CRISPR/Cas9技术敲减Ku80基因对人乳头状甲状腺癌细胞生物学功能的影响

Effect of Ku80 gene knockdown by CRISPR/Cas9 technique on biological function of human papillary thyroid carcinoma cell

目的探讨Ku80基因敲减对人乳头状甲状腺癌(papillary thyroid carcinoma,PTC)细胞功能的影响。方法采用real time PCR方法检测Ku80在PTC癌细胞株K1、B-CPAP中m RNA的表达水平;以PTC癌细胞株K1、B-CPAP为研究对象,分为KO(Knockout)组(慢病毒感染Ku80)、NC组(慢病毒感染Ku80无关序列)和MOCK组(未进行病毒感染)。应用CRISPR/Cas9(一种能够对任何物种基因组的特定位点进行精确编辑的技术)行Ku80基因敲减,采用MTT、Transwell、克隆形成、流式细胞等实验方法检测Ku80敲减对癌细胞增殖、侵袭、克隆形成能力及凋亡等生物学功能的影响。结果 RT-PCR结果证实Ku80基因在2株乳头状甲状腺癌细胞中均高表达。CRISPR/Cas9技术Ku80敲除效率分别为95. 6%和56%。与NC组相比较,K1细胞中KO组细胞增殖活力、侵袭及克隆形成能力均显著降低,而细胞凋亡率明显上升(P <0. 05);与NC组相比较,BCPAP细胞中KO组细胞增殖活力及侵袭力均呈下降趋势,但无统计学差异(P> 0. 05);克隆形成能力显著降低,细胞凋亡数增加(P <0. 05)。结论 Ku80基因敲减促进乳头状甲状腺癌细胞凋亡、抑制细胞增殖、侵袭。

Objective To investigate the effect of Ku80 gene knockdown on the cell function of human papillary thyroid carcinoma(PTC).Methods The mRNA levels of Ku80 in PTC cell lines K1 and B-CPAP were detected by real time PCR.K1 and B-CPAP cells were used as research subjects,and each cell line was divided into KO group(transfected with Ku80-sgRNA virus),NC group(transfected with Ku80 unrelated sequence virus)and MOCK group(no virus infection).Then Ku80 gene was knocked down by CRISPR/Cas9 technique,and then the effects of Ku80 knockdown on cell proliferation,invasion,colony formation and apoptosis were evaluated by MTT,Transwell,colony formation and cell apoptosis assays.Results Ku80 mRNA were found high expression both in K1 and B-CPAP cell lines by RT-PCR.The Ku80 knochdown efficiency was 95.6%in K1 and 56%in B-CPAP,respectively.Compared with NC group,the proliferation,invasion,and tumorigenic ability of K1 cells in KO group were all decreased significantly after Ku80 gene knockdown,while the apoptosis rate increased significantly(P<0.05).Compared with NC group,the ability of proliferation and invasiveness of B-CPAP cells in KO group showed a decreasing trend,but there was no statistical difference(P>0.05).Similarly,compared with NC group,the ability of colony formation was significantly decreased in KO group,while the number of apoptosis was increased(P<0.05).Conclusion Ku80 gene knockdown by CRISPR/Cas9 technique may inhibit proliferation,invasion ability,and promote apoptosis ability of PTC cells.