HMGA2及let-7d miRNA对糖尿病肾脏病肾小管上皮细胞转分化及肾脏纤维化的调控

发布时间：2017-12-27 22:32

本文关键词：HMGA2及let-7d miRNA对糖尿病肾脏病肾小管上皮细胞转分化及肾脏纤维化的调控　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：背景和目的TGFβ1诱导的肾小管上皮间质转化(EMT)在糖尿病肾脏病(DKD)肾纤维化中起关键作用,HMGA2被报道通过多重机制参与TGFβ 1诱导的EMT的发生过程。微小RNA(miRNA)作为重要的转录后水平调控因子也参与EMT的发生。既往对TGF β 1/HMGA2在EMT中的研究多集中于肿瘤或其他器官领域,而在DKD背景下,尤其是在肾小管上皮细胞EMT及纤维化进程中的作用目前并不明确。本研究拟探讨HMGA2对TGFβ 1诱导的肾小管上皮细胞EMT及纤维化过程的影响和作用,并进一步针对靶基因HMGA2,研究其上游miRNA对HMGA2的影响及参与DKD肾纤维化的潜在作用机制。通过上述研究,将有助于我们据此针对有效的信号通路发现重要的干预和治疗靶点,从而为DKD肾纤维化的治疗提供新的思路。方法1.建立TGFβ 1刺激大鼠肾小管上皮细胞(NRK-52E)的细胞模型,利用qRT-PCR及western blot的方法检测TGFβ 1刺激前后HMGA2及EMT相关基因的表达变化。2.利用siRNA转染的方法验证HMGA2在TGF β 1诱导的EMT及肾纤维化中的作用,用qRT-PCR及western blot检测siRNA沉默HMGA2基因前后其下游转录因子的表达变化。3.通过生物信息学方法,针对靶基因HMGA2筛选有价值的上游mmiRNA,用qRT-PCR检测TGF β 1诱导NRK-52E细胞前后各miRNA的表达差异,同时进一步于UUO小鼠模型中探索HMGA2及其上游miRNA的表达变化。4.通过过表达(转染miRNA mimics)和抑制(转染miRNA inhibitors)的功能实验进一步验证筛选出的miRNA对TGFβ1诱导的EMT和肾纤维化的影响和作用。用qRT-PCR及western blot检测HMGA2及EMT相关因子于miRNA过表达及抑制前后的表达情况。结果1.qRT-PCR 及 western blot 结果表明:给予 10ng/mL TGFβ1 刺激 NRK-52E细胞 48h 后,与 Control 组比较,TGFβ 1(48h)组中 HMGA2、α-SMA、vimentin、Col-I及FN的表达均明显升高,E-Cad的表达明显降低。2.qRT-PCR及western blot结果表明:转染siRNA-HMGA2能明显抑制TGFβ1诱导的NRK-52E细胞中HMGA2及α-SMA、vimentin表达的上调以及E-Cad表达的下调;转染siRNA-HMGA2对其下游转录因子Snail,Slug和Twist的表达无明显影响。3.针对靶基因HMGA2,筛选出有价值的上游miRNA:miRNA-let-7d、miRNA-98、miRNA-33;qRT-PCR 结果显示:miRNA-let-7d 在 TGFβ1诱导的 NRK-52E 细胞及UUO小鼠肾脏中的表达均明显降低,而HMGA2的表达则呈反向升高;TGFβ1的刺激对miRNA-98及miRNA-33的表达无明显影响。4.qRT-PCR及western blot结果表明:转染let-7d mimic能明显抑制TGFβ1诱导的NRK-52E细胞中HMGA2及α-SMA、Col-I、FN表达的上调以及E-Cad表达的下调;转染let-7d inhibitor能明显增强TGFβ 1诱导的HMGA2及Col-I、FN表达的上调以及E-Cad表达的下调。结论1.TGFβ 1可诱导NRK-52E细胞中HMGA2的高表达,并使细胞发生EMT及纤维化改变。2.对HMGA2进行基因沉默(siRNA-HMGA2)可逆转TGFβ1诱导的EMT及纤维化改变,它是一个重要的靶基因。3.miRNA-let-7d 为 HMGA2 重要的上游 miRNA,miRNA-let-7d/HMGA2 在 TGFβ 1诱导的EMT及肾纤维化进程中可能发挥作用。4.过表达miRNA-let-7d可下调HMGA2的表达,进而抑制TGF β 1诱导的EMT及肾纤维化改变;抑制miRNA-let-7d可上调HMGA2的表达,进而加快TGFβ1诱导的EMT及肾纤维化进展。5.miRNA-let-7d可能通过调节HMGA2的表达从而调控肾小管上皮细胞转分化及纤维化,干预这一过程可能是一个预防和治疗DKD肾纤维化的新途径。
[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.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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