| BackgroudRenal fibrosis is a common pathological pathway of various chronic kidney disease progressed to end-stage renal disease.Its characteristics mainly include tubular atrophy,the accumulation of inflammatory cells,the fibroblasts/myofibroblasts activation,accumulation and dysregulated remodeling of extracellular matrix(ECM)such as collagen type I(Col-I),III(Col-III),and fibronectin(FN).Furthermore,epithelial-mesenchymal transition(EMT)is closely involved with the pathogenesis and progression of renal fibrosis.It has been reported that renal fibrosis is closely correlated with a decline in renal function and is to be the most important prognostic marker.Preventing renal fibrosis could be an effective treatment approach to ameliorate,or even reverse,chronic kidney disease.However,the etiology of renal fibrosis is complex,and no effective medication is currently available to manage its progression.The pathophysiological mechanisms of its occurrence and development remain unclear and still need to be investigated.EMT is considered to be a process in which epithelial cells lose apical-basal polarity,and acquire the migratory and invasive properties of mesenchymal cells.Reports indicated that the biomarkers of these cells exhibit alterations over timeincluding the loss of the epithelial cell marker protein E-cadherin,the acquisition of mesenchymal features such as ?-smooth muscle actin(?-SMA),and the production of interstitial matrix proteins such as fibronectin and collagens.In addition,numerous growth factors mediates EMT,such as cytokines,hormones and extracellular cues in various ways.Reports indicate that the EMT,ECM accumulation and renal fibrosis progression under pathological conditions are mainly mediated via transforming growth factor-?1(TGF-?1)/ Smad signaling pathway.TGF-?1 binds to its receptor,and could form complexes with Smad2,Smad3,and Smad4.This complex translocates from the cytoplasm to the nucleus,and regulates various target gene expression such as collagens.But,smad7 is part of a negative feedback loop in TGF-?1 signaling.N-Myc downstream-regulated gene-2(NDRG2)is a member of the NDRG family,which comprises four members,NDRG1-4.NDRG2 regulates cell differentiation and cell proliferation in different tissues such as the brain,heart,liver,and kidney.Reports have shown that NDRG2,as a tumor suppressor,inhibits the EMT in breast cancer and gallbladder carcinoma.In addition,NDRG2 ameliorates liver fibrosis by inhibiting the TGF-?1/Smad pathway in rats.It has been reported that NDRG2 was significantly upregulated in the normal kidney proximal tubular cells when compared with renal cell carcinoma,and can inhibit the proliferation of renal carcinoma cells.However,NDRG2 functions in TGF-?1-induced human renal tubular epithelial cells have not yet been investigated.In this study,we examined the expression of NDRG2 in TGF-?1-induced human renal tubular epithelial cells(HK-2).We further evaluated the biological function of NDRG2 and explored its potential underlying mechanism.Our results showed that TGF-?1 down-regulated NDRG2 mRNA and protein expression in HK-2 cells.Then,NDRG2 could regulate EMT process and secretion of the extracellular matrix,which is associated with TGF-?1/Smad3 pathway.Objective(1)Real-time fluorescent quantitative PCR and Western Blotting were used todetect the expression of NDRG2 in different concentrations of TGF-?1-induced HK-2cell line.(2)Construction of three kinds of NDRG2-induced HK-2 cell sublines and their control groups,Western blotting and real-time quantitative PCR were used to detect the expression of protein and mRNA in EMT markers in different HK-2 cell sublines The expression levels of extracellular matrix in different HK-2 cell sublines were detected by ELISA and real-time fluorescence quantitative PCR.(3)The expression levels of Smad2(p-Smad2),Smad3(p-Smad3)and Smad7 in different cell sublines were detected by Western Blotting.The expression of Smad2(p-Smad2)and Smad3 were detected by Western Blotting.The expression of protein and mRNA in EMT marker was detected by NDRG2-siRNA + SIS3(SelleckChem)and the expression of extracellular matrix was detected by ELISA and real-time fluorescence quantitative PCR.Materials and Methods1.HK-2 cell line: An immortalized human renal proximal tubular cell line,purchased from ATCC(American Type Culture Collection,VA,USA).2.Methods: Different concentrations of TGF-?1(0ng / ml,2.5 ng / ml,5 ng /ml,10 ng / ml,20 ng / ml)were added to HK-2 cell lines for 48 h The expression level of NDRG2 in different concentrations of TGF-?1-induced HK-2 cell line was detected by real-time fluorescence quantitative PCR and Western Blotting.The expression level of protein and mRNA of EMT markers in different HK-2 cell sublines was detected by Western Blotting and real-time fluorescence quantitative PCR.The expression of protein and mRNA in HK-2 cells were detected by ELISA,The expression levels of extracellular matrix in different HK-2 cell sublines were detected by real-time fluorescence quantitative PCR.The expression levels of Smad2(p-Smad2),Smad3(p-Smad3)and Smad7(p-Smad7)in different cell sublines were detected by Western Blotting.Western blotting was used to detect the expression of Smad2(p-Smad3)Blotting method and real-time fluorescence quantitative PCR were used to detect the expression of protein and mRNA of EMT marker underNDRG2-siRNA + SIS3(SelleckChem)and the expression of extracellular matrix was detected by ELISA and real-time fluorescence quantitative PCR.3.All data are presented as the mean ± standard deviation(SD).Statistical analysis was performed using SPSS 17.0 software.Comparisons among more than 2groups involved were tested with the Student's t-test or one-way analysis of variance(ANOVA)with the Bonferroni post-hoc test.P<0.05 was considered significant.Results(I)Expression and significance of NDRG2 in different concentrations of TGF-?1-induced HK-2 cell line1.The mRNA expression of NDRG2 in HK-2 cell line induced by different concentrations of NDRG2 was detected by RT-PCR.The expression of NDRG2 in A1(0ng / ml TGF-?1),B1(2.5ng / ml TGF-The expression levels of D1(10ng / ml TGF-?1)and E1(20ng / ml TGF-?1)HK-2 cells were decreased in B1 group,C1 group,D1 group and Compared with A1 group,(P = 0.031),P1 group and C1 group compared with(P = 0.013),E1 group and D1 group compared with(P = 0.031),C1 group and A1 group compared(P = 0.012,0.011,0.003,0.016,0.013),the difference was statistically significant.2.Western blot was used to detect the expression of NDRG2 in different concentrations of TGF-?1-induced HK-2 cell line.NDRG2 in A2(0ng / ml TGF-?1),B2(2.5ng / ml TGF-?1),C2(5ng The expression levels of D2(10ng / ml TGF-?1)and E2(20ng / ml TGF-?1)HK-2 cells were decreased in group B2,C2,D2 and E2 The P value of the group was 0.011,0.001,0.004 and 0.017 respectively compared with that of the A2 group,P = 0.021 in the C2 group and P= 0.014 in the D2 group compared with the C2 group,and P = 0.003 in the E2 group compared with the D2 group,The differences were statistically significant.(II)Effects of NDRG2 and TGF-?1 on EMT markers and extracellular matrix and its significance1.TGF-?1 + pcDNA3.1-NDRG2 HK-2 cell sub-line and its control group TGF-?1 + pc-control,TGF-?1 + NDRG2-siRNA HK-2 cell sub-line and its Theexpression of TGF-?1 + si-NC and TGF-?1 HK-2 cells in the control group were detected by Cell Counting Kit-8(CCK-8)(P> 0.05),indicating that the cell viability of each cell line was not affected by pcDNA3.1-NDRG2,NDRG2-siRN,TGF-?1 and corresponding to the control group The effect of the control.2.Western blot was used to detect the expression of NDRG2 in HK-2 cell subunits transfected with pcDNA3.1-NDRG2 and NDRG2-si RN.NDRG2 in HK-2cells containing only TGF-?1(group B1 / group B2)(P = 0.011;P = 0.009);NDRG2was transfected into HK-2 cells transfected with pcDNA3.1-NDRG2(group D1)compared with control group A1(group A1 / group A2)(P = 0.024);the relative expression of NDRG2 in the subtype of HK-2 cells transfected with NDRG2-siRNA(F1 group)was significantly higher than that in control group(P <0.05).The difference was statistically significant(P <0.05).The difference was statistically significant(P <0.05).3.RT-PCR and Western blot were used to detect the expression of mRNA and protein in E-cadherin(A-SMA,Vimentin,Snail)in different HK-2 cell sublines.The relative expression of E-cadherin in HK-2 cells with TGF-?1(group B2)was significantly lower than that in control group(P <0.05),while a-SMA,Vimentin,Snail,and the expression level was significantly higher than that of the control group(P <0.05);The relative expression of E-cadherin in HK-2 cell sub-lines transfected with pcDNA3.1-NDRG2(D2)was significantly higher than that in control group(P<0.05),while a-SMA,Vimentin(P <0.05).The expression level of E-cadherin in HK-2 cell sub-line transfected with NDRG2-siRNA(F2 group)was significantly higher than that in control group(P <0.05),and the expression level of Snail was significantly lower than that of control group)Compared with the control group were significantly higher than the control group,the difference was statistically significant.4.ELISA and RT-PCR were used to detect the expression of protein and mRNA in the extracellular matrix(COI-I,COI-III,FN)in different HK-2 cell sublines.The relative expression levels of COI-I,COI-III and FN protein and mRNA in HK-2 cell sublines containing TGF-?1(group B4)were significantly higher than those in control group(P < 0.05).The expression of TGF-?1 + pcDNA3.1-NDRG2 and TGF-?1 + pc-control COI-I,COI-III and FN were significantly decreased(P <0.05).The expression of COI-I,COI-III,FN protein and mRNA in F4 group(TGF-?1 +NDRG2-siRNA)and E4 group(TGF-?1 + si-NC)were significantly increased(P<0.05).The above differences were statistically significant.(III)Effects of NDRG2 and TGF-?1 on Smad expression in HK-2 cell line and its significance1.Western blot was used to detect the expression of Smad2(p-Smad2),Smad3(p-Smad3)and Smad7 in HK-2 cell subfamilies transfected with pcDNA3.1-NDRG2 and NDRG2-siRN.The expression levels of t-Smad2 and p-Smad2(phosphorylation)in group B1(TGF-?1)were significantly higher than those in control group A1(P<0.05),while in group D1(pc-DNA)(P <0.05),and there was no significant difference between the two groups(P> 0.05),but there was no significant difference between the two groups(P> 0.05).The expression of t-Smad3 and p-Smad3 in group B1(TGF-?1)was significantly higher than that in control group(P <0.05).There was no significant difference in the protein expression of t-Smad3 between D1 group(pcDNA3.1-NDRG2)and F1 group(NDRG2-siRNA)compared with control group C1(pc-control)and E1 group(si-NC)(P> 0.05),p-Smad3 in the D1 group(pcDNA3.1-NDRG2)compared with the control group C1(pc-control)compared to the protein expression was significantly reduced(P <0.05).The expression of Smad7 in group B1(TGF-?1)was significantly higher than that in control group,and the expression of Smad7 was significantly higher than that of control group(NDRG2-siRNA)(P = 0.011),while in group D1(pcDNA3.1-NDRG2)and F1(NDRG2-siRNA)were significantly lower than those in control group C1(P> 0.05)respectively.There was no significant difference between the two groups(P> 0.05).The above differences were statistically significant.2.Western blot was used to detect the protein expression levels of Smad2(p-Smad2),Smad3(p-Smad3)and Smad7 in HK-2 cell subunits transfected with NDRG2-siRN(SIS3).The expression levels of Smad2,p-Smad2,Smad3 and Smad7 in SIS group(NDRG2-siRNA + SIS3)were not significantly different from those in TGF group(P> 0.05).The protein expression level of p-Smad3 in SIS group(NDRG2-siRNA + SIS3)was significantly lower than that in TGF group(NDRG2-siRNA)(P <0.05).3.RT-PCR and Western blot were used to detect the expression of E-cadherin(a-SMA,Vimentin,Snail)mRNA and protein in different HK-2 cell sublines.The mRNA expression of E-cadherin,a-SMA,Vimentin,Snail in HK-2 cell sublines of TGF-?1 + si-NC(NC group)was significantly lower than that of TGF-?1(Con group)(P> 0.05).The relative expression of E-cadherin in TGF-?1 + NDRG2-siRNA(TGF)HK-2 cell,while the expression of a-SMA,Vimentin and Snail mRNA was significantly higher than that of the control group(P <0.05);The mRNA expression of E-cadherin in TGF-?1 + NDRG2-siRNA + SIS3(SIS group)HK-2 cell sub-line was significantly higher than that in control group(TGF-?1 + NDRG2-si RNA)(P<0.05),while the mRNA expression of a-SMA,Vimentin and Snail was significantly lower than that of the control group(P <0.05).The above differences were statistically significant.4.ELISA and RT-PCR were used to detect the expression of protein and mRNA in the extracellular matrix(COI-I,COI-III,FN)in different HK-2 cell sublines.The relative expression levels of protein and mRNA in Co-I,COI-III and FN in HK-2 cell sublines of TGF-?1 + si-NC(NC group)were significantly lower than those in TGF-?1(Con group)(TGF-?1 + NDRG2-siRNA)and NC group(TGF-?1 + si-NC)COI-I,COI-III,FN protein(TGF-?1 + NDRG2-siRNA)were significantly different(P = 2.050,P = 1.240,P was significantly higher than that in NC group(TGF-?1 +si-NC)COI-I(P> 0.05).,and the protein and mRNA expression of COI-III and FN were significantly decreased.The above differences were statistically significant.ConclusionsNDRG2 regulates TGF-?1-induced HK-2 cell fibrosis and may be achieved by TGF-?1 / Smad3 signaling pathway.(I)The mRNA and protein expression levels of NDRG2 were down-regulated in different concentrations of TGF-?1-induced HK-2 cells in a dose-dependent manner,especially TGF-?1(> 10 ng / mL),the most significant(P <0.05).These results indicate that TGF-?1 down-regulates the expression of NDRG2 mRNA and protein in HK-2 cells,indicating that it may play a role in the regulation of renal fibrosis.(II)According to the expression of mRNA and protein in E-cadherin,a-SMA,Vimentin and Snail in different HK-2 cell sublines,it was revealed that at the mRNA and protein levels,E-cadherin contained HK-2 Cell sublines and NDRG2-siRNA HK-2 cell sublines,and overexpressed in pcDNA3.1-NDRG2 HK-2 cell sublines,a-SMA,Vimentin,Snail overexpression in HK-2 cell sublines and NDRG2-siRNA HK-2 cell sublines that only contained TGF-?1,whereas expression in pcDNA3.1-NDRG2 HK-2 cell sublines,Suggesting that NDRG2 regulates protein and mRNA expression of EMT markers in HK-2 cells induced by TGF-?1,and there is a potential regulatory mechanism for NDRG2 / TGF-?1 during EMT.(III)According to the expression levels of COI-I,COI-III and FN in protein and mRNA in different HK-2 cell sublines,it was revealed that at the mRNA and protein levels,COI-I,COI-III and FN were expressed in TGF-Of HK-2 cell sublines and NDRG2-siRNA HK-2 cell sublines,indicating that NDRG2 regulates TGF-?1-induced HK-2 cells in the subtypes of pcDNA3.1-NDRG2 HK-2 cells In the secretion of extracellular matrix,in the process of ECM NDRG2 / TGF-?1 also exist in the potential regulatory mechanism.(IV)NDRG2 inhibits the phosphorylation of Smad3 in TGF-?1-induced HK-2cells;NDRG2 regulates TGF-?1-induced fibrosis of HK-2 cells and can be expressed by Smad3 phosphorylated specific inhibitor(SIS3)NDRG2 regulates the secretion of extracellular matrix in HK-2 cells induced by TGF-?1 and reverses the regulation of NDRG2 by the specific inhibitor of Smad3 phosphorylation(SIS3);NDRG2regulates TGF-?1 Induced HK-2 cell fibrosis,and may be achieved through the TGF-?1 / Smad3 signaling pathway.If the drug can be developed to increase the expression of NDRG2 drugs or inhibition of TGF-?1 / Smad3 signaling pathway of drugs,to a certain extent,can delay or terminate the development of renal fibrosis,thereby alleviating the incidence of chronic kidney disease. |
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