【摘要】：目的探討miR-9對轉(zhuǎn)化生長因子β(TGF-β)誘導的HK-2腎小管上皮細胞纖維化過程中的作用。方法永生化的HK-2細胞給予5 ng/m L TGF-β刺激0、72 h,應用實時熒光定量PCR檢測上皮鈣黏素(E-cadherin)、α平滑肌肌動蛋白(α-SMA)、1型膠原蛋白(Col1)、3型膠原蛋白(Col3)、纖連蛋白(fibronectin)、結(jié)締組織生長因子(CTGF)的mRNA和miR-9水平;Western blot法檢測E-cadherin、α-SMA、Col1、Col3、fibronectin、CTGF的表達變化;給予5 ng/m L TGF-β刺激或不用TGF-β刺激的HK-2細胞,培養(yǎng)48 h,轉(zhuǎn)染miR-9的模擬物、模擬物陰性對照,采用實時定量PCR及Western blot法檢測上述指標的變化。生物信息學方法預測miR-9的靶基因,熒光素酶報告基因法進行驗證。結(jié)果 TGF-β刺激72 h后,HK-2細胞中的miR-9、α-SMA、Col1、Col3、fibronectin、CTGF的水平增加,而E-cadherin水平下降;給予HK-2細胞轉(zhuǎn)染miR-9模擬物后,miR-9、α-SMA、Col1、Col3、fibronectin、CTGF含量增加,E-cadherin含量下降;給予HK-2細胞TGF-β刺激48 h并轉(zhuǎn)染miR-9抑制劑后,miR-9、α-SMA、Col1、Col3、fibronectin、CTGF含量下降,E-cadherin含量增加。通過Target Scan進行生物學信息預測,結(jié)果顯示E-cadherin可能為miR-9的靶基因,并通過熒光素酶報告基因確證。結(jié)論 miR-9在人HK-2細胞轉(zhuǎn)分化中起重要作用,并且通過抑制E-cadherin的表達而促進HK-2細胞轉(zhuǎn)分化及纖維化。miR-9抑制劑可逆轉(zhuǎn)TGF-β刺激HK-2細胞轉(zhuǎn)分化及纖維化。
[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, 偽 -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, 偽 -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, 偽 -SMA-Col1 and Col3fifiectinin CTGF increased, but E-cadherin decreased, and the levels of miR-9, 偽 -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 偽 -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.
【作者單位】： 第四軍醫(yī)大學西京醫(yī)院腎臟內(nèi)科;西安市楊凌示范區(qū)醫(yī)院;
【基金】：國家自然科學基金(81270768,81270849)
【分類號】：R692

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