miR-149-3p在先天性心脏病胎鼠心脏组织中的表达及其对P19细胞分化的影响

doi:10.11958/20220707

天津医药 ›› 2023, Vol. 51 ›› Issue (1): 45-49.doi: 10.11958/20220707

miR-149-3p在先天性心脏病胎鼠心脏组织中的表达及其对P19细胞分化的影响

胡丹慧¹(), 罗慧臣²^,^∆(), 刘海英¹

1 南华大学衡阳医学院附属第一医院新生儿科（邮编421001）
2 南华大学衡阳医学院附属第一医院风湿免疫科（邮编421001）

收稿日期:2022-05-09 修回日期:2022-08-05 出版日期:2023-01-15 发布日期:2023-01-17
通讯作者: 罗慧臣 E-mail:hudanhui723@163.com;ftvc2679@163.com
作者简介:胡丹慧（1983），女，主治医师，主要从事新生儿常见疾病的发病机制与治疗方面研究。E-mail：hudanhui723@163.com
基金资助:
湖南省卫生健康委科研立项课题(202106010111)

The expression of miR-149-3p in heart tissue of newborn mice with congenital heart disease and its effect on differentiation of P19 cells

HU Danhui¹(), LUO Huichen²^,^∆(), LIU Haiying¹

1 Department of Neonatology, the First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang 421001, China
2 Department of Rheumatism Immunology, the First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang 421001, China

Received:2022-05-09 Revised:2022-08-05 Published:2023-01-15 Online:2023-01-17
Contact: LUO Huichen E-mail:hudanhui723@163.com;ftvc2679@163.com

胡丹慧, 罗慧臣, 刘海英. miR-149-3p在先天性心脏病胎鼠心脏组织中的表达及其对P19细胞分化的影响[J]. 天津医药, 2023, 51(1): 45-49.

HU Danhui, LUO Huichen, LIU Haiying. The expression of miR-149-3p in heart tissue of newborn mice with congenital heart disease and its effect on differentiation of P19 cells[J]. Tianjin Medical Journal, 2023, 51(1): 45-49.

摘要/Abstract

摘要：

目的探讨miR-149-3p在先天性心脏病（CHD）胎鼠心脏组织中的表达及其对小鼠畸胎瘤P19细胞分化的影响。方法建立CHD室间隔缺损胎鼠模型，于妊娠第19天，HE染色观察心脏组织病理学变化，实时荧光定量PCR（qPCR）检测心脏组织中miR-149-3p和热休克蛋白蛋白家族B成员6（HSPB6）mRNA表达水平。二甲基亚砜（DMSO）诱导P19细胞向心肌细胞分化，并收集分化第0、5、10天的细胞，qPCR检测诱导分化过程中心肌分化标志物GATA结合蛋白4（GATA4）、心肌肌钙蛋白T（cTnT）、心房利钠尿多肽（ANP）的mRNA表达水平以及HSPB6 mRNA和miR-149-3p表达水平。miR-149-3p过表达慢病毒和空载慢病毒感染P19细胞，DMSO诱导分化第10天，免疫荧光染色检测细胞中cTnI蛋白表达情况，qPCR检测心肌分化相关标志物mRNA表达水平。双荧光素酶报告基因实验验证miR-149-3p与HSPB6之间的靶向关系。结果与正常组比较，CHD组胎鼠出现心脏室间隔缺损，心脏组织中miR-149-3p高表达，而HSPB6低表达（P<0.01）。P19细胞在向心肌细胞分化的过程中，GATA4、cTnT、ANP和HSPB6 mRNA水平均逐渐上升，而miR-149-3p表达水平逐渐下降（P<0.05）。诱导分化第10天，miR-149-3p过表达可明显降低心肌细胞分化率，并下调GATA4、cTnT、ANP mRNA表达水平（P<0.01）。双荧光素酶报告基因实验证实，miR-149-3p可以靶向调控HSPB6表达。结论 miR-149-3p可能通过靶向下调HSPB6抑制P19细胞向心肌细胞分化。

关键词: 心脏缺损，先天性, 微RNAs, HSP20热休克蛋白质类, 细胞分化, miR-149-3p, P19细胞

Abstract:

Objective To explore the expression of miR-149-3p in heart tissues of newborn mice with congenital heart disease (CHD) and its effect on differentiation of mouse embryonic teratoma P19 cells. Methods The newborn mouse model of ventricular septal defect CHD was established. On the 19th day of pregnancy, HE staining was used to observe pathological changes of fetal heart. Then qPCR was used to detect the expression levels of miR-149-3p and heat shock protein family B in fetal mouse heart tissue member 6 (HSPB6) mRNA in heart tissue of fetal mice. Dimethyl sulfoxide (DMSO) was used to induce P19 cells to differentiate into cardiomyocytes, and cells on the 0 th, 5 th and 10 th days of differentiation were collected. qPCR was used to detect markers of myocardial differentiation GATA4, cTnT, the mRNA expression level of atrial natriuretic peptide (ANP) and HSPB6 mRNA and miR-149-3p expression levels during the induction of differentiation. P19 cells were infected with miR-149-3p overexpression lentivirus and empty lentivirus. After induction for 10 days by DMSO, the expression of cTnI protein in cells was detected by immunofluorescence staining. mRNA expression levels of myocardial differentiation markers were detected by qPCR. The dual-luciferase reporter gene experiment was used to verify the targeting relationship between miR-149-3p and HSPB6. Results Compared with the normal group, the newborn mice in the CHD group developed cardiac ventricular septal defect, with high expression of miR-149-3p and low expression of HSPB6 in cardiac tissue (P<0.01). During the differentiation of P19 cells into cardiomyocytes, the expression levels of GATA4, cTnT, ANP and HSPB6 mRNA were gradually increased, while the expression level of miR-149-3p was gradually decreased (P<0.05). After 10 days of differentiation, the overexpression of miR-149-3p significantly reduced the differentiation rate of cardiomyocytes, and down-regulated the mRNA expression levels of GATA4, cTnT and ANP (P<0.01). The dual luciferase reporter gene experiment confirmed that miR-149-3p could target the expression of HSPB6. Conclusion miR-149-3p might inhibit the differentiation of P19 cells into cardiomyocytes through targeted down- regulation of HSPB6.

Key words: heart defects, congenital, microRNAs, HSP20 heat-shock proteins, cell differentiation, miR-149-3p, P19 cells

中图分类号:

R541.1

图/表 9

表1 实时荧光定量PCR引物序列

Tab.1 The primer sequences of real-time fluorescent quantitative PCR

基因名称	引物序列（5'→3'）	产物大小（bp）
U6	上游：CTCGCTT CGGCAGCACA	89
	下游：AACGCTTCACGAATTTGCGT
	RT引物：AACGCTTCACGAATTTGCGT
miR-149-3p	上游：CGGGCGAGGGAGGGACGGGG	89
	下游：CAGCCACAAAA GAGCACAAT
	RT引物：CCTGTTGTCTCCAGCCACAAA AGAGCACAATATTTCAGGAGACAACAGG
HSPB6	上游：CCATGTGGAGGTCCATGCTCG	78
HSPB6	下游：GCAGGTGGTGACGGAAGTGAG	78
GAPDH	上游：ACTAGGCGCTCACTGTTCTC	87
GAPDH	下游：ATCCGTTGACTCCGACCTTC	87
GATA4	上游：CCAACTGCCAGACTACCAC	479
GATA4	下游：GGACAGGCTGTTCCAAGA	479
cTnT	上游：CAGAGGAGGCCAACGTAGAAG	138
cTnT	下游：CTCCATCGGGGATCTTGGGT	138
ANP	上游：GCTTCCAGGCCATATTGGAG	152
ANP	下游：GGGGGCATGACCTCATCTT	152

图1 胎鼠心脏组织病理学变化（HE染色，×400）

Fig.1 Histopathological changes of fetal mouse heart tissue(HE staining, ×400)

表2 诱导分化过程中GATA4、cTnT、ANP mRNA和miR-149-3p表达变化

Tab.2 Changes in expression levels of GATA4, cTnT and ANP mRNA and miR-149-3p during the process of inducing differentiation

时间	GATA4	cTnT	ANP	miR-149-3p
第0天	0.973±0.023	0.950±0.030	0.987±0.086	1.009±0.076
第5天	3.367±0.751^a	4.600±0.614^a	3.500±0.755^a	0.449±0.120^a
第10天	7.333±0.651^b	9.167±0.777^b	8.233±0.666^b	0.216±0.071^b
F	94.072^＊＊	155.374^＊＊	119.370^＊＊	59.349^＊＊

表3 miR-149-3p过表达对P19细胞向心肌细胞分化过程中miR-149-3p表达和心肌细胞分化率的影响

Tab.3 Effects of miR-149-3p overexpression on miR-149-3p expression and cardiomyocyte differentiation during P19 cell differentiation into cardiomyocytes

组别	miR-149-3p	心肌细胞分化率（%）
blank组	1.007±0.021	67.977±5.234
Vector组	1.033±0.025	66.403±6.330
miR-149-3p组	5.277±0.677^ab	21.840±6.719^ab
F	118.404^＊＊	54.832^＊＊

图2 cTnI蛋白的表达情况（细胞免疫荧光，×400）

Fig.2 The expression of cTnI protein (cellular immunofluorescence, ×400)

表4 miR-149-3p过表达对P19细胞向心肌细胞分化过程中GATA4、cTnT和ANP mRNA表达的影响

Tab.4 Effects of miR-149-3p overexpression on GATA4, cTnT and ANP mRNA expressions during the differentiation of P19 cells into cardiomyocytes

组别	GATA4	cTnT	ANP
blank组	0.993±0.088	1.009±0.087	0.969±0.043
Vector组	1.007±0.089	1.028±0.085	1.002±0.099
miR-149-3p组	0.364±0.062^ab	0.290±0.075^ab	0.305±0.099^ab
F	62.128^＊＊	77.285^＊＊	65.468^＊＊

图3 HSPB6蛋白的表达情况

Fig.3 The expression of HSPB6 protein

表5 miR-149-3p过表达对P19细胞向心肌细胞分化过程中HSPB6 mRNA和蛋白表达的影响

Tab.5 Effects of miR-149-3p overexpression on HSPB6 mRNA and protein expression during the differentiation of P19 cells into cardiomyocytes

组别	HSPB6 mRNA	HSPB6蛋白
blank组	1.010±0.084	1.098±0.180
Vector组	1.000±0.116	1.136±0.059
miR-149-3p组	0.217±0.083^ab	0.458±0.099^ab
F	67.815^＊＊	28.391^＊＊

图4 miR-149-3p与HSPB6的靶向结合位点

Fig.4 The targeting site of miR-149-3p and HSPB6

参考文献 16

[1]	WILLIAMS K, CARSON J, LO C. Genetics of congenital heart disease[J]. Biomolecules, 2019, 9(12):879. doi:10.3390/biom9120879.
[2]	WU X L, LI R, FU F, et al. Chromosome microarray analysis in the investigation of children with congenital heart disease[J]. BMC Pediatr, 2017, 17(1):117. doi:10.1186/s12887-017-0863-3.
[3]	王维. 新生儿先天性心脏病防控措施研究进展[J]. 武警后勤学院学报(医学版), 2021, 30(1):80-84.
	WANG W. Prevention and control measures for congenital heart disease in newborns[J]. Journal of Logistics University of PAP (Medical Sciences),2021, 30(1):80-84. doi:10.16548/j.2095-3720.2021.01.025.
[4]	NAGY O, BARÁTH S, UJFALUSI A. The role of microRNAs in congenital heart disease[J]. EJIFCC, 2019, 30(2):165-178.
[5]	BASTAMI M, CHOUPANI J, SAADATIAN Z, et al. miRNA polymorphisms and risk of cardio-cerebrovascular diseases:A systematic review and meta-analysis[J]. Int J Mol Sci, 2019, 20(2):293. doi:10.3390/ijms20020293.
[6]	LU M, XU L, WANG M, et al. miR-149 promotes the myocardial differentiation of mouse bone marrow stem cells by targeting Dab2[J]. Mol Med Rep, 2018, 17(6):8502-8509. doi:10.3892/mmr.2018.8903.
[7]	ULLAH M, QIAN N P M, YANNARELLI G, et al. Heat shock protein 20 promotes sirtuin 1-dependent cell proliferation in induced pluripotent stem cells[J]. World J Stem Cells, 2021, 13(6):659-669. doi:10.4252/wjsc.v13.i6.659.
[8]	FANG X, BOGOMOLOVAS J, TREXLER C, et al. The BAG3- dependent and -independent roles of cardiac small heat shock proteins[J]. JCI Insight, 2019, 4(4):e126464. doi:10.1172/jci.insight.126464.
[9]	张国明, 何丽芸, 马松峰. 不同方法在先天性心脏病室间隔缺损动物模型建立中的对比研究[J]. 中国心血管病研究, 2020, 18(11):1026-1029.
	ZHANG G M, HE L Y, MA S F. Comparative study of different methods in establishing animal models of ventricular septal defec[J]. Chinese Journal of Cardiovascular Research, 2020, 18(11):1026-1029. doi:10.3969/j.issn.1672-5301.2020.11.013.
[10]	CHEN T, LI S J, CHEN B, et al. Akt3 is a target of miR-29c-3p and serves an important function in the pathogenesis of congenital heart disease[J]. Int J Mol Med, 2019, 43(2):980-992. doi:10.3892/ijmm.2018.4008.
[11]	HUANG G J, XIE X L, ZOU Y. MiR-23b targets GATA6 to down-regulate IGF-1 and promote the development of congenital heart disease[J]. Acta Cardiol, 2021:1-10. doi:10.1080/00015385.2021.1948207.
[12]	HU C, HUANG S, WU F, et al. MicroRNA-219-5p participates in cyanotic congenital heart disease progression by regulating cardiomyocyte apoptosis[J]. Exp Ther Med, 2021, 21(1):36. doi:10.3892/etm.2020.9468.
[13]	REN Z H, KE Z P, LUO M, et al. Icariin protects against ischemia-reperfusion injury in H9C2 cells by upregulating heat shock protein 20[J]. Mol Med Rep, 2018, 17(2):3336-3343. doi:10.3892/mmr.2017.8251.
[14]	GOMEZ C R. Role of heat shock proteins in aging and chronic inflammatory diseases[J]. Geroscience, 2021, 43(5):2515-2532. doi:10.1007/s11357-021-00394-2.
[15]	LI W, LIU J, ZOU D, et al. Exploration of bladder cancer molecular mechanisms based on miRNA-mRNA regulatory network[J]. Oncol Rep, 2017, 37(3):1461-1468. doi:10.3892/or.2017.5433.
[16]	CHEN Y F, HU F, WANG X G, et al. MicroRNA-23a-5p is involved in the regulation of lipopolysaccharide-induced acute lung injury by targeting HSP20/ASK1[J]. Oxid Med Cell Longev, 2021, 2021:9942557. doi:10.1155/2021/9942557.

miR-149-3p在先天性心脏病胎鼠心脏组织中的表达及其对P19细胞分化的影响

The expression of miR-149-3p in heart tissue of newborn mice with congenital heart disease and its effect on differentiation of P19 cells

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