高糖通过miR-192调控CAV-1/EGF影响肾小球系膜细胞增殖、迁移及细胞外基质形成的机制研究

doi:10.11958/20220268

天津医药 ›› 2022, Vol. 50 ›› Issue (10): 1020-1025.doi: 10.11958/20220268

高糖通过miR-192调控CAV-1/EGF影响肾小球系膜细胞增殖、迁移及细胞外基质形成的机制研究

吴玮熙(), 刘帅辉, 周赛君, 刘红岩, 张睿, 于珮()

天津医科大学朱宪彝纪念医院,天津市内分泌研究所,国家卫健委激素与发育重点实验室,天津市代谢性疾病重点实验室（邮编300134）

收稿日期:2022-02-19 修回日期:2022-04-11 出版日期:2022-10-15 发布日期:2022-10-20
通讯作者: 于珮 E-mail:15849588283@163.com;peiyu@tmu.edu.cn
作者简介:吴玮熙（1996）,女,硕士在读,主要从事糖尿病肾病方面研究。E-mail： 15849588283@163.com
基金资助:
天津市卫生健康委科技人才培育项目(RC20175);森美中华糖尿病科研基金(Z-2017-26-1902)

Effects of high glucose on the proliferation and migration of mesangial cells and extracellular matrix formation by regulating the CAV-1/EGF through miR-192

WU Weixi(), LIU Shuaihui, ZHOU Saijun, LIU Hongyan, ZHANG Rui, YU Pei()

NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin 300134, China

Received:2022-02-19 Revised:2022-04-11 Published:2022-10-15 Online:2022-10-20
Contact: YU Pei E-mail:15849588283@163.com;peiyu@tmu.edu.cn

吴玮熙, 刘帅辉, 周赛君, 刘红岩, 张睿, 于珮. 高糖通过miR-192调控CAV-1/EGF影响肾小球系膜细胞增殖、迁移及细胞外基质形成的机制研究[J]. 天津医药, 2022, 50(10): 1020-1025.

WU Weixi, LIU Shuaihui, ZHOU Saijun, LIU Hongyan, ZHANG Rui, YU Pei. Effects of high glucose on the proliferation and migration of mesangial cells and extracellular matrix formation by regulating the CAV-1/EGF through miR-192[J]. Tianjin Medical Journal, 2022, 50(10): 1020-1025.

摘要/Abstract

摘要：

目的探讨高糖条件下miR-192对肾小球系膜细胞增殖、迁移及细胞外基质形成的影响及可能的作用机制。方法体外培养人肾小球系膜细胞并分为6组：正糖（NG,5.6 mmol/L葡萄糖）组、高糖（HG,25 mmol/L葡萄糖）组、正糖加miR-192模拟物（NG+mimics）组、高糖加miR-192模拟物（HG+mimics）组、正糖加miR-192抑制剂（NG+inhibitor）组、高糖加miR-192抑制剂（HG+inhibitor）组。干预24 h后,CCK-8法检测细胞增殖活性,划痕实验检测细胞迁移能力,实时荧光定量PCR（qPCR）技术检测miR-192、小窝蛋白1（CAV-1）、表皮生长因子（EGF）、1型胶原蛋白（COLⅠ）、纤连蛋白（FN）mRNA水平,Western blot检测相关蛋白表达。结果与NG组相比,HG组细胞增殖率和划痕愈合率增加,EGF、FN、COLⅠ mRNA和蛋白表达升高,CAV-1 mRNA和蛋白表达降低（均P<0.05）。在NG组和HG组分别加入miR-192 inhibitor后,细胞增殖率和划痕愈合率减少,EGF、FN、COLⅠmRNA和蛋白表达降低,CAV-1 mRNA和蛋白表达升高（均P<0.05）,HG组加入miR-192 inhibitor变化更加明显。在NG组和HG组分别加入miR-192 mimics后,细胞增殖率和划痕愈合率增加,EGF、FN、COLⅠ mRNA和蛋白表达升高,CAV-1 mRNA和蛋白表达降低（均P<0.05）。结论高糖通过miR-192-CAV-1-EGF途径增加肾小球系膜细胞增殖和迁移,导致细胞外基质的形成。

关键词: 糖尿病肾病, 肾小球系膜细胞, 表皮生长因子, 细胞外基质, 细胞增殖, 微小RNA-192, 小窝蛋白1

Abstract:

Objective To investigate the effect of miR-192 on proliferation, migration and extracellular matrix of mesangial cells under high glucose condition. Methods Human mesangial cells were cultured in vitro for 24 h and divided into six groups: the normal glucose concentration (NG, 5.6 mmol/L glucose) group, the high glucose concentration (HG, 25 mmol/L glucose) group, the normal glucose concentration+miR-192 mimics (NG+mimics) group, the high glucose concentration+miR-192 mimics (HG+ mimics) group, the normal glucose concentration+miR-192 inhibitor (NG+inhibitor) group and the high glucose concentration+miR-192 inhibitor (HG+inhibitor) group. After 24 h of intervention, cell viability was detected by CCK-8 assay, the cell migration ability was detected by Scratch healing test, and expression levels of miR-192, caveolin 1 (CAV-1), epidermal growth factor (EGF), collagen type 1 (COLⅠ) and fibronectin (FN) mRNA and related proteins were detected by qPCR and Western blot assay. Results Compared with the NG group, the proliferation and migration rates were increased, the expression levels of EGF, FN and COLⅠmRNA and protein were increased, and the expression of CAV-1 mRNA and protein decreased in the HG group (all P<0.05). When miR-192 inhibitor was added to the NG group and the HG group, the cell proliferation and migration were decreased, the expression of EGF, FN and COLⅠ mRNA and protein were decreased, while the expression levels of CAV-1 mRNA and protein were increased (all P<0.05). Changes were more significant in the HG group after the addition of miR-192 inhibitor. After adding miR-192 mimics to the NG group and the HG group, the cell proliferation and migration increased, expression levels of EGF, FN and COLⅠ mRNA and protein increased, and the expression of CAV-1 decreased (all P<0.05). Conclusion High glucose increases the proliferation and migration of glomerular mesangial cells through miR-192-CAV-1-EGF pathway, leading to the formation of extracellular matrix and participating in the occurrence and development of diabetic nephropathy.

Key words: diabetic nephropathies, mesangial cells, epidermal growth factor, extracellular matrix, cell proliferation, micro RNA-192, caveolin-1

中图分类号:

R737.9

图/表 7

参考文献 23

[1]	INGELSSON E, MCCARTHY M I. Human genetics of obesity and type 2 diabetes mellitus: Past,present,and future[J]. Circ Genom Precis Med, 2018, 11(6):e002090. doi: 10.1161/CIRCGEN.118.002090.
[2]	SUGAHARA M, PAK W, TANAKA T, et al. Update on diagnosis,pathophysiology,and management of diabetic kidney disease[J]. Nephrology (Carlton), 2021, 26(6):491-500. doi: 10.1111/nep.13860.
[3]	CHUNG A C. MicroRNAs in diabetic kidney disease[J]. Adv Exp Med Biol, 2015, 888:253-269. doi: 10.1007/978-3-319-22671-2_13.
[4]	YU F N, HU M L, WANG X F, et al. Effects of microRNA-370 on mesangial cell proliferation and extracellular matrix accumulation by binding to canopy 1 in a rat model of diabetic nephropathy[J]. J Cell Physiol, 2019, 234(5):6898-6907. doi: 10.1002/jcp.27448.
[5]	PUTTA S, LANTING L, SUN G, et al. Inhibiting microRNA-192 ameliorates renal fibrosis in diabetic nephropathy[J]. J Am Soc Nephrol, 2012, 23(3):458-469. doi: 10.1681/ASN.2011050485.
[6]	YANG X Y, LIU S H, ZHANG R, et al. Microribonucleic acid-192 as a specific biomarker for the early diagnosis of diabetic kidney disease[J]. J Diabetes Investig, 2017, 9(3):602-609. doi: 10.1111/jdi.12753.
[7]	HU C, SUN L, XIAO L, et al. Insights into the mechanisms involved in the expression and regulation of extracellular matrix proteins in diabetic nephropathy[J]. Curr Med Chem, 2015, 22(24):2858-2870. doi: 10.2174/0929867322666150625095407.
[8]	RICCIARDI C A, GNUDI L. Kidney disease in diabetes:From mechanisms to clinical presentation and treatment strategies[J]. Metabolism, 2021, 124:154890. doi: 10.1016/j.metabol.2021.154890.
[9]	BüLOW R D, BOOR P. Extracellular matrix in kidney fibrosis:More than just a scaffold[J]. J Histochem Cytochem, 2019, 67(9):643-661. doi: 10.1369/0022155419849388.
[10]	RAYEGO-MATEOS S, CAMPILLO S, RODRIGUES-DIEZ R R, et al. Interplay between extracellular matrix components and cellular and molecular mechanisms in kidney fibrosis[J]. Clin Sci (Lond), 2021, 135(16):1999-2029. doi: 10.1042/CS20201016.
[11]	CHANDRASEKARAN K, KAROLINA D S, SEPRAMANIAM S, et al. Role of microRNAs in kidney homeostasis and disease[J]. Kidney Int, 2012, 81(7):617-627. doi: 10.1038/ki.2011.448.
[12]	李栋, 林珊. microRNAs在糖尿病肾病发病机制中的作用[J]. 天津医药, 2015, 43(6):698-702.
	LI D, LIN S. The role of microRNAs in the pathogenesis of diabetic nephropathy[J]. Tianjin Med J, 2015, 43(6):698-702. doi: 10.11958/j.issn.0253-9896.2015.06.032.
[13]	REN H, WANG Q. Non-coding RNA and diabetic kidney disease[J]. DNA Cell Biol, 2021, 40(4):553-567. doi: 10.1089/dna.2020.5973.
[14]	KRUPA A, JENKINS R, LUO D D, et al. Loss of microRNA-192 promotes fibrogenesis in diabetic nephropathy[J]. J Am Soc Nephrol, 2010, 21(3):438-447. doi: 10.1681/ASN.2009050530.
[15]	WANG B, KOMERS R, CAREW R, et al. Suppression of microRNA-29 expression by TGF-β1 promotes collagen expression and renal fibrosis[J]. J Am Soc Nephrol, 2012, 23(2):252-265. doi: 10.1681/ASN.2011010055.
[16]	KATO M, ZHANG J, WANG M, et al. MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-beta-induced collagen expression via inhibition of E-box repressors[J]. Proc Natl Acad Sci U S A, 2007, 104(9):3432-3437. doi: 10.1073/pnas.0611192104.
[17]	WILLIAMS T M, LISANTI M P. The caveolin genes: From cell biology to medicine[J]. Ann Med, 2004, 36(8):584-595. doi: 10.1080/07853890410018899.
[18]	FUJITA Y, MARUYAMA S, KOGO H, et al. Caveolin-1 in mesangial cells suppresses MAP kinase activation and cell proliferation induced by bFGF and PDGF[J]. Kidney Int, 2004, 66(5):1794-1804. doi: 10.1111/j.1523-1755.2004.00954.x.
[19]	CHEN P, FENG Y, ZHANG H, et al. MicroRNA‑192 inhibits cell proliferation and induces apoptosis in human breast cancer by targeting caveolin 1[J]. Oncol Rep, 2019, 42(5):1667-1676. doi: 10.3892/or.2019.7298.
[20]	PALMER B F. Proteinuria as a therapeutic target in patients with chronic kidney disease[J]. Am J Nephrol, 2007, 27(3):287-293. doi: 10.1159/000101958.
[21]	PERKINS B A, FICOCIELLO L H, OSTRANDER B E, et al. Microalbuminuria and the risk for early progressive renal function decline in type 1 diabetes[J]. J Am Soc Nephrol, 2007, 18(4):1353-1361. doi: 10.1681/ASN.2006080872.
[22]	TANG S, WANG X, DENG T, et al. Identification of C3 as a therapeutic target for diabetic nephropathy by bioinformatics analysis[J]. Sci Rep, 2020, 10(1):13468. doi: 10.1038/s41598-020-70540-x.
[23]	ORLICHENKO L, WELLER S G, CAO H, et al. Caveolae mediate growth factor-induced disassembly of adherens junctions to support tumor cell dissociation[J]. Mol Biol Cell, 2009, 20(19):4140-4152. doi: 10.1091/mbc.e08-10-1043.

基因名称	引物序列（5'→3'）
miR-192	上游：CGCGCGCGCTGACCTATGAATTG
miR-192	下游：ATCCAGTGCAGGGTCCGAGG
CAV-1	上游：ACGTAGACTCGGAGGGACATC
CAV-1	下游：GCAGACAGCAAGCGGTAAA
EGF	上游：GTAGCCAGCTCTGCGTTCCT
EGF	下游：TGGTTGTGGTCCTGAAGCTG
FN	上游：TAGCCCTGTCCAGGAGTTCA
FN	下游：CTGCAAGCCTTCAATAGTCA
COLⅠ	上游：GCAGGAGGTTTCGGCTAAGT
COLⅠ	下游：GCAACAAAGTCCGCGTATCC
β-actin	上游：CTATCGGCAATGAGCGGTTC
β-actin	下游：CTTAGGAGTTGGGGGTGGCT
miR-192 inhibitor	GGCUGUCAAUUCAUAGGUCAG
miR-192 mimics	CUGACCUAUGAAUUGACAGCC

基因名称	引物序列（5'→3'）
miR-192	上游：CGCGCGCGCTGACCTATGAATTG
miR-192	下游：ATCCAGTGCAGGGTCCGAGG
CAV-1	上游：ACGTAGACTCGGAGGGACATC
CAV-1	下游：GCAGACAGCAAGCGGTAAA
EGF	上游：GTAGCCAGCTCTGCGTTCCT
EGF	下游：TGGTTGTGGTCCTGAAGCTG
FN	上游：TAGCCCTGTCCAGGAGTTCA
FN	下游：CTGCAAGCCTTCAATAGTCA
COLⅠ	上游：GCAGGAGGTTTCGGCTAAGT
COLⅠ	下游：GCAACAAAGTCCGCGTATCC
β-actin	上游：CTATCGGCAATGAGCGGTTC
β-actin	下游：CTTAGGAGTTGGGGGTGGCT
miR-192 inhibitor	GGCUGUCAAUUCAUAGGUCAG
miR-192 mimics	CUGACCUAUGAAUUGACAGCC

组别		miR-192		CAV-1
NG组		1.01±0.03		1.07±0.06
HG组		1.45±0.01^a		0.83±0.01^a
NG+mimics组		1.23±0.01^a		0.51±0.01^a
HG+mimics组		1.56±0.01^bc		0.48±0.01^bc
NG+inhibitor组		0.21±0.01^a		1.43±0.01^a
HG+inhibitor组		0.18±0.02^bd		1.66±0.01^bd
F		2 943.628^＊＊		1 001.858^＊＊
组别	EGF		FN		COLⅠ
NG组	1.01±0.03		1.01±0.03		1.00±0.03
HG组	1.53±0.01^a		1.49±0.06^a		1.63±0.02^a
NG+mimics组	1.37±0.01^a		1.52±0.03^a		1.52±0.01^a
HG+mimics组	1.62±0.00^bc		1.65±0.02^bc		1.72±0.08^bc
NG+inhibitor组	0.74±0.01^a		0.72±0.02^a		0.62±0.02^a
HG+inhibitor组	0.61±0.04^bd		0.68±0.01^bd		0.57±0.01^bd
F	1 302.088^＊＊		1 244.431^＊＊		619.000^＊＊

组别		miR-192		CAV-1
NG组		1.01±0.03		1.07±0.06
HG组		1.45±0.01^a		0.83±0.01^a
NG+mimics组		1.23±0.01^a		0.51±0.01^a
HG+mimics组		1.56±0.01^bc		0.48±0.01^bc
NG+inhibitor组		0.21±0.01^a		1.43±0.01^a
HG+inhibitor组		0.18±0.02^bd		1.66±0.01^bd
F		2 943.628^＊＊		1 001.858^＊＊
组别	EGF		FN		COLⅠ
NG组	1.01±0.03		1.01±0.03		1.00±0.03
HG组	1.53±0.01^a		1.49±0.06^a		1.63±0.02^a
NG+mimics组	1.37±0.01^a		1.52±0.03^a		1.52±0.01^a
HG+mimics组	1.62±0.00^bc		1.65±0.02^bc		1.72±0.08^bc
NG+inhibitor组	0.74±0.01^a		0.72±0.02^a		0.62±0.02^a
HG+inhibitor组	0.61±0.04^bd		0.68±0.01^bd		0.57±0.01^bd
F	1 302.088^＊＊		1 244.431^＊＊		619.000^＊＊

组别	CAV-1	EGF	FN	COLⅠ
NG组	0.95±0.01	0.71±0.02	0.67±0.01	0.81±0.01
HG组	0.93±0.01^a	0.78±0.11^a	0.95±0.02^a	0.84±0.01^a
NG+mimics组	0.80±0.01^a	0.88±0.11^a	0.73±0.05^a	0.93±0.01^a
HG+mimics组	0.55±0.01^bc	0.88±0.11^b	1.07±0.11^bc	1.08±0.08^bc
NG+inhibitor组	1.06±0.01^a	0.66±0.02^a	0.58±0.02^a	0.78±0.08^a
HG+inhibitor组	1.20±0.01^bd	0.48±0.01^bd	0.41±0.01^bd	0.54±0.01^bd
F	1 740.498^＊＊	509.618^＊＊	68.769^＊＊	76.459^＊＊

高糖通过miR-192调控CAV-1/EGF影响肾小球系膜细胞增殖、迁移及细胞外基质形成的机制研究

Effects of high glucose on the proliferation and migration of mesangial cells and extracellular matrix formation by regulating the CAV-1/EGF through miR-192

引用本文

RichHTML

PDF (PC)

可视化

摘要/Abstract

使用本文

图/表 7

参考文献 23

相关文章 15

编辑推荐

Metrics

本文评价

[1]	张晋玮, 王燕, 王通. miR-107对口腔鳞癌细胞系CAL27增殖、侵袭及迁移的影响[J]. 天津医药, 2024, 52(9): 897-899.
[2]	侯维玲, 乔云阳, 吴小芸, 施会敏, 曲高婷, 张爱青. 锌指蛋白281抑制高糖诱导的肾小管上皮细胞上皮间质转化和细胞外基质合成[J]. 天津医药, 2024, 52(7): 720-726.
[3]	王娴, 刘霞明, 陈曼玉, 赵君, 王立东. 基于机器学习对2型糖尿病肾病预测模型的构建及验证[J]. 天津医药, 2024, 52(7): 775-780.
[4]	夏雨薇, 乔云阳, 刘雪薇, 施会敏, 曲高婷, 张爱青, 甘卫华. tRF-1:30对高糖诱导的肾小管上皮细胞炎性因子表达的影响[J]. 天津医药, 2024, 52(6): 561-566.
[5]	刘丹阳, 李永涛, 张海燕, 李林, 刘洋, 沈雷. 乳腺癌细胞条件培养基对骨髓间充质干细胞生物学行为的影响[J]. 天津医药, 2024, 52(5): 454-458.
[6]	林瑶, 刘从娜, 王世霞, 张志勇. 金合欢素调节HMGB1/TLR4信号通路对脂多糖诱导牙髓细胞凋亡的影响[J]. 天津医药, 2024, 52(12): 1238-1243.
[7]	谷巍, 张惠娜, 侯丽萍, 于敏, 程黎蓉. 脂质相关指数与糖尿病肾病相关性研究[J]. 天津医药, 2024, 52(12): 1308-1312.
[8]	张贵婷, 何超. oxLDL/β2GPⅠ/aβ2GPⅠ复合物通过TLR4/MyD88/NF-κB途径促进血管内皮细胞血管生成[J]. 天津医药, 2024, 52(11): 1131-1136.
[9]	郝凯凯, 王晓敏, 刘峥, 刘东洋, 李静. 藁本内酯调节RhoA/ROCK信号通路对食管癌细胞生物学行为的影响[J]. 天津医药, 2024, 52(11): 1164-1170.
[10]	孙创新, 李刚. NID1在肾透明细胞癌血管生成中的作用研究[J]. 天津医药, 2024, 52(10): 1009-1013.
[11]	徐桂颖, 李玉, 李雪, 刘怡萌, 陈怀永. 气道类器官研究哮喘中Lkb1调控上皮再生的机制[J]. 天津医药, 2024, 52(1): 11-15.
[12]	张琳琳, 赵唐明, 黄婵, 李善文, 甘卫华. 新型多肽AMPP2在TGF-β1诱导的系膜细胞增殖中的作用及其机制[J]. 天津医药, 2024, 52(1): 50-55.
[13]	王倩倩, 李婷芳, 王峰. CHD4调控端粒功能促进宫颈癌HeLa细胞生长的机制研究[J]. 天津医药, 2023, 51(9): 909-914.
[14]	黄冠又, 葛学成, 甘鸿川, 郝淑煜, 吴震. CCL18在胶质母细胞瘤中的表达及其对U87MG细胞增殖和迁移的影响[J]. 天津医药, 2023, 51(9): 915-921.
[15]	岳巾晶, 曾莹, 郭晓珮, 董越, 姬若楠, 彭瑞, 罗晓华. miR-155通过调控PKG1影响滋养细胞生物学功能并参与子痫前期的机制探讨[J]. 天津医药, 2023, 51(9): 928-934.