CRISPR/Cas9介导的IL-6基因敲除对结肠癌SW1116细胞化疗敏感性的影响

Effect of CRISPR/Cas9-mediated IL-6 knockout on chemo-sensitivity in human colorectal cancer SW1116 cell line

目的采用成簇的、规律间隔的短回文重复序列/核酸内切酶9(CRISPR/Cas9)基因组编辑技术构建白细胞介素6(IL-6)基因敲除结肠癌SW1116细胞系,探讨其对化疗药物顺铂(CDDP)的敏感性。方法采用CRISPR/Cas9基因组编辑技术,构建IL-6基因敲除的SW1116细胞系;CCK-8法检测细胞增殖能力;流式细胞术检测细胞周期及细胞凋亡;Western blot法检测细胞IL-6、cyclin E、Bax、Bcl-2、p-STAT3和STAT3表达。结果成功构建了IL-6-/+SW1116和IL-6-/-SW1116细胞系;IL-6-/+SW1116和IL-6-/-SW1116细胞与野生型SW1116细胞相比,其生长状态、细胞增殖能力、细胞周期及细胞凋亡率均无明显差异(P>0.05);5mmol/mL CDDP干预后,IL-6-/-SW1116细胞凋亡率明显增加、细胞增殖受到明显抑制、细胞内Bax表达明显增高,Bcl-2、p-STAT3和STAT3表达明显降低,差异均有统计学意义(P<0.05)。结论成功构建了对CDDP敏感,且生长增殖周期稳定的IL-6-/-SW1116细胞系,可为IL-6介导的结肠癌化疗耐药机制研究提供实验基础。

Objective IL-6 knockout colorectal cancer SW1116 cell line was constructed using CRISPR/Cas9 genomic editing techniques and the effect on chemo-sensitivity to cisplatin(CDDP)was explored.Methods The CRISPR/Cas9 genome editing technique was used to construct the IL-6 knockout SW1116 cell line.The cell proliferation was detected by CCK-8 assay.The cell cycle and apoptosis were detected by flow cytometry.The expressions of IL-6,cyclin E,Bax,Bcl-2,p-STAT3 and STAT3 were detected by Western blot.Results IL-6-/+SW1116 and IL-6-/-SW1116 cell lines were successfully constructed.Compared with the wild type SW1116 cells,the cell growth status,proliferation,cell cycle and apoptosis rate of IL-6-/+SW1116 and IL-6-/-SW1116 cells showed no significant difference(P>0.05).After treated with 5 mmol/mL CDDP,the apoptosis rate of IL-6-/-SW1116 cells increased,the proliferation inhibited,the expressions of Bcl-2,p-STAT3 and STAT3 reduced,but Bax increased,there was statistical significantly difference(P<0.05).Conclusion IL-6-/-SW1116 cell line that is sensitive to CDDP and stable in growth,is successfully constructed,which provided an experimental basis for the study of drug resistance mechanism of chemotherapy mediated by IL-6.