Circ_0000502靶向调节miR-1231促进口腔鳞状细胞癌HSC-3细胞增殖和抑制细胞凋亡的体外研究

doi:10.3969/j.issn.1005-4979.2022.02.004

摘要/Abstract

摘要：

目的: 探讨环状RNA 0000502（circ_0000502）是否靶向微小RNA-1231（microRNA-1231，miR-1231）调控口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）细胞HSC-3的增殖和凋亡。方法: 实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）分析OSCC组织、癌旁组织中circ_0000502和miR-1231的表达水平。四甲基偶氮唑盐微量酶反应比色法（MTT法）、流式细胞术分析circ_0000502和miR-1231表达对HSC-3细胞增殖和凋亡的影响。双荧光素酶报告实验和RT-qPCR用于确定circ_0000502和miR-1231调控关系。结果: OSCC组织中，circ_0000502呈高表达，miR-1231呈低表达（P<0.05）。抑制circ_0000502或过表达miR-1231能显著降低HSC-3的细胞活力，提高细胞凋亡率（P<0.05）。与单独抑制circ_0000502比较，同时抑制circ_0000502和miR-1231后，HSC-3细胞活力显著升高，凋亡率显著降低（P<0.05）。结论: circ_0000502靶向负调控miR-1231能促进OSCC细胞HSC-3增殖，抑制细胞凋亡。Circ_0000502/miR-1231分子轴是OSCC的潜在治疗靶点。

关键词: 环状RNA 0000502, 微小RNA-1231, 口腔鳞状细胞癌, 细胞增殖, 凋亡

Abstract:

Objective: In this article, we explore whether circular RNA 0000502(circ_0000502) targets miR-1231 and regulates the proliferation and apoptosis of oral squamous cell carcinoma(OSCC) cell line HSC-3. Methods: The expression levels of circ_0000502 and miR-1231 in OSCC tissues and adjacent tissues were analyzed by real-time quantitative polymerase chain reaction(RT-qPCR). MTT method and flow cytometry were used to analyze the effects of circ_0000502 and miR-1231 expression on the proliferation and apoptosis of HSC-3. The dual luciferase reporter assay and RT-qPCR were performed to determine the regulatory relationship between circ_0000502 and miR-1231. Results: Circ_0000502 was intensely expressed and miR-1231 was weakly expressed in OSCC tissues(P<0.05). Inhibition of circ_0000502 or overexpression of miR-1231 significantly reduced HSC-3 cell viability and increased cell apoptosis rate(P<0.05). Compared with the inhibition of circ_0000502, the cell viability of HSC-3 cells was significantly increased, and the apoptosis rate was significantly reduced after both circ_0000502 and miR-1231 were inhibited(P<0.05). Conclusion: The above findings suggest that circ_0000502 promotes the proliferation and inhibits cell apoptosis of OSCC HSC-3 cells by negatively regulating miR-1231. The circ_0000502/miR-1231 molecular axis is a potential therapeutic target for OSCC.

Key words: circ_0000502, miR-1231, oral squamous cell carcinoma, cell proliferation, apoptosis

马素伟, 张娟, 杨晋, 靳昊, 王焱, 高杰. Circ_0000502靶向调节miR-1231促进口腔鳞状细胞癌HSC-3细胞增殖和抑制细胞凋亡的体外研究[J]. 《口腔颌面外科杂志》, 2022, 32(2): 93-99.

MA Suwei, ZHANG Juan, YANG Jin, JIN Hao, WANG Yan, GAO Jie. Circ_0000502 promotes the proliferation and inhibites apoptosis of oral squamous cell carcinoma HSC-3 cells by targeting miR-1231 in vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2022, 32(2): 93-99.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2022/V32/I2/93

图/表 13

表1 Circ_0000502和miR-1231在OSCC组织中的相对表达量

Table 1 The relative expression levels of circ_0000502 and miR-1231 in OSCC tissues

组别	circ_0000502	miR-1231
癌旁组织（n=43）	1.00±0.06	1.00±0.05
OSCC组织（n=43）	3.13±0.22^①	0.40±0.03^①
t值	61.251	67.475
P值	<0.05	<0.05

图1 癌旁组织和OSCC组织病理图片（×100） A. OSCC组织；B. 癌旁组织。

Figure 1 Histopathological features of OSCC and OSCC adjacent tissues (×100)

表2 抑制circ_0000502表达对HSC-3细胞增殖的影响

Table 2 Effect of inhibiting circ_0000502 on the proliferation of HSC-3 cells

组别	circ_0000502	吸光度值	Ki-67蛋白
si-NC组（n=9）	1.00±0.05	0.67±0.05	0.52±0.03
si-circ_0000502组（n=9）	0.25±0.03^①	0.33±0.02^①	0.17±0.02^①
t值	38.587	18.941	29.122
P值	<0.05	<0.05	<0.05

图2 Ki-67蛋白表达

Figure 2 Expression of Ki-67 protein

表3 抑制circ_0000502表达对HSC-3细胞凋亡的影响

Table 3 Effect of inhibiting circ_0000502 on the apoptosis of HSC-3 cells

组别	凋亡率/%	Bcl-2蛋白	Bax蛋白
si-NC组（n=9）	4.83±0.36	0.74±0.05	0.22±0.02
si-circ_0000502组（n=9）	22.29±2.26^①	0.29±0.03^①	0.68±0.04^①
t值	22.888	23.152	30.858
P值	<0.05	<0.05	<0.05

图3 抑制circ_0000502表达对HSC-3细胞凋亡的影响 A. 凋亡相关蛋白的表达；B. 细胞凋亡流式图。

Figure 3 Effect of circ_0000502 inhibition on the apoptosis of HSC-3 cells

图4 Circ_0000502的序列中含有与miR-1231互补的核苷酸序列红色字母表示突变序列。

Figure 4 The sequence of circ_0000502 contains a nucleotide sequence complementary to miR-1231

表4 双荧光素酶报告实验

Table 4 Double luciferase reporter assay

组别	WT-circ_0000502	MUT-circ_0000502
miR-NC组（n=9）	1.00±0.06	0.96±0.04
miR-1231组（n=9）	0.49±0.04^①	0.98±0.05
t值	21.217	1.200
P值	<0.05	0.248

表5 Circ_0000502调控miR-1231的表达

Table 5 Circ_0000502 regulates the expression of miR-1231

组别	miR-1231
pcDNA组（n=9）	1.00±0.07
pcDNA-circ_0000502组（n=9）	0.59±0.05^①
si-NC组（n=9）	1.02±0.08
si-circ_0000502组（n=9）	2.96±0.25^②
F值	533.768
P值	<0.05

表6 miR-1231过表达对HSC-3细胞增殖和凋亡的影响

Table 6 Effects of overexpressing miR-1231 on proliferation and apoptosis of HSC-3 cells

组别	miR-1231	吸光度值	凋亡率/%	Ki-67蛋白	Bcl-2蛋白	Bax蛋白
miR-NC组（n=9）	1.00±0.06	0.69±0.04	4.77±0.33	0.54±0.04	0.77±0.05	0.21±0.02
miR-1231组（n=9）	2.65±0.24^①	0.43±0.03^①	18.31±1.37^①	0.22±0.02^①	0.35±0.03^①	0.64±0.04^①
t值	20.009	15.600	28.825	21.466	21.609	28.845
P值	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05

图5 miR-1231过表达对HSC-3细胞增殖和凋亡的影响 A. 增殖和凋亡相关蛋白的表达；B. 细胞凋亡流式图。

Figure 5 Effects of overexpressing miR-1231 on proliferation and apoptosis of HSC-3 cells

表7 抑制miR-1231表达逆转了抑制circ_0000502表达对HSC-3细胞增殖和凋亡的作用

Table 7 Inhibiting the expression of miR-1231 reversed the effect of inhibiting circ_0000502 on the proliferation and apoptosis of HSC-3 cells

组别	miR-1231	吸光度值	凋亡率/%	Ki-67蛋白	Bcl-2蛋白	Bax蛋白
si-circ_0000502+anti-miR-NC组（n=9）	1.00±0.07	0.32±0.02	23.86±2.09	0.16±0.02	0.27±0.02	0.69±0.06
si-circ_0000502+anti-miR-1231组（n=9）	0.28±0.03^①	0.57±0.04^①	10.53±0.62^①	0.42±0.03^①	0.62±0.05^①	0.31±0.02^①
t值	28.362	16.771	18.344	21.633	19.498	18.025
P值	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05

图6 抑制miR-1231表达逆转了抑制circ_0000502表达对HSC-3细胞增殖和凋亡的作用 A. 增殖和凋亡相关蛋白的表达；B. 细胞凋亡流式图。

Figure 6 Inhibiting the expression of miR-1231 reversed the effect of inhibiting circ_0000502 on the proliferation and apoptosis of HSC-3 cells

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