本文關(guān)鍵詞： miR-141 HIPK2 腎纖維化 EMT　出處：《南方醫(yī)科大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：背景：隨著社會(huì)經(jīng)濟(jì)的發(fā)展和人們生活方式的改變,人類疾病譜正在發(fā)生變化,慢性腎臟病(chronic kidney disease, CKD)已呈現(xiàn)流行特征。CKD具有患病率高、醫(yī)療費(fèi)用巨大、易合并心血管疾病而導(dǎo)致病死率和致殘率高等特點(diǎn),目前已成為影響我國(guó)國(guó)民健康的主要疾病之一。腎小管間質(zhì)纖維化是各種原因引起的CKD發(fā)展到終末期腎衰竭的共同歸路,而上皮細(xì)胞間充質(zhì)轉(zhuǎn)分化(epithelial-mesenchymal transition, EMT)是導(dǎo)致腎小管間質(zhì)纖維化中心環(huán)節(jié)之一。尋找一種新的有效、可行、副作用小的CKD治療方法,將在很大程度上改變CKD患者的治療現(xiàn)狀。隨著分子生物學(xué)和遺傳學(xué)的發(fā)展,CKD的靶向治療成為目前研究的焦點(diǎn)。MicroRNA (miRNA)是一類存在于病毒和高等生物中,進(jìn)化上高度保守的內(nèi)源性非編碼RNA。miRNA廣泛參與調(diào)控包括個(gè)體生長(zhǎng)發(fā)育、細(xì)胞凋亡、增殖及分化在內(nèi)的許多生命過程。近來發(fā)現(xiàn)miRNA與纖維化有非常密切的關(guān)系。 目的：目前,已在多種纖維化中發(fā)現(xiàn)miR-141家族表達(dá)異常,其中有報(bào)道m(xù)iR-141在多種纖維化中顯著低表達(dá)并具有抑制TGFβ1誘導(dǎo)的EMT進(jìn)程的作用。通過軟件預(yù)測(cè)和熒光定量PCR驗(yàn)證,發(fā)現(xiàn)miR-141可靶向調(diào)控HIPK2表達(dá),同時(shí),有報(bào)道指出,HIPK2在人腎纖維化的EMT過程中起重要作用,而靶向調(diào)控HIPK2的miRNA卻鮮有報(bào)道。本課題擬研究miR-141通過靶向調(diào)控HIPK2抑制腎小管上皮細(xì)胞HK2中TGFβ1誘導(dǎo)的EMT進(jìn)程,以期為尋找腎纖維化的防治靶標(biāo)提供堅(jiān)實(shí)理論基礎(chǔ)。 方法：1、用不同濃度的TGFβ1處理HK2細(xì)胞48h,用real-time PCR檢測(cè)miR-141的表達(dá)以及HIPK2mRNA的表達(dá),Western blot檢測(cè)HIPK蛋白的表達(dá)；2、用2ng/ml的TGFβ1處理HK2細(xì)胞不同的時(shí)間,以real-time PCR檢測(cè)miR-141的表達(dá)以及HIPK2mRNA的表達(dá),Western blot檢測(cè)HIPK蛋白的表達(dá)；3、利用miR-141模擬物以及miR-141Sponge載體轉(zhuǎn)染HK2細(xì)胞,用real-time PCR檢測(cè)miR-141的表達(dá)以及HIPK2mRNA的表達(dá),Western blot分析HIPK蛋白的表達(dá)；4、利用雙熒光素酶報(bào)告基因進(jìn)一步驗(yàn)證miR-141對(duì)HIPK2的靶向調(diào)控作用；5、TGFβ1與miR-141聯(lián)合處理HK2細(xì)胞, Western blot檢測(cè)EMT標(biāo)志因子Vimentin、FSP-1和E-cadherin的表達(dá)；6、構(gòu)建HIPK2過表達(dá)載體,與miR-141共轉(zhuǎn)染HK2細(xì)胞,Western blot檢測(cè)EMT標(biāo)志因子Vimentin、FSP-1和E-cadherin的表達(dá)。 結(jié)果：1、隨著TGFβ1處理濃度和處理時(shí)間的增加,real-time PCR結(jié)果顯示,miR-141表達(dá)逐漸下調(diào),HIPK2mRNA表達(dá)逐漸上調(diào),HIPK且Western blot結(jié)果顯示,HIPK2蛋白表達(dá)逐漸上調(diào)；2、隨著TGFβ1處理時(shí)間的增加,real-timePCR結(jié)果顯示,miR-141表達(dá)逐漸下調(diào),HIPK2mRNA表達(dá)逐漸上調(diào),且Western blot結(jié)果顯示,HIPK2蛋白表達(dá)逐漸上調(diào)。當(dāng)TGFβ1濃度為2ng/mL處理時(shí)間為2天時(shí),HIPK2mRNA和蛋白與未處理細(xì)胞相比分別上調(diào)1.4和1.7倍,通過t檢驗(yàn),p值分別為0.0018和0.0001,而miR-141下調(diào)40%,t檢驗(yàn)得到p值為0.0054,均有統(tǒng)計(jì)學(xué)意義；3、miR-141模擬物轉(zhuǎn)染HK2細(xì)胞時(shí),miR-141過表達(dá),HIPK2表達(dá)收到抑制；而miR-141Sponge載體轉(zhuǎn)染HK2細(xì)胞時(shí),miR-141被沉默,HIPK2表達(dá)上調(diào)。經(jīng)t檢驗(yàn),p值分別為0.0006和0.0005,具有統(tǒng)計(jì)學(xué)意義；4、雙熒光素酶報(bào)告基因?qū)嶒?yàn)結(jié)果顯示miR-141可靶向抑制HIPK2的表達(dá),與對(duì)照組相比,miR-141轉(zhuǎn)染組和miR-141Sponge轉(zhuǎn)染組均有顯著變化,p值均小于0.0001；5、TGFβ1可上調(diào)HIPK2, Vimentin和FSP1的表達(dá),與對(duì)照組相比,p值分別為0.0002,0.0003和0.0011,下調(diào)E-cadherin的表達(dá),與對(duì)照組相比,p值為0.0002；miR-141可抑制HIPK2, Vimentin和FSP1的表達(dá),與對(duì)照組相比,p值分別為0.0001,0.0001和0.0002；上調(diào)E-cadherin的表達(dá),與對(duì)照組相比,p值為0.0007。TGFβ1和miR-141聯(lián)合作用可逆轉(zhuǎn)TGFβ1對(duì)HIPK2,Vimentin和FSP1的上調(diào)作用,與TGFβ1組相比,p值分別為0.0030,0.0004和0.0013；以及可逆轉(zhuǎn)對(duì)E-cadherin的抑制作用,與TGFβ1組相比,p值分別為0.0005;6、HIPK2可上調(diào)Vimentin和FSP1的表達(dá),與對(duì)照組相比,p值分別為0.0113和0.0087,下調(diào)E-cadherin的表達(dá),與對(duì)照組相比,p值為0.0001；miR-141可抑制Vimentin和FSP1的表達(dá),與對(duì)照組相比,p值分別為0.0002和0.0003；上調(diào)E-cadherin的表達(dá),與對(duì)照組相比,p值為0.0021。miR-141和HIPK2聯(lián)合作用可逆轉(zhuǎn)miR-141對(duì)Vimentin和FSP1的抑制作用,與miR-141轉(zhuǎn)染組相比,p值分別為0.0003和0.0001；以及可逆轉(zhuǎn)對(duì)E-cadherin的上調(diào)作用,與miR-141轉(zhuǎn)染組相比,p值為0.0022。 結(jié)論：1、TGFβ1可下調(diào)miR-141而上調(diào)HIPK2表達(dá)；2、miR-141可靶向抑制HIPK2的表達(dá)；3、TGFβ1可通過抑制miR-141而調(diào)控EMT進(jìn)程；4、miR-141可通過抑制HIPK2而調(diào)控EMT進(jìn)程；5、miR-141通過靶向抑制而HIPK2調(diào)控TGFβ1介導(dǎo)的腎纖維化EMT進(jìn)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692

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