miR-21-5p靶向调控TIMP3抑制2型糖尿病肾病小鼠肾脏系膜细胞增殖及细胞外基质堆积

doi:10.6040/j.issn.1671-7554.0.2019.1007

摘要/Abstract

摘要： 目的探讨miR-21-5p对2型糖尿病肾病(T2DN)小鼠肾组织中组织金属蛋白酶组织抑制因子3(TIMP3)表达以及对肾脏系膜细胞增殖及细胞外基质堆积的影响。方法选取10只雄性db/m小鼠作为正常对照(Control)组,另选30只SPF级雄性db/db T2DN小鼠随机分成模型(T2DN)组、miR-21-5p激动剂(miR-21-5p agomir)组和miR-21-5p拮抗剂(miR-21-5p antagomir)组,每组10只。分别给予生理盐水、miR-21-5p agomir、miR-21-5p antagomir尾静脉注射,每3 d注射1次,共7次。qRT-PCR检测各组小鼠肾脏组织中miR-21-5p表达水平;ELISA法检测各组小鼠24 h尿蛋白(Upro/24 h)、肌酐(Scr)和尿素氮(BUN)的表达水平;HE染色观察各组小鼠肾脏病理变化;PAS染色观察小鼠肾小球细胞外基质堆积情况;Western bloting法检测肾组织TIMP3、Col IV及FN蛋白的表达水平;双荧光素酶报告基因实验检测miR-21-5p和TIMP3的靶向关系。结果与Control组比较,T2DN组小鼠Upro/24 h、Scr和BUN水平均升高(t_{Upro/24 h}=84.67,P<0.001;t_Scr=16.81,P<0.001;t_BUN=19.26,P<0.001),miR-21-5p agomir组小鼠Upro/24 h、Scr和BUN水平均升高(t_{Upro/24 h}=100.44,P<0.001;t_Scr=36.76,P<0.001;t_BUN=52.42,P<0.001),肾脏肾小球系膜增生,基底膜增厚,系膜基质相对面积增加(t=9.10,P<0.001;t=14.16,P<0.001),TIMP3蛋白异常低表达(t=8.51,P=0.001;t=12.66,P<0.001),纤维化蛋白Col IV(t=10.04,P<0.001;t=23.54,P<0.001)和FN(t=11.49,P<0.001;t=22.34,P<0.001)异常高表达;与T2DN组比较,miR-21-5p antagomir组小鼠Upro/24 h、Scr和BUN水平均降低(t_{Upro/24 h}=20.31,P<0.001;t_Scr=7.90,P<0.001;t_BUN=8.91,P<0.001),肾脏肾小球系膜增生和基底膜增厚缓解,系膜基质相对面积降低(t=7.96,P=0.001),TIMP3蛋白升高(t=11.71,P<0.001),纤维化蛋白Col IV和FN降低(t_{Col IV}=6.58,P=0.003;t_FN=6.27,P=0.003);miR-21-5p agomir组小鼠与miR-21-5p antagomir组小鼠生化指标和肾脏肾小球的病症相反;双荧光素酶结果显示,miR-21-5p能靶向调控TIMP3基因的表达。结论 miR-21-5p通过靶向调控TIMP3抑制T2DN小鼠肾脏系膜细胞的增殖及细胞外基质的堆积。

关键词: miR-21-5p, 组织金属蛋白酶组织抑制因子3, 2型糖尿病肾病, 细胞增殖, 细胞外基质

Abstract: Objective To investigate the effects of miR-21-5p on the expression of tissue inhibitor of metalloproteinases-3(TIMP3)in kidney tissue of type 2 diabetic nephropathy(T2DN)mice, and the proliferation and extracellular matrix accumulation of mesangial cells. Methods Ten db/m mice were selected as control group, and other 30 SPF male db/db T2DN mice were randomly divided into model(T2DN)group, miR-21-5p agomir group and miR-21-5p antagomir group with 10 mice in each group. Normal saline, miR-21-5p agomir and miR-21-5p antagomir were injected respectively into tail vein once every three days, 7 times in total. The expression of miR-21-5p was detected by qRT-PCR in kidney tissues of mice. The expression levels of Upro/24 h, Scr and BUN in urine of mice were detected by ELISA assay. The kidney pathological changes were observed by HE staining. The extracellular matrix accumulation was observed by PAS staining. The expression levels of TIMP3, Col IV and FN protein in renal tissue were detected by Western blotting method. Dual luciferase reporter system was used to detect the targeting relationship between miR-21-5p and TIMP3. Results In the T2DN group and miR-21-5p agomir group, the levels of Upro/24 h(t=84.67, P<0.001; t=100.44, P<0.001), Scr(t=16.81, P<0.001; t=36.76, P<0.001), and BUN(t=19.26, P<0.001; t=52.42, P<0.001)increased, mesangial proliferated, basement membrane thickened, relative area of mesangial matrix increased(t=9.10,P<0.001;t=14.16,P<0.001), TIMP3 expression was abnormally lower(t=8.51,P=0.001;t=12.66,P<0.001), and fibrosis protein Col IV expression(t=10.04,P<0.001;t=23.54,P<0.001)and FN expression(t=11.49,P<0.001;t=22.34,P<0.001)was abnormally higher than those in the control group. Compared with T2DN group, the levels of Upro/24 h, Scr and BUN decreased(t_{Upro/24 h}=20.31,P<0.001;t_Scr=7.902,P<0.001;t_BUN=8.913,P<0.001), mesangial hyperplasia and basement membrane thickening alleviated, the relative area of mesangial matrix decreased(t=7.96,P=0.001), TIMP3 expression increased(t=11.71,P<0.001), fibrosis proteins Col IV expression(t_{Col IV}=6.58,P=0.003)and FN expression(t_FN=6.27,P=0.003)decreased in the miR-21-5p antagomir group. The biochemical indexes and symptom of glomerular diseases in miR-21-5p antagomir group and miR-21-5p agomir group were opposite. Dual luciferase assay showed that miR-21-5p could target the expression of TIMP3 gene. Conclusion miR-21-5p targeting TIMP3 inhibits the proliferation of mesangial cells and the extracellular matrix accumulation in T2DN mice.

Key words: miR-21-5p, Tissue inhibitor of metalloproteinases-3, Type II diabetic nephropathy, Cell proliferation, Extracellular matrix

中图分类号:

R574

张宝文,雷香丽,李瑾娜,罗湘俊,邹容. miR-21-5p靶向调控TIMP3抑制2型糖尿病肾病小鼠肾脏系膜细胞增殖及细胞外基质堆积[J]. 山东大学学报 (医学版), 2020, 58(7): 7-14.

ZHANG Baowen, LEI Xiangli, LI Jinna, LUO Xiangjun, ZOU Rong. miR-21-5p targeted TIMP3 to inhibit proliferation and extracellular matrix accumulation of mesangial cells in Type II diabetic nephropathy mice[J]. Journal of Shandong University (Health Sciences), 2020, 58(7): 7-14.

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