MicroRNA-34a Regulates Mesangial Proliferation Via Targeting PDGF Receptor β

Objective: Mesangial proliferative glomerulonephritis (MsPGN) is one of the kidney diseases with the most commen incidence in China, with main pathological changes including mesangial cells (MC) proliferation and mesangial matrix increase, which may eventually lead to the irreversible glomerulosclerosis.To inhibit the mesangial proliferation is an important treatment strategy of the treatment of glomerular diseases. In recent years the field of life science research is a major breakthrough in a large number of eukaryotic cells that regulate the function of non-coding RNA-microRNA (miRNA). The broad participation of small RNA regulation of biological transcription, not only participate in a series of biological functions, but also, and is closely related to human diseases. Mature miRNA regulate the expression of target genes both to direct degradation mRNA and inhibition of protein translation , the role of a miRNA can have a common target sequence in a variety of mRNA. Conversely, a target gene mRNA may have multiple binding sites for miRNA target by more than the regulation of miRNA, miRNA silencing effect of this multi-faceted nature of the cells in a variety of signaling pathways can be quickly converted. Present study shows that, miR-34a may be involved in regulation of cell proliferation signaling pathway thereby affecting multiple cells. The topics to miR-34a as the starting point of its downstream target genes, miR-34a clear of rat mesangial cell proliferation mechanism. The topics to miR-34a as the starting point of its downstream target genes, clear of regulate rat mesangial cell proliferation by miR-34a.Methods: Use of retained Thy1 antibody, inject tail vein of SD rats to establish anti-Thy1 mesangial proliferative glomerulonephritis model. The miR-34a mimic (mimics) or miR negative control transfected RMC. 1 MTT detection method used to detect different time points (24h, 48h, 72h) of the proliferation rate changes between miR-34a and the control group level. 2 Using flow cytometry for analysis cell cycle changes. 3 Using online software miRGen predict downstream target genes of miR-34a ,selected target genes associated with proliferation and to identify target genes of interest. 4 Detection of PDGFR-βmRNA and protein expression. According to flow cytometry to detect cell cycle results, select the corresponding changes in the cell cycle as a research target. 5 RMC transfected with miR-34a mimics or miR-34a negative control transfection, using real time PCR and western blot detection of cell cycle-related proteins (CDK, CKI) of the mRNA and protein expression changes. 6 Finally, to find the possible impact of cell cycle signaling pathways, and to detect the key of signaling pathway on the expression of regulatory factors to change.Results:1 Thy1 mesangial proliferative glomerulonephritis rats model successfully prepared by using Thy1 anti body. We have detected that Thy1 animal models of renal tissue at each time point the expression of miR-34a changes, the results show that, miR-34a decreased with increased proliferation, and with the pathological outcome of the recovery(p<0.05).2 Detected rat mesangial cells (RMC) by MTT and flow cytometry which transfected by miR-34a mimics or miR negative control. The results showed that the transfected miR-34a mimics compared with the control group, proliferative activity decreased from the beginning 48h (p <0.05), and that mainly affect the cell cycle arrest of the G0/G1 phase (making the G0 / G1 period is extended), and ultimately shorten the S/G2/M.3 Using bioinformatics software miRGEN to predicted target genes of miR-34a, according to mesangial proliferative glomerulonephritis in the pathophysiology of features, the research focus chosen PDGFR-β(platelet-derived growth factor-β). By dual luciferase experiments show, PDGFR-βmay be a direct miR-34a target gene.4 RMC cells transfected with the miR-34a mimics or miR negative control, 48h after the test, miR-34a mimics group and miR negative control group, the former PDGFR-βand p-PDGFR-βprotein expression also decreased, with statistical Significance (p <0.05), while the mRNA levels of PDGFR-βdid not change, no statistical significance (p> 0.05), that, miR-34a may be through the transcriptional regulation of PDGFR-βregulation.5 Subsequently, we selected the cycle-related proteins,CDK proteins and CKI proteins which are control cell G0G1 phase, were determined. The RMC transfected miR-34a mimics or miR negative control, 48h test, compared with control, miR-34a mimics group, cyclin D1, cyclin E, CDK2, CDK4, CDK6 protein levels were decreased (p <0.05). p21 protein levels decreased (p <0.05), mRNA levels increased (p <0.05),and p27 protein levels increased (p <0.05), mRNA levels increased (p <0.05)6 We detected RAS / MAPK pathway. The phosphorylation of ERK, MEK protein expression levels are changed in order to prove miR-34a control whether the expression of PDGFR-βthrough the RAS / MAPK signaling pathway downstream of its cell cycle-related proteins (CDK Protein, CKI protein) to control changes, the results show that the ERK protein expression did not change (p> 0.05), while the MEK protein levels decreased (p <0.05). The p-ERK and p-MEK, miR-34a mimics compared with the control group decreased significantly (p <0.05)Conclusions:1 Anti Thy1 mesangial proliferative glomerulonephritis rat model, miR-34a change with the proliferation of RMC.2 miR-34a can decrease cell proliferation, mainly by extending the G0/G1.3 miR-34a to lower the level of PDGFR-βprotein expression, and by dual-luciferase PDGFR-βexperiments show that miR-34a may be a direct target gene.4 miR-34a stimulate PDGFR-βby RAS / MAPK pathway decreased G0/G1 cell cycle protein cyclin D1, CDK protein CDK2, CDK4, CDK6 expression, etc., but no change in mRNA levels except for CDK4, possibly because of miR-34a in Target genes not only PDGFR-β, and there is cyclin D1, CDK4, CDK6, and CDK2 may be because of synergies, all mRNA levels did not change. Finally, miR-34a inhibition of PDGFR-βby G0/G1 phase arrest and thus to influence cell proliferation, the regulation of cell proliferation provides a new way of thinking.