Identification of miR-200a as a novel suppressor of connexin 43?in breast cancer cells

Both miRNAs (miRs) and connexin 43 (Cx43) were important regulators of the metastasis of breast cancer, whereas the miRs regulating Cx43 expression in breast cancer cells were still obscure. In the present study, we scanned and found miR-1, miR-206, miR-200a, miR-381, miR-23a/b and miR-186 were functional suppressors of human Cx43 mRNA and protein expression. Specially, we demonstrated that only miR-200a could directly target the 3′-untranslated region (3′-UTR) of human Cx43 gene. Functionally, overexpression of Cx43 in MCF cells potentiated the migration activity, whereas additional miR-200a treatment notably prevented this effect. Finally, we demonstrated that decreased levels of miR-200a and elevated expression of Cx43 in the metastatic breast cancer tissues compared with the primary ones. Thus, we are the first to identify miR-200a as a novel and direct suppressor of human Cx43, indicating that miR200a/Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.     

Aldose reductase regulates miR-200a-3p/141-3p to coordinate Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling in renal mesangial cells and the renal cortex of diabetic mice

Aberrant regulation in oxidative stress, fibrogenesis, and the epithelial–mesenchymal transition (EMT) in renal cells under hyperglycemic conditions contributes significantly to the onset and progression of diabetic nephropathy. The mechanisms underlying these hyperglycemia-induced dysregulations, however, have not been clearly elucidated. Herein, we report that aldose reductase is capable of regulating the expression of miR-200a-3p/141-3p negatively in renal mesangial cells. MiR-200a-3p/141-3p, in turn, act to target Keap1, Tgfβ2, fibronectin, and Zeb2 directly and regulate Tgfβ1 and Nrf2 indirectly under high-glucose conditions, resulting in profound dysregulations in Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling. In vivo in streptozotocin-induced diabetic mice, we found that aldose reductase deficiency caused significant elevations in miR-200a-3p/141-3p in the renal cortex, which were accompanied by a significant downregulation of Keap1, Tgfβ1/2, and fibronectin but significant upregulation of Nrf2. Moreover, in vivo administration of inhibitors of miR-200a-3p in diabetic animals significantly exacerbated cortical and glomerular fibrogenesis and increased urinary albumin excretion, tightly linking dysregulated miR-200a-3p with the development of diabetic nephropathy. Collectively, our results reveal a novel mechanism whereby hyperglycemia induces aldose reductase to regulate renal expression of miR-200a-3p/141-3p to coordinately control hyperglycemia-induced renal oxidative stress, fibrogenesis, and the EMT. Our novel findings also suggest that inhibition of aldose reductase and in vivo renal cortical restoration of miR-200a-3p/141-3p or their combination are very promising avenues for the development of therapeutic strategies or drugs against diabetic nephropathy.     

Knockdown of UCA1 attenuated the progression of alcoholic fatty disease by sponging miR-214

Alcoholic fatty liver (AFL) is the initial manifestation of Alcoholic liver disease which can develop into alcoholic cirrhosis even extensive necrosis of liver cells, which induces liver failure finally. This study aims to focus on the role of long noncoding RNA UCA1 in AFL and further explored possible mechanism of this disease. We first downloaded GSE28619 to identify the expression of UCA1 in patients with AFL and use lncRNAs microarray to confirm UCA1 expression in serum of patients with AFL. Then we established ethanol-induced L02 cell model to mimic hepatocyte injury condition. By conducting qRT-PCR, we measured the expression of LncRNA UCA1 and miR-214 in serum of patients and ethanol-induced L02 cell. MTT assay, transwell migration, ELISA, qRT-PCR, and western blotting analysis were applied to evaluating the effect of UCA1 on ethanol-induced L02 cell. The bioinformatics analysis and the rescue experiment were devoted to the underlying mechanism. In this study, we first detected the expression of UCA1 was up-regulated in serum of patients with AFL and ethanol-induced L02 cells. And knockdown of UCA1 reversed the inhibiting effect of ethanol on the biological behavior of L02 cells including cell proliferation, migration, and apoptosis. Besides, lncRNA UCA1 regulated the expression of KLF5 by sponging miR-214. LncRNA UCA1 regulated the biological behavior of ethanol-induced L02 cells by sponging miR-214, which may provide novel therapeutic strategies for alcoholic fatty liver.

     

microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.     

Intestine-specific ablation of the Hepatocyte Nuclear Factor 4a (Hnf4a) gene in mice has minimal impact on serum lipids and ileum gene expression profile due to upregulation of its paralog Hnf4g

Ablation of the gene encoding the nuclear receptor Hepatocyte Nuclear Factor 4a (Hnf4a) in the liver strongly affects HDL concentration, structure and functionality but the role of this receptor in the intestine, the second organ contributing to serum HDL levels, has been overlooked. In the present study we show that mice with intestine-specific ablation of Hnf4a (H4IntKO) had undetectable levels of ΗΝF4A in ileum, proximal and distal colon but normal expression in liver. H4IntKO mice presented normal serum lipid levels, HDL-C and particle size (α1-α3). The expression of the major HDL biogenesis genes Apoa1, Abca1, Lcat was not affected but there was significant increase in Apoc3 as well as in Hnf4g, a paralog of Hnf4a. RNA-sequencing identified metabolic pathways significantly affected by Hnf4a ablation such as type II diabetes, glycolysis, gluconeogenesis and p53 signaling. Chromatin immunoprecipitation assays showed that HNF4G bound to various apolipoprotein gene promoters in control mice but its binding affinity was reduced in the ileum of H4IntKO mice suggesting a redundancy but also a cooperation between the two factors. In the distal colon of H4IntKO mice, where both HNF4A and HNF4G are absent and in a mouse model of DSS-induced colitis presenting decreased levels of HNF4A, most lipoprotein genes were strongly downregulated. In conclusion, Hnf4a ablation in mice does not significantly affect serum lipid levels or lipoprotein gene expression in ileum possibly due to compensatory effects by its paralog Hnf4g in this tissue.     

miR-200c is upregulated by oxidative stress and induces endothelial cell apoptosis and senescence via ZEB1 inhibition

We examined the effect of reactive oxygen species (ROS) on MicroRNAs (miRNAs) expression in endothelial cells in vitro, and in mouse skeletal muscle following acute hindlimb ischemia. Human umbilical vein endothelial cells (HUVEC) were exposed to 200 μM hydrogen peroxide (H₂O₂) for 8 to 24 h; miRNAs profiling showed that miR-200c and the co-transcribed miR-141 increased more than eightfold. The other miR-200 gene family members were also induced, albeit to a lower level. Furthermore, miR-200c upregulation was not endothelium restricted, and occurred also on exposure to an oxidative stress-inducing drug: 1,3-bis(2 chloroethyl)-1nitrosourea (BCNU). miR-200c overexpression induced HUVEC growth arrest, apoptosis and senescence; these phenomena were also induced by H₂O₂ and were partially rescued by miR-200c inhibition. Moreover, miR-200c target ZEB1 messenger RNA and protein were downmodulated by H₂O₂ and by miR-200c overexpression. ZEB1 knockdown recapitulated miR-200c-induced responses, and expression of a ZEB1 allele non-targeted by miR-200c, prevented miR-200c phenotype. The mechanism of H₂O₂-mediated miR-200c upregulation involves p53 and retinoblastoma proteins. Acute hindlimb ischemia enhanced miR-200c in wild-type mice skeletal muscle, whereas in p66^{ShcA −/−} mice, which display lower levels of oxidative stress after ischemia, upregulation of miR-200c was markedly inhibited. In conclusion, ROS induce miR-200c and other miR-200 family members; the ensuing downmodulation of ZEB1 has a key role in ROS-induced apoptosis and senescence.     

Reciprocal inhibition between miR-26a and NF-κB regulates obesity-related chronic inflammation in chondrocytes

Obesity is causally linked to osteoarthritis (OA), with the mechanism being not fully elucidated. miRNAs (miRs) are pivotal regulators of various diseases in multiple tissues, including inflammation in the chondrocytes. In the present study, we for the first time identified the expression of miR-26a in mouse chondrocytes. Decreased level of miR-26a was correlated to increased chronic inflammation in the chondrocytes and circulation in obese mouse model. Mechanistically, we demonstrated that miR-26a attenuated saturated free fatty acid-induced activation of NF-κB (p65) and production of proinflammatory cytokines in chondrocytes. Meanwhile, NF-κB (p65) also suppressed miR-26a production by directly binding to a predicted NF-κB binding element in the promoter region of miR-26a. Finally, we observed a negative correlation between NF-κB and miR-26a in human patients with osteoarthritis. Thus, we identified a reciprocal inhibition between miR-26a and NF-κB downstream of non-esterified fatty acid (NEFA) signalling in obesity-related chondrocytes. Our findings provide a potential mechanism linking obesity to cartilage inflammation.     

Role of PPARα and HNF4α in Stress-Mediated Alterations in Lipid Homeostasis

Stress is a risk factor for several cardiovascular pathologies. PPARα holds a fundamental role in control of lipid homeostasis by directly regulating genes involved in fatty acid transport and oxidation. Importantly, PPARα agonists are effective in raising HDL-cholesterol and lowering triglycerides, properties that reduce the risk for cardiovascular diseases. This study investigated the role of stress and adrenergic receptor (AR)-related pathways in PPARα and HNF4α regulation and signaling in mice following repeated restraint stress or treatment with AR-antagonists administered prior to stress to block AR-linked pathways. Repeated restraint stress up-regulated Pparα and its target genes in the liver, including Acox, Acot1, Acot4, Cyp4a10, Cyp4a14 and Lipin2, an effect that was highly correlated with Hnf4α. In vitro studies using primary hepatocyte cultures treated with epinephrine or AR-agonists confirmed that hepatic AR/cAMP/PKA/CREB- and JNK-linked pathways are involved in PPARα and HNF4α regulation. Notably, restraint stress, independent of PPARα, suppressed plasma triglyceride levels. This stress-induced effect could be attributed in part to hormone sensitive lipase activation in the white adipose tissue, which was not prevented by the increased levels of perilipin. Overall, this study identifies a mechanistic basis for the modification of lipid homeostasis following stress and potentially indicates novel roles for PPARα and HNF4α in stress-induced lipid metabolism.     

miR-141 and miR-200c as Markers of Overall Survival in Early Stage Non-Small Cell Lung Cancer Adenocarcinoma

Background

Several treatments in non-small cell lung cancer (NSCLC) are histology-dependent, and the need for histology-related markers is increasing. MicroRNAs (miRNAs) are promising molecular markers in multiple cancers and show differences in expression depending on histological subtype. The miRNA family miR-200 has been associated with the regulation of epithelial-mesenchymal (EMT)/mesenchymal-epithelial transition (MET). EMT involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties that facilitates metastasis. A dual role for the miR-200 family in the prognosis of several tumors has been related to tumor cell origin. However, the prognostic role and function of miR-200 family in early-stage NSCLC adenocarcinoma and squamous cell carcinoma (SCC) have not been well established.

Methods

miRNA expression was determined using TaqMan assays in 155 tumors from resected NSCLC patients. Functional studies were conducted in three NSCLC cell lines: H23, A-549 and HCC-44.

Results

High miR-200c expression was associated with shorter overall survival (OS) in the entire cohort (p = 0.024). High miR-200c (p = 0.0004) and miR-141 (p = 0.009) expression correlated with shorter OS in adenocarcinoma – but not in SCC. In the multivariate analysis, a risk score based on miR-141 and miR-200c expression emerged as an independent prognostic factor for OS in the entire cohort (OR, 2.787; p = 0.033) and in adenocarcinoma patients (OR, 10.649; p = 0.002). Functional analyses showed that miR-200c, was related to mesenchymal-epithelial transition (MET) and affected cell migration and E-cadherin levels, while overexpression of miR-141 reduced KLF6 protein levels and produced an increase of secretion of VEGFA in vitro (H23, p = 0.04; A-549, p = 0.03; HCC-44, p = 0.02) and was associated with higher blood microvessel density in patient tumor samples (p<0.001).

Conclusion

High miR-141 and miR-200c expression are associated with shorter OS in NSCLC patients with adenocarcinoma through MET and angiogenesis.     

MicroRNA-217 promotes ethanol-induced fat accumulation in hepatocytes by down-regulating SIRT1

Ethanol-mediated inhibition of hepatic sirtuin 1 (SIRT1) plays a crucial role in the pathogenesis of alcoholic fatty liver disease. Here, we investigated the underlying mechanisms of this inhibition by identifying a new hepatic target of ethanol action, microRNA-217 (miR-217). The role of miR-217 in the regulation of the effects of ethanol was investigated in cultured mouse AML-12 hepatocytes and in the livers of chronically ethanol-fed mice. In AML-12 hepatocytes and in mouse livers, chronic ethanol exposure drastically and specifically induced miR-217 levels and caused excess fat accumulation. Further studies revealed that overexpression of miR-217 in AML-12 cells promoted ethanol-mediated impairments of SIRT1 and SIRT1-regulated genes encoding lipogenic or fatty acid oxidation enzymes. More importantly, miR-217 impairs functions of lipin-1, a vital lipid regulator, in hepatocytes. Taken together, our novel findings suggest that miR-217 is a specific target of ethanol action in the liver and may present as a potential therapeutic target for treating human alcoholic fatty liver disease.