circRASSF2靶向miR-1343-3p对乳腺癌MDA-MB-231细胞增殖和凋亡的影响

doi:10.11958/20221254

摘要/Abstract

摘要：

目的探讨circRASSF2对乳腺癌MDA-MB-231细胞增殖和凋亡的影响及分子机制。方法逆转录荧光定量聚合酶链反应（RT-qPCR）检测37例乳腺癌及癌旁组织中circRASSF2和miR-1343-3p表达水平；将乳腺癌MDA-MB-231细胞分为si-circRASSF2组、si-NC组、miR-NC组、miR-1343-3p组、si-circRASSF2+anti-miR-1343-3p组、si-circRASSF2+anti-miR-NC组；四甲基偶氮唑盐比色法（MTT）检测细胞活性；克隆形成实验检测细胞克隆形成数；Annexin V-FITC/PI双染法检测细胞凋亡情况；Western blot检测Cleaved-caspase 3、Cleaved-caspase 9蛋白相对表达水平；双荧光素酶报告实验检测circRASSF2和miR-1343-3p的靶向关系。结果与癌旁组织比较，乳腺癌组织中circRASSF2表达水平升高，miR-1343-3p表达水平降低（P<0.01）。与si-NC组比较，si-circRASSF2组MDA-MB-231细胞OD值降低，细胞克隆形成数减少，而凋亡率升高（P<0.05）。与miR-NC组比较，miR-1343-3p组MDA-MB-231细胞OD值降低，细胞克隆形成数减少，而凋亡率升高（P<0.05）。与miR-NC组比较，miR-1343-3p组WT-circRASSF2荧光素酶活性降低（P<0.05）。与si-circRASSF2+anti-miR-NC组比较，si-circRASSF2+anti-miR-1343-3p组MDA-MB-231细胞OD值、克隆形成数升高，凋亡率降低（P<0.05）。结论抑制circRASSF2表达可上调miR-1343-3p，进而抑制乳腺癌MDA-MB-231细胞增殖，促进其凋亡。

关键词: 乳腺肿瘤, 细胞增殖, 细胞凋亡, 基因表达调控, circRASSF2, miR-1343-3p

Abstract:

Objective To explore the effect of circRASSF2 on the proliferation and apoptosis of breast cancer MDA-MB-231 cells and its molecular mechanism. Methods Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect circRASSF2 and miR-1343-3p expression levels in 37 breast cancer tissue samples and adjacent tissue samples. Breast cancer MDA-MB-231 cells were divided into the si-circRASSF2 group, the si-NC group, the miR-1343-3p group, the miR-NC group, the si-circRASSF2+anti-miR-1343-3p group and the si-circRASSF2+anti-miR-NC group. Tetramethylazolium salt colorimetry method (MTT) was used to detect cell viability. Clone formation experiment was used to detect the number of cell clones. Annexin V-FITC/PI double staining method was used to detect cell apoptosis. The relative expression levels of Cleaved-caspase 3 and Cleaved-caspase 9 proteins were detected by Western blot assay. Dual luciferase reporter experiment was used to detect targeting relationship between circRASSF2 and miR-1343-3p. Results Compared with paracancer tissue samples, the expression level of circRASSF2 was increased and the expression level of miR-1343-3p was decreased in breast cancer tissue samples (P<0.01). Compared with the si-NC group, the OD value of MDA-MB-231 cells was decreased in the si-circRASSF2 group, the number of cell clone formation was decreased, and the apoptosis rate was increased (P<0.05). Compared with the miR-NC group, the OD value of MDA-MB-231 cells was decreased in the miR-1343-3p group, the number of cell clone formation was decreased, and the apoptosis rate was increased (P<0.05). Compared with the miR-NC group, WT-circRASSF2 luciferase activity decreased in the miR-1343-3p group (P<0.05). Compared with the si-circRASSF2+anti-miR-NC group, the OD value and the number of cell clone formation were increased, and the apoptosis rate was decreased in the si-circRASSF2+anti-miR-1343-3p group (P<0.05). Conclusion The inhibition of circRASSF2 expression can up-regulate miR-1343-3p, thus inhibiting the proliferation of breast cancer MDA-MB-231 cells and promoting apoptosis.

Key words: breast neoplasms, cell proliferation, apoptosis, gene expression regulation, circRASSF2, miR-1343-3p

中图分类号:

R737.9

图/表 14

表1 乳腺癌组织和癌旁组织中circRASSF2和miR-1343-3p的表达水平比较 (（n=37，）$\bar{x}±s$)

Tab.1 Comparison of circRASSF2 and miR-1343-3p expression levels between breast cancer tissue and paracancer tissue

组织	circRASSF2	miR-1343-3p
癌旁组织	1.00±0.02	1.00±0.05
乳腺癌组织	4.03±0.21	0.37±0.03
t	89.518^＊＊	60.709^＊＊

图1 si-NC组和si-circRASSF2组细胞克隆形成能力

Fig.1 Cell clonogenesis ability in the si-NC group and the si-circRASSF2 group

表2 si-NC组和si-circRASSF2组细胞增殖水平比较 (n=3, $\bar{x}±s$)

Tab.2 Comparison of cell proliferation between the si-NC group and the si-circRASSF2 group

组别	circRASSF2	OD₄₉₀	克隆形成数/个
si-NC组	1.00±0.06	0.71±0.06	101.25±8.96
si-circRASSF2组	0.34±0.02	0.34±0.03	48.79±2.34
t	19.219^＊＊	9.992^＊＊	9.815^＊＊

图2 si-NC组和si-circRASSF2组细胞凋亡情况

Fig.2 Apoptosis of the si-NC group and the si-circRASSF2 group

图3 si-NC组和si-circRASSF2组细胞凋亡相关蛋白表达

Fig.3 Expression levels of apoptosis related proteins in the si-NC group and the si-circRASSF2 group

表3 si-NC组和si-circRASSF2组细胞凋亡率及凋亡相关蛋白表达比较 (n=3, $\bar{x}±s$)

Tab.3 Comparison of apoptosis rate and related protein expression levels between the si-NC group and the si-circRASSF2 group

组别	细胞凋亡率/%	Cleaved-caspase 3	Cleaved-caspase 9
si-NC组	8.75±0.13	0.27±0.04	0.20±0.02
si-circRASSF2组	23.89±3.21	0.75±0.09	0.54±0.06
t	8.156^＊＊	8.486^＊＊	10.304^＊＊

图4 circRASSF2与miR-1343-3p的结合序列

Fig.4 Binding sequence of circRASSF2 and miR-1343-3p

图5 circRASSF2靶向调控miR-1343-3p的表达＊P<0.05；A：各组细胞相对荧光素酶活性比较；B：过表达或沉默circRASSF2后miR-1343-3p表达水平比较。

Fig.5 circRASSF2 targeting regulates the expression of miR-1343-3p

图6 miR-NC组和miR-1343-3p组细胞克隆形成和凋亡

Fig.6 Clonal formation and apoptosis in the miR-NC group and the miR-1343-3p group

图7 miR-NC组和miR-1343-3p组细胞凋亡相关蛋白表达

Fig.7 Expression of apoptosis related proteins in the miR-NC group and the miR-1343-3p group

表4 miR-NC组和miR-1343-3p组细胞增殖和凋亡比较（n=3，$\bar{x}±s$）

Tab.4 Comparison of proliferation and apoptosis between the miR-NC group and the miR-1343-3p group

组别	miR-1343-3p	OD₄₉₀	克隆形成数/个	细胞凋亡率/%	Cleaved-caspase 3	Cleaved-caspase 9
miR-NC组	1.01±0.02	0.70±0.05	99.87±8.43	7.48±0.51	0.25±0.03	0.19±0.02
miR-1343-3p组	3.21±0.13	0.42±0.03	50.23±2.45	19.96±2.03	0.73±0.08	0.48±0.05
t	28.357^＊＊	9.058^＊＊	9.794^＊＊	10.317^＊＊	9.821^＊＊	10.793^＊＊

表5 si-circRASSF2+anti-miR-NC组和si-circRASSF2+anti-miR-1343-3p组细胞增殖和凋亡 (n=3, $\bar{x}±s$)

Tab.5 Proliferation and apoptosis in the si-circRASSF2+anti-miR-NC group and the si-circRASSF2+anti-miR-1343-3p group

组别	miR-1343-3p	OD₄₉₀	克隆形成数/个	细胞凋亡率/%	Cleaved-caspase 3	Cleaved-caspase 9
si-circRASSF2+anti-miR-NC组	1.00±0.03	0.39±0.02	40.78±3.51	27.45±3.11	0.75±0.09	0.52±0.06
si-circRASSF2+anti-miR-1343-3p组	0.28±0.02	0.65±0.04	82.63±8.74	12.13±0.98	0.32±0.04	0.24±0.02
t	35.040^＊＊	10.373^＊＊	7.696^＊＊	8.140^＊＊	7.460^＊＊	7.545^＊＊

图8 si-circRASSF2+anti-miR-NC组和si-circRASSF2+anti-miR-1343-3p组细胞克隆形成和凋亡

Fig.8 Clonal formation and apoptosis in the si-circRASSF2+anti-miR-NC group and the si-circRASSF2+anti-miR-1343-3p group

图9 si-circRASSF2+anti-miR-NC组和si-circRASSF2+anti-miR-1343-3p组细胞凋亡相关蛋白表达 A：si-circRASSF2+anti-miR-NC组；B：si-circRASSF2+anti-miR-1343-3p组。

Fig.9 Expression of apoptosis related proteins in the si-circRASSF2+anti-miR-NC group and the si-circRASSF2+anti-miR-1343-3p group

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