本文關(guān)鍵詞：HMGA2及l(fā)et-7d miRNA對糖尿病腎臟病腎小管上皮細(xì)胞轉(zhuǎn)分化及腎臟纖維化的調(diào)控　出處：《南方醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Let-7d 腎小管上皮細(xì)胞 HMGA2 上皮間質(zhì)轉(zhuǎn)化 腎纖維化

【摘要】：背景和目的TGFβ1誘導(dǎo)的腎小管上皮間質(zhì)轉(zhuǎn)化(EMT)在糖尿病腎臟病(DKD)腎纖維化中起關(guān)鍵作用,HMGA2被報(bào)道通過多重機(jī)制參與TGFβ 1誘導(dǎo)的EMT的發(fā)生過程。微小RNA(miRNA)作為重要的轉(zhuǎn)錄后水平調(diào)控因子也參與EMT的發(fā)生。既往對TGF β 1/HMGA2在EMT中的研究多集中于腫瘤或其他器官領(lǐng)域,而在DKD背景下,尤其是在腎小管上皮細(xì)胞EMT及纖維化進(jìn)程中的作用目前并不明確。本研究擬探討HMGA2對TGFβ 1誘導(dǎo)的腎小管上皮細(xì)胞EMT及纖維化過程的影響和作用,并進(jìn)一步針對靶基因HMGA2,研究其上游miRNA對HMGA2的影響及參與DKD腎纖維化的潛在作用機(jī)制。通過上述研究,將有助于我們據(jù)此針對有效的信號通路發(fā)現(xiàn)重要的干預(yù)和治療靶點(diǎn),從而為DKD腎纖維化的治療提供新的思路。方法1.建立TGFβ 1刺激大鼠腎小管上皮細(xì)胞(NRK-52E)的細(xì)胞模型,利用qRT-PCR及western blot的方法檢測TGFβ 1刺激前后HMGA2及EMT相關(guān)基因的表達(dá)變化。2.利用siRNA轉(zhuǎn)染的方法驗(yàn)證HMGA2在TGF β 1誘導(dǎo)的EMT及腎纖維化中的作用,用qRT-PCR及western blot檢測siRNA沉默HMGA2基因前后其下游轉(zhuǎn)錄因子的表達(dá)變化。3.通過生物信息學(xué)方法,針對靶基因HMGA2篩選有價(jià)值的上游mmiRNA,用qRT-PCR檢測TGF β 1誘導(dǎo)NRK-52E細(xì)胞前后各miRNA的表達(dá)差異,同時(shí)進(jìn)一步于UUO小鼠模型中探索HMGA2及其上游miRNA的表達(dá)變化。4.通過過表達(dá)(轉(zhuǎn)染miRNA mimics)和抑制(轉(zhuǎn)染miRNA inhibitors)的功能實(shí)驗(yàn)進(jìn)一步驗(yàn)證篩選出的miRNA對TGFβ1誘導(dǎo)的EMT和腎纖維化的影響和作用。用qRT-PCR及western blot檢測HMGA2及EMT相關(guān)因子于miRNA過表達(dá)及抑制前后的表達(dá)情況。結(jié)果1.qRT-PCR 及 western blot 結(jié)果表明:給予 10ng/mL TGFβ1 刺激 NRK-52E細(xì)胞 48h 后,與 Control 組比較,TGFβ 1(48h)組中 HMGA2、α-SMA、vimentin、Col-I及FN的表達(dá)均明顯升高,E-Cad的表達(dá)明顯降低。2.qRT-PCR及western blot結(jié)果表明:轉(zhuǎn)染siRNA-HMGA2能明顯抑制TGFβ1誘導(dǎo)的NRK-52E細(xì)胞中HMGA2及α-SMA、vimentin表達(dá)的上調(diào)以及E-Cad表達(dá)的下調(diào);轉(zhuǎn)染siRNA-HMGA2對其下游轉(zhuǎn)錄因子Snail,Slug和Twist的表達(dá)無明顯影響。3.針對靶基因HMGA2,篩選出有價(jià)值的上游miRNA:miRNA-let-7d、miRNA-98、miRNA-33;qRT-PCR 結(jié)果顯示:miRNA-let-7d 在 TGFβ1誘導(dǎo)的 NRK-52E 細(xì)胞及UUO小鼠腎臟中的表達(dá)均明顯降低,而HMGA2的表達(dá)則呈反向升高;TGFβ1的刺激對miRNA-98及miRNA-33的表達(dá)無明顯影響。4.qRT-PCR及western blot結(jié)果表明:轉(zhuǎn)染let-7d mimic能明顯抑制TGFβ1誘導(dǎo)的NRK-52E細(xì)胞中HMGA2及α-SMA、Col-I、FN表達(dá)的上調(diào)以及E-Cad表達(dá)的下調(diào);轉(zhuǎn)染let-7d inhibitor能明顯增強(qiáng)TGFβ 1誘導(dǎo)的HMGA2及Col-I、FN表達(dá)的上調(diào)以及E-Cad表達(dá)的下調(diào)。結(jié)論1.TGFβ 1可誘導(dǎo)NRK-52E細(xì)胞中HMGA2的高表達(dá),并使細(xì)胞發(fā)生EMT及纖維化改變。2.對HMGA2進(jìn)行基因沉默(siRNA-HMGA2)可逆轉(zhuǎn)TGFβ1誘導(dǎo)的EMT及纖維化改變,它是一個(gè)重要的靶基因。3.miRNA-let-7d 為 HMGA2 重要的上游 miRNA,miRNA-let-7d/HMGA2 在 TGFβ 1誘導(dǎo)的EMT及腎纖維化進(jìn)程中可能發(fā)揮作用。4.過表達(dá)miRNA-let-7d可下調(diào)HMGA2的表達(dá),進(jìn)而抑制TGF β 1誘導(dǎo)的EMT及腎纖維化改變;抑制miRNA-let-7d可上調(diào)HMGA2的表達(dá),進(jìn)而加快TGFβ1誘導(dǎo)的EMT及腎纖維化進(jìn)展。5.miRNA-let-7d可能通過調(diào)節(jié)HMGA2的表達(dá)從而調(diào)控腎小管上皮細(xì)胞轉(zhuǎn)分化及纖維化,干預(yù)這一過程可能是一個(gè)預(yù)防和治療DKD腎纖維化的新途徑。
[Abstract]:Background and objective renal tubular epithelial mesenchymal transition (EMT) induced by TGF beta 1 plays a key role in renal fibrosis in diabetic nephropathy (DKD). HMGA2 is reported to be involved in the pathogenesis of EMT induced by TGF 1 through multiple mechanisms. Small RNA (miRNA), as an important post transcriptional regulatory factor, also participates in the occurrence of EMT. Previous studies on TGF beta 1/HMGA2 in EMT are mostly concentrated in the field of tumor or other organs, but in the context of DKD, especially in the role of EMT and fibrosis in renal tubular epithelial cells, it is not clear. The aim of this study was to investigate the effect and role of HMGA2 on TGF and beta 1 induced EMT and fibrosis in renal tubular epithelial cells, and to further investigate the effect of upstream miRNA on HMGA2 and the underlying mechanisms involved in DKD renal fibrosis based on target gene HMGA2. These studies will help us to find important intervention and therapeutic targets for effective signaling pathways, so as to provide new ideas for the treatment of DKD renal fibrosis. Methods 1.. A cell model was established to stimulate rat renal tubular epithelial cells (NRK-52E) stimulated by TGF beta 1. The expression of HMGA2 and EMT related genes before and after stimulation of TGF beta 1 was detected by qRT-PCR and Western blot. 2., we used siRNA transfection to verify the role of HMGA2 in TGF beta 1 induced EMT and renal fibrosis. We used qRT-PCR and Western blot to detect the expression of downstream transcription factors before and after siRNA silencing HMGA2 gene. 3., bioinformatics method was used to screen valuable upstream mmiRNA for target gene HMGA2. QRT-PCR TGF was used to detect TGF 1 and induce miRNA expression difference before and after NRK-52E cells. Meanwhile, HMGA2 and its upstream miRNA expression were further explored in UUO mice model. 4., through overexpression (transfection of miRNA mimics) and inhibition (transfection of miRNA inhibitors), we further verify the effect of miRNA on EMT and renal fibrosis induced by TGF beta 1. QRT-PCR and Western blot were used to detect the expression of HMGA2 and EMT related factors before and after miRNA overexpression and inhibition. Results 1.qRT-PCR and Western blot results showed that: compared with Control group, the expression of TGF, beta 1, 48h and TGF increased significantly in the TGF beta 1 (48h) group after 10ng/mL TGF beta 1 stimulation. 2.qRT-PCR and Western blot results showed that the transfection of siRNA-HMGA2 could inhibit the expression of TGF 1 in NRK-52E cells induced by HMGA2 and alpha -SMA, vimentin upregulation and downregulation of E-Cad expression; siRNA-HMGA2 transfection on its downstream transcription factor Snail, expression of Slug and Twist had no significant impact. 3. target genes were screened with HMGA2, the value of miRNA:miRNA-let-7d, miRNA-98, miRNA-33 upstream of qRT-PCR; the results showed that the expression of miRNA-let-7d in TGF beta 1 induced NRK-52E cells and UUO in mouse kidney were significantly decreased, while the expression of HMGA2 is negatively increased; no obvious effect to stimulate the expression of TGF beta 1 on miRNA-98 and miRNA-33 the. 4.qRT-PCR and Western blot results showed that the transfection of let-7d mimic inhibited TGF beta 1 induced NRK-52E cells and expression of HMGA2 -SMA, Col-I, FN expression and down-regulation of E-Cad expression; transfection of let-7d inhibitor significantly increased TGF beta 1 induced by HMGA2 and Col-I, FN expression and E-Cad expression. Conclusion 1.TGF beta 1 can induce the high expression of HMGA2 in NRK-52E cells and make EMT and fibrosis changes in cells. 2. gene silencing (siRNA-HMGA2) on HMGA2 can reverse the EMT and fibrosis changes induced by TGF beta 1, which is an important target gene. 3.miRNA-let-7d is an important upstream miRNA of HMGA2, and miRNA-let-7d/HMGA2 may play a role in the process of TGF beta 1 induced EMT and renal fibrosis. 4., over expression of miRNA-let-7d can down regulate the expression of HMGA2 and further inhibit the change of EMT and renal fibrosis induced by TGF beta 1. Inhibition of miRNA-let-7d can upregulate the expression of HMGA2, and accelerate the progression of EMT and renal fibrosis induced by TGF 1. 5.miRNA-let-7d may regulate transdifferentiation and fibrosis of renal tubular epithelial cells by regulating the expression of HMGA2. This intervention may be a new way to prevent and treat DKD renal fibrosis.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R692.9

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