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目的 验证miRNA-200能否通过调控snail来诱导肾小管上皮向间充质转化，从而在糖尿病肾病间质纤维化中发挥作用。方法 将来源北纳生物的NRK52E细胞分为六组：正常葡萄糖(5 mM)组；等渗对照(25 mM甘露醇+正常葡萄糖5 mM)组，高葡萄糖(30 mM)组，高葡萄糖(30 mM)+NC组，高葡萄糖(30 mM)+miRNA-200模拟物组，高葡萄糖(30 mM)+miRNA抑制剂组。给予高葡萄糖(30 mM)组，高葡萄糖(30 mM)+NC组，高葡萄糖(30 mM)+miRNA-200模拟物组，高葡萄糖(30 mM)+miRNA抑制剂组30mM葡萄糖处理，对高葡萄糖(30 mM)+miRNA-200模拟物组，高葡萄糖(30 mM)+miRNA抑制剂组进行过表达质粒转染。qRT-PCR及Western blot法检测各组中snail、Vimentin、Collagen IV、α-SMA、E-cadherin mRNA和蛋白的表达水平；Transwell检测各组细胞的侵袭能力；划痕实验检测各组细胞的迁移能力；双荧光素酶报告基因实验验证靶定效果。结果 与正常葡萄糖(5 mM)组相比，高葡萄糖(30 mM)组Vimentin、Collagen IV、α-SMA mRNA和蛋白的表达量增加，E-cadherin mRNA和蛋白的表达量下降(P<0.05);但与高葡萄糖(30 mM)组相比，高葡萄糖(30 mM)+miRNA-200模拟物组Vimentin、Collagen IV、α-SMA mRNA和蛋白的表达量降低，E-cadherin mRNA和蛋白的表达量增加(P<0.05);Transwell检测中，与正常葡萄糖(5 mM)组相比，高葡萄糖(30 mM)组侵袭能力降低，而高葡萄糖(30 mM)+miRNA-200模拟物组与其相比，细胞的迁移能力增强，microRNA-200抑制剂组细胞的迁移能力降低(P<0.01);划痕实验(48 h),与高葡萄糖(30 mM)组相比，高葡萄糖剂组(30 mM)+miRNA-200模拟物组使迁移能力明显增加，高葡萄糖(30 mM)+miRNA抑制剂组迁移能力明显减弱(P<0.01)。双荧光素酶报告基因实验验证microRNA-200和野生型snail1共转染细胞的荧光素酶活性显著降低，而突变型snail1的荧光无明显变化，证明snail1是miR-200b-3p的直接靶点(P<0.01)。结论 miRNA-200可以通过snail1调控肾小管上皮上皮细胞间充质转化诱导糖尿病肾病间质纤维化。
Abstract：Objective To determine whether miRNA-200 could induce renal tubular epithelial cells to undergo mesenchymal transformation by regulating Snail, thereby playing a role in the interstitial fibrosis of diabetic nephropathy.Methods Six groups of NRK52E cells from Northa Biotech were divided: normal glucose(5 mM) group; isotonic control group(25 mM mannitol +5 mM glucose),high glucose(30 mM) group, high glucose(30 mM) + NC group, high glucose(30 mM) + miRNA-200 mimic group, and high glucose(30 mM) + miRNA inhibitor group.The high glucose group, high glucose+ NC group, high glucose+ miRNA-200 mimic group, and high glucose+ miRNA inhibitor group were treated with 30 mM glucose, and the high glucose + miRNA-200 mimic group and high glucose + miRNA inhibitor group were transfected with overexpression plasmids.qRT-PCR and Western blot were used to detect the expression levels of Snail-1,Vimentin, Collagen IV,α-SMA,and E-cadherin in each group.Transwell assay was used to detect the invasive ability of cells.The scratch assay was used to detect the migratory ability of cells, and the dual luciferase reporter gene assay was used to verify the targeting effect.Results Compared with normal glucose group, the expression of Vimentin, Collagen IV and α-SMA in high-glucose group were increased, with decreased expression of E-cadherin(P<0.05).However, compared with the high glucose group, the expression levels of Vimentin, Collagen IV and α-SMA in the high glucose + miRNA-200 mimic group were decreased, while the expression level of E-cadherin was increased(P<0.05).Transwell assays revealed that the invasion ability of the high-glucose group was reduced compared to the normal glucose group, while the high-glucose + miRNA-200 mimic group demonstrated enhanced invasion ability, and the miRNA-200 inhibitor group showed reduced invasion(P<0.01).Scratch assays at 48 hours post-treatment showed that the high-glucose + miRNA-200 mimic group significantly improved migratory ability, while the high-glucose + miRNA inhibitor group significantly reduced(P<0.01).Dual luciferase reporter gene assay confirmed that co-transfection of miRNA-200 and wild-type snail1 significantly reduced luciferase activity, whereas mutant snail1 showed no significant change, indicating that snail1 was the direct target of miR-200b-3p(P<0.01).Conclusion miRNA-200 can regulate renal tubular epithelial-mesenchymal transition-induced interstitial fibrosis in diabetic nephropathy through Snail-1.
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基本信息:
DOI：10.13799/j.cnki.mdjyxyxb.2024.05.006
中图分类号:R587.2;R692.9
引用信息:
[1]李婷婷,高志辉,董传令,等.miRNA-200调控snail1诱导上皮间质转化改善糖尿病肾间质纤维化的研究[J].牡丹江医学院学报,2024,45(05):1-7.DOI:10.13799/j.cnki.mdjyxyxb.2024.05.006.
基金信息:
黑龙江省省属高等学校基本科研业务费攀登项目(2022-KYYWFMY-0002); 黑龙江省医药卫生科研项目(20220101040735)