Effects And Mechanism Of MiR-377-3p Regulating SGK3 In Diabetic Nephropathy

Objective Diabetes mellitus is expected to be the 3rd most serious disease in our country,after cancer and cardiovascular diseases.Diabetic nephropathy is the major cause of the diabetes mellitus related death,which is a severe threat to human health.miRNAs may involve in many physiological and pathological processes by the regulation of its target gene,such as cell proliferation,apoptosis and cancer initiation and progression.It had been shown in previous studies that miRNAs also involved in the biological regulation of diabetic nephropathy.The purpose of our study is to identify the miRNAs that implicated in the dysregulation of diabetic nephropathy,and the mechanism miRNAs play its role.It may provides an evidence for clinical application of diabetic nephropathy.Methods In our study,miRNA microarray was used to screen the differentially expressed miRNAs.miR-377-3p was chosen for further experiments according to the result of microarray,and the biological function were also considered.The expression levels of miR-377-3p in HK-2 cells and circulating micro RNA in diabetic patient were tested by q RT-PCR.The animal model of diabetic nephropathy was constructed to conduct functional study in vivo in the next step.Next,the effect of miR-377-3p on HK-2 cell viability was examined by MTT assay,and the effect of miR-377-3p on HK-2 cell apoptosis or apoptosis-related protein was detected by flow cytometry or western blot.In the animal study,we confirmed the influence of miR-377-3p on the proliferation and apoptosis of renal tubular epithelial cells by tunel and immunohistochemistry assay.To identify the potential downstream target gene regulated by miR-377-3p,we predicted the targets by bioinformatics algorithms,and confirmed the relationship between miR-377-3p and its target by fluorescent reporter experiment.q RT-PCR and western blot was used to determine the influence of miR-377-3p on the m RNA and protein expression of target gene.Lastly,we used the similar way to investigate the function of target gene in cell and animal model.Results The results of microarray and q RT-PCR showed that miR-377-3p was highly expressed in the HK-2 cells which were cultured in high-glucose environment.But miR-377-3p was lowly expressed in the serum of diabetic patient.The animal model of diabetic nephropathy was constructed successfully,and can be used in functional study in vivo in the next step.The results of MTT showed that the cell viability of HK-2 cells was obviously decreased after overexpressing miR-377-3p.miR-377-3p promote apoptosis of HK-2 cells by upregulating Bax and downregulating Bcl-2 expression.On the other hand,we conducted experiments in the mouse model,and found that miR-377-3p could suppress the proliferation and promote the apoptosis of renal tubular epithelial cells of mouse suffering from diabetic nephropathy.Base-pairing complementation revealed that the 3' untranslated region of SGK3 contain a putative binding site which has significant complementarity with miR-377-3p.The result of fluorescent reporter experiment revealed that miR-377-3p bind directly to the 3'UTR of SGK3 m RNA.SGK3 inhibits apoptosis of HK-2 cells by downregulating Bax and upregulating Bcl-2 expression,and can counteract the suppressing effect of miR-377-3p on cell viability.Meanwhile,SGK3 enhanced cell viability and inhibited apoptosis of renal tubular epithelial cells in mouse model.Otherwise,miR-377-3p also influence the expresson of the molecules involved in PI3 K / AKT signal pathway.Conclusions miR-377-3p was highly expressed in the HK-2 cells which were cultured in high-glucose environment while was lowly expressed in the serum of diabetic patient.miR-377-3p could damage the renal tubular epithelial cells and resulting in diabetic nephropathy by suppressing cell growth and accelerating apoptosis.miR-377-3p targets SGK3 and negatively regulates its expression.Contrary to miR-377-3p,SGK3 plays a positive role in cell growth.Collectively,our findings characterize miR-377-3p/ SGK3 as an important regulation pathway associated with diabetic nephropathy,potentially providing new biomarkers for diabetes mellitus.