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

doi:10.11958/20220268

Abstract

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

CLC Number:

R737.9

Figures/Tables 7

References 23

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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^＊＊