miR-155对肾小管上皮细胞转分化的影响及其机制研究

Study on the effects of miR-155 on epithelial-mesenchymal transition in renal tubular epithelial cells and relevant mechanisms

目的·研究miR-155在慢性肾脏疾病(CKD)患者血清中的表达情况,探讨miR-155是否通过下调Kriippel样因子4(KLF4)影响肾小管上皮细胞转分化(EMT)。方法·收集126例CKDⅠ期患者、163例CKDⅢ期患者以及130例正常体检人员的血清,通过实时荧光定量PCR(RT-PCR)检测各组患者血清中miR-155的表达情况。在细胞实验中,用10μg/L TGF-β诱导肾小管上皮细胞EMT,通过转染miR-155 mimic和miR-NC至离体培养的肾小管上皮细胞中,光学显微镜下观察细胞形态变化。Transwell试验检测细胞的迁移侵袭能力,免疫印迹试验(Westernblotting)检测细胞中平滑肌α-肌动蛋白(SMα-actin)、胶原蛋白Ⅰ(collagenⅠ)、胶原蛋白Ⅳ(collagenⅣ)、钙黏着蛋白-E(E-cadherin)的表达情况。生物信息学预测miR-155潜在靶基因为KLF4,利用荧光素酶试验验证KLF4是miR-155的靶基因;再通过转染过表达KLF4的质粒至肾小管上皮细胞,检测细胞中SMα-actin、collagenⅠ、collagenⅣ、E-cadherin、KLF4的表达情况。结果·相比于正常人群组和CKDⅠ期患者,miR-155在CKDⅢ期患者血清中的表达降低。在细胞实验中,相比于空质粒组,转染miR-155 mimic后,肾小管上皮细胞中SMα-actin、collagenⅠ、collagenⅣ、KLF4的表达减少,E-cadherin表达增加;荧光素酶试验结果显示,miR-155能够显著降低含KLF4-3'-UTR序列质粒的荧光素酶活性;转染过表达KLF4质粒后,肾小管上皮细胞中SMα-actin、collagenⅠ、collagenⅣ表达增加,E-cadherin表达减少。结论·在CKD患者血清中,miR-155低表达;miR-155可以通过下调KLF4从而抑制肾小管上皮细胞EMT。

Objective · To investigate serum miR-155 expression in patients with chronic kidney disease （CKD） and to explore whether miR-155 influences epithelial-mesenchymal transition （EMT） in renal tubular epithelial cells via down-regulating Krüppel-like factor 4 （KLF4）. Methods · Serum samples from 126 CKD Ⅰ stage patients, 163 CKD Ⅲ stage patients, and 170 healthy individuals were collected. RT-PCR was used to test the serum miR-155 expression. In cell experiment, EMT in renal tubular epithelial cells was induced with 10 μg/L TGF-β. miR-155 mimic and miR-NC were transfected to renal tubular epithelial cells cultured in vitro. Changes in cell morphology were observed with optical microscope. Transwell assay was used to test the cell migration ability. Western blotting was used to examine the expressions of SMα-actin, Collagen Ⅰ, Collagen Ⅳ, E-cadherin, and KLF4. Bioinformatics predicted that the potential target gene for miR-155 was KLF4, which was confirmed with luciferase assay. Plasmids with over-expressed KLF4 were transfected to renal tubular epithelial cells and expressions of SMα-actin, collagen Ⅰ, collagen Ⅳ, E-cadherin, and KLF4 were detected. Results · CKD Ⅲ stage patients had lower serum miR-155 expression compared with healthy individuals and CKD Ⅰ stage patients. In cell experiment, the expression of SMα-actin, collagen Ⅰ, collagen Ⅳ and KLF4 renal tubular epithelial cells decreased and the expression of E-cadherin increased after being transfected with miR-155 mimic, as compared with the control group. Results of the luciferase assay showed that miR-155 significantly decreased the luciferase activity of KLF4-3＇-UTR plasmid. Expressions of SMα-actin, collagen Ⅰ, and collagen Ⅳ in renal tubular epithelial cells were increased and the expression E-cadherin was decreased after being transfected with plasmids with over-expressed KLF4. Conclusion · CKD patients have low serum miR-155 expression. miR-155 can inhibit EMT in renal tubular epithelial cells by down-regulating KLF4.