BRIT1基因过表达对人宫颈癌HeLa细胞凋亡的影响

Effect of over-expression of BRIT1 gene on apoptosis of cervical cancer HeLa cells

目的探讨BRIT1基因过表达对人宫颈癌HeLa细胞凋亡的影响。方法真核表达质粒pcDNA3.1(-)/BRIT1经双酶切和测序鉴定后转染HeLa细胞,并设空载体转染组(阴性对照)和未处理组。转染后48 h,采用RT-PCR和Real timePCR法检测细胞中BRIT1基因mRNA的转录和表达情况,Western blot检测细胞中BRIT1蛋白的表达水平,流式细胞术分析细胞的凋亡情况。结果重组真核表达质粒pcDNA3.1(-)/BRIT1经双酶切和测序证实构建正确;转染48 h后,可有效上调HeLa细胞中BRIT1基因mRNA和蛋白的表达,细胞凋亡率[(12.37±0.19)%]较阴性对照组[(1.81±0.22)%]和未处理组[(2.06±0.10)%]明显增加,且差异均有统计学意义(P<0.05)。结论 BRIT1基因过表达能诱导人宫颈癌HeLa细胞体外凋亡,为进一步研究BRIT1基因在宫颈癌细胞凋亡中的作用及其机制奠定了基础。

Objective To investigate the effect of over-expression of BRIT1 gene on apoptosis of cervical cancer HeLa cells. Methods Eukaryotic expression plasmid pcDNA3. 1 （-）/BRIT1 was identified by restriction analysis and sequencing and transfected to HeLa cells, using the cells transfected with empty plasmid and those untransfected as control. Forty-eight hours after transfection, the cells were determined for transcription and expression of BRIT1 mRNA by RT-PCR and real-time PCR, for expression of BRIT1 protein by Western blot, and for apoptosis by flow cytometry. Results Restriction analysis and sequencing proved that recombinant plasmid pcDNA3. 1 （-）/BRIT1 was constructed correctly. Both the expressions of BRIT1 mRNA and protein were up-regulated effectively in HeLa cells 48 h after transfection with the constructed plasmid. However, the apoptosis rate of HcLa cells transfected with plasmid peDNA3. 1 （-）/BRIT1 [ （12. 37 -＋ O. 19）% ] was significantly higher than those transfected with empty plasmid[ （1.81 ~ 0. 22）% ~ and those untransfected [（2. 06 ＋ 0. 10）% ] （P 〈 0. 05）. Conclusion Over-expression of BRIT1 gene induced the apoptosis of HeLa cells in vitro, which laid a foundation of further study on the role of BRIT1 gene in apoptosis of cervical cancer cells and the relevant mechanism.