miR-19b在人主动脉瓣间质细胞钙化中的作用

doi:10.3969/j.issn.1673-8640.2022.05.014

摘要/Abstract

摘要： 目的探讨miR-19b在人主动脉瓣间质细胞（hVIC）钙化中的作用和机制。方法使用含骨形态发生蛋白2（BMP-2）、地塞米松、抗维生素C和β-甘油磷酸的钙化培养液诱导hVIC细胞钙化（钙化模型组）,通过Von Kossa染色鉴定模型是否构建成功,以未做处理的hVIC细胞作为对照组。根据转染质粒的不同,将hVIC细胞分为miR-19b空白对照（miR-NC）组、miR-19b过表达组、SMAD4过表达组、miR-19b和SMAD4同时过表达（miR-19b+SMAD4过表达）组。采用荧光定量聚合酶链反应（PCR）检测各组hVIC细胞miR-19b、转录受体相关转录因子-2（RUNX2）mRNA、骨钙素（OC）mRNA、SMAD1 mRNA、骨桥蛋白（OPN）mRNA和SMAD4 mRNA的相对表达量。采用免疫印迹法检测SMAD4蛋白的相对表达量。采用StarBase数据库预测miR-19b的下游靶基因,采用双荧光素酶报告基因实验进行验证。结果钙化诱导后的hVIC细胞互相重叠生长,并表现出明显的聚集样生长趋势,细胞内钙化结节的数目明显增多（P<0.05）,成骨相关因子RUNX2 mRNA、OC mRNA、SMAD1 mRNA和OPN mRNA的相对表达量明显升高（P<0.05）。钙化模型组miR-19b表达下调（P<0.05）。过表达miR-19b后hVIC细胞内RUNX2 mRNA、OC mRNA、SMAD1 mRNA和OPN mRNA相对表达量显著降低（P<0.05）。双荧光素酶报告基因实验结果证实SMAD4是miR-19b的下游靶基因。过表达SMAD4后hVIC细胞内RUNX2 mRNA、OC mRNA、SMAD1 mRNA和OPN mRNA相对表达量显著上调（P<0.05）,同时过表达miR-19b和SMAD4的hVIC细胞内成骨相关因子表达无变化（P>0.05）。结论 hVIC细胞钙化模型中miR-19b表达显著下调。miR-19b通过与下游靶基因SMAD4的靶向结合调控hVIC的钙化。miR-19b和SMAD4或可作为治疗主动脉瓣钙化疾病的潜在治疗靶标。

关键词: miR-19b, 主动脉瓣间质细胞钙化, 成骨分化

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

杨濛, 丁辉. miR-19b在人主动脉瓣间质细胞钙化中的作用[J]. 检验医学, 2022, 37(5): 466-471.

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

图/表 6

参考文献 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^**