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[目的]通过RNA干扰技术致基因沉默和基因过表达的方法研究IER5基因与宫颈癌SiHa细胞系辐射敏感性的相关性,探讨IER5基因不同表达的细胞系对辐射敏感性的差异。[方法]构建IER5沉默及过表达的真核表达载体,转染细胞并筛选出稳定表达的细胞系,经Western Blot和Real-time PCR鉴定成功。观察IER5基因不同表达的3种细胞系的形态;对细胞系进行4Gy的辐射后使用CCK-8检测细胞生长曲线;对细胞系进行10Gy的辐射后使用流式细胞仪检测细胞凋亡情况;对细胞系进行4Gy的辐射后使用流式细胞仪检测不同时间点细胞周期的情况。[结果]IER5基因沉默使SiHa细胞略有增大,照射后的细胞增殖速度加快;而其过表达可使SiHa细胞缩小呈圆形,并且细胞增殖速度有所减慢。IER5沉默导致辐射诱导的SiHa细胞凋亡率降低,过表达可促进辐射诱导的SiHa细胞凋亡,其凋亡在照射后48h最为显著。照射后细胞周期的G2-M期阻滞效应随IER5表达含量的降低而升高,IER5基因沉默促进细胞的增殖分裂。[结论 ]IER5基因与宫颈癌SiHa细胞的增殖、凋亡和细胞周期的变化及对辐射的反应有明显相关性,IER5基因可能可提高宫颈癌细胞对放射的敏感性。
[Objective] To study the correlation between IER5 gene and radiation sensitivity of cervical cancer SiHa cell line by gene silencing and gene overexpression induced by RNAi, and to explore the difference of radiosensitivity of cell lines with different expression of IER5 gene. [Method] The eukaryotic expression vector with silence and overexpression of IER5 was constructed. The transfected cells were screened and stably expressed. The cell lines were identified by Western Blot and Real-time PCR. The morphology of the three cell lines with different expression of IER5 gene was observed. The cell growth curve was detected by CCK-8 after the cell line was irradiated with 4Gy. The cell apoptosis was detected by flow cytometry after the cell line was irradiated with 10Gy. The cell lines were irradiated with 4Gy and the flow cytometry was used to detect the cell cycle at different time points. [Result] The silencing of IER5 caused a slight increase of SiHa cells, and accelerated the proliferation of cells after irradiation. However, overexpression of SiRNA caused the rounding of SiHa cells and the rate of cell proliferation slowed down. Silencing of IER5 resulted in the decrease of apoptosis rate of SiHa cells induced by radiation. Overexpression of IER5 promoted the apoptosis of SiHa cells induced by radiation. The apoptosis of SiHa cells was the most significant at 48h after irradiation. G2-M phase arrest of cell cycle increased with the decrease of IER5 expression, while silencing of IER5 promoted cell division. [Conclusion] The IER5 gene has a significant correlation with the proliferation, apoptosis and cell cycle of cervical cancer SiHa cells and the response to radiation. IER5 gene may enhance the sensitivity of cervical cancer cells to radiation.