MiR-579-3p靶向肌动蛋白相关蛋白3B调控口腔鳞状细胞癌细胞增殖和凋亡的机制研究

doi:10.3969/j.issn.1005-4979.2020.03.006

摘要/Abstract

摘要：

目的: 探讨miR-579-3p对口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）细胞增殖和凋亡的影响及作用机制。方法: 培养正常人口腔角质形成细胞（normal human oral keratinocyte，NHOK）和OSCC细胞系CAL27、CAL33、SCC15，实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测细胞中miR-579-3p和肌动蛋白相关蛋白3B（actin-related protein 3B，ACTR3B）信使RNA（mRNA）的表达水平，蛋白质印迹法（western blot）检测ACTR3B蛋白表达水平。以CAL33细胞为研究对象，构建过表达miR-579-3p或沉默ACTR3B表达的CAL33细胞，四甲基噻唑蓝染色法（methylthiazoletrazolium，MTT）检测细胞增殖，流式细胞仪检测细胞凋亡，western blot检测细胞周期蛋白D1（CyclinD1）和活化的半胱天冬酶-3（C-caspase-3）蛋白水平。Starbase生物信息学软件预测ACTR3B可能是miR-579-3p的靶基因，双荧光素酶报告基因实验验证miR-579-3p和ACTR3B调控关系。结果: 与NHOK细胞比较，OSCC细胞系CAL27、CAL33和SCC15中miR-579-3p表达水平降低（P<0.05），ACTR3B mRNA和蛋白表达水平升高（P<0.05）。过表达miR-579-3p或沉默ACTR3B表达均可降低CAL33细胞存活率和CyclinD1蛋白表达水平（P<0.05），提高CAL33细胞凋亡率和C-caspase-3蛋白表达水平（P<0.05）。miR-579-3p靶向负调控ACTR3B表达。过表达ACTR3B降低了过表达miR-579-3p对CAL33细胞存活率、凋亡率及CyclinD1和C-caspase-3蛋白表达的影响。结论: 过表达miR-579-3p可能通过下调ACTR3B表达抑制OSCC细胞增殖，并诱导细胞凋亡。

关键词: miR-579-3p, 肌动蛋白相关蛋白3B, 口腔鳞状细胞癌, 细胞增殖, 凋亡

Abstract:

Objective: To investigate the effect and mechanism of miR-579-3p on proliferation and apoptosis of oral squamous cell carcinoma (OSCC) cells. Methods: The normal human oral keratinocytes (NHOK) and oral squamous carcinoma cell lines CAL27, CAL33 and SCC15 were cultured. The expression levels of miR-579-3p and actin-related protein 3B (ACTR3B) mRNA were detected by quantitative real-time PCR(qRT-PCR). The expression level of ACTR3B protein was detected by western blot. Taking CAL33 cells as the research object, CAL33 cells with miR-579-3p overexpressed or ACTR3B expression silenced were constructed. Then cell proliferation was detected by methylthiazoletrazolium(MTT) assay, apoptosis was detected by flow cytometry, and the expression levels of CyclinD1 and C-caspase-3 protein were detected by western blot. Starbase Bioinformatics software predicts that ACTR3B may be the target gene of miR-579-3p, and the dual luciferase reporter gene assay validates the regulatory relationship between miR-579-3p and ACTR3B. Results: Compared with NHOK cells, the expression levels of miR-579-3p in oral squamous cell carcinoma cell lines CAL27, CAL33 and SCC15 were decreased (P<0.05), and the expression levels of ACTR3B mRNA and protein were increased (P<0.05). Overexpression of miR-579-3p or silencing ACTR3B could decrease the survival rate of CAL33 cells and the expression level of CyclinD1 protein (P<0.05), and increase the apoptosis rate of CAL33 cells and the expression level of C-caspase-3 protein (P<0.05). MiR-579-3p targets negative regulation of ACTR3B expression. Overexpression of ACTR3B reduced the effect of overexpression of miR-579-3p on CAL33 cell viability, apoptotic rate, and the expression level of CyclinD1 and C-caspase-3 protein. Conclusion: Overexpression of miR-579-3p may inhibit the proliferation of oral squamous carcinoma cells and induce apoptosis by down-regulating the expression of ACTR3B.

Key words: miR-579-3p, actin-related protein 3B (ACTR3B), oral squamous cell carcinoma, cell proliferation, apoptosis

中图分类号:

R782

高功杰, 王海业, 李晓彦. MiR-579-3p靶向肌动蛋白相关蛋白3B调控口腔鳞状细胞癌细胞增殖和凋亡的机制研究[J]. 《口腔颌面外科杂志》, 2020, 30(3): 156-162.

GAO Gongjie, WANG Haiye, LI Xiaoyan. Mechanism of MiR-579-3p Regulating Proliferation and Apoptosis of Oral Squamous Cell Carcinoma Cells by Targeting ACTR3B[J]. 《Journal of Oral and Maxillofacial Surgery》, 2020, 30(3): 156-162.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2020/V30/I3/156

图/表 11

图1 Western blot检测ACTR3B蛋白表达

Figure 1 Western blot detected the expression of ACTR3B protein

表1 OSCC细胞系中 miR-579-3p和ACTR3B表达水平（$\bar{x}\pm s$，n=9）

Table 1 Expression levels of miR-579-3p and ACTR3B in oral squamous carcinoma cell lines ($\bar{x}\pm s$, n=9)

组别	miR-579-3p	ACTR3B mRNA	ACTR3B蛋白
NHOK	1.00±0.10	1.00±0.09	0.43±0.04
CAL27	0.25±0.03^*	2.01±0.20^*	0.98±0.10^*
CAL33	0.30±0.04^*	3.24±0.32^*	0.88±0.09^*
SCC15	0.42±0.05^*	2.86.±0.29^*	0.76±0.08^*
F	286.940	151.881	78.931
P	0.000	0.000	0.000

图2 过表达miR-579-3p对CAL33细胞增殖和凋亡的影响注：A. 流式细胞仪检测细胞凋亡；B. Western blot检测CyclinD1、C-caspase-3蛋白的表达。

Figure 2 Effect of overexpression of miR-579-3p on the proliferation and apoptosis of CAL33 cells

表2 过表达miR-579-3p对CAL33细胞增殖和凋亡的影响（$\bar{x}\pm s$，n=9）

Table 2 Effects of overexpression of miR-579-3p on the proliferation and apoptosis of CAL33 cells ($\bar{x}\pm s$, n=9)

组别	miR-579-3p	CyclinD1	C-caspase-3	细胞存活率/%	凋亡率/%
miR-NC组	1.00±0.11	1.10±0.12	0.36±0.04	100.79±10.08	7.35±0.73
miR-579-3p组	2.79±0.28^*	0.42±0.05^*	0.89±0.09^*	53.16±5.30^*	20.08±2.01^*
t	17.850	15.692	16.144	12.547	17.859
P	0.000	0.000	0.000	0.000	0.000

表3 沉默ACTR3B对CAL33细胞增殖和凋亡的影响（$\bar{x}\pm s$，n=9）

Table 3 Effect of silencing ACTR3B on the proliferation and apoptosis of CAL33 cells ($\bar{x}\pm s$, n=9)

组别	ACTR3B	CyclinD1	C-caspase-3	细胞存活率/%	凋亡率/%
si-NC组	0.86±0.09	1.12±0.13	0.34±0.03	100.77±10.07	7.24±0.72
si-ACTR3B组	0.40±0.04^*	0.35±0.05^*	0.78±0.08^*	61.08±6.10^*	19.48±1.95^*
t	14.012	16.585	15.449	10.113	17.665
P	0.000	0.000	0.000	0.000	0.000

图3 Western blot检测ACTR3B、CyclinD1、C-caspase-3蛋白的表达

Figure 3 Western blot detected the expression of ACTR3B, CyclinD1, C-caspase-3 protein

图4 miR-579-3p靶向调控ACTR3B表达注：A. ACTR3B的3′UTR与miR-579-3p互补的核苷酸序列；B. Western blot检测ACTR3B蛋白的表达。

Figure 4 miR-579-3p targets ACTR3B expression

表4 双荧光素酶活性检测结果（$\bar{x}\pm s$，n=9）

Table 4 Test results of double luciferase activity ($\bar{x}\pm s$, n=9)

组别	ACTR3B	CyclinD1	C-caspase-3	细胞存活率/%	凋亡率/%
miR-579-3p+pcDNA-NC组	0.38±0.04	0.45±0.05	0.88±0.09	100.37±10.05	21.04±2.10
miR-579-3p+pcDNA-ACTR3B组	0.76±0.08^*	1.01±0.11^*	0.42±0.04^*	134.27±13.42^*	10.86±1.09^*
t	12.746	13.904	14.012	6.066	12.908
P	0.000	0.000	0.000	0.000	0.000

表5 miR-579-3p对ACTR3B蛋白表达的影响（$\bar{x}\pm s$，n=9）

Table 5 Effect of miR-579-3p on the expression of ACTR3B protein ($\bar{x}\pm s$, n=9)

组别	荧光素酶活性
组别	WT-ACTR3B	MUT-ACTR3B
miR-NC组	1.00±0.10	1.04±0.11
miR-579-3p 组	0.36±0.04^*	1.08±0.12
t	17.827	0.737
P	0.000	0.472

表6 过表达ACTR3B降低了miR-579-3p过表达对CAL33细胞增殖和凋亡的影响（$\bar{x}\pm s$，n=9）

Table 6 Overexpression of ACTR3B reduced the effect of miR-579-3p overexpression on the proliferation and apoptosis of CAL33 cells ($\bar{x}\pm s$, n=9)

组别	ACTR3B
miR-NC组	0.85±0.09
miR-579-3p组	0.36±0.04^*
anti-miR-NC组	0.89±0.10
anti-miR-579-3p组	1.22±0.12^#
F	132.669
P	0.000

图5 Western blot检测ACTR3B、CyclinD1、C-caspase-3蛋白表达

Figure 5 Western blot detected the expression of ACTR3B, CyclinD1, C-caspase-3 protein

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