with the negative control. In vitro cell migration analysis demonstrated that depletion of c‐fos or overexpression of miR‐139 in MHCC97H cells reduced cell migration, whereas overexpression of c‐fos or depletion of miR‐139 in MHCC97L cells increased cell migration. Thus, we got the conclusion that miR‐139 expression is down‐regulated in human HCC cell sublines with high spontaneous metastatic potentials (MHCC97H). Derepression of c‐Fos caused by miR‐139 down‐regulation contributes to the metastasis of HCC. Copyright © 2012 John Wiley & Sons, Ltd.     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.     

miR‐18a promotes Mycobacterial survival in macrophages via inhibiting autophagy by down‐regulation of ATM

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of leading causes of global deaths. This study aimed to explore the role of miR‐18a in RAW264.7 cells response to Mtb infection. Exosomes derived from Mtb‐infected cells were isolated and further validated by size, transmission electron microscopy and Western blot. RT‐PCR was utilized to measure miR‐18a expression. Cell viability and ultrastructure were examined by CFU counting, CCK‐8 and electron microscope, respectively. Potential target genes of miR‐18a were predicted with bioinformatics and further confirmed using RT‐PCR, Western blot and laser confocal microscope analysis, respectively. LC3, AMPK and mTOR were measured using Western blot. We found that miR‐18a was induced both in Mtb‐infected RAW264.7 cells and its derived exosomes compared with the controls. In addition, up‐regulation of miR‐18a promoted intracellular Mtb survival, attenuated cell viability and reduced LC3‐II level, while its down‐regulation had the opposite effect. miR‐18a overexpression suppressed level of ATM, one possible target of miR‐18a, while its underexpression enhanced ATM. We also found that inhibition of ATM induced LC3‐II decrease in Mtb‐infected cells and could reverse the increase of LC3‐II caused by inhibition of miR‐18a. Moreover, down‐regulation of miR‐18a increased p‐AMPK level while reduction of ATM could reverse the change. Taken together, our results suggest that miR‐18a is up‐regulated in macrophages response to Mtb infection, and it promotes intracellular Mtb survival through repressing autophagic process by down‐regulation of ATM pathway. This provides new thought for TB pathogenesis, diagnosis and treatment.     

Alcohol‐mediated miR‐34a modulates hepatocyte growth and apoptosis

MicroRNAs (miRs) have been recently shown to be heavily involved in the development of alcoholic liver disease (ALD) and suggested as a potential therapeutic target in ALD. The miR‐34a was consistently reported to be significantly elevated in several ALD rodent models, but it remains unclear how miR‐34a modulates the cellular behaviours of hepatocytes in ALD development and progression. This study aims to characterize alcohol‐induced miR‐34a impact on hepatocytes growth and apoptosis. The miRNA array was performed to assess changes in miRNA after chronic alcohol feeding. Liver and blood samples were used to examine ALD progression. The miR‐34a was overexpressed in human hepatocytes to evaluate its impact on cell growth and apoptosis. Real‐time quantitative PCR and Western blot were used to determine the growth and apoptosis molecular signalling pathways associated with miR‐34a. Alcohol feeding significantly promoted fatty liver progression, serum ALT levels, apoptosis and miR‐34a expression in rat liver. Overexpression of miR‐34a in human hepatocytes suppressed cell growth signallings, including c‐Met, cyclin D1 and cyclin‐dependent kinase 6 (CDK6). The miR‐34a might also inhibit the expression of sirtuin 1 (Sirt1) and its target, B‐cell lymphoma 2. Interestingly, the expression of miR‐34a reverses the suppressive effects of ethanol on cell growth. But, miR‐34a promotes hepatocyte senescence and apoptosis. Although the miR‐34a‐mediated down‐regulation of cell growth‐associated genes may contribute to cell growth retardation, other miR‐34a targets, such as Sirt1, may reverse this phenotype. Future studies will be needed to clarify the role of miR‐34a in ALD progression.     

MicroRNA Profile of Tumorigenic Cells During Carcinogenesis of Lung Adenocarcinoma

To obtain microRNA (miRNA) profile and clarify their biological function in tumorigenic Sca‐1⁺CD34⁺ cells during carcinogenesis of lung adenocarcinoma. After intranasal infection with recombinant Adeno‐Cre viruses (AdV‐Cre), lung adenocarcinoma was identified pathologically in Lox‐stop‐lox Kras (LSL‐Kras) G12D mice. Sca‐1⁺CD34⁺ cells were sorted by flow cytometry and tested for tumor‐initiating ability, self‐renewal and tumorigenicity. MiRNA profiles were obtained using microarray and further confirmed by real‐time RT‐PCR (qRT‐PCR). MiRNA functions were predicted bioinformatically, and miR‐294 function was verified to explore its role in tumor migration and invasion. Lung adenocarcinoma was induced in LSL‐Kras G12D mice within 30 days. In vivo, the tumorigenicity of Sca‐1⁺CD34⁺ cells was 25 times stronger than Sca‐1^?CD34^? cells. During tumorigenesis of lung adenocarcinoma, the expression of 145 miRNAs in Sca‐1⁺CD34⁺ cells increased and 72 miRNAs decreased (P < 0.01). Four successively up‐regulated miRNAs (miR‐15a*, miR‐203, miR‐294 and miR‐295*) and three successively down‐regulated ones (miR‐19b, miR‐483 and miR‐615–5p) were identified. Among them, miR‐294 could constitutively bind to 3'‐UTR of matrix metalloproteinase 3 (MMP3), and down‐regulate MMP3 protein expression. MiR‐294 also significantly inhibited migration and invasion of Lewis lung cancer cells. MiRNAs are characteristically expressed in tumor‐initiating Sca‐1⁺CD34⁺ cells of lung adenocarcinoma, and may play important roles during the carcinogenesis of lung adenocarcinoma. J. Cell. Biochem. 116: 458–466, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.