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

doi:10.11958/20220707

Abstract

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

CLC Number:

R541.1

Figures/Tables 9

References 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.

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

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

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^＊＊

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^＊＊

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^＊＊