TGF—β1诱导肾小管上皮细胞间充质转化中ILK对MMP-9／TIMP-1的影响

Effect of integrin-linked kinase towards matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 in the process of TGF-β1-induced epithelial-mesenchymal transition of renal tubular cell

目的观察肾小管上皮细胞间充质转化（EMT）过程中，整合素连接激酶（ILK）的变化对基质金属蛋白酶-9/金属蛋白酶组织抑制剂-1（MMP-9/TIMP-1）比值的影响。方法培养人肾皮质近曲小管上皮细胞（HK-2），并随机分为正常组、转化生长因子-β1（TGF-β1）刺激组（以TGF-β1诱导HK-2细胞间充质转化）、TGF-β1＋ILK-siRNA组[使用小干扰RNA （siRNA）技术抑制ILK基因表达]、TGF-β1＋无序siRNA组（使用无序小干扰RNA转染）。后两组采用脂质体转染技术，将小干扰RNA转染入HK-2细胞，而后用10 ng/ml TGF-β1刺激细胞，分别提取不同组别细胞的mRNA和蛋白质，采用Real-time RT-PCR和Western blot检测细胞ILK、E-钙粘蛋白（E-cadherin）、α-平滑肌肌动蛋白（α-SMA）、MMP-9、TIMP-1的mRNA和蛋白质的表达，计算MMP-9、TIMP-1 mRNA和蛋白质表达的相对比值，比较其统计学差异。结果TGF-β1刺激的HK-2细胞E-cadherin表达减少，α-SMA表达增加，ILK表达显著增加，MMP-9/TIMP-1比值升高；转染ILK-siRNA可大幅度抑制HK-2细胞ILK基因的表达；且其E-cadherin表达增加，α-SMA表达减少，同时可减小MMP-9/TIMP-1比值升高的程度。结论ILK可通过影响MMP-9/TIMP-1比值，促进EMT进程。

Objective To investigate the possible regulating effect of integrin-linked kinase （ILK） towards matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 （ MMP-9/TIMP-1 ） ratio in the process of transforming growth factor-β1 （ TGF-β1 ） -induced epithelial-mesenchymal transition （EMT） in human kidney proximal tubular epithelial （HK-2） ceils. Methods HK-2 ceils were cultured and stimulated with 10 ng/ml TGF-β1. Specific ILK-small interfering RNA （ILK-siRNA） was used to inhibit ILK expression. The characteristic epithelial marker （E-cadherin） and mesenchymal marker （α-SMA） of EMT were examined by Real-time reverse transeription polymerase chain reaction （real-time RT-PCR） and Western blot. The expressions of ILK, MMP-9, and TIMP-1 were also examined, to determine the regulating effect of ILK towards MMP-9/TIMP-1 ratio. Results In the HK-2 ceils cultured with TGF-β1, the expression of E-cadherin decreased, and α-SMA expression increased; overexpression of ILK and an abnormal changing of MMP-9/TIMP-1 ratio were observed. ILK inhibition by ILK-siRNA could adjust MMP-9/TIMP-1 ratio to near normal. Meanwhile, the overexpressed ILK and α-SMA were decreased. Conclusions Our data indicates that ILK-siRNA successfully inhibits ILK expression, which regulates the MMP-9/TIMP-1 ratio in HK-2 cells. The inhibition of ILK expression suppresses TGF-β1-induced EMT partially.