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山东大学学报 (医学版) ›› 2020, Vol. 1 ›› Issue (7): 7-14.doi: 10.6040/j.issn.1671-7554.0.2019.1007

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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

张宝文1,雷香丽1,李瑾娜2,罗湘俊1,邹容1   

  • 出版日期:2020-07-20 发布日期:2020-07-10
  • 通讯作者: 雷香丽. E-mail:zhangmingyan5918@163.com
  • 基金资助:
    衡阳市科学技术发展计划项目(2015KS15);衡阳市社会发展科技支撑计划项目(2014KS50)

miR-21-5p targeted TIMP3 to inhibit proliferation and extracellular matrix accumulation of mesangial cells in Type II diabetic nephropathy mice

ZHANG Baowen1, LEI Xiangli1, LI Jinna2, LUO Xiangjun1, ZOU Rong1   

  1. 1. Department of Nephrology, Affiliated Nanhua Hospital, University of South China, Hengyang 421002, Hunan, China;
    2. Department of Nephrology, Hengyang First Peoples Hospital, Hengyang 421002, Hunan, China
  • Online:2020-07-20 Published:2020-07-10

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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(tUpro/24 h=84.67,P<0.001;tScr=16.81,P<0.001;tBUN=19.26,P<0.001),miR-21-5p agomir组小鼠Upro/24 h、Scr和BUN水平均升高(tUpro/24 h=100.44,P<0.001;tScr=36.76,P<0.001;tBUN=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(tUpro/24 h=20.31,P<0.001;tScr=7.90,P<0.001;tBUN=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(tCol IV=6.58,P=0.003;tFN=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(tUpro/24 h=20.31,P<0.001;tScr=7.902,P<0.001;tBUN=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(tCol IV=6.58,P=0.003)and FN expression(tFN=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
[1] 裘静英, 董志春, 王璟. 参芪降糖颗粒联合阿托伐他汀对2型糖尿病合并代谢综合征胰岛β细胞功能、胰岛素抵抗和血管内皮细胞功能的影响[J]. 中华中医药学刊, 2019, 37(7): 1725-1728. QIU Jingying, DONG Zhichun, WANG Jing. Effects of shenqi jiangtang granule combined with atorvastatin on islet β cell function, insulin resistance and vascular endothelial cell function in patients with type 2 diabetes combined with metabolic syndrome[J]. Chinese Archives of Traditional Chinese Medicine, 2019, 37(7): 1725-1728.
[2] Kirkman MS, Mahmud H, Korytkowski MT. Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes mellitus[J]. Endocrinol Metab Clin North Am, 2018, 47(1): 81-96.
[3] Zang L, Shimada Y, Nakayama H, et al. Therapeutic silencing of centromere protein X ameliorates hyperglycemia in zebrafish and mouse models of type 2 diabetes mellitus[J]. Front Genet, 2019, 10: 693. doi: 10.3389/fgene. 2019.00693.
[4] Boussageon R, Bejan-Angoulvant T, Saadatian-Elahi M, et al. Effect of intensive glucose lowering treatment on all cause mortality, cardiovascular death, and microvascular events in type 2 diabetes: meta-analysis of randomised controlled trials[J]. BMJ, 2011, 343: d4169. doi: 10.1136/bmj. d4169.
[5] 宋孟晓, 王燕, 刘进忠. miR-222-5p在人根尖乳头干细胞成骨/成牙本质向分化中的作用[J]. 山东大学学报(医学版), 2020, 58(3): 87-93. SONG Mengxiao, WANG Yan, LIU Jinzhong. MiR-222-5p promotes osteo/odontogenic differentiation of stem cells from human apical papilla[J]. Journal of Shandong University(Health Sciences), 2020, 58(3): 87-93.
[6] Zhu H, Leung SW. Identification of microRNA biomarkers in type 2 diabetes: a meta-analysis of controlled profiling studies[J]. Diabetologia, 2015, 58(5): 900-911.
[7] Zampetaki A, Kiechl S, Drozdov I, et al. Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes[J]. Circ Res, 2010, 107(6): 810-817.
[8] Ong J, Woldhuis RR, Boudewijn IM, et al. Age-related gene and miRNA expression changes in airways of healthy individuals[J]. Sci Rep, 2019, 9(1):1-8.
[9] Feng Y, Ge Y, Wu M, et al. Long non-coding RNAs regulate inflammation in diabetic peripheral neuropathy by acting as ceRNAs targeting miR-146a-5p[J]. Diabetes Metab Syndr Obes, 2020, 13: 413-422. doi: 10.2147/DMSO.S242789.
[10] 林海燕, 王红梅, 祝诚. 转化生长因子-β对基质金属蛋白酶及其组织抑制因子调控的研究进展[J]. 生物化学与生物物理进展, 2003, 30(1): 7-12. LIN Haiyan, WANG Hongmei, ZHU Cheng. Regulation of matrix metalloproteinases and their tissue inhibitors by transforming growth factor-β[J]. Prog Biochem Biophys, 2003, 30(1): 7-12.
[11] GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013[J]. Lancet, 2015, 385(9963): 117-171.
[12] Pourghasem M, Shafi H, Babazadeh Z. Histological changes of kidney in diabetic nephropathy[J]. Caspian J Intern Med, 2015, 6(3): 120-127.
[13] 金海燕,张炎,马小莉,等. miR-122与miR-33a在2型糖尿病合并冠心病患者中的表达[J]. 山东大学学报(医学版), 2020, 58(3): 94-98. JIN Haiyan, ZHANG Yan, MA Xiaoli, et al. Expression of miR-122 and miR-33a in patients with type 2 diabetes complicated with coronary artery disease[J]. Journal of Shandong University(Health Sciences), 2020, 58(3): 94-98.
[14] Lu S, Dong L, Jing X, et al. Abnormal lncRNA CCAT1/microRNA-155/SIRT1 axis promoted inflammatory response and apoptosis of tubular epithelial cells in LPS caused acute kidney injury[J]. Mitochondrion, 2020: S1567-7249(19)30066-2. doi: 10.1016/j.mito.2020.03.010.
[15] Geng X, Song N, Zhao S, et al. LncRNA GAS5 promotes apoptosis as a competing endogenous RNA for miR-21 via thrombospondin 1 in ischemic AKI[J]. Cell Death Discov, 2020, 6: 19. doi: 10.1038/s41420-020-0253-8.
[16] Hennino MF, Buob D, Van der Hauwaert C, et al. miR-21-5p renal expression is associated with fibrosis and renal survival in patients with IgA nephropathy[J]. Sci Rep, 2016, 6: 27209. doi: 10.1038/srep27209.
[17] Faragalla H, Youssef YM, Scorilas A, et al. The clinical utility of miR-21 as a diagnostic and prognostic marker for renal cell carcinoma[J]. J Mol Diagn, 2012, 14(4): 385-392.
[18] Srivastava SP, Goodwin JE, Kanasaki K, et al. Inhibition of angiotensin-converting enzyme ameliorates renal fibrosis by mitigating DPP-4 level and restoring antifibrotic microRNAs[J]. Genes(Basel), 2020, 11(2): E211. doi: 10.3390/genes11020211.
[19] Li N, Wang Z, Gao F, et al. Melatonin ameliorates renal fibroblast-myofibroblast transdifferentiation and renal fibrosis through miR-21-5p regulation[J]. J Cell Mol Med, 2020. doi: 10.1111/jcmm.15221.
[20] Dey N, Das F, Mariappan MM, et al. MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes[J]. J Biol Chem, 2011, 286(29): 25586-25603.
[21] Baker MA, Davis SJ, Liu P, et al. Tissue-specific microRNA expression patterns in four types of kidney disease[J]. J Am Soc Nephrol, 2017, 28(10): 2985-2992.
[22] Wang JY, Gao YB, Zhang N, et al. miR-21 overexpression enhances TGF-β1-induced epithelial-to-mesenchymal transition by target smad7 and aggravates renal damage in diabetic nephropathy[J]. Mol Cell Endocrinol, 2014, 392(1-2): 163-172.
[23] Wang X, Khalil RA. Matrix metalloproteinases, vascular remodeling, and vascular disease[J]. Adv Pharmacol, 2018, 81:241-330. doi: 10.1016/bs.apha.2017.08.002.
[24] Schrimpf C, Xin C, Campanholle G, et al. Pericyte TIMP3 and ADAMTS1 modulate vascular stability after kidney injury[J]. J Am Soc Nephrol, 2012, 23(5): 868-883.
[25] Kassiri Z, Defamie V, Hariri M, et al. Simultaneous transforming growth factor β-tumor necrosis factor activation and cross-talk cause aberrant remodeling response and myocardial fibrosis in Timp3-deficient heart[J]. J Biol Chem, 2009, 284(43): 29893-29904.
[26] Fiorentino L, Cavalera M, Mavilio M, et al. Regulation of TIMP3 in diabetic nephropathy: a role for micro-RNAs[J]. Acta Diabetologica, 2013, 50(6): 965-969.
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