The change of mRNA expression in murine immortalized podocyte after miR-26a silencing
ABSTRACT: The change of mRNA expression in murine immortalized podocyte were analyzed after miR-26a silencing. These results provide a basical information of molecular pathology in podocyte biology. Mouse podocytes immortalized by temperature sensitive SV40 were used. Podocyte cultures grown at 33 °C were trypsinized and then cultured with RPMI-1640 without antibiotics in 24-well plates at 60–70% confluence for 2 days. On day 3, an anti-miR negative control (40 pmol) or the miR-26a miRNA inhibitor (40 pmol) was transfected to podocytes. The cells were analyzed after culturing for 24 hour.
Project description:MicroRNAs contribute to the pathogenesis of certain diseases and may serve as biomarkers. We analyzed glomerular microRNA expression in B6.MRLc1, which serve as a mouse model of autoimmune glomerulonephritis. We found that miR-26a was the most abundantly expressed microRNA in the glomerulus of normal C57BL/6 and that its glomerular expression in B6.MRLc1 was significantly lower than that in C57BL/6. In mouse kidneys, podocytes mainly expressed miR-26a, and glomerular miR-26a expression in B6.MRLc1 mice correlated negatively with the urinary albumin levels and podocyte-specific gene expression. Puromycin-induced injury of immortalized mouse podocytes decreased miR-26a expression, perturbed the actin cytoskeleton, and increased the release of exosomes containing miR-26a. Although miR-26a expression increased with differentiation of immortalized mouse podocytes, silencing miR-26a decreased the expression of genes associated with the podocyte differentiation and formation of the cytoskeleton. In particular, the levels of vimentin and actin significantly decreased. In patients with lupus nephritis and IgA nephropathy, glomerular miR-26a levels were significantly lower than those of healthy controls. In B6.MRLc1 and patients with lupus nephritis, miR-26a levels in urinary exosomes were significantly higher compared with those for the respective healthy control. These data indicate that miR-26a regulates podocyte differentiation and cytoskeletal integrity, and its altered levels in glomerulus and urine may serve as a marker of injured podocytes in autoimmune glomerulonephritis.
Project description:To evaluate involvement of miR-221 and miR-222 in lung cancer, we investigated the effects of miR-221 and miR-222 overexpression on six lung cancer cell lines as well as one immortalized normal human bronchial epithelial cell line. Two cell lines, H3255 and H1299 with no replicates were studied. Cells were transfected with miR-221, miR-222, or miR control. Microarray analysis was done to identify genes differentially expressed in lung cancer cells after the transfection of miR-221 or miR-222.
Project description:So far the pathomechanism of preeclampsia in pregnancy is focussed on increased circulating levels of soluble fms-like tyrosin kinase-1 (sFLT-1) that neutralizes glomerular VEGF-A expression and prevents its signaling at the glomerular endothelium. As a result of changed glomerular VEGF-A levels endotheliosis and podocyte foot process effacement are typical morphological features of preeclampsia. Recently, microRNA-26a-5p (miR-26a-5p) was described to be also upregulated in the preeclamptic placenta. We found that miR-26a-5p targets VEGF-A expression by means of PIK3C2? in cultured human podocytes and that miR-26a-5p overexpression in zebrafish causes proteinuria, edema, glomerular endotheliosis and podocyte foot process effacement. Interestingly, recombinant zebrafish Vegf-Aa protein could rescue glomerular changes induced by miR-26a-5p. In a small pilot study, preeclamptic patients with podocyte damage identified by podocyturia, expressed significantly more urinary miR-26a-5p compared to healthy controls. Thus, functional and ultrastructural glomerular changes after miR-26a-5p overexpression can resemble the findings seen in preeclampsia and indicate a potential pathophysiological role of miR-26a-5p in addition to sFLT-1 in this disease.
Project description:Recent studies have demonstrated that micro (mi)RNA molecules can be detected in the circulation and can serve as potential biomarkers of various diseases. This study used microarray analysis to identify aberrantly expressed circulating miRNAs in patients with type 1 autoimmune hepatitis (AIH) compared with healthy controls. Patients with well-documented and untreated AIH were selected from the National Hospital Organization (NHO)-AIH-liver-network database. They underwent blood sampling and liver biopsy with inflammation grading and fibrosis staging before receiving treatment. To further confirm the microarray data, circulating expression levels of miR-21 and miR-122 were quantified by real-time quantitative polymerase chain reaction in 46 AIH patients, 40 patients with chronic hepatitis C (CHC), and 15 healthy controls. Consistent with the microarray data, serum levels of miR-21 were significantly elevated in AIH patients compared with CHC patients and healthy controls. miR-21 and miR-122 serum levels correlated with alanine aminotransferase levels. Circulating levels of miR-21 and miR-122 were significantly reduced in AIH patients with liver cirrhosis, and were inversely correlated with increased stages of fibrosis. By contrast, levels of circulating miR-21 showed a significant correlation with the histological grades of inflammation in AIH. We postulate that aberrantly expressed serum miRNAs are potential biomarkers of AIH and could be implicated in AIH pathogenesis. Alternations of miR-21 and miR-122 serum levels could reflect their putative roles in the mediation of inflammatory processes in AIH. Case-control study, steroid treatment
Project description:We newly identified skeletal muscle differentiation-associated miRNAs by comparing miRNA expression profile between C2C12 cell and Wnt4-overexpressing C2C12 cell. miR-487b, miR-3963 and miR-6412 are significantly down-regulated in differentiating C2C12 cells, and transfection of their mimics resulted in reduced expression of myogenic differentiation markers including Troponin T, myosin heavy chain fast and slow type. Single analysis for each condition (proliferating C2C12 cells, differentiating C2C12 cells, proliferating Wnt4-overexpressing C2C12 subline cells
Project description:Podocyte apoptosis is a typical early feature of diabetic nephropathy (DN), with loss of nephrin integrity contributing to increased proteinuria in patients with DN. Emerging evidence shows that microRNAs (miRNAs) play vital roles in the pathogenesis of DN. Thus, we aimed to further elucidate the role of miRNAs in podocyte apoptosis in DN. We used db/db and db/m mice maintained under a continuous feeding regime for 12 weeks. Using microarray analysis, we found several miRNAs potentially related to podocyte apoptosis. In addition, we cultured a conditionally immortalized human podocyte cell line in 30 mM D-glucose and found that miR-134-5p was upregulated in both db/db mice and high-glucose (HG)-treated podocytes. Upregulation of miR-134-5p was accompanied by podocyte apoptosis and downregulation of nephrin. Inhibition of miR-134-5p produced the opposite effect. Dual-luciferase reporter assays showed that miR-134-5p directly targeted the 3'-untranslated region of the B-cell lymphoma-2 gene (BCL2), and further study confirmed an increase in bcl-2 protein level in HG-treated podocytes transfected with anti-miR-134-5p. Knockdown of BCL2 impeded the antiapoptotic effect of anti-miR-134-5p. Finally, we found that miR-134-5p might regulate apoptosis in db/db mice and podocytes by targeting BCL2. Taken together, our findings suggest that miR-134-5p promotes podocyte apoptosis under HG conditions by targeting BCL2. Our study provides a meaningful approach to interpret the mechanisms of action of miRNAs involved in DN.
Project description:Global gene expression patterns of the iCMs shift from a MEF state toward a cardiac-like phenotype by Gata4/Mef2c/Tbx5 (GMT) or GMT/miR-133 transduction at 3, 7 and 18 days after transduction (D3, D7 and D18) MiR-133 silenced fibroblast signatures in parallel with cardiac gene activation, and Snai1 overexpression inhibited the effects of miR-133-mediated cardiac reprogramming. MEFs were used for negative control, mouse heart tissue for positive control. Gene expression profiles were compared among MEFs, iCMs and heart. 23474 probes were analyzed in each experiment.
Project description:Global gene expression profile of total 24460 probes in the iCMs. The gene expression shifts from a fibroblast state toward a cardiac-like phenotype by Gata4/Mef2c/Tbx5/Mesp1/Myocd (GMTMM) or GMTMM/miR-133 transduction at 7 days after transduction. MiR-133 silenced fibroblast signatures in parallel with cardiac gene activation, and Snai1 overexpression inhibited the effects of miR-133-mediated cardiac reprogramming. HCFs were used for negative control, human heart tissue for positive control. Gene expression profiles were compared among HCFs, iCMs and heart. 24460 probes were analyzed in each experiment.
Project description:MicroRNAs (miRNAs) are essential for podocyte homeostasis, and the miR-30 family may be responsible for this action. However, the exact roles and clinical relevance of miR-30s remain unknown. In this study, we examined the expression of the miR-30 family in the podocytes of patients with FSGS and found that all members are downregulated. Treating cultured human podocytes with TGF-?, LPS, or puromycin aminonucleoside (PAN) also downregulated the miR-30 family. Podocyte cytoskeletal damage and apoptosis caused by treatment with TGF-? or PAN were ameliorated by exogenous miR-30 expression and aggravated by miR-30 knockdown. Moreover, we found that miR-30s exert their protective roles by direct inhibition of Notch1 and p53, which mediate podocyte injury. In rats, treatment with PAN substantially downregulated podocyte miR-30s and induced proteinuria and podocyte injury; however, transfer of exogenous miR-30a to podocytes of PAN-treated rats ameliorated proteinuria and podocyte injury and reduced Notch1 activation. Finally, we demonstrated that glucocorticoid treatment maintains miR-30 expression in cultured podocytes treated with TGF-?, LPS, or PAN and in the podocytes of PAN-treated rats. Glucocorticoid-sustained miR-30 expression associated with reduced Notch1 activation and alleviated podocyte damage. Taken together, these findings demonstrate that miR-30s protect podocytes by targeting Notch1 and p53 and that the loss of miR-30s facilitates podocyte injury. In addition, sustained miR-30 expression may be a novel mechanism underlying the therapeutic effectiveness of glucocorticoids in treating podocytopathy.
Project description:Alport syndrome (AS) is a genetic disorder involving mutations in the genes encoding collagen IV ?3, ?4 or ?5 chains, resulting in the impairment of glomerular basement membrane. Podocytes are responsible for production and correct assembly of collagen IV isoforms; however, data on the phenotypic characteristics of human AS podocytes and their functional alterations are currently limited. The evident loss of viable podocytes into the urine of patients with active glomerular disease enables their isolation in a non-invasive way. We here isolated, immortalized and subcloned podocytes from the urine of three different AS patients for molecular and functional characterization. AS podocytes expressed a typical podocyte signature and showed a collagen IV profile reflecting each patient's mutation. Furthermore, RNA-sequencing analysis revealed 348 genes differentially expressed in AS podocytes compared with control podocytes. Gene Ontology analysis underlined the enrichment in genes involved in cell motility, adhesion, survival, and angiogenesis. In parallel, AS podocytes displayed reduced motility. Finally, a functional permeability assay, using a podocyte-glomerular endothelial cell co-culture system, was established and AS podocyte co-cultures showed a significantly higher permeability of albumin compared to control podocyte co-cultures, in both static and dynamic conditions under continuous perfusion. In conclusion, our data provide a molecular characterization of immortalized AS podocytes, highlighting alterations in several biological processes related to extracellular matrix remodelling. Moreover, we have established an in vitro model to reproduce the altered podocyte permeability observed in patients with AS. This article is protected by copyright. All rights reserved.