过表达环状RNA孤儿核受体4A1(circNR4A1/Hsa-circ-0026352)抑制MCF-7乳腺癌细胞增殖并促进其凋亡

Over-expression of circNR4A1/Hsa-circ-0026352 inhibits proliferation and induces apoptosis in MCF-7 cells

目的环状RNA Hsa-circ-0026352(circNR4A1)在乳腺癌患者组织、外周血、细胞系中的相对表达及其对细胞增殖、凋亡的影响。方法通过人circRNA芯片筛选在乳腺癌组织及癌旁组织中差异表达的circRNA,通过实时定量PCR验证circNR4A1在15对乳腺癌组织及癌旁正常组织、40例患者及40例健康人群外周血以及MCF-7、HCC-1937、MDA-MB-231、T-47D人乳腺癌细胞及MCF-10A人乳腺上皮细胞的表达。使用circPrimer 1.2软件绘制circRNA结构图,使用环状RNA交互作用组(Circular RNA interactome)数据库预测circRNA结合的miRNA和RNA-蛋白结合位点,通过京都基因与基因组百科全书(KEGG)数据库进行circRNA生物学功能预测。构建circNR4A1过表达载体并转染MCF-7细胞,采用CCK-8法和克隆形成实验检测乳腺癌细胞增殖、Hoechst33258染色结合流式细胞技术检测细胞凋亡和细胞周期,实时定量PCR检测癌细胞丝裂原激活蛋白激酶激酶/细胞外信号调节激酶(MEK/ERK)信号通路因子K-RAS、MEK、ERK,凋亡相关基因B细胞白血病/淋巴瘤因子2(Bcl2)、B细胞白血病/淋巴瘤因子2关联凋亡因子X(BAX)、B细胞白血病/淋巴瘤因子2细胞死亡激活子(BAD)、胱天蛋白酶3(caspase-3)等mRNA水平,Western blot法检测MEK、ERK、Bcl2、BAD、caspase-3蛋白水平。结果circNR4A1(Hsa-circ-0026352)在乳腺癌组织、外周血、乳腺癌细胞系中表达显著低于癌旁组织、健康人群外周血、人乳腺上皮细胞。在MCF-7细胞中过表达circNR4A1能够抑制癌细胞增殖,促进细胞凋亡,阻滞细胞周期于G1/S期,并抑制MEK、ERK、Bcl2蛋白表达,促进BAD、caspase-3蛋白表达。结论circNR4A1/Hsa-circ-0026352在乳腺癌细胞中低表达,过表达circNR4A1能够显著抑制MCF-7细胞增殖,诱导细胞凋亡。

Objective To investigate the expression of circNR4A1/Hsa-circ-0026352 in breast tumor tissue,peripheral blood,cell lines and its effect on the proliferation and apoptosis of MCF-7 breast cancer cells.Methods Using circRNA microarray,different expression of human circRNAs was found out between the breast cancer tissues and adjacent normal tissues.The expression of circNR4A1 in breast cancer tissues,peripheral blood and breast cancer cell lines including MCF-7,HCC-1937,MDA-MB-231,T-47D and MCF-10A were detected by quantitative real-time PCR(qRT-PCR).Circular RNA interactome software and KEGG software were used to predict its target miRNA and bio-function.The circNR4A1 over-expression vector was constructed and transfected into MCF-7 cells.The effect of circNR4A1 on the proliferation of MCF-7 cells was detected by CCK-8 assay and clone formation assay.The cell apoptosis and cell cycle distribution were analyzed by flow cytometry and Hoechst 33258 stainning.The qRT-PCR was performed to detect the mRNA expression levels of MEK/ERK signal pathway genes(K-RAS,MEK,ERK)and apoptosis-related genes(Bcl2,BAX,BAD,caspase-3).Western blotting was used to detect the expression of MEK,ERK,Bcl2,BAD and caspase-3 proteins.Results The expression of circNR4A1(Hsa-circ-0026352)was lower in breast cancer tissues,peripheral blood and breast cancer cell lines than in adjacent normal tissues,healthy individuals′peripheral blood and human breast epithelial cells.Over-expression of circNR4A1 in MCF-7 cells inhibited the cell proliferation,promoted cell apoptosis and blocked the cell cycle in G1/S phase.The mRNA and protein expression of MEK,ERK,Bcl2 were significantly depressed,while the expression of BAD,caspase-3 were enhanced in the MCF-7 cells over-expressing circNR4A1.Conclusion The expression of circNR4A1/Hsa-circ-0026352 is lower in breast cancer tissues,peripheral blood and breast cancer cells.Over-expression of circNR4A1 can inhibit cell proliferation and induce cell apoptosis in MCF-7 cells.