The effect of miR-942-5p on viral myocarditis cell injury by targeting TRAF6

doi:10.3877/cma.j.issn.2095-1221.2021.04.004

Abstract

Abstract:

Objective

To investigate the effect of miR-942-5p on viral myocarditis cell injury by targeting tumor necrosis receptor related factor 6 (TRAF6) .

Method

The cardiomyocytes of BALB/c mice were isolated and cultured in vitro, and Coxsackievirus B3 (CVB3) was used to infect cardiomyocytes to establish a viral myocarditis cell model, the cells were divided into Con group (no CVB3 virusinfected) and CVB3 group (CVB3 virusinfected) , CVB3+miR-NC group (CVB3 virusinfected and miR-NC transfected) , CVB3+miR-942-5p group (CVB3 virusinfected and miR-942-5ptransfected) , CVB3+si-NC group (CVB3 virusinfectedandsi-NC transfected) , CVB3+si-TRAF6 group (CVB3 virusinfected and si-TRAF6 transfected) , CVB3+miR-942-5p+ pcDNA group (CVB3 virusinfected, miR-942-5p and pcDNA transfected) and CVB3+miR-942-5p+ TRAF6 group (CVB3 virusinfected, miR-942-5p and TRAF6 transfected) . Then Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-942-5p and TRAF6 mRNA. Western blot detected the expression of TRAF6, B-cell lymphoma/leukemia-2 (Bcl-2) and B-cell lymphoma/leukemia-2 related protein X (Bax) protein. Thiazole blue colorimetry (MTT) detected cell viability. Apoptosis was assessed by flow cytometry. ELISA was used to detect tumor necrosis factor (TNF-α) , interleukin-1β (IL-1β) expression and dual luciferase report experiment verified the relationship between miR-942-5p and TRAF6. The independent sample t test was used for the comparison between the two groups.

Result

Compared with the Con group, the expression of miR-942-5p (0.97±0.06 vs 0.36±0.03) , cell viability[ (98.10±8.67) ﹪ vs (44.61±5.30) ﹪, (97.51±8.89) ﹪ vs (46.69±5.42) ﹪], and Bcl-2 (0.93±0.06 vs 0.38±0.03, 0.92±0.06 vs 0.41±0.03) protein expression in CVB3 group were significantly reduced. TRAF6 mRNA (0.96±0.08 to 2.14±0.21) and protein expression (0.86±0.07 vs 1.69±0.17) , apoptosis rate [ (8.92±1.13) ﹪ vs (26.27±2.24) ﹪, (7.20±0.94) ﹪ vs (24.58±2.12) ﹪], Bax (0.94±0.06 vs 1.67±0.15, 0.98±0.07 vs 1.77±0.16) protein expression, TNF-α (56.70±5.56 vs 97.62±8.89, 48.52±4.68 vs 81.24±7.67) , IL-1β (38.42±4.01 vs 86.91±8.09, 33.21±4.06 vs 77.39±7.21) were significantly increased. Overexpression of miR-942-5p or interference with TRAF6 can significantly increase cell viability [ (41.24±4.71) ﹪ vs (65.32±5.81) ﹪, (50.23±4.94) ﹪ vs (68.32±6.44) ﹪], Bcl-2 (0.41±0.04 vs 0.64±0.05, 0.39±0.04 vs 0.63±0.05) protein expression, and reduce cell apoptosis rate[ (25.67±1.91) ﹪ vs (14.22±1.46) ﹪, (23.86±1.82) ﹪ vs (16.34±1.27) ﹪], Bax (1.70±0.16 vs 1.29±0.12, 1.73±0.15 vs 1.26±0.15) protein expression, TNF-α (103.68±9.82 vs 78.42±6.68, 86.08±8.04 vs 63.42±5.89) , IL-1β (81.27±7.81 vs 57.43±4.78, 74.48±6.88 vs 55.64±5.18) . miR-942-5p can target the expression of TRAF6, and overexpression of TRAF6 can reverse the miR-942-5p overexpression effecton the viability, apoptosis and inflammatory response of viral myocarditis cells induced by CVB3.

Conclusion

miR-942-5p can target apoptosis and inflammation in CVB3 infected cardiomyocytes by targeting TRAF6 expression.

Key words: miR-942-5p, TRAF6, Viral myocarditis, Apoptosis, Inflammatory response

Ting Yang. The effect of miR-942-5p on viral myocarditis cell injury by targeting TRAF6[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(04): 215-221.

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References 20

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引物名称	序列（5'→3'）	预期扩增产物长度（bp）
miR-942-5p	上游AGGGTCTTCTCTGTTTTGGC	129
	下游GTTGTGGTTGGTTGGTTTGT
	RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACC ACAT
U6	上游CTCGCTTCGGCAGCACA	105
	下游AACGCTTCACGAATTTGCGT
TRAF6	上游GCCCATGCCGTATGAAGAGA	212
	下游ACTGAATGTGCAGGGGACTG
β-actin	上游CACCCGCGAGTACAACCTTC	154
	下游CCCATACCCACCCATCACACC

引物名称	序列（5'→3'）	预期扩增产物长度（bp）
miR-942-5p	上游AGGGTCTTCTCTGTTTTGGC	129
	下游GTTGTGGTTGGTTGGTTTGT
	RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACC ACAT
U6	上游CTCGCTTCGGCAGCACA	105
	下游AACGCTTCACGAATTTGCGT
TRAF6	上游GCCCATGCCGTATGAAGAGA	212
	下游ACTGAATGTGCAGGGGACTG
β-actin	上游CACCCGCGAGTACAACCTTC	154
	下游CCCATACCCACCCATCACACC

分组	实验次数	miR-942-5p	TRAF6 mRNA	TRAF6蛋白
对照	3	0.97±0.06	0.96±0.08	0.86±0.07
CVB3	3	0.36±0.03	2.14±0.21	1.69±0.17
t值		15.750	9.095	7.820
P值		<0.001	0.001	0.001

分组	实验次数	miR-942-5p	TRAF6 mRNA	TRAF6蛋白
对照	3	0.97±0.06	0.96±0.08	0.86±0.07
CVB3	3	0.36±0.03	2.14±0.21	1.69±0.17
t值		15.750	9.095	7.820
P值		<0.001	0.001	0.001

分组	实验次数	miR-942-5p	细胞活力（﹪）	细胞凋亡率（﹪）	Bcl-2	Bax	TNF-α	IL-1β
对照	3	0.99±0.07	98.10±8.67	8.92±1.13	0.93±0.06	0.94±0.06	56.70±5.56	38.42±4.01
CVB3	3	0.40±0.04	44.61±5.30	26.27±2.24	0.38±0.03	1.67±0.15	97.62±8.89	86.91±8.09
t值		12.675	9.117	11.978	14.201	7.826	6.759	9.302
P值		< 0.001	0.001	< 0.001	< 0.001	< 0.001	0.002	0.001
CVB3+miR-NC	3	0.38±0.03	41.24±4.71	25.67±1.91	0.41±0.04	1.70±0.16	103.68±9.82	81.27±7.81
CVB3+miR-942-5p	3	0.62±0.05	65.32±5.81	14.22±1.46	0.64±0.05	1.29±0.12	78.44±6.68	57.43±4.78
t值		7.129	5.576	8.249	6.222	3.551	3.681	4.510
P值		0.002	0.005	0.001	0.003	0.024	0.021	0.011

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