柯萨奇病毒A16型3C蛋白对人横纹肌肉瘤细胞凋亡的诱导作用及其机制

doi:10.13481/j.1671-587X.20210408

摘要/Abstract

摘要： 目的

探讨柯萨奇病毒A16型（CA16）3C蛋白对横纹肌肉瘤RD细胞凋亡的影响，并阐明其可能的作用机制。

方法

体外培养RD细胞，分为对照组（转染0.4 μg空载体质粒pCMV-HA）、0.1 μg pCMV-HA-3C组（转染0.1 μg pCMV-HA-3C和0.3 μg pCMV-HA）、0.2 μg pCMV-HA-3C组（转染0.2 μg pCMV-HA-3C和0.2 μg pCMV-HA）、0.4 μg pCMV-HA-3C组（转染0.4 μg pCMV-HA-3C）和蛋白激酶B （AKT）激活剂SC79处理组（先用4 mg·L^－1 SC79处理细胞4 h，然后转染0.2 μg pCMV-HA-3C和0.2 μg pCMV-HA）。MTT法检测各组细胞存活率，流式细胞术检测各组细胞凋亡率，实时荧光定量 PCR（RT-qPCR）法检测各组RD细胞中Bcl-2同源拮抗剂/杀伤剂（Bak）、Bcl-2相关的细胞死亡激动剂（Bad）和p53上调凋亡调节因子（PUMA）mRNA表达水平，Western blotting 法检测各组 RD细胞中Bad、Bak、PUMA、聚ADP核糖聚合酶（PARP）、裂解聚ADP核糖聚合酶（cleaved-PARP）、裂解含半胱氨酸的天冬氨酸蛋白水解酶3（cleaved-caspase-3）、AKT和磷酸化AKT（p-AKT）蛋白表达水平。

结果

与对照组比较，0.4 μg pCMV-HA-3C组细胞存活率明显降低（P<0.01）。与对照组比较，不同剂量pCMV-HA-3C组细胞凋亡率明显升高（P<0.01）。与对照组比较，0.2 μg pCMV-HA-3C组细胞中Bak、Bad和PUMA mRNA和蛋白表达水平均明显升高（P<0.05或P<0.01）。与对照组比较，不同剂量 pCMV-HA-3C组细胞中cleaved-PARP和cleaved-caspase-3蛋白表达水平明显升高（P<0.05或P<0.01）。SC79处理实验中，与对照组比较，0.2 μg pCMV-HA-3组细胞中cleaved-PARP、cleaved-caspase-3和AKT蛋白表达水平明显升高（P<0.05），p-AKT蛋白表达水平明显降低（P<0.01）；与0.2 μg pCMV-HA-3C组比较，SC79处理组细胞中cleaved-PARP、cleaved-caspase-3和AKT蛋白表达水平明显降低（P<0.05或P<0.01），p-AKT蛋白表达水平明显升高（P<0.01）。

结论

CA16型3C蛋白可能通过抑制AKT信号通路诱导RD细胞凋亡。

关键词: 柯萨奇病毒A16型, 3C蛋白, 蛋白激酶B信号通路, 细胞凋亡

Abstract: Objective

To investigate the effect of Coxsackievirus A16 3C protein on the apoptosis of rhabdomyosarcoma RD cells， and to illuminate its possible mechanism.

Methods

The RD cells were cultured in vitro and divided into control group （transfected with 0.4 μg pCMV-HA）， 0.1 μg pCMV-HA-3C group （transfected with 0.1 μg pCMV-HA-3C and 0.3 μg pCMV-HA）， 0.2 μg pCMV-HA-3C group （transfected with 0.2 μg pCMV-HA-3C and 0.2 μg pCMV-HA），0.4 μg pCMV-HA-3C group （transfected with 0.4 μg pCMV-HA-3C） and protein kinase B（AKT） activator SC79 treatment group （pretreated with 4 mg·L^－1　SC79 for 4 h， then transfected with 0.2 μg pCMV-HA-3C and 0.2 μg pCMV-HA）.The survival rates of cells in various groups were detected by MTT assay，the apoptotic rates were detected by flow cytometry， the expression levels of Bcl-2 homologous antagonist /killer （Bak），Bcl-2 associated agonist of cell death （Bad） and p53 up-regulated modulator of apoptosis （PUMA） mRNA were detected by Real-time fluorescence quantitative PCR （RT-qPCR），and the expression levels of Bad， Bak， PUMA， PARP， cleaved-PARP， cleaved-caspase-3 and phosphorylated AKT （p-AKT） proteins in the RD cells in various groups were detected by Western blotting method.

Results

Compared with control group， the survival rate of the cells 0.4 μg pCMV-HA-3C group was significantly decreased （P<0.01）. Compared with control group， the apoptotic rate of the cells in pCMV-HA-3C group was significantly increased （P<0.01）. Compared with control group，the mRNA and protein expression levels of Bak， Bad and PUMA in 0.2 μg PCMV-HA-3C group were significantly increased （P<0.05）， and the expression levels of cleaved-PARP and cleaved-caspase-3 proteins in different doses of pCMV-HA-3C groups were significantly increased （P<0.05）.Compared with control group， the expression levels of cleaved-PARP and cleaved-caspase-3 proteins in 0.2 μg pCMV-HA-3C group were significantly increased （P<0.05），and the expression level of p-AKT protein was significantly decreased （P<0.01）. Compared with 0.2 μg pCMV-HA-3C group，the the expression levels of cleaved-PARP，cleaved-caspase-3 and AKT proteins in the cells in SC79 treatment group were significantly decreased（P<0.05），and the p-AKT protein expression level was significantly increased （P<0.01）.

Conclusion

Coxsackievirus A16 3C protein may induce the apoptosis of RD cells by inhibiting AKT signaling pathway.

Key words: Coxsackievirus A16, 3C protein, protein kinase B signaling pathway, apoptosis

中图分类号:

R373.23

史颖颖,陈仕同,胡波,沈铎,朱旷,叶思玮,冯金梅. 柯萨奇病毒A16型3C蛋白对人横纹肌肉瘤细胞凋亡的诱导作用及其机制[J]. 吉林大学学报(医学版), 2021, 47(4): 874-881.

Yingying SHI,Shitong CHEN,Bo HU,Duo SHEN,Kuang ZHU,Siwei YE,Jinmei FENG. Induction of 3C protein of Coxsackievirus A16 in apoptosis of human rhabdomyosarcoma cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2021, 47(4): 874-881.

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Gene	Primer sequence（5'－3'）
Bak	Forward AGGGCTTAGGACTTGGTTTG
Bak	Reverse GGGATTCCTAGTGGTGTTGATAG
Bad	Forward GGAGGATGAGTGACGAGTTTG
Bad	Reverse GGAGCTTTGCCGCATCT
PUMA	Forward GTGACCACTGGCATTCATTTG
PUMA	Reverse TCCTCCCTCTTCCGAGATTT
β-actin	Forward CACGATGGAGGGGCCGGACTCATC
β-actin	Reverse TAAAGACCTCTATGCCAACACAGT

Group	Survival rate
Control	90.00±5.98
pCMV-HA-3C 0.1 μg	85.83±5.71
0.2 μg	82.68±6.02
0.4 μg	65.09±3.78^*

Group	Bak		Bad		PUMA
Group	mRNA	Protein	mRNA	Protein	mRNA	Protein
Control	1.02±0.14	0.69±0.01	1.02±0.12	0.77±0.01	1.01±0.10	0.80±0.01
0.2 μg pCMV-HA-3C	1.54±0.11^*	0.78±0.01^**	1.72±0.05^**	0.83±0.01^*	1.73±0.19^*	0.85±0.01^*

Group	PARP	Cleaved- PARP	Cleaved- caspase-3
Control pCMV-HA-3C	0.88±0.01	0.71±0.01	0.55±0.01
0.1 μg	0.86±0.01	0.75±0.01^*	0.60±0.01^*
0.2 μg	0.82±0.07	0.77±0.01^*	0.68±0.01^*
0.4 μg	0.70±0.02^**	0.99±0.03^**	0.88±0.03^**

Group	Cleaved-PARP	Cleaved-caspase-3	AKT	p-AKT
Control	0.82±0.01	0.81±0.01	1.02±0.02	0.98±0.01
0.2 μg pCMV-HA-3C	1.07±0.01^*	1.16±0.02^*	1.10±0.01^*	0.82±0.01^**
SC79 treatment	0.84±0.01^ΔΔ	0.97±0.04^Δ	0.94±0.01^ΔΔ	1.10±0.01^ΔΔ