miR-135a targets IRS2 and regulates insulin signaling and glucose uptake in the diabetic gastrocnemius skeletal muscle

Although aberrant miRNA signatures are associated with diabetes, yet, the status and role of altered miRNAs in the diabetic skeletal muscle is currently poorly understood. Here, we report that 41 miRNAs are altered in the diabetic gastrocnemius skeletal muscle and of these, miR-135a that is identified as a critical regulator of myogenesis, is significantly up-regulated. IRS2 is predicted as its potential putative target and its levels are down-regulated in the diabetic gastrocnemius skeletal muscle. In C2C12 cells, while miR-135a levels decreased during differentiation, IRS2 levels were up-regulated. miR-135a significantly reduced IRS2 protein levels and its 3′UTR luciferase reporter activity and these were blunted by the miR-135a inhibitor and mutation in the miR-135a binding site. Knock-down of endogenous miR-135a levels increased IRS2 at the mRNA and protein levels. miR-135a also attenuated insulin stimulated phosphorylation and activation of PI3Kp85α and Akt and glucose uptake. miR-135a levels were also found to be elevated in the human diabetic skeletal muscle. In-vivo silencing of miR-135a alleviated hyperglycemia, improved glucose tolerance and significantly restored the levels of IRS2 and p-Akt in the gastrocnemius skeletal muscle of db/db mice without any effect on their hepatic levels. These suggest that miR-135a targets IRS2 levels by binding to its 3′UTR and this interaction regulates skeletal muscle insulin signaling.     

miR-15b and miR-16 regulate TNF mediated hepatocyte apoptosis via BCL2 in acute liver failure

Acute liver failure (ALF) still has an unacceptable high mortality rate, despite substantial improvements with multidisciplinary care. The precise underlying mechanism of ALF remains to be explored. It has been reported that microRNAs (miRNAs) are novel regulators in a number of liver diseases, but the role of miRNAs in the development of ALF is not fully understood. An ALF murine model was generated by ip injection of D: -GalN/LPS, which was confirmed with histopathology and biochemistry. The hepatic miRNA expression profile in ALF was determined by microarray and verified by qRT-PCR. The functions and signal pathways of the targeted genes of these deregulated miRNAs were predicted, using bioinformatics analysis. The possible underlying mechanism was investigated by exploring the relationship between miRNA modification and hepatocyte apoptosis. There were a total of 95 significantly changed miRNAs in ALF compared to mock-treated (P < 0.01). Among these 95 miRNAs, 20 were up-regulated and 26 were down-regulated at both 5 and 7 h time points. Bioinformatics analysis predicted that some of these 46 miRNAs were involved in apoptosis. Among the up-regulated miRNAs involved in apoptosis, miR-15b and miR-16 showed the highest enrichment and targeted the common anti-apoptotic gene, BCL2. Our in vitro data demonstrated that miR-15b and/or miR-16 regulated BCL2 at the protein level. Inhibition of miR-15b and/or miR-16 reduced hepatic apoptosis and TNF production. These data suggest that miR-15b and miR-16 regulate TNF mediated hepatic apoptosis via BCL2 during ALF, and may shed light on the development of a therapeutic strategy for treatment of ALF.     

胃癌多药耐药相关microRNA的筛选和鉴定

目的:研究胃癌多药耐药相关microRNA并对其进行鉴定、靶基因预测和预测靶基因的生物信息学分析。方法:运用microRNA芯片对胃癌多药耐药细胞SGC7901/ADR和其亲本细胞SGC7901进行microRNA表达谱分析;采用实时定量PCR的方法对差异表达的miRNA进行验证;再运用生物信息学方法对差异表达的miRNA进行靶基因预测;再对预测的靶基因进行GO和KEGG通路分析。结果:与SGC7901相比SGC7901/ADR表达上调超过2倍的miRNA有6个,表达下调超过2倍的有11个。实时定量PCR对共同差异表达的microRNA进行验证显示与芯片结果的一致性。对这17个差异表达的miRNA进行靶基因预测,再对预测得到的靶基因进行GO和KEGG通路分析显示预测的靶基因参与了肿瘤相关通路、MAPK通路、Focal Adhesion通路等。结论:我们初步筛选得到了胃癌多药耐药相关miRNA并对其进行了生物信息学分析,为进一步地探索miRNA在胃癌多药耐药中的作用及其分子机制奠定了基础。     

Differential expression of microRNAs in decidua-derived mesenchymal stem cells from patients with pre-eclampsia

Background

Mesenchymal stem cells (MSCs) at maternal-fetal interface are considered to play an important role in the pathogenesis of pre-eclampsia (PE). microRNAs (miRNAs) also have an important influence on differentiation, maturation, and functions of MSCs. Our aim in this study was to determine the differential expression of miRNAs in decidua-derived MSCs (dMSCs) from severe PE and normal pregnancies.

Results

miRNA expression profiles in dMSCs from five patients with severe PE and five healthy pregnant women were screened using microarray. Then, bioinformatic analysis of the microarray results was performed. Out of 179 differentially expressed miRNAs, 49 miRNAs had significant (p < 0.05) differential expression of ≥ 2.0-fold changes, including 21 up-regulated and 28 down-regulated. miRNA-Gene-network and miRNA-Gene ontology (GO) -network analyses were performed. Overall, 21 up-regulated and 15 down-regulated miRNAs showed high degrees in these analyses. Moreover, the significantly enriched signaling pathways and GOs were identified. The analyses revealed that pathways associated with cell proliferation, angiogenesis, and immune functions were highly regulated by the differentially expressed miRNAs, including Wnt signaling pathway, mitogen-activated protein kinase signaling pathway, transforming growth factor beta signaling pathway, T-cell receptor signaling pathway, and B cell receptor signaling pathway. Four miRNA predicted target genes, vascular endothelial growth factor A (VEGFA), indoleamine 2,3-dioxygenase, suppression of cytokine signaling 3, and serine/threonine protein phosphatase 2A 55 kDa regulatory subunit B α isoform (PPP2R2A) were all decreased in dMSCs from patients with PE. Furthermore, the physiological roles of miR-16 and miR-136 in the down-regulation of VEGFA and PPP2R2A, respectively, were confirmed through reporter assays.

Conclusions

These findings suggest that miRNAs in dMSCs may be important regulatory molecules in the development of PE.     

Differential expression of microRNAs in mouse liver under aberrant energy metabolic status

Despite years of effort, exact pathogenesis of nonalcoholic fatty liver disease (NAFLD) remains obscure. To gain an insight into the regulatory roles of microRNAs (miRNAs) in aberrant energy metabolic status and pathogenesis of NAFLD, we analyzed the expression of miRNAs in livers of ob/ob mice, streptozotocin (STZ)-induced type 1 diabetic mice, and normal C57BL/6 mice by miRNA microarray. Compared with normal C57BL/6 mice, ob/ob mice showed upregulation of eight miRNAs and downregulation of four miRNAs in fatty livers. Upregulation of miR-34a and downregulation of miR-122 was found in livers of STZ-induced diabetic mice. These results demonstrate that distinct miRNAs are strongly dysregulated in NAFLD and hyperglycemia. Comparison between miRNA expressions in livers of ob/ob mice and STZ-administered mice further revealed upregulation of four miRNAs and downregulation of two miRNAs in livers of ob/ob mice, indicating that these miRNAs may represent a molecular signature of NAFLD. A distinctive miRNA expression pattern was identified in ob/ob mouse liver, and hierarchical clustering of this pattern could clearly discriminate ob/ob mice from either normal C57BL/6 mice or STZ-administered mice. These findings suggest an important role of miRNAs in hepatic energy metabolism and implicate the participation of miRNAs in the pathophysiological processes of NAFLD.     

Differential microRNA Profiles and Their Functional Implications in Different Immunogenetic Subsets of Chronic Lymphocytic Leukemia

Critical processes of B-cell physiology, including immune signaling through the B-cell receptor (BcR) and/or Toll-like receptors (TLRs), are targeted by microRNAs. With this in mind and also given the important role of BcR and TLR signaling and microRNAs in chronic lymphocytic leukemia (CLL), we investigated whether microRNAs could be implicated in shaping the behavior of CLL clones with distinct BcR and TLR molecular and functional profiles. To this end, we examined 79 CLL cases for the expression of 33 microRNAs, selected on the following criteria: (a) deregulated in CLL versus normal B-cells; (b) differentially expressed in CLL subgroups with distinct clinicobiological features; and, (c) if meeting (a) + (b), having predicted targets in the immune signaling pathways. Significant upregulation of miR-150, miR-29c, miR-143 and miR-223 and downregulation of miR-15a was found in mutated versus unmutated CLL, with miR-15a showing the highest fold difference. Comparison of two major subsets with distinct stereotyped BcRs and signaling signatures, namely subset 1 [IGHV1/5/7-IGKV1(D)-39, unmutated, bad prognosis] versus subset 4 [IGHV4-34/IGKV2-30, mutated, good prognosis] revealed differences in the expression of miR-150, miR-29b, miR-29c and miR-101, all down-regulated in subset 1. We were also able to link these distinct microRNA profiles with cellular phenotypes, importantly showing that, in subset 1, miR-101 downregulation is associated with overexpression of the enhancer of zeste homolog 2 (EZH2) protein, which has been associated with clinical aggressiveness in other B-cell lymphomas. In conclusion, specific miRNAs differentially expressed among CLL subgroups with distinct BcR and/or TLR signaling may modulate the biological and clinical behavior of the CLL clones.     

Grouping Pentylenetetrazol-Induced Epileptic Rats According to Memory Impairment and MicroRNA Expression Profiles in the Hippocampus

Previous studies have demonstrated a close relationship between abnormal regulation of microRNA (miRNA) and various types of diseases, including epilepsy and other neurological disorders of memory. However, the role of miRNA in the memory impairment observed in epilepsy remains unknown. In this study, a model of temporal lobe epilepsy (TLE) was induced via pentylenetetrazol (PTZ) kindling in Sprague-Dawley rats. First, the TLE rats were subjected to Morris water maze to identify those with memory impairment (TLE-MI) compared with TLE control rats (TLE-C), which presented normal memory. Both groups were analyzed to detect dysregulated miRNAs in the hippocampus; four up-regulated miRNAs (miR-34c, miR-374, miR-181a, and miR-let-7c-1) and seven down-regulated miRNAs (miR-1188, miR-770-5p, miR-127-5p, miR-375, miR-331, miR-873-5p, and miR-328a) were found. Some of the dysregulated miRNAs (miR-34c, miR-1188a, miR-328a, and miR-331) were confirmed using qRT-PCR, and their blood expression patterns were identical to those of their counterparts in the rat hippocampus. The targets of these dysregulated miRNAs and other potentially enriched biological signaling pathways were analyzed using bioinformatics. Following these results, the MAPK, apoptosis and hippocampal signaling pathways might be involved in the molecular mechanisms underlying the memory disorders of TLE.     

Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs

In order to understand the role of microRNAs (miRNAs) in vascular physiopathology, we took advantage of deep-sequencing techniques to accurately and comprehensively profile the entire miRNA population expressed by endothelial cells exposed to hypoxia. SOLiD sequencing of small RNAs derived from human umbilical vein endothelial cells (HUVECs) exposed to 1% O₂ or normoxia for 24 h yielded more than 22 million reads per library. A customized bioinformatic pipeline identified more than 400 annotated microRNA/microRNA* species with a broad abundance range: miR-21 and miR-126 totaled almost 40% of all miRNAs. A complex repertoire of isomiRs was found, displaying also 5′ variations, potentially affecting target recognition. High-stringency bioinformatic analysis identified microRNA candidates, whose predicted pre-miRNAs folded into a stable hairpin. Validation of a subset by qPCR identified 18 high-confidence novel miRNAs as detectable in independent HUVEC cultures and associated to the RISC complex. The expression of two novel miRNAs was significantly down-modulated by hypoxia, while miR-210 was significantly induced. Gene ontology analysis of their predicted targets revealed a significant association to hypoxia-inducible factor signaling, cardiovascular diseases, and cancer. Overexpression of the novel miRNAs in hypoxic endothelial cells affected cell growth and confirmed the biological relevance of their down-modulation. In conclusion, deep-sequencing accurately profiled known, variant, and novel microRNAs expressed by endothelial cells in normoxia and hypoxia.     

Differentially expressed miR-20, miR-21, miR-100, miR-125a and miR-146a as a potential biomarker for prostate cancer

Prostate cancer is the leading cause of death among men worldwide. Deregulation of microRNAs has been reported in many cancers. Expression of microRNAs miR-20a-5p, miR-21-5p, miR-100-5p, miR-125a-5p and miR-146a-5p in tissue blocks of histologically confirmed prostate cancer patients compared with BPH patients, to identify potential microRNA biomarker for prostate cancer. MicroRNA was isolated and expression was quantified by qRT-PCR using Taqman Advanced microRNA assay kits. The interactions between the microRNA:target mRNA were predicted by using bioinformatics tools such as miRwalk and miRTargetlink. The experimentally validated targets were analysed using gprofiler to identify their molecular function, biological process and related pathways. The expression analysis revealed that miR-21 and miR-100 were significantly down-regulated whereas miR-125a was up-regulated in prostate cancer patients. Comparative analysis of the expression levels with tumor grading reveal that miR-100 was significantly down-regulated (p?<?0.05) in high grade tumor, indicating that miR-100 associated with prostate cancer. ROC analysis revealed that combined analysis of down-regulated miRNAs (miR-21 and miR-100) shown AUC of 0.72 (95% CI 0.65–0.79). The combined analysis of all five miRNAs showed AUC of 0.87 (95% CI 0.81–0.92). The targets prediction analysis revealed several validated targets including BCL2, ROCK1, EGFR, PTEN, MTOR, NAIF1 and VEGFA. Our results provide evidence that combined analysis of all the five miRNAs as a panel can significantly improve the prediction level of the presence of prostate cancer and may be used as a potential diagnostic biomarker.

     

Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy

Diabetic nephropathy, as a severe microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. However, no effective therapeutic strategy has been developed to prevent renal damage progression to end stage renal disease. Hence, the present study evaluated the protective effects of grape seed procyanidin B2 (GSPB2) and explored its molecular targets underlying diabetic nephropathy by a comprehensive quantitative proteomic analysis in db/db mice. Here, we found that oral administration of GSPB2 significantly attenuated the renal dysfunction and pathological changes in db/db mice. Proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified 53 down-regulated and 60 up-regulated proteins after treatment with GSPB2 in db/db mice. Western blot analysis confirmed that milk fat globule EGF-8 (MFG-E8) was significantly up-regulated in diabetic kidney. MFG-E8 silencing by transfection of MFG-E8 shRNA improved renal histological lesions by inhibiting phosphorylation of extracellular signal-regulated kinase1/2 (ERK1?2), Akt and glycogen synthase kinase-3beta (GSK-3β) in kidneys of db/db mice. In contrast, over-expression of MFG-E8 by injection of recombinant MFG-E8 resulted in the opposite effects. GSPB2 treatment significantly decreased protein levels of MFG-E8, phospho-ERK1/2, phospho-Akt, and phospho-GSK-3β in the kidneys of db/db mice. These findings yield insights into the pathogenesis of diabetic nephropathy, revealing MFG-E8 as a new therapeutic target and indicating GSPB2 as a prospective therapy by down-regulation of MFG-E8, along with ERK1/2, Akt and GSK-3β signaling pathway.     

miR-1-2 gets to the heart of the matter

Although many microRNAs (miRNAs) and their targets have been identified, the importance of miRNAs in vivo is still unclear. In this issue, Zhao et al. (2007) generate mice deficient in a cardiac-specific miRNA, miR-1-2, and reveal that this microRNA plays a crucial role in heart development and physiology.     

The rno-miR-34 family is upregulated and targets ACSL1 in dimethylnitrosamine-induced hepatic fibrosis in rats

The mechanisms whereby hepatic fibrosis develops in chronic liver diseases remain incompletely defined. Here, we sought to examine whether microRNA (miRNA) became dysregulated in dimethylnitrosamine-induced hepatic fibrosis in rats. Our microarray analysis revealed that the miR-34 family was upregulated along with other miRNAs in liver fibrotic tissues. Six miRNAs, such as rno-miR-878, were downregulated. The findings were confirmed by RT-PCR assays. Gene ontology analysis further showed that many of these dysregulated miRNAs were involved in lipid/fatty acid metabolism. The acyl-CoA synthetase long-chain family member 1 (ACSL1) gene contained specific binding sites for miR-34a/miR-34c. Additional enhanced green fluorescence protein reporter activity assays indicated that the miR-34 family targeted ACSL1. Our RT-PCR and immunoblotting assays further demonstrated that both the mRNA and protein levels of ACSL1 were markedly reduced in fibrotic liver tissues. Our findings suggest that miRNA becomes dysregulated during hepatic fibrosis, and that the miR-34 family may be involved in the process by targeting ACSL1.     

MicroRNA-122a functions as a novel tumor suppressor downstream of adenomatous polyposis coli in gastrointestinal cancers

Aberrant regulation of APC/β-catenin signaling pathway is common in the pathogenesis of colorectal and other cancers. Targets regulated by APC/β-catenin signaling pathway play crucial roles in cancer development. In the current study, we aimed to illustrate the influence of APC/β-catenin signaling pathway on expression of microRNAs, one new group of players important to carcinogenesis. Restoration of APC function in colorectal cancer cells led to the deregulation of several cancer-related microRNAs, such as miR-122a which was recognized as the liver-specific microRNA. MiR-122a was down-regulated in gastrointestinal cancer cell lines as well as primary carcinoma tissues. Inhibition of miR-122a could reverse wild-type APC-induced growth inhibition of gastrointestinal cancer cells while miR-122a mimic inhibited cell growth. In summary, we identified some cancer-related microRNAs regulated by APC/β-catenin signaling pathway. The down-regulation of miR-122a mediated by aberrant APC/β-catenin signaling is important to the pathogenesis of gastrointestinal cancers.     

MiR-21 plays an important role in radiation induced carcinogenesis in BALB/c mice by directly targeting the tumor suppressor gene Big-h3

Dysregulation of certain microRNAs (miRNAs) in cancer can promote tumorigenesis, metastasis and invasion. However, the functions and targets of only a few mammalian miRNAs are known. In particular, the miRNAs that participates in radiation induced carcinogenesis and the miRNAs that target the tumor suppressor gene Big-h3 remain undefined. Here in this study, using a radiation induced thymic lymphoma model in BALB/c mice, we found that the tumor suppressor gene Big-h3 is down-regulated and miR-21 is up-regulated in radiation induced thymic lymphoma tissue samples. We also found inverse correlations between Big-h3 protein and miR-21 expression level among different tissue samples. Furthermore, our data indicated that miR-21 could directly target Big-h3 in a 3'UTR dependent manner. Finally, we found that miR-21 could be induced by TGFβ, and miR-21 has both positive and negative effects in regulating TGFβ signaling. We conclude that miR-21 participates in radiation induced carcinogenesis and it regulates TGFβ signaling.