Zinc finger protein 281 inhibits high glucose-induced epithelial-mesenchymal transition and extracellular matrix synthesis in renal tubular epithelial cells

doi:10.11958/20240031

Abstract

Abstract:

Objective To investigate the role and mechanism of zinc finger protein 281 (ZNF281) in high glucose (HG)-induced epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) synthesis in renal tubular epithelial cells (RTECs). Methods HG induced RTECs were used to construct a diabetic kidney disease cell model, and cells were divided into the control group, the HG group and the mannitol group. Cell proliferation viability was detected by CCK-8. The expression of ZNF281 was knocked down in HG-treated RTECs using small interfering RNA (siRNA). HG-induced RTECs after knockdown of ZNF281 were divided into the control group, the HG group, the HG+ZNF281 siRNA group and the HG+ZNF281 vector group. Adenosine monophosphate-activated protein kinase (AMPK) was activated using AMPK agonist, acadexin (AICAR), and then cells were divided into the control group, the HG group, the HG+AICAR group and the HG+dimethyl sulfoxide group. The expression levels of ZNF281, EMT and ECM synthesis-related indexes were detected by qPCR and Western blot assay. Results Compared with the control group, the protein and mRNA expression levels of vimentin, α-smooth muscle actin (α-SMA), fibronectin (FN) and collagen Ⅰ (Col Ⅰ) were significantly higher, and the expression of E-cadherin was significantly lower in the HG group. Compared with the HG group, the protein and mRNA expression levels of EMT and ECM synthesis-related indexes were significantly changed in the HG+ZNF281 siRNA group and the HG+AICAR group. The protein and mRNA expression levels of ZNF281 were significantly reduced in the HG+AICAR group compared with the HG group. In cells co-treated with AICAR and transfected with ZNF281 plasmid, the expression levels of vimentin, α-SMA, FN and Col Ⅰ were significantly higher in the AICAR+ZNF281 group, and E-cadherin was significantly lower compared with that of the vector group. Conclusion AMPK inhibits EMT and ECM synthesis in HG-treated RTECs by negatively regulating the expression level of ZNF281.

Key words: diabetic nephropathies, tristetraprolin, epithelial-mesenchymal transition, extracellular matrix, AMP-activated protein kinases, tubulointerstitial fibrosis, zinc finger protein 281

CLC Number:

R587.24，R34

Figures/Tables 12

References 24

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基因名称	引物序列（5′→3′）	产物大小/bp
ZNF281	上游：GCCATCCTCTCCCCAAGTC 下游：GAGCTTCGGAAAGCAGCACTA	65
E-cadherin	上游：TCGGAAGACTCCCGATTCAAA 下游：CGGACGAGGAAACTGGTCTC	95
vimentin	上游：CGTCCACACGCACCTACAG 下游：GGGGGATGAGGAATAGAGGCT	74
α-SMA	上游：CAGCAAACAGGAATACGACGAA 下游：AACCACGAGTAACAAATCAAAGC	168
FN	上游：GCAAGAAGGACAACCGAGGAAA 下游：GGACATCAGTGAAGGAGCCAGA	126
ColⅠ	上游：GCTCCTCTTAGGGGCCACT 下游：ATTGGGGACCCTTAGGCCAT	91
β-actin	上游：CATCCGTAAAGACCTCTATGCCAAC 下游：ATGGAGCCACCGATCCACA	171

基因名称	引物序列（5′→3′）	产物大小/bp
ZNF281	上游：GCCATCCTCTCCCCAAGTC 下游：GAGCTTCGGAAAGCAGCACTA	65
E-cadherin	上游：TCGGAAGACTCCCGATTCAAA 下游：CGGACGAGGAAACTGGTCTC	95
vimentin	上游：CGTCCACACGCACCTACAG 下游：GGGGGATGAGGAATAGAGGCT	74
α-SMA	上游：CAGCAAACAGGAATACGACGAA 下游：AACCACGAGTAACAAATCAAAGC	168
FN	上游：GCAAGAAGGACAACCGAGGAAA 下游：GGACATCAGTGAAGGAGCCAGA	126
ColⅠ	上游：GCTCCTCTTAGGGGCCACT 下游：ATTGGGGACCCTTAGGCCAT	91
β-actin	上游：CATCCGTAAAGACCTCTATGCCAAC 下游：ATGGAGCCACCGATCCACA	171

组别	mRNA（n=4）
组别	ZNF281	E-cadherin	vimentin	α-SMA	FN	ColⅠ
Control组	1.04±0.32	1.02±0.21	1.07±0.45	1.02±0.22	1.01±0.13	1.03±0.30
HG组	2.37±0.52^a	0.49±0.08^a	2.39±0.58^a	1.97±0.29^a	2.18±0.45^a	1.75±0.08^a
Mannitol组	1.02±0.30	0.86±0.05	0.96±0.26	1.18±0.22	0.96±0.30	0.94±0.35
F	15.580^＊＊	17.020^＊＊	12.560^＊＊	17.180^＊＊	18.780^＊＊	10.890^＊＊
组别	蛋白（n=3）
组别	ZNF281	E-cadherin	vimentin	α-SMA	FN	Col Ⅰ
Control组	1.00±0.18	1.00±0.21	1.00±0.18	1.00±0.13	1.00±0.09	1.00±0.13
HG组	2.11±0.15^a	0.33±0.11^a	1.71±0.06^a	1.60±0.10^a	1.38±0.07^a	1.33±0.10^a
Mannitol组	0.92±0.33	0.98±0.13	1.01±0.39	0.92±0.18	0.97±0.08	0.78±0.17
F	23.900^＊＊	17.420^＊＊	8.034^＊	21.650^＊＊	22.990^＊＊	12.290^＊＊

组别	mRNA（n=4）
组别	ZNF281	E-cadherin	vimentin	α-SMA	FN	ColⅠ
Control组	1.04±0.32	1.02±0.21	1.07±0.45	1.02±0.22	1.01±0.13	1.03±0.30
HG组	2.37±0.52^a	0.49±0.08^a	2.39±0.58^a	1.97±0.29^a	2.18±0.45^a	1.75±0.08^a
Mannitol组	1.02±0.30	0.86±0.05	0.96±0.26	1.18±0.22	0.96±0.30	0.94±0.35
F	15.580^＊＊	17.020^＊＊	12.560^＊＊	17.180^＊＊	18.780^＊＊	10.890^＊＊
组别	蛋白（n=3）
组别	ZNF281	E-cadherin	vimentin	α-SMA	FN	Col Ⅰ
Control组	1.00±0.18	1.00±0.21	1.00±0.18	1.00±0.13	1.00±0.09	1.00±0.13
HG组	2.11±0.15^a	0.33±0.11^a	1.71±0.06^a	1.60±0.10^a	1.38±0.07^a	1.33±0.10^a
Mannitol组	0.92±0.33	0.98±0.13	1.01±0.39	0.92±0.18	0.97±0.08	0.78±0.17
F	23.900^＊＊	17.420^＊＊	8.034^＊	21.650^＊＊	22.990^＊＊	12.290^＊＊

组别	mRNA（n=4）
组别	ZNF281	E-cadherin	vimentin	α-SMA	FN	Col Ⅰ
Control组	1.02±0.25	1.04±0.31	1.03±0.27	1.02±0.21	1.01±0.15	1.01±0.19
HG组	2.17±0.63^a	0.43±0.07^a	2.08±0.21^a	2.09±0.19^a	2.18±0.15^a	2.06±0.18^a
HG+ZNF281 siRNA组	0.42±0.10^b	0.85±0.08^b	1.55±0.32^b	1.39±0.37^b	1.45±0.22^b	1.60±0.21^b
HG+ZNF281 vector组	2.06±0.50	0.51±0.07	2.29±0.24	2.29±0.46	2.26±0.32	2.07±0.14
F	15.830^＊＊	11.750^＊＊	18.320^＊＊	13.230^＊＊	29.910^＊＊	30.500^＊＊
组别	蛋白（n=3）
组别	ZNF281	E-cadherin	vimentin	α-SMA	FN	Col Ⅰ
Control组	1.00±0.27	1.00±0.09	1.00±0.17	1.00±0.11	1.00±0.08	1.00±0.10
HG组	2.39±0.33^a	0.58±0.03^a	1.74±0.15^a	1.62±0.13^a	1.44±0.13^a	1.32±0.03^a
HG+ZNF281 siRNA组	1.24±0.57^b	0.76±0.08^b	1.17±0.25^b	1.09±0.24^b	1.10±0.14^b	0.97±0.10^b
HG+ZNF281 vector组	2.71±0.43	0.43±0.07	1.57±0.20	1.46±0.37	1.35±0.07	1.20±0.06
F	12.220^＊＊	36.510^＊＊	9.089^＊＊	4.662^＊	10.870^＊＊	13.220^＊＊