CTCF过表达对乳腺癌细胞凋亡因子Bax和Bcl-2表达的影响

Effects of overexpression of CTCF on apoptosis factors Bax and Bcl-2 in breast cancer cells

目的探讨CTCF过表达对人乳腺癌细胞系MDA-MB-231中细胞凋亡因子Bax和Bcl-2表达的影响。方法用逆转录聚合酶链式反应(RT-PCR)检测人乳腺癌细胞系MDA-MB-231中CTCF、Bax和Bcl-2的表达。构建CTCF/pEGFP-N1过表达载体,用慢病毒转染法分别将CTCF/p EGFP-N1和空载质粒转入MDA-MB-231中,记作CTCF组和对照组。经RT-PCR鉴定CTCF成功转染MDA-MB-231后,采用实时定量PCR(Q-PCR)检测CTCF组和对照组MDA-MB-231中Bax和Bcl-2的mRNA表达,Western blot和酶联免疫吸附试验(ELISA)检测Bax和Bcl-2的蛋白表达。结果 MDA-MB-231中不表达CTCF,表达Bax和Bcl-2。Q-PCR结果显示,CTCF组、对照组中Bax的mRNA表达水平分别为4.63±1.08和2.27±0.16,2组间差异有统计学意义(t=27.50,P<0.05);Bcl-2的mRNA表达水平分别为1.39±0.14和3.56±0.97,2组间差异有统计学意义(t=39.00,P<0.05)。Western blot结果显示,CTCF组中Bax蛋白表达水平高于对照组,Bcl-2蛋白水平低于对照组。ELISA结果显示,CTCF组和对照组中Bax蛋白表达水平分别为15.25±2.17和6.24±1.78,2组间差异有统计学意义(t=26.84,P<0.05);Bcl-2蛋白表达水平分别为4.59±0.97和10.68±1.93,2组间差异有统计学意义(t=21.72,P<0.05)。结论 CTCF过表达可促进乳腺癌细胞中凋亡因子的表达,抑制抗凋亡因子的表达。

Objective To investigate the effect of the over-expressed CTCF on apoptosis factors Bax and Bcl-2 inhuman breast cancer cell line MDA-MB-231. Methods Reverse transcription-polymerase chain reaction(RT-PCR) wereused to detect the expressions of CTCF,Bax and Bcl-2 in MDA-MB-231. The overexpression vector of CTCF/p EGFP-N1 was constructed. The overexpression plasmid CTCF/pEGFP-N1 and the empty vector plasmid p EGFP-N1 were transfectedinto breast cancer cell line MDA-MB-231 by lentivirus transfection, and the MDA-MB-231 cells were divided into CTCFgroup and control group. After successfully transfection of MDA-MB-231 identified by RT-PCR, real time quantitative PCR(Q-PCR) was used to detect the mRNA levels of Bax and Bcl-2 in MDA-MB-231 of the CTCF group and the control group.The protein levels of Bax and Bcl-2 were detected by Western blot assay and enzyme-linked immunosorbent assay(ELISA).Results The expression of CTCF was not found in MDA-MB-231, and expressions of Bax and Bcl-2 were found in MDA-MB-231. Results of Q-PCR showed that the mRNA levels of Bax were 4.63±1.08 and 2.27±0.16 in CTCF group and controlgroup, respectively, and they were statistically significant(t=27.50, P<0.05). The mRNA levels of Bcl-2 were 1.39±0.14 and3.56± 0.97 in CTCF group and control group, and there was significant difference between two groups(t=39.00, P<0.05).Results of Western blot assay showed that the protein level of Bax was higher in CTCF group compared with that of controlgroup. The protein level of Bcl-2 was lower in CTCF group compared with that of control group. Results of ELISA showedthat the protein levels of Bax were 15.25±2.17 and 6.24±1.78 in CTCF group and control group, respectively, and there wassignificant difference between the two groups(t=26.84, P<0.05). The protein levels of Bcl-2 were 4.59±0.97 and 10.68±1.93, and there was significant difference between the two groups(t=21.72, P<0.05). Conclusion The over-expressedCTCF can promote the expression of apoptotic factors and inhibit the expression of anti-apoptotic factors in breast cancer cells.