TGFβ1抑制miR-141上调HIPK2促进肾小管上皮细胞转分化

发布时间：2018-02-27 22:47

本文关键词： miR-141 HIPK2 肾纤维化 EMT　出处：《南方医科大学》2014年博士论文　论文类型：学位论文

【摘要】：背景：随着社会经济的发展和人们生活方式的改变,人类疾病谱正在发生变化,慢性肾脏病(chronic kidney disease, CKD)已呈现流行特征。CKD具有患病率高、医疗费用巨大、易合并心血管疾病而导致病死率和致残率高等特点,目前已成为影响我国国民健康的主要疾病之一。肾小管间质纤维化是各种原因引起的CKD发展到终末期肾衰竭的共同归路,而上皮细胞间充质转分化(epithelial-mesenchymal transition, EMT)是导致肾小管间质纤维化中心环节之一。寻找一种新的有效、可行、副作用小的CKD治疗方法,将在很大程度上改变CKD患者的治疗现状。随着分子生物学和遗传学的发展,CKD的靶向治疗成为目前研究的焦点。MicroRNA (miRNA)是一类存在于病毒和高等生物中,进化上高度保守的内源性非编码RNA。miRNA广泛参与调控包括个体生长发育、细胞凋亡、增殖及分化在内的许多生命过程。近来发现miRNA与纤维化有非常密切的关系。目的：目前,已在多种纤维化中发现miR-141家族表达异常,其中有报道miR-141在多种纤维化中显著低表达并具有抑制TGFβ1诱导的EMT进程的作用。通过软件预测和荧光定量PCR验证,发现miR-141可靶向调控HIPK2表达,同时,有报道指出,HIPK2在人肾纤维化的EMT过程中起重要作用,而靶向调控HIPK2的miRNA却鲜有报道。本课题拟研究miR-141通过靶向调控HIPK2抑制肾小管上皮细胞HK2中TGFβ1诱导的EMT进程,以期为寻找肾纤维化的防治靶标提供坚实理论基础。方法：1、用不同浓度的TGFβ1处理HK2细胞48h,用real-time PCR检测miR-141的表达以及HIPK2mRNA的表达,Western blot检测HIPK蛋白的表达；2、用2ng/ml的TGFβ1处理HK2细胞不同的时间,以real-time PCR检测miR-141的表达以及HIPK2mRNA的表达,Western blot检测HIPK蛋白的表达；3、利用miR-141模拟物以及miR-141Sponge载体转染HK2细胞,用real-time PCR检测miR-141的表达以及HIPK2mRNA的表达,Western blot分析HIPK蛋白的表达；4、利用双荧光素酶报告基因进一步验证miR-141对HIPK2的靶向调控作用；5、TGFβ1与miR-141联合处理HK2细胞, Western blot检测EMT标志因子Vimentin、FSP-1和E-cadherin的表达；6、构建HIPK2过表达载体,与miR-141共转染HK2细胞,Western blot检测EMT标志因子Vimentin、FSP-1和E-cadherin的表达。结果：1、随着TGFβ1处理浓度和处理时间的增加,real-time PCR结果显示,miR-141表达逐渐下调,HIPK2mRNA表达逐渐上调,HIPK且Western blot结果显示,HIPK2蛋白表达逐渐上调；2、随着TGFβ1处理时间的增加,real-timePCR结果显示,miR-141表达逐渐下调,HIPK2mRNA表达逐渐上调,且Western blot结果显示,HIPK2蛋白表达逐渐上调。当TGFβ1浓度为2ng/mL处理时间为2天时,HIPK2mRNA和蛋白与未处理细胞相比分别上调1.4和1.7倍,通过t检验,p值分别为0.0018和0.0001,而miR-141下调40%,t检验得到p值为0.0054,均有统计学意义；3、miR-141模拟物转染HK2细胞时,miR-141过表达,HIPK2表达收到抑制；而miR-141Sponge载体转染HK2细胞时,miR-141被沉默,HIPK2表达上调。经t检验,p值分别为0.0006和0.0005,具有统计学意义；4、双荧光素酶报告基因实验结果显示miR-141可靶向抑制HIPK2的表达,与对照组相比,miR-141转染组和miR-141Sponge转染组均有显著变化,p值均小于0.0001；5、TGFβ1可上调HIPK2, Vimentin和FSP1的表达,与对照组相比,p值分别为0.0002,0.0003和0.0011,下调E-cadherin的表达,与对照组相比,p值为0.0002；miR-141可抑制HIPK2, Vimentin和FSP1的表达,与对照组相比,p值分别为0.0001,0.0001和0.0002；上调E-cadherin的表达,与对照组相比,p值为0.0007。TGFβ1和miR-141联合作用可逆转TGFβ1对HIPK2,Vimentin和FSP1的上调作用,与TGFβ1组相比,p值分别为0.0030,0.0004和0.0013；以及可逆转对E-cadherin的抑制作用,与TGFβ1组相比,p值分别为0.0005;6、HIPK2可上调Vimentin和FSP1的表达,与对照组相比,p值分别为0.0113和0.0087,下调E-cadherin的表达,与对照组相比,p值为0.0001；miR-141可抑制Vimentin和FSP1的表达,与对照组相比,p值分别为0.0002和0.0003；上调E-cadherin的表达,与对照组相比,p值为0.0021。miR-141和HIPK2联合作用可逆转miR-141对Vimentin和FSP1的抑制作用,与miR-141转染组相比,p值分别为0.0003和0.0001；以及可逆转对E-cadherin的上调作用,与miR-141转染组相比,p值为0.0022。结论：1、TGFβ1可下调miR-141而上调HIPK2表达；2、miR-141可靶向抑制HIPK2的表达；3、TGFβ1可通过抑制miR-141而调控EMT进程；4、miR-141可通过抑制HIPK2而调控EMT进程；5、miR-141通过靶向抑制而HIPK2调控TGFβ1介导的肾纤维化EMT进程。
[Abstract]:Background: with the development of social economy and the change of people's lifestyle, changing the spectrum of human diseases, chronic kidney disease (chronic kidney disease, CKD) has presented the epidemic characteristics of.CKD have high prevalence rate, huge medical costs, easy to complicated with cardiovascular disease caused mortality rate and disability rate higher characteristic, has become one of the main diseases of our national health. Renal tubulointerstitial fibrosis is caused by various reasons CKD to the final common return stage of renal failure, and epithelial mesenchymal transition (epithelial-mesenchymal transition EMT) is one of the leading center of renal tubule interstitial fibrosis. To find a new effective, feasible, vice effect of CKD treatment of small, to change the status quo in the treatment of CKD to a great extent. With the development of molecular biology and genetics, the research into CKD targeted therapy The focus of.MicroRNA (miRNA) is a kind of virus and in higher organisms, evolutionarily highly conserved endogenous non encoding RNA.miRNA is widely involved in the regulation of individual growth, cell apoptosis, proliferation and differentiation, many life processes. Recently there is a very close relationship with miRNA fiber.
Objective: at present, the abnormal expression of miR-141 family have been found in a variety of fibrosis, which miR-141 has been reported in a variety of fibrosis significantly low expression and inhibit TGF beta 1 EMT process induced effect. Through the software prediction and quantitative PCR validation, found that miR-141 targeted regulation of HIPK2 expression, at the same time, there are reports that HIPK2 plays an important role in renal fibrosis in EMT process, and targeted regulation of HIPK2 miRNA has been reported. This paper intends to study the miR-141 inhibition of renal tubular epithelial cells in HK2 TGF beta to HIPK2 through regulation target 1 induced by the EMT process, in order to provide a solid theoretical basis for prevention and treatment of target for renal fibrosis.
Methods: 1, with different concentrations of TGF beta 1 in HK2 cells treated with 48h, with the expression of real-time PCR to detect miR-141 and HIPK2mRNA expression, Western protein expression was detected by HIPK blot; 2, 2ng/ml TGF 1 beta HK2 cells treated with different time, the real-time PCR detected the expression of miR-141 and HIPK2mRNA expression. To detect the expression of HIPK protein of Western blot; 3, miR-141Sponge mimics and vector transfected HK2 cells with miR-141 expression by real-time PCR detection of miR-141 and HIPK2mRNA expression, Western blot analysis of HIPK protein expression; 4, further validation of miR-141 targeted regulation of HIPK2 by dual luciferase reporter gene; 5, the combined treatment of HK2 cell TGF beta 1 and miR-141, Western blot EMT mark detection factor Vimentin, expression of FSP-1 and E-cadherin; 6, HIPK2 over expression vector was constructed, and miR-141 Western BL were transfected into HK2 cells. OT was used to detect the expression of EMT marker factors Vimentin, FSP-1 and E-cadherin.
Results: 1, with the increase of TGF beta 1 treatment concentration and treatment time, real-time PCR showed that the expression of miR-141 decreased gradually, the expression of HIPK2mRNA is increased, HIPK and Western blot showed that the expression of HIPK2 protein increased gradually; 2, with the increase of TGF beta 1 treatment time, the results of real-timePCR showed that the expression of miR-141 decreased gradually the expression of HIPK2mRNA is increased, and the Western blot results showed that the expression of HIPK2 protein increased gradually. When the TGF concentration is 2ng/mL beta 1 processing time for 2 days, HIPK2mRNA and protein were up-regulated by 1.4 and 1.7 times compared with the untreated cells, by t test, P values were 0.0018 and 0.0001, miR-141 by 40%, t test the value of P was 0.0054, there was statistically significant; 3, miR-141 mimics was transfected into HK2 cells, overexpression of miR-141, HIPK2 and miR-141Sponge expression is suppressed; vector was transfected into HK2 cells, miR-141 knockdown, HIPK2 Expression. By t test, P values were 0.0006 and 0.0005, with statistical significance; 4, dual luciferase reporter assay showed that miR-141 can inhibit the expression of HIPK2, compared with the control group, significant changes in the miR-141 transfected group and miR-141Sponge transfected group, P values were less than 0.0001; 5, TGF beta 1 can up regulate the expression of Vimentin and HIPK2, FSP1, compared with the control group, P values were 0.0002,0.0003 and 0.0011 respectively, down regulating the expression of E-cadherin, compared with the control group, P = 0.0002; miR-141 can inhibit the HIPK2 expression of Vimentin and FSP1, compared with the control group, the P values were 0.0001,0.0001 and 0.0002 up-regulated; E-cadherin, compared with the control group, the p value of the combined effects of 0.0007.TGF beta 1 and miR-141 could reverse TGF beta 1 of HIPK2, up regulation of Vimentin and FSP1, compared with group TGF beta 1, P = 0.0030,0.0004 and 0.0013 respectively; and the reversal of The inhibition of E-cadherin, compared with group TGF beta 1, P = 0.0005; 6, HIPK2 can up regulate the expression of Vimentin and FSP1, compared with the control group, the P values were 0.0113 and 0.0087, down regulating the expression of E-cadherin, compared with the control group, the p value is 0.0001; the expression of miR-141 can inhibit Vimentin and FSP1, compared with the control group, the P values were 0.0002 and 0.0003; the upregulation of the expression of E-cadherin, compared with the control group, the value of P can inhibit the reversed the effect of miR-141 on Vimentin and FSP1 for the combined effects of 0.0021.miR-141 and HIPK2, compared with miR-141 group, the P values were 0.0003 and 0.0001; and the reversal of E-cadherin to investigate the effects of miR-141 transfection group, compared with the value of 0.0022., P
Conclusion: 1, TGF beta 1 expression of miR-141 and overexpression of HIPK2; 2, miR-141 can inhibit the expression of HIPK2; 3, TGF beta 1 can inhibit miR-141 and EMT regulation process; 4, miR-141 can inhibit HIPK2 and EMT process control; 5, miR-141 inhibition by targeting and regulation of TGF beta HIPK2 1 renal fibrosis mediated by EMT process.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

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