| Part one The expression of DNMT3A in radial artery calcified tissues of chronic kidney disease and its relationship with phenotypic transformation and autophagyObjective:DNA methyltransferase 3A(DNMT3A)is an important epige-netic regulatory factor,which play a critical role in regulating cell autophagy,apoptosis,differentiation and other cell biological processes.The transdifferentiation and autophagy of vascular smooth muscle cells are the pathogenesis of vascular calcification.This study was aimed to observe the expression of DNA methyltransferase 3A(DNMT3A)in calcified radial artery tissues of patients with chronic kidney disease,and investigate the differential expression of Runt-related transcription factor 2(Runx2),Smooth muscle 22α(SM22α),autophagy index microtubule associated protein light chain 3(LC3),P62 in different radial artery tissue groups,in order to explore the relationship between DNMT3A and phenotype transformation and autophagy.Methods:24 Patients diagnosed with end stage renal disease(ESRD)proposed operation venous internal fistula in the Department of Nephrology of Cangzhou Central Hospital from October 2019 to October2020 were selected.End-to-end anastomosis was used to obtain full-layer radial artery tissue with a width of about 1mm parallel to the arterial stump during the operation.According to the results of silver nitrate staining of radial artery tissue,they were divided into calcified group and non-calcified group.Image Pro Plus(IPP)image analysis software was used to conduct semi-quantitative analysis of calcification expression between two groups.DNA methylation index,osteogenic phenotype Runx2 and contractile phenotype SM22α,autophagy related indexes LC3 and P62 was detected by immunohistochemistry in the significantly calcified radial artery tissue.Osteogenic phenotype Runx2 and contractile phenotype SM22α,autophagy related indices LC3 and P62,DNA methylation indices DNMT3A were analyzed according to integral optical density(IOD)of IPP software.Spearman correlation analysis was used to investigate the relationship between calcium,phosphorus,intact parathyroid hormone(i PTH)and calcification level,the relationship between DNMT3A and calcium,phosphorus,i PTH,the relationship between DNMT3A,Runx2 and LC3.Results:1.General information of patients with radial artery tissue takenA total of 24 patients were selected in this study with the average age of73.54±7.85 years.The primary disease composition of the patients included 5cases of chronic glomerulonephritis(20.8%),9 cases of diabetic nephropathy(37.5%),and 5 cases of hypertensive nephropathy(20.8%).The other 5 cases(20.8%).According to the results of silver nitrate staining,the enrolled patients were divided into 2 groups:12 patients in the calcification group and 12patients in the non-calcification group by age and sex.The results of comparison between the two groups showed that there were no statistical differences in body mass index and primary diseases(including primary glomerular disease,hypertension and diabetes)between the two groups,while the levels of blood phosphorus,blood calcium and parathyroid hormone in the medium-calcification group were significantly higher than those in the non-calcification group,with statistical significance(P<0.05).Spearman correlation analysis showed that calcium was positively correlated with the semi-quantitative integral of silver nitrate staining(r~2=0.47,p=0.0002),phosphorus was positively correlated with the semi-quantitative integral of silver nitrate staining(r~2=0.42,p=0.0006),parathyroid hormone level was positively correlated with the semi-quantitative integral of silver nitrate staining(r~2=0.62,p<0.001).2.The expression of DNMT3A was elevated in peripheral vascular calcified tissuesCompared with non-calcifying group,the expression level of DNMT3A in calcifying group was increased,and the difference was statistically significant(P<0.05).Immunohistochemical results showed that the expression of Runx2,a phenotypic transformation indicator,was increased,while the expression of SM22α,a contractile phenotypic indicator,was decreased in calcified bone.LC3 expression was increased in the calcified group,while P62expression was decreased in the calcified group,indicating autophagy activity was increased in the calcified group.Spearman correlation analysis showed that DNMT3A was positively correlated with calcium(r~2=0.84,p=0.009),phosphorus(r~2=0.88,p=0.005)and also positively correlated with parathyroid hormone(r~2=0.85,p=0.007).3.Correlation analysis of DNMT3A with Runx2 and LC3Spearman correlation analysis showed that DNMT3A was positively correlated with Runx2(r~2=0.99,p<0.001)and also positively correlated with LC3(r~2=0.98,p=0.001).Conclusions:The expression level of DNMT3A was increased in calcified radial artery tissues,along with the expression levels of osteogenic phenotype markers and autophagy markers.The semi-quantitative integral of silver nitrate staining was positively correlated with serum calcium,phosphorus and parathyroid hormone levels.DNMT3A was positively correlated with serum calcium,phosphorus and parathyroid hormone levels.DNMT3A was also positively correlated with Runx2 and LC3.Part Two DNMT3A regulated phenotypic transformation and autophagy in vascular smooth muscle cell calcification induced by hig-h phosphorusObjective:Previous studies have shown that DNMT3A plays an import-ant regulatory role in cell transdifferentiation and autophagy.The calcification model of vascular smooth muscle cells(VSMCs)induced by high phosphorus was established to investigate the mechanism of DNMT3A regulating phenotypic transformation and autophagy in this study.Methods:1.Primary rat VSMCs were isolated and cultured by the adherent culture method of rat aortic tissue blocks,then passed through,cryopreservation and resuscitation.The experimental group(high phosphate,P)and the control group(normal phosphate,N)were cultured with VSMCs stimulated by high phosphorus for 2 days(D2),7 days(D7),and 9 days(D9).There are 6 groups in total:N2,P2,N7,P7,N9 and P9.Alizarin red staining and intracellular calcium content were used to detect the calcification status of VSMCs treated with high phosphorus at different times.Western blot was used to detect the protein expressions of DNMT3A,Runx2,SM22α,LC3 and P62 in cells of each group.Quantitative real time reverse transcription polymerase chain reaction(q RT-PCR)was used to verify the changes of m RNA expression of DNMT3A,Runx2,SM22α.2.A plasmid interfering with the expression of DNMT3A RNA was constructed,and 3 generations of rats were given VSMCs to transfect sh RNA plasmid targeting DNMT3A gene in high phosphorus environment.The rats were divided into 4 groups:Normal Phosphate(N),High Phosphate(β-GP),β-GP+DNMT3A empty plasmid group(β-GP+sh RNA-NC group),β-GP+DNMT3A RNA interference group(β-GP+sh RNA-DNMT3A group).Western blot was used to detect the protein expressions of Runx2,SM22α,LC3(LC3II,LC3I),and P62 in each group,and q RT-PCR was used to verify the m RNA expressions of DNMT3A,Runx2,and SM22αafter transfection of sh RNA plasmid targeting DNMT3A gene.Results:1.The expression of DNMT3A was increased in calcification of VSMCsAlizarin red staining showed more red calcified nodules in the high phosphorus group compared with the normal group.After 2d,7d and 9d of high phosphorus treatment,the expression of intracellular calcium content in VSMCs was significantly increased compared with that in normal group,and with the extension of time,calcium nodules and intracellular calcium content in alizarin red staining in high phosphorus group gradually increased,reaching a peak at day 9.It was suggested that high phosphorus could induce calcification of VSMCs.Compared with the normal group,Western blot and q RT-PCR showed the expression of DNMT3A protein and m RNA in the high phosphorus calcification group increased in a time dependent manner.2.The expression of osteogenic phenotypic indicators and autophagy rela-ted indicators were increased in calcification of VSMCsCompared with the normal group,Western blot and q RT-PCR showed the expression of Runx2 protein and m RNA was increased in VSMCs calcified group,the expression of SM22αprotein and m RNA was decreased in VSMCs calcified group,which was in a time dependent manner.In the VSMCs calcification group,Western blot showed LC3II/LC3I protein expression was increased,and P62 expression was decreased and presented in a time manner.3.Knock-down of DNMT3A could significantly down-regulate osteogen-ic phenotype and up-regulate autophagy related indexes,and inhibited VSMCs calcificationCompared with DNMT3A empty plasmid transfection group,Western blot and q RT-PCR showed that transfection of DNMT3A RNA interference plasmid was significantly down regulated the expression of DNMT3A protein and m RNA,Runx2 protein and the m RNA of these genes were decreased,SM22αprotein and m RNA were increased.Compared with DNMT3A empty plasmid transfection group,Western blot showed that LC3II/LC3I protein expression was increased,P62 protein expression was decreased.Compared with the sh RNA-NC group,the red calcium nodes and intracellular calcium content in alizarine red staining were decreased in the sh RNA-DNMT3A group,suggesting that DNMT3A could affect VSMCs calcification through mediating osteogenic phenotype and autophagy related indicators.Conclusions:DNMT3A mediated phenotypic transformation and autophagy related indicators affecting VSMCs calcification induced by high phosphorus.Part Three DNMT3A mediated phenotypic transformation and autoph-agy of vascular smooth muscle cells by ERK signaling path-wayObjective:Extracellular regulated proteinkinases(ERK)signaling path-way plays a key role in vascular calcification,which is regulated cell function and activity.This study was aimed to observe the expression and role of ERK signaling pathway in VSMCs calcification,and to explore how DNMT3A mediates phenotypic transformation and autophagy related indicators by regulating ERK signaling pathway in VSMCs calcification model.Methods:1.Rat VSMC was isolated and cultured in vitro,and the cells cultured to the fourth generation were randomly divided into normal group and high phosphorus group.The normal control group was cultured on medium containing 10%fetal bovine serum,and the high-phosphorus group was cultured on high-phosphorus medium containing 10 mmol/Lβ-glycerophosphate for 2,7 and 9 days.Accordingly,they were divided into 6groups:N2,P2,N7,P7,N9 and P9.The protein expression of P-ERK and ERK in each group was detected by Western blot.2.The third-generation VSMCs stimulated by high phosphorus for 48h were given specific inhibitors of P-ERK,that is PD0325901.The protein expressions of Runx2,SM22α,LC3 and P62 in each group were measured by Western blot.3.The third generation of VSMCs was transfected with DNMT3A RNA interfering plasmid,according to this they were divided into 4 groups:N,β-GP,β-GP+DNMT3A empty plasmid(β-GP+sh RNA-NC group),β-GP+DNMT3A interfering RNA group(β-GP+sh RNA-DNMT3A group),the expression of P-ERK and ERK protein in each group after transfection was measured by Western blot.Results:1.P-ERK signaling pathway was increased in calcified VSMCs model induced by hyperphosphorusIn the VSMCs calcification group,the expression of P-ERK/ERK was gradually increased with the time of high phosphorus stimulation.2.ERK signaling pathway could regulate phenotypic transformation and autophagy indicators in the VSMCs calcification modelCompared with P+DMSO group,Runx2 protein expression was decrea-sed and SM22αprotein expression was increased in P+PD0325901 group,suggesting that ERK signaling pathway regulated phenotypic transformation related indicators.Compared with P+DMSO group,the expression of LC3II/LC3I was increased and the expression of P62 was decreased in the P+PD0325901 group,suggesting that ERK signaling pathway related proteins could regulate autophagy related indicators.3.ERK signaling pathway proteins were regulated by DNMT3A in the model of VSMCsAfter transfection with DNMT3A,the ERK signaling pathway was inhibited and P-ERK/ERK expression was decreased.This suggests that ERK signaling pathway related proteins were regulated by DNMT3A.Conclusions:DNMT3A modulated phenotypic transformation and autophagy-related markers through ERK signaling pathway in VSMCs.Summary:DNMT3A was highly expressed in radial artery tissues of patients with chronic renal disease and was significantly correlated with the degree of calcification.DNMT3A regulated the phenotypic transformation and autophagy of high phosphorus induced VSMCs,and one of the possible mechanisms was that DNMTA modulated phenotypic transformation and autophagy-related makers through ERK signaling pathway. |
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