敲低NPHP1表达诱导犬肾集合管上皮细胞发生上皮间质转分化

NPHP1 knockdown induces epithelial-mesenchymal transition in Madin-Darby canine kidney cells

目的研究敲低NPHP1基因表达对。肾小管上皮细胞特性的影响。方法靶向siRNA干扰技术敲低犬肾集合管上皮细胞（MDCK细胞）NPHP1的表达。实验分为空白对照组、阴性对照组、siRNA干扰组。显微镜下观察细胞形态和迁移能力；实时定量PCR和明胶酶谱法分别检测基质金属蛋白酶2和9（MMP2、MMP9）的mRNA表达和酶活性；实时定量PCR、Western印迹和细胞免疫荧光检测上皮钙黏素（E-cadherin）、β联蛋白（β-catenin）、闭锁小带蛋白1（ZO-1）、ZO-1相关核酸结合蛋白（ZONAB）及成纤维细胞标志蛋白仅平滑肌肌动蛋白（仪．SMA）的表达和分布。结果与对照组比较，靶向siRNA干扰24h，MDCK细胞迁移能力增强，E-cadherin、β-catenin、ZO-1的mRNA表达下降（均P〈0．01），α-SMA、MMP2、MMP9及ZONAB的mRNA表达均升高（均P〈0．05）；干扰48h见细胞变梭长形，E-cadherin、B-catenin、ZO-1的蛋白表达均下降（均P〈0．01），ZONAB、d．SMA蛋白表达升高（均P〈0.05），MMP2、MMP9活性升高（均P〈0．01）；干扰72h，可见细胞膜上β-catenin、E-cadherin表达明显减少，呈现断续改变或完全缺失，部分细胞可见α-SMA表达，ZONAB在细胞质和细胞核显著表达。结论敲低NPHPl表达可诱导MDCK细胞发生上皮间质转分化，激活ZO-1／ZONAB信号通路。这些改变可能与I型肾单位肾痨肾间质纤维化发生有关。

Objective To explore the impacts of NPHP1 knockdown on the phenotype of Madin-Darby canine kidney （MDCK） cells. Methods The expression of NPHP1 in MDCK cells was knockdown by siRNA interference. Cells were divided into normal control group, negative control group and siRNA group. The cellular morphology and migration were observed by light microscope. The mRNA expressions and activities of matrix metalloproteinases 2 and 9 （MMP2 and MMP9） were detected by real time PCR and gelatin zymography. The mRNA and protein expressions of E- cadherin, β- eatenin, zonula oecluden- 1 （ZO- 1）, ZO- 1- associated nucleic acid binding protein （ZONAB） and ct-smooth muscle actin （or-SMA） were analyzed by real time PCR, Western blotting and immunocytochemistry. Results Compared with those in normal control group, in siRNA group the mRNA expressions of E - cadherin, β - catenin and ZO- 1 decreased, and MMP9, MMP2, α - SMA and ZONAB increased after interfering NPHP1 24 h （all P 〈 0.05）; the protein expressions of E-cadherin, -catenin and ZO- 1 decreased and ZONAB and α- SMA increased after 48 h （all P 〈 0.05）, and MDCK cells became elongated with enhanced migration capacity; siRNA cells had decreased expressions of E- cadherin and α- catenin on the membrane, but increased expression of ZONAB in cytoplasm and nucleoplasm after 72 h, and α- SMA was also observed in some interfered ceils. Conclusions NPHP1 knockdown induces epithelial-mesenchymal transition in MDCK ceils, and ZO-1/ ZONAB signaling pathway was activated. These changes may associate with renal interstitial fibrosis of Nephronophthisis type I.