Role of miR-19b in human aortic valve interstitial cell calcification

doi:10.3969/j.issn.1673-8640.2022.05.014

Abstract

Abstract: Objective To investigate the role of miR-19b in human aortic valve interstitial cell（hVIC） calcification and its mechanism. Methods A calcification medium containing bone morphogenetic protein 2（BMP-2）,dexamethasone,anti-vitamin C and β-glycerophosphate was used to induce the calcification of hVIC,and Von Kossa staining was used to determine whether the model was constructed successfully. Untreated hVIC cells were used as control group. According to different transfection plasmids,hVIC cells were classified into miR-19b blank control（miR-NC） group,miR-19b overexpression group,SMAD4 overexpression group and miR-19b and SMAD4 overexpression（miR-19b+SMAD4 overexpression） group. The mRNA expression levels of miR-19b,Runt-related gene 2（RUNX 2）,osteocalcin（OC）,SMAD1,osteopontin（OPN） and SMAD4 in hVIC of each group were determined by fluorescent quantitative polymerase chain reaction（PCR）. The expression level of SMAD4 protein was determined by western blotting. The downstream target genes of miR-19b were predicted by StarBase database and verified by dual-luciferase reporter gene experiments. Results The hVIC after calcification induced overlap each other and showed an obvious aggregation-like growth trend,and the number of intracellular calcified nodules was increased（P<0.05）. The expression levels of RUNX2,OC,SMAD1 and OPN mRNA were increased（P<0.05）. The expression of miR-19b was down-regulated in the calcified hVIC model（P<0.05）,and the expression levels of osteogenic factors,hVIC intracell RUNX2 mRNA,OC mRNA,SMAD1 mRNA and OPN mRNA,in the model was decreased after the overexpression of miR-19b（P<0.05）. The results of dual luciferase reporter gene experiments confirmed that SMAD4 was the downstream target gene of miR-19b. The overexpression of SMAD4 up-regulated the expression of osteogenesis-related factors in calcified hVIC cells（P<0.05）,while the overexpression of miR-19b reversed the up-regulation effect of overexpression of SMAD4 on the expression of osteogenesis-related factors（P>0.05）. Conclusions The expression of miR-19b is down-regulated in the calcified hVIC model. By targeting the downstream target gene SMAD4 to regulate the calcification of hVIC,miR-19b and SMAD4 are potential therapeutic targets for treating aortic valve calcification diseases.

Key words: MiR-19b, Aortic valve calcification, Osteogenic differentiation

YANG Meng, DING Hui. Role of miR-19b in human aortic valve interstitial cell calcification[J]. Laboratory Medicine, 2022, 37(5): 466-471.

Figures/Tables 6

References 21

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基因	正向引物（5'~3'）	反向引物（5'~3'）	长度/bp
RUNX2	AGCTTCTGTCTGTGCCTTCTGG	GGAGTAGAGAGGCAAGAGTTT	22/21
OC	CTTTGTGTCCAAGCAGGA	CTGAAAGCCGATGTGGTCAG	18/20
SMAD1	TTCCATGCCTCCTCCACAAG	AGGCATTCGGCATACACCTC	20/20
OPN	TTCCAAGTAAGTCCAACGAAAG	GTGACCAGTTCATCAGATTTCAT	22/23
SMAD4	GATACGTGGACCCTTCTGGA	CCTTTGCCTATGTGCAACCT	20/20
β-actin	AACAGCCGCCTAGAAGCAC	CGTTGACATCCGTAAAGACC	19/20
miR-19b	CAGTGCAATGTTAAAAGGGCAT	TAGAGTGAGTGTAGCGAGCA	22/20
U6	GCTTCGGCAGCACATATACTAAAAT	CGCTTCACGAATTTGCGTGTCAT	25/23

基因	正向引物（5'~3'）	反向引物（5'~3'）	长度/bp
RUNX2	AGCTTCTGTCTGTGCCTTCTGG	GGAGTAGAGAGGCAAGAGTTT	22/21
OC	CTTTGTGTCCAAGCAGGA	CTGAAAGCCGATGTGGTCAG	18/20
SMAD1	TTCCATGCCTCCTCCACAAG	AGGCATTCGGCATACACCTC	20/20
OPN	TTCCAAGTAAGTCCAACGAAAG	GTGACCAGTTCATCAGATTTCAT	22/23
SMAD4	GATACGTGGACCCTTCTGGA	CCTTTGCCTATGTGCAACCT	20/20
β-actin	AACAGCCGCCTAGAAGCAC	CGTTGACATCCGTAAAGACC	19/20
miR-19b	CAGTGCAATGTTAAAAGGGCAT	TAGAGTGAGTGTAGCGAGCA	22/20
U6	GCTTCGGCAGCACATATACTAAAAT	CGCTTCACGAATTTGCGTGTCAT	25/23

组别	RUNX2 mRNA	OC mRNA	SMAD1 mRNA	OPN mRNA
miR-19b过表达组	0.72±0.11	0.65±0.08	0.51±0.06	0.54±0.06
钙化模型组	1.00±0.13	1.00±0.10	1.00±0.12	1.00±0.16
t值	2.848	4.734	6.326	4.663
P值	<0.05	<0.01	<0.01	<0.01

组别	RUNX2 mRNA	OC mRNA	SMAD1 mRNA	OPN mRNA
miR-19b过表达组	0.72±0.11	0.65±0.08	0.51±0.06	0.54±0.06
钙化模型组	1.00±0.13	1.00±0.10	1.00±0.12	1.00±0.16
t值	2.848	4.734	6.326	4.663
P值	<0.05	<0.01	<0.01	<0.01

组别	RUNX2 mRNA	OC mRNA	SMAD1 mRNA	OPN mRNA
SMAD4过表达组	2.75±0.21	2.13±0.17	1.36±0.11	1.54±0.16
钙化模型组	1.00±0.13^***	1.00±0.10^***	1.00±0.12^*	1.00±0.16^**
miR-19b+SMAD4过表达组	1.03±0.13^***	0.97±0.11^***	1.11±0.15^*	1.07±0.09^**