miR-9在TGF-β诱导的人肾小管上皮细胞纤维化中的作用

发布时间：2018-07-17 05:43

【摘要】：目的探讨miR-9对转化生长因子β(TGF-β)诱导的HK-2肾小管上皮细胞纤维化过程中的作用。方法永生化的HK-2细胞给予5 ng/m L TGF-β刺激0、72 h,应用实时荧光定量PCR检测上皮钙黏素(E-cadherin)、α平滑肌肌动蛋白(α-SMA)、1型胶原蛋白(Col1)、3型胶原蛋白(Col3)、纤连蛋白(fibronectin)、结缔组织生长因子(CTGF)的mRNA和miR-9水平;Western blot法检测E-cadherin、α-SMA、Col1、Col3、fibronectin、CTGF的表达变化;给予5 ng/m L TGF-β刺激或不用TGF-β刺激的HK-2细胞,培养48 h,转染miR-9的模拟物、模拟物阴性对照,采用实时定量PCR及Western blot法检测上述指标的变化。生物信息学方法预测miR-9的靶基因,荧光素酶报告基因法进行验证。结果 TGF-β刺激72 h后,HK-2细胞中的miR-9、α-SMA、Col1、Col3、fibronectin、CTGF的水平增加,而E-cadherin水平下降;给予HK-2细胞转染miR-9模拟物后,miR-9、α-SMA、Col1、Col3、fibronectin、CTGF含量增加,E-cadherin含量下降;给予HK-2细胞TGF-β刺激48 h并转染miR-9抑制剂后,miR-9、α-SMA、Col1、Col3、fibronectin、CTGF含量下降,E-cadherin含量增加。通过Target Scan进行生物学信息预测,结果显示E-cadherin可能为miR-9的靶基因,并通过荧光素酶报告基因确证。结论 miR-9在人HK-2细胞转分化中起重要作用,并且通过抑制E-cadherin的表达而促进HK-2细胞转分化及纤维化。miR-9抑制剂可逆转TGF-β刺激HK-2细胞转分化及纤维化。
[Abstract]:Objective to investigate the effect of miR-9 on transforming growth factor 尾 (TGF- 尾) -induced fibrosis of HK-2 renal tubular epithelial cells. Methods immortalized HK-2 cells were stimulated with 5 ng/m L TGF- 尾 for 0 72 h. E-cadherin, 伪 -SMA, collagen type 1 (Col3) and connective tissue growth factor (CTGF) of fibronectin (fibronectin), were detected by real-time fluorescence quantitative PCR. The expression of E-cadherin, 伪 -SMA-Col1and Col3fibronectinine CTGF was detected by Western blot. HK-2 cells were incubated with 5 ng/m L TGF- 尾 or without TGF- 尾 for 48 h. The mimics of miR-9 were transfected. The changes of the above indexes were detected by real-time quantitative PCR and Western blot. The target gene of miR-9 was predicted by bioinformatics and verified by luciferase reporter gene method. Results after 72 h of TGF- 尾 stimulation, the levels of miR-9, 伪 -SMA-Col1 and Col3fifiectinin CTGF increased, but E-cadherin decreased, and the levels of miR-9, 伪 -SMA-Col1, Col3fibronectin CTGF increased and E-cadherin decreased after transfection of HK-2 cells. After stimulation with TGF- 尾 for 48 h and transfection of miR-9 inhibitor, the content of 伪 -SMA-Col1 and Col3fibronectinine CTGF decreased and the content of E-cadherin increased. The results showed that E-cadherin might be the target gene of miR-9 and confirmed by luciferase reporter gene. Conclusion miR-9 plays an important role in the transdifferentiation of HK-2 cells and can reverse the transdifferentiation and fibrosis of HK-2 cells stimulated by TGF- 尾 by inhibiting the expression of E-cadherin.
【作者单位】：第四军医大学西京医院肾脏内科;西安市杨凌示范区医院;
【基金】：国家自然科学基金(81270768,81270849)
【分类号】：R692

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