Effect Of TET2 On The Regulation Of Transform Growth Factor β1 Expression Of Mesangial Cells Induced By High Glucose

Diabetic nephropathy(DN)is one of the major microvascular complications of diabetes,and is also the primary underlying diseases of end-stage renal failure(ESRF)in western developed countries.Previous studise suggested that hyperglycemia is thought to contribute to the development of DN though a variety of mechanisms.In recent years,the epigenetics have been recognized by the people,that is,the change of the genetic and reverse changes which occur under the premise of not changing the DNA sequence.Recently,epigenetics as a considerable mechanism which can explain the persistence effects of high glucose on risk of diabetic complications,often referred to as metabolic memory.DNA methylation as an important mechanism of genetics played a key role in regulation of gene expression.The DNA methyltransferases(DNMTs)include DNMT1,DNMT3 A,DNMT3B which catalyzing passive demethylation and TET methylcytosine dioxygenases(TETs)family include TET1、TET2、TET3 which catalyzing positive demethylation were all played an important role in the epigenetic mechanism of mammalian genes.ObjectiveThis paper intends to dynamic observate the expression of DNMTs、TETs、α-SMA 、TGF-β1and it’s methylation status of Cp G island also include cell proliferation in human mesangial cells(HMCs)stimulated by high glucose and the renal cortex of db/db mice.Subsequently,after interference of TET2 expression by shRNA in mesangial cells and by inhibitor of PARP in db/db mice,we observed the corresponding expression changes of TGF-β1 and it’s methylation status on Cp G island,also include the expression of α-smooth muscle action(α-SMA)and cell proliferation of mesangial cells,so as to explore the function of TET2 on high expression of transforming growth factor-β1(TGF-β1)and phenotype transformation of mesangial cells induced by high glucose.Method1.The cultured human mesangial cells(HMCs)were randomlydivided for five groups in accordance with the glucose concentration: normal glucose control group(NG,5.5 mmol / L),high glucose group(HG,30 mmol / L);and the high glucose group were cultured with 12h、24h、48h and 72 h.2.We used the shRNA to interfere the gene expression of TET2 in HMCs cultured with high glucose and divided into 4 groups:blank control group(control B),control group of transfection reagent(control M),negtive control group(control N)and shRNA interference group(sh RNA).3.There were 20 female C57BLKS/J mice including:7 weeks old db/db mice、11 weeks old db/db mice、11 weeks old db/m mice and15 weeks old db/db mice with five animals of each group.all mice were adaptively fed for one week,and divided into 4 groups: db / m group: db / m mice aged 12 weeks,db / db-8w group :db/db mice aged 8 weeks,db / db-12 w group: db/db mice aged 12 weeks,db / db-16 w group: db/db mice aged 16 weeks.4.There were 15 female C57BLKS/J mice aged 5 weeks including:10 db/db mice and 5 db/m mice.The db/db mice were divided into two groups with completely random method: db/db control group and db/db+PJ-34 group.The PARP inhibitor PJ-34 were dissolved in distilled water to 2.4 g / L,and added right amount of aspartame for convenient feeding.Mice of db/db+PJ-34 group were fed daily with the amount of 30 mg per kg of body weight per day till 16 weeks,and the rest of the db / m control group and db/db control group were fed daily with the same amount of distilled water which added right amount of aspartame per day till 16 weeks.5.The m RNA and protein expression of DNMT1、DNMT3a、DNMT3b、TGF-β1、TET1~3 and α-smooth muscle actin(α-SMA)were all detected respectively by quantitative real-time PCR(qRT-PCR)and Western blotting.The methylation status of Cp G island located in TGF-β1 gene region was detected through bisulfite sequencing PCR(BSP).MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide)colorimetric assay was used to detect proliferation of humanmesangial cells.Immunohistochemistrywas used detect the expression of α-SMA in mouse kidney cortex.The pathological changes of renal cortex in the mouse kidney were observed b y PAS staining.Result1.high glucose could stimulate the expression of TGF-β1 and the demethylation of CpG island located in TGF-β1 gene region appeared to demethylation.Inthisstudy,we startedto observe the effect of high glucose on TGF-β1expressionlevel in human mesangial cells.The study found that compared with the control groupafterstimulate with 24 hours by high glucose,the mRNA and protein expression of TGF-β1 were all increased significantly,and there was a time-dependent effect.we also found that: compared with the control groupafter stimulate with 24 hours by high glucose,the methylation status of 4 CG sides in Cp G island located in the first exon of TGF-β1 appeared to demethylation obviously.2.The expression ofTET2 was also stimulated by high glucose.Thus,we found: compared with the control group,after12 h induced by high glucose the expression of TET2 which participated in the gene demethylation was increaseed in HMCs with a time-dependent effect,and it’s consistent with the effect of the demethylation change of CpG island located in TGF-β1 gene region.However,there were no significant change in expression of TET1,TET3,DNMT1,DNMT3 A.3.The using of the shRNA which proved can suppress the expression of TET2 could reverse the effect that the high expression of TGF-β1 and the demethylation of CpG island located TGF-β1 gene region induced by high glucose.Compared with the control group,the use of(short hairpin RNA)shRNA could significantly reduce the gene and protein expression of TET2 induced by high glucose.Furthermore,the application of shRNA could significantly reduce the expression of TGF-β1 induced by high glucose.Meanwhile,the application of shRNA could reverse the demethylation status in Cp G island located gene regulation region of TGF-β1induced by high glucose.4.Further study found that the using of shRNA which proved can suppress the expression of TET2 could reverse the effect that the high expression of α-SMA and the increased of cell proliferation in HMCs.5.With the occurrence and progression of diabetic nephropathy in db/db mice,the expression levels of TET2 and TGF-β1 in renal cortex were gradually increased,and the CpG islands in the gene regulation regionof TGF-β1 were significantly hypomethylation.Our experimental study further observed the expression of TGF-β1 in renal cortex of db/db mice at different ages revealed that: compared with db/m control group,the mRNA and protein expression of TGF-β1 were graduallyincreased in renal cortex.The test of BSP sequencing found: compared with db/m control group,the db/db mice from the age of 8 weeks to 16 weeks,whose CG sides continued to hypomethylation in promoter region and first exon of TGF-β1 in renal cortex.Thus,we further study the expression change of enzymesrelated to DNA demethylation in renal cortex of db / db mice,we found: compared with db/m control group,the db/db mice with increased weeks of age,the mRNA and protein expression of TET2 were obviously increased with the age increasing in renal cortex.However,the expression of TET1,TET3,DNMT1,DNMT3 A showed no significant change.6.The using of PJ-34 which proved can suppress the expression of TET2 could reverse the high expression of TGF-β 1 and the demethylation of Cp G island located in regulative region of TGF-β1 in renal cortex of db/db mice.In order to investigate the interrelation between TET2 with PARP inhibitors in vivo,we fed the db / db mice of the same age with(poly ADP-ribose polymerase)PARP inhibitors and same dose of distilledwater and found:compared with db/m control group and db / db control group,the TET2 mRNA and protein expression of db / db+pj-34 group were significantly down-regulated.With the same feeding method,we further observed the expression of TGF-β1 and found that: compared with db/db control group,the mRNA and protein expression of db / db+pj-34 group were significantly down-regulated in renal cortex;and compared with db/m control group,the expression of TGF-β1 were still higher.7.Inhibited the expression of TET2 in the renal cortex of db/db mice,the proliferation of glomerular mesangial cells and the synthesis of-SMA were also significantly inhibited in db/db mice.We observed the renal cortex through PAS staining:compared with db/db control group,the pathological changes including the glomerular hypertrophy、mesangial matrix、department of membrane area and capillary basement membrane thickness and other pathological changes were significantly reduced.The deposition of α-SMA in renal cortex was observed by immunohistochemistry and the results showed that the expression of α-SMA in db/db+PJ-34 group was significantly decreased compared with db/db control group.ConclusionTET2 which mediated the demethylation of the Cp G island of TGF-β1 may play an important role in the expression of TGF-β1 induced by high glucose.Interference of TET2 expression can inhibit the phenotype transformation of HMC induced by high glucose and the development of diabetic nephropathy.