miR-125b Is an adhesion-regulated microRNA that protects mesenchymal stem cells from anoikis

Mesenchymal stem cells (MSCs) have the capacity for multilineage differentiation and are being explored as a source for stem cell-based therapies. Previous studies have shown that adhesion to extracellular matrix plays a critical role in guiding MSC differentiation to distinct lineages. Here, we conducted a focused screen of microRNAs to reveal one microRNA, miR-125b, whose expression changes as a function of cell adhesion. miR-125b expression was upregulated by limiting cell-matrix adhesion using micropatterned substrates, knocking down beta5 integrin or placing cells in suspension culture. Interestingly, we noted that suspending human MSCs (hMSCs) did not induce substantial apoptosis (anoikis) as is typically observed in adherent cells. Although miR-125b appeared to have some effects on hMSC differentiation, we demonstrated a striking role for miR-125b in protecting hMSCs from anoikis. Knockdown of miR-125b increased anoikis while expressing a mimic protected cells. Mechanistic studies demonstrated that miR-125b protected against anoikis by increasing ERK phosphorylation and by suppressing p53. Lastly, we found that miR-125b expression is quite limited in endothelial cells and mouse embryonic fibroblasts (MEFs). The rapid anoikis normally observed in endothelial cells was antagonized by transfection of a miR-125b mimic, suggesting that miR-125b can confer resistance to anoikis in multiple cell types. We also found that endogenous miR-125b was significantly upregulated during reprogramming of MEFs to induced pluripotent cells, suggesting that miR-125b expression may be associated with stem cell populations. Collectively, these observations demonstrate a novel link between cell-matrix adhesion, miR-125b expression, and a stem cell-specific survival program triggered in adhesion-limited contexts such as might occur in early development and wound healing.     

Increased microRNA-155 expression in the serum and peripheral monocytes in chronic HCV infection

ABSTRACT: BACKGROUND: Hepatitis C Virus (HCV), a single stranded RNA virus, affects millions of people worldwide and leads to chronic infection characterized by chronic inflammation in the liver and in peripheral immune cells. Chronic liver inflammation leads to progressive liver damage. MicroRNAs (miRNA) regulate inflammation (miR-155, -146a and -125b) as well as hepatocyte function (miR-122). METHODS: Here we hypothesized that microRNAs are dysregulated in chronic HCV infection. We examined miRNAs in the circulation and in peripheral monocytes of patients with chronic HCV infection to evaluate if specific miRNA expression correlated with HCV infection. RESULTS: We found that monocytes from chronic HCV infected treatment-naive (cHCV) but not treatment responder patients showed increased expression of miR-155, a positive regulator of TNFalpha, and had increased TNFalpha production compared to monocytes of normal controls. After LPS stimulation, miR-155 levels were higher in monocytes from cHCV patients compared to controls. MiR-125b, which has negative regulatory effects on inflammation, was decreased in cHCV monocytes compared to controls. Stimulation of normal monocytes with TLR4 and TLR8 ligands or HCV core, NS3 and NS5 recombinant proteins induced a robust increase in both miR-155 expression and TNFalpha production identifying potential mechanisms for in vivo induction of miR-155. Furthermore, we found increased serum miR-155 levels in HCV patients compared to controls. Serum miR-125b and miR-146a levels were also increased in HCV patients. Serum levels of miR-122 were elevated in cHCV patients and correlated with increased ALT and AST levels and serum miR-155 levels. CONCLUSION: In conclusion, our novel data demonstrate that miR-155, a positive regulator of inflammation, is upregulated both in monocytes and in the serum of patients with chronic HCV infection. Our study suggests that HCV core, NS3, and NS5 proteins or TLR4 and TLR8 ligands can mediate increased miR-155 and TNFalpha production in chronic HCV infection. The positive correlation between serum miR-155 and miR-122 increase in cHCV may be an indicator of inflammation-induced hepatocyte damage.     

The altered expression of inflammation-related microRNAs with microRNA-155 expression correlates with Th17 differentiation in patients with acute coronary syndrome

MicroRNAs (miRNAs) are a novel class of small, non-coding RNAs that play a significant role in both inflammatory and cardiovascular diseases. Immune cells, especially T helper (Th) cells, are critical in the development of atherosclerosis and the onset of acute coronary syndrome (ACS). To assess whether inflammation-related miRNAs (such as miR-155, 146a, 21, 125a-5p, 125b, 31) are involved in the imbalance of Th cell subsets in patients with ACS, we measured the expression of related miRNAs in patients with acute myocardial infarction (AMI), unstable angina (UA), stable angina (SA) and chest pain syndrome (CPS); analyzed the relationship between miRNA expression and the frequency of Th cell subsets; and observed the co-expression of miR-155 and IL-17A in peripheral blood mononuclear cells (PBMCs) of patients with ACS. The results showed that the expression of miR-155 in the PBMCs of patients with ACS was decreased by approximately 60%, while the expression of both miR-21 and miR-146a was increased by approximately twofold. The expression patterns of miRNAs in plasma correlated with those in PBMCs, except for miR-21, which was increased by approximately sixfold in the AMI group and showed no significant difference between the UA group and the CPS group. We also found that the expression of miR-155 inversely correlated with the frequency of Th17 cells (r=-0.896, P<0.01) and that miR-155 was co-expressed with IL-17A in patients with ACS. In conclusion, our study revealed the expression patterns of inflammation-related miRNAs in patients with ACS and found that miR-155 may be associated with Th17 cell differentiation.     

Expression profile of microRNAs in fetal lung development of Sprague-Dawley rats

As well-known regulators of gene expression, microRNAs (miRNAs) play an important role not only in cell proliferation and differentiation, but also in tumorigenesis and organ development. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Simultaneously, in the clinic, with advances in neonatal care, a larger number of premature infants are being saved, and thus diseases of lung development, including bronchopulmonary dysplasia (BPD) have become more and more common. However, only a few miRNA studies have studied their connection with diseases of lung development. In our study, we used a miRNA microarray including more than 1891 capture probes to profile the expression of miRNAs at three time points of rat lung development [embryonic (E) Day 16 (E16), E19, E21]. miRNAs found to have consistent fold-changes (fold-change>2.0) during all three time points were selected and validated by real-time PCR. As a result, 167 differentially expressed miRNAs were found during rat lung organogenesis, including 81 upregulated and 86 downregulated miRNAs. Seven miRNAs were selected and characterized by having a consistent >2-fold changes between all three groups. Among these 7 miRNAs, except for let-7a, the other 6 miRNAs (miR-1949, miR-125b-5p, miR-296, miR-93, miR-146b, miR-3560) are all first reported for the first time in lung development. Finally, due to the fact that they demonstrated higher fold changes, from these 7 miRNAs we selected miR-125b-5p, miR-296, miR-93, miR-146b and miR-3560 for real-time PCR. We hypothesized that these newly identified miRNAs may play an important role in fetal lung development, and this experimental result could help us to further clarify the mechanism of normal lung development including the development of type?II pneumocytes. This may provide a physiological basis for future research on diseases of lung development.     

Down-regulation of microRNA-126 and microRNA-133b acts as novel predictor biomarkers in progression and metastasis of non small cell lung cancer

Objective: MiRNAs play crucial roles in progression of cancer. However, the underlying mechanisms of miRNAs in non small cell lung cancer are still poorly understood. The aim of this study was to investigate the expression level of microRNA-126 (miR-126) and microRNA-133b (miR-133b) and also their association with clinicopathological features in patients with non small cell lung cancer (NSCLC). Methods: Total RNA was purified from NSCLC tissues and adjacent non-tumor tissues and then quantitative real-time PCR (qRT-PCR) was used to evaluate the expression rate of microRNAs. Furthermore, the association of miR-126 and miR-133b level with clinicopathological features and prognosis were evaluated. Results: Our findings showed that expression of miR-126 was decreased in NSCLC tissues compared with adjacent non-tumor tissues. On the other hand, a lower expression of miR-133b was seen in NSCLC tissues when compared with adjacent non-tumor tissues. In term of miR-126, our results showed that miR-126 was associated with tumor stage and lymph nodes metastasis (P<0.05). In term of miR-133b, our finding indicated that decreased expression of miR-133b was correlated with advanced tumor stage and lymph nodes metastasis (P<0.05). Kaplan-Meier analysis and log-rank test indicated that patients with low expression of miR-126 and miR-133b had a shorter overall survival (log-rank test; P<0.05). Multivariate Cox proportional hazards model revealed that low expression of miR-126 and miR-133b, advanced tumor stage and lymph nodes metastasis were independent prognostic factors for overall survival of NSCLC patients. Conclusions: These findings suggested that miR-126 and miR-133b might play a key role in the progression and metastasis of NSCLC and would be applied as a novel therapeutic agent.     

Association between active pulmonary tuberculosis and circulating microRNAs: a preliminary study from Turkey

Background/aim Tuberculosis is a public health problem that still remains significant. For prevention, diagnosis, and treatment of tuberculosis more effective novel biomarkers are needed. MicroRNAs can regulate innate and adaptive immune responses, alter host-pathogen interactions, and affect progression of diseases. The relationship between microRNA expression and active pulmonary tuberculosis (APT) has not yet been investigated in the Turkish population. We aimed to test the potential diagnostic value of some microRNAs whose levels were previously reported to be altered in APT patients.Materials and methods Using two different references (U6 and miR-93), we compared the expression levels of potentially important microRNAs in serum of APT patients with healthy individuals using quantitative polymerase chain reaction (qPCR).Results miR-144 expression level was down-regulated in APT patients when either U6 or miR-93 was used for normalization. When data was normalized with miR-93, a statistically significant decrease in miR-125b (0.8 fold) and miR-146a (0.7 fold) expression levels were observed, while no differences were detected for U6. The receiver operating characteristic suggested that miR-144 may be a candidate biomarker for discriminating APT patients and controls (p < 0.05) both for U6 and miR-93.Conclusion These findings suggest that miR-144 can have potential as a biomarker for APT. Using a single reference may be misleading in evaluation of microRNA expression. U6 and miR-93 can be used in combination as references for normalization of serum microRNA expression data.     

HCV核心蛋白对HepG2 细胞microRNA表达谱的影响

 摘要：目的 分析HCV核心蛋白（HCV-Core）对HepG2细胞microRNA表达谱的影响。方法 将构建好的重组真核表达质粒pCDNA3.1（+）/HCV-Core用脂质体转染HepG2细胞,经G418筛选得到稳定转染HCV-Core的HepG2细胞,命名为HepG2-HCV Core细胞株。然后用反转录聚合酶链反应（RT-PCR）,细胞免疫荧光和Western blot对HCV核心蛋白在HepG2细胞里mRNA和蛋白水平的表达情况进行验证。利用博奥生物公司的miRNA Array基因芯片,分析稳定表达HCV核心蛋白的HepG2细胞和转染空质粒pCDNA3.1（+）的HepG2细胞二者microRNA表达谱的差异,最后用Taqman探针荧光定量PCR法进行验证。 结果 感染HCV-Core后,HepG2细胞表达有差异的microRNA有8种,用Taqman探针荧光定量PCR法进行分析,发现HCV核心蛋白可以上调miR-29、 miR-146a、miR-149、miR-192、miR-221、miR-222和miR-193b;下调miR-196a。结论 HCV核心蛋白可以诱导肝细胞microRNA表达谱发生改变,在肝癌发生发展过程中发挥重要作用。     

Angiotensin II induced differentially expressed microRNAs in adult rat cardiac fibroblasts

Angiotensin II (Ang II) plays a pivotal role in cardiac fibrosis, and microRNAs (miRNAs) have been shown to participate in diverse pathological processes. Our aim is to identify the Ang II-induced miRNAs in cardiac fibroblasts (CFs). The miRNA array was used to analyze the miRNA expression profile in CFs treated by Ang II and control cells. Stem-loop real-time PCR was performed to re-measure the levels of the differentially expressed miRNAs. Analysis of miRNA arrays showed that 33 miRNAs were differentially expressed (13 up- and 20 downregulated) in response to Ang II (100 nM) for 24 h as compared to control cells. Quantitative PCR revealed that Ang II upregulated the levels of miR-132, -125b-3p and miR-146b but downregulated the levels of miR-300-5p, -204* and miR-181b in CFs. The trend of miRNA change is consistent with microarray and qRT-PCR. Bioinformatic analysis revealed that MMP9 as the target of miR-132, MMP16 as the target of miR-146b and TIMP3 as the target of miR-181b have been listed in the miR database with experimentally validated targets, indicating the potential role of those miRNAs in cardiac fibrosis. Our results demonstrated that we did identify a subset of miRNAs that was differentially expressed in Ang II-treated CFs, which provide a starting point to explore their potential roles in cardiac fibrosis and hypertension.     

HPV-16 E6 promotes cell growth of esophageal cancer via downregulation of miR-125b and activation of Wnt/β-catenin signaling pathway

High-risk human papillomavirus (HPV) is a possible cause of esophageal cancer. However, the molecular pathogenesis of HPV-infected esophageal cancer remains unclear. The expression levels of some microRNAs including miR-125b have been negatively correlated with HPV infection, and miR-125b downregulation is associated with tumorigenesis. In addition, Wnt/β-catenin signaling pathway has been suggested to play an important role in esophageal cancer (EC). We examined miR-125b and Wnt/β-catenin signaling pathway in HPV-16 E6 promoted tumor progression in EC. HPV-16 E6 transfection decreased markedly the expression levels of miR-125b and promoted the colony formation in the Eca 109 and Kyse 150 cell lines, and restoration of miR-125b expression level antagonized the increased colony formation in HPV-16 E6 transfected cell lines. We also demonstrated that overexpression of E6 upregulated the Wnt/β-catenin signaling activity via modulating the multiple regulators including TLE1, GSK3β, and sFRP4. Overexpression of miR-125b restored the expression levels of these proteins. Expression of miR-125b was lower in HPV-16 E6 positive esophageal cancer tissues, and was negatively correlated with E6 mRNA levels. Our results indicate that HPV-16 E6 promotes tumorigenesis in EC via down-regulation of miR-125b, and this underlying mechanism may be involved in the activation of the Wnt/β-catenin signaling pathway.     

类风湿关节炎患者外周血miR-146a、miR-16的表达水平及临床指标与中医证型的关系探讨

目的研究类风湿关节炎（RA）患者外周血miR-146a、miR-16的表达及ESR、CRP、RF、抗CCP抗体与中医证型的关系,为RA辨证分型提供客观依据。方法根据《中药新药临床研究指导原则》有关RA的中医证候诊断标准,主要分为两个证型：风湿夹瘀型（n=20）和肝肾亏损型（n=16）;16名健康体检者作为健康对照组。Real-timePCR检测RA患者及健康对照组miR-146a、miR-16的表达,记录ESR、CRP、RF、抗CCP抗体等临床指标。结果风湿夹瘀型miR-146a、miR-16的表达水平高于肝肾亏损型和对照组（P均〈0.05）,肝肾亏损型miR-146a、miR-16的表达水平与健康对照组相比差异无统计学意义（P均〉0.05）,风湿夹瘀型RA患者ESR及CRP高于肝肾亏损型（P〈0.01和P〈0.05）,RF低于肝肾亏损型（P〈0.05）,抗CCP抗体在两种证型之间差异无统计学意义（P〉0.05）。结论 miR-146a、miR-16的表达水平和ESR、CRP、RF等临床指标可能为RA风湿夹瘀及肝肾亏损型的辨证分型提供参考。     

MiR-146a modulates macrophage polarization in systemic juvenile idiopathic arthritis by targeting INHBA

Monocytes from patients with systemic juvenile idiopathic arthritis (SJIA) have both features of classical activated M1 and alternatively activated M2 macrophages. An increasing number of studies have indicated that microRNAs (miRNAs) are critical regulators of monocyte polarization. Here, we focused on miR-146a expression in SJIA and investigated the function of miR-146a in monocyte polarization. We found that miR-146a expression was highly up-regulated in SJIA monocytes and correlated with the systemic features. miR-146a was expressed at a higher level in monocytes polarized with M2 conditions than those polarized with M1 conditions. miR-146a overexpression significantly decreased the production of M1 phenotype markers such as IL-6, IL-12, TNF-α, CD86 and iNOS in M1 macrophages, but increased the production of M2 marker genes such as Arg1, CCL17, CCL22 and CD206 in M2 macrophages. Conversely, knockdown of miR-146a promoted M1 macrophage polarization but diminished M2 macrophage polarization. We subsequently demonstrated that miR-146a targeted the 3′-untranslated region (UTR) of INHBA to inhibit its expression. Additionally, INHBA overexpression rescued the reduced IL-6, IL-12, and TNF-α levels induced by miR-146a overexpression in M1 macrophages, and rescued the increased Arg1, CCL17, and CCL22 levels induced by miR-146a overexpression in M2 macrophages. Similarly, the effects of miR-146a inhibition in monocyte polarization were all partly reversed by INHBA inhibition. Taken together, the data suggest that miR-146a serves as a molecular regulator in monocyte polarization and might play an important role in monocytes from patients with SJIA.     

FMR1基因敲除小鼠海马组织中microRNA-125b和microRNA-132的表达

目的 检测FMR1基因敲除小鼠海马组织中microRNA-125b(miR-125b)和miR-132表达水平,探讨脆性X智力低下蛋白(FMRP)缺失是否通过改变其转录后表达影响树突棘发育.方法 取FVB近交系雄性1周龄FMR1基因敲除型(K0)和同龄野生型(WT)小鼠各3只,取海马组织标本,应用microRNA芯片技术和荧光定量实时PCR检测miR-125b和miR-132的表达.结果 microRNA芯片检测显示,1周龄WT与KO小鼠海马组织miR-125b和miR-132的荧光值比较,差异无统计学意义(miR-125b:4 919.295±431.981比4 997.578±141.402;miR- 132:244.289±31.125比238.517±62.275,均P＞0.05);荧光定量实时PCR检测显示,miR-125b和miR-132的相对表达量差异亦无统计学意义(miR-125b:11.45±0.32比11.55±0.43;miR- 132:18.28±0.34比18.50±0.40,均P＞0.05).结论 脆性X综合征树突棘发育不良与miR-125b和miR-132的转录后表达水平无关.     

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin,and ALPL gene expression

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.     

MicroRNA-125b在胃癌组织中的高表达并促进胃癌细胞系HGC-27和MGC-803的增殖

 目的 探讨microRNA-125b (miR-125b)基因在胃癌患者组织中的表达改变情况,及其对胃癌细胞系增殖和凋亡的影响。方法 使用real-time PCR方法检测miR-125b在40例临床诊断为胃癌患者的癌组织与癌旁对照组织中的表达情况。随后使用miR-125b mimic转染胃癌细胞系HGC-27和MGC-803,确认过表达成功后,分别使用CCK-8试剂盒和流式细胞仪检测过表达miR-125b对细胞增殖和凋亡的影响。结果 证实在胃癌患者组织中miR-125b的表达水平显著高于癌旁对照组（P<0.01）。在胃癌细胞系HGC-27和MGC-803中过表达miR-125b后,细胞增殖明显增加：转染72h,HGC-27（scramble组：1.632±0.09,mimic组：2.473±0.08）,MGC-803（scramble组：1.603±0.05,mimic组：2.554±0.07））,同时细胞凋亡也受到抑制。结论 miR-125b可能作为癌基因在胃癌中发挥作用,并对细胞增殖和凋亡具有显著影响。     

microRNA-146 up-regulation predicts the prognosis of non-small cell lung cancer by miRNA in situ hybridization

Non-small cell lung cancer (NSCLC) accounts for approximately 70% of all lung cancer-related deaths worldwide. Prognostic markers are essential for the early detection of lung cancer in patients. In this study, we first identified microRNA146 (miR-146) expression in cancer cell lines using miRNA in situ hybridization (MISH) and confirmed the accuracy of MISH using q-RT-PCR. In addition, two different systems, BCIP/NBT and ELF, were used to detect the signal for a comparative analysis of the specificity of MISH. Compared to the BCIP/NBT system, the ELF detection system was more effective for MISH. Furthermore we detected the expression of miR-146 in NSCLC tissues (43 cases) and normal tissues (32 cases). Based on our results, we can conclude that miR-146 is more highly expressed in cancer tissue than normal tissue (t-test, P < 0.05) and that miR-146 can predict the prognosis of NSCLC by MISH. Our findings preliminary demonstrate that MISH can be applied as a molecular diagnostic tool to determine the expression and localization of miRNAs in cancer tissues and that miR-146, determined by MISH, predicts the prognosis of NSCLC patients.     

The occurrence of cervical cancer in Uygur women in Xinjiang Uygur Autonomous Region is correlated to microRNA-146a and ethnic factor

Aims: The present study is to investigate the effect of microRNA-146a (miR-146a) and ethnic factor in the occurrence of cervical cancer in Uygur women in Xinjiang Uygur Autonomous Region. Methods: A total of 620 pieces of cervical tissues were obtained between September 2010 and September 2013, including 208 cases of cervicitis, 207 cases of cervical intraepithelial neoplasia, and 205 cases of cervical cancer. The relative expression of miR-146a in tissues was measured using quantitative real-time polymerase chain reaction. Polymerase chain reaction - restriction fragment length polymorphism was used to determine the genotypes of miR-146a (rs2910164). Differences between two groups and multiple groups were compared using t-test and one-factor analysis of variance, respectively. Comparison of genotype compositions and genetic balance examinations were performed using χ² test. Results: Uygur women had earlier age of marriage, more times of pregnancy, and more childbirths than Han women. The miR-146a (rs2910164) genotype composition was significantly different between Uygur and Han, with the ratio of GG genotype in Uygur being higher than that in Han. Logistic regression analysis showed that miR-146a (rs2910164) genotypes were significantly correlated to ethnic factor and tumor sizes. The expression of miR-146a was elevated in cervical intraepithelial neoplasia and cervical cancer, especially for Uygur women, with the GG genotype being the most highly expressed. Conclusions: The miR-146a (rs2910164) polymorphism is significantly correlated to ethnic factor and tumor diameters. miR-146a has differential expression in cervical tissues. Allele G of miR-146a (rs2910164) is related to the high expression of miR-146a, and the progression of cervical cancer.