Metformin Reduces The Senescence Of Renal Tubular Epithelial Cells In Diabetic Nephropathy Via The MBNL1/miR-130a-3p/STAT3 Pathway

Diabetes is a metabolic disorder characterized by elevated blood glucose levels.The increasing morbidity of diabetes exposes more patients to diabetic complications,e.g.,diabetic nephropathy,which is the major contributor to end-stage renal disease(ESRD)and involves renal glomerular,vascular and tubular injuries.Studies have revealed that renal tubular epithelial cells present premature senescence in type II diabetic nephropathy,indicating that senescence of renal tubular epithelial cells is one of the mechanisms involved in the progression of diabetic nephropathy.Metformin is a biguanide derivative and a first-line oral therapeutic drug for type II diabetes.Metformin has several hypoglycemic effects.Studies have shown that metformin can also partially reverse the renal damage caused by diabetic nephropathy.RNA binding proteins(RBPs)can directly bind to RNA,thus forming a ribonucleoprotein complex and,in this way,they regulate the biological functions of RNA.MBNL1(muscleblind like splicing regulator 1)is an RBP consisting of 343 amino acids and located at chromosome 3q25.1-q25.2,and its location imbalance in cells is an important pathogenic factor for myotonic dystrophy.MBNL1 can bind to several RNAs to regulate their functions including stability.However,there are currently no reports about the effects of metformin or MBNL1 on diabetic nephropathy-associated senescence.miRNAs are non-coding RNAs with conservative sequences,and formed of 21-25 nucleotides;miRNAs inhibit the expression of target genes by binding with the corresponding mRNA 3'UTR,thus regulating several cellular biological activities including cell differentiation,proliferation,apoptosis and migration.miR-130a-3p is located at chromosome 11q12.a,and it has been demonstrated that miR-130a-3p expression was reduced in the blood and liver of elderly mice.By analysis and prediction using RBPDB software in this study,we found that MBNL1 and miR-130a-3p are potential binding partners.STAT3(signal transmission and transcription activation factor 3)is located at chromosome 17 q21.2 and can act on polypeptide receptors on the cell surface,resulting in the activation of various biological pathways.STAT3 plays an important role in the pathogenesis of diabetic nephropathy.A potential binding site of miR-130a-3p and STAT3 was predicted with targetScan and miRanda software in our study,but the action mechanism of miR-130a-3p and STAT3 has not yet been reported.This study aimed to provide a new theoretical and experimental basis for the pathogenesis of diabetic nephropathy and the relevance of treatment with metformin for this disease.We firstly explored the endogenous expression of MBNL1,miR-130a-3p and STAT3 in diabetic nephropathy and their relation with the senescence of renal tubular epithelial cells.We then determined the regulation patterns among MBNL1,mi R-130a-3p and STAT3,and further investigated whether metformin was able to protect renal tubular epithelial cells from senescence via the MBNL1/mi R-130a-3p/STAT3 pathway.Method: Part1: The HK-2 human renal proximal tubular epithelial cell line was purchased from the Shanghai Institute for Biological Sciences Cell Resource Center.The HK-2 cells were cultured in normal-glucose(D-glucose 5.56mmol/L),high-glucose(D-glucose 30mmol/L)and hypertonic(D-glucose 5.56mmol/L and mannitol 24mmol/L)medium.Senescencerelated beta-galactosidase activity(SA-beta-gal)staining,immunohistochemistry and Western Blot of senescence related marker P21 were used to detect senescence level of different cells.Lentiviral vector and puromycin were used to generate MBNL1,STAT3 stable overexpressed and knocked down cell lines.Lipofectamine 2000 was used to transfect miR-130a-3p mimics and miR-130a-3p inhibitor.Real-time PCR and Western blot were used to test transfection efficiency.Western Blot were used to detect the influence of MBNL1,miR-130a-3p,STAT3 and their relationship on senescence.RIP and nascent RNA capture experiments were used to verify the binding between MBNL1 and miR-130a-3p.Luciferase reporter assay was used to verify the regulation of STAT3 by miR-130a-3p.Part2: For treatment with metformin,HK-2 cells were pretreated with metformin(1?mmol/L)for 2 h after serum starvation for 24 h.SA-beta-gal staining and Western Blot were used to detect senescence level of cells.Through real-time PCR,western blot and the cell lines in Part1,we verified the effect of metformin on senescence through MBNL1,miR-130a-3p and STAT3.Part3: Male db/m and db/db mice were used for in vivo experiments.Mice were sacrificed at 8,16,24 weeks old,separately.Kidneys of mice were taken out,embedded and sectioned.HE,PAS,Masson staining were used to test pathological changes.SA-beta-gal staining was used to detect level of senescence.Expression of MBNL1 and STAT3 were tested by immunohistochemistry and miR-130a-3p expression was tested by in situ hybridization.Results: Part1: At 24 and 48 h,there was no statistically significant difference between the highglucose(HG)group and the mannitol hypertonic control group or the normal-glucose group on level of senescence.At 72 h,the level of senescence was significantly higher in the high-glucose group than in the mannitol hypertonic control group or the normalglucose group.Thus,subsequent HG group was performed under high glucose condition for 72 h.Compared with normal glucose group,HG group showed decreased MBNL1 and miR-130a-3p expression,and enhanced STAT3 expression.Under HG condition,MBNL1,miR-130a-3p and knocking down STAT3 could inhibit cell senescence.RIP and nascent RNA capture experiments showed that MBNL1 could bind with miR-130a-3p and thus increase its stability.Luciferase reporter assay showed miR-130a-3p could negative regulate STAT3 by binding with the 3'UTR region of its mRNA.Part2: Metformin treatment could significantly reduce P21 expression and SA-beta-gal staining positive rate in HG group.Metformin could enhance the expression of MBNL1,miR-130a-3p and decrease STAT3 expression under HG condition.Metformin could increase the stability of miR-130a-3p by up-regulating MBNL1,and thus reduces the senescence of cells.Metformin enhances the negative regulation of STAT3 by upregulating miR-130a-3p and thus reduced the cell senescence.Part3: Compared with db/m mice,db/db diabetic mice showed increased serum creatinine and urine ACR.Moreover,compared with db/m mice,renal HE staining showed glomerular hypertrophy,PAS staining indicated increased glomerular mesangial matrix,and Masson trichrome staining revealed aggravated interstitial fibrosis of renal tubules in db/db group.We also found enhanced senescence level combined with low expression of MBNL1,miR-130a-3p and high expression of STAT3 compared with db/m control mice during nephropathy development.Meanwhile,metformin could alleviate above mentioned phenomenon,and increase the expression of MBNL1,mi R-130a-3p as well as decreased STAT3 expression.Conclusion 1.High-glucose could induce senescence of renal tubular epithelial cells.2.MBNL1 and miR-130a-3p were low expressed in high-glucose could induced renal tubular epithelial cells.STAT3 was high expressed in high-glucose could induced renal tubular epithelial cells.3.MBNL1,miR-130a-3p and knocking down STAT3 could inhibit high-glucose induced senescence in renal tubular epithelial cells.4.MBNL1 could bind with miR-130a-3p and thus increase its stability.STAT3 is the target gene of miR-130a-3p.5.High-glucose could induce senescence of renal tubular epithelial cells through MBNL1/miR-130a-3p/STAT3 pathway.6.Metformin reduced the high-glucose-induced senescence of renal tubular epithelial cells.7.Metformin increased the stability of miR-130a-3p by up-regulating MBNL1,and thus reduced the high-glucose-induced senescence of renal tubular epithelial cells.8.Metformin enhanced the negative regulation of STAT3 by up-regulating mi R-130a-3p and thus reduced the high-glucose-induced senescence of renal tubular epithelial cells 9.Metformin reduced the senescence of renal tubular epithelial cells via the MBNL1/miR-130a-3p/STAT3 pathway 10.MBNL1 and miR-130a-3p were low expressed in senescent renal tubular epithelial cells of db/db mice.STAT3 was high expressed in senescent renal tubular epithelial cells of db/db mice.11.Metformin reduced the senescence of renal tubular epithelial cells via the MBNL1/miR-130a-3p/STAT3 pathway in db/db mice.