Cep63表达及其与甲状腺乳头状癌细胞系TPC-1凋亡功能的研究

目的研究中心体蛋白Cep63在甲状腺乳头状癌(papillary thyroid carcinoma,PTC)细胞系TPC-1凋亡过程中的作用及相关机制。方法收集PTC病例的癌组织和癌旁组织,通过实时荧光定量PCR(qRT-PCR)检测组织Cep63的表达情况并分析其与临床病理因素的关系。本实验随机分为阴性对照组(NC)、低表达组Cep63(-)]和过表达组Cep63(+)],将Cep63慢病毒转染野生型TPC-1细胞系,并通过蛋白免疫印迹法(WB)和qRT-PCR验证Cep63的干扰效果。通过平板克隆实验与MTT法检测细胞增殖能力变化;流式细胞术检测细胞的凋亡率,免疫组织化学(immunohistochemistry,IHC)和WB检测转染前后细胞凋亡相关蛋白表达差异。2组间均数比较采用t检验,单因素方差分析进行多组间均数差异的比较,病理因素关联分析采用卡方检验。结果相比NC组,Cep63(-)组细胞增殖受到抑制(3.18±0.07比2.14±0.09,t=8.54,P<0.01),Cep63(+)组细胞的增殖能力明显提高(3.18±0.07比3.58±0.10,t=3.21,P<0.05);NC组、Cep63(-)组和Cep63(+)组凋亡率分别为3.03%±0.24%、8.66%±0.44%和1.17%±0.44%,流式结果表明Cep63的低表达使TPC-1细胞的凋亡水平明显提高(F=157.7,P<0.001);NC组、Cep63(-)组和Cep63(+)组Bcl-2表达量分别为1.07±0.03、0.49±0.01和1.99±0.09,BAX表达量分别为0.64±0.02、1.06±0.01和0.21±0.03,WB结果表明,Cep63的低表达使TPC-1细胞Bcl-2蛋白的表达量明显降低(F=183.2,P<0.001),同时BAX表达明显上调(F=283.7,P<0.001)。结论Cep63可能通过Bcl-2/BAX通路调控了PTC细胞系TPC-1的凋亡过程,Cep63可能是PTC的一个潜在癌基因。

Study on Cep63 expression and apoptosis of thyroid papillary carcinoma cell lines TPC-1

Objective To investigate the effect of centrosomal protein Cep63 on the apoptosis of papillary thyroid carcinoma(PTC)cell lines TPC-1 and underlying mechanism.Methods With collected PTC tissues and adjacent tissues,Cep63 expression was detected by RT-qPCR and its relationship with clinicopathological factors was analyzed.The experiment included negative control group(NC),low expression group(Cep63(-))and overexpression group(Cep63(+)),and wild-type TPC-1 cells were transfected with Cep63 lentivirus.The efficiency of Cep63 was detected by western blot(WB)and qRT-PCR.Cell proliferation ability was detected by plate cloning experiment and MTT assay.Cell apoptotic rate was detected by flow cytometry,and expression levels of apoptosis-related proteins were detected by immunohistochemistry and WB.The t-test was used to compare the differences in the means between the two groups,the one-way analysis of variance was used to compare multiple groups,and the chi-square test was used to analyze the association between gene expression levels and pathological factors.Results Compared with NC group,cell proliferation ability was significantly decreased in Cep63(-)group(3.18±0.07 vs.2.14±0.09,t=8.54,P<0.01)and significantly increased in Cep63(+)group(3.18±0.07 vs.3.58±0.10,t=3.21,P<0.05).Apoptotic rates in NC,Cep63(-)and Cep63(+)groups were respectively 3.03%±0.24%,8.66%±0.44%and 1.17%±0.44%,and the flow cytometry showed that the low expression of Cep63 significantly increased the apoptosis TPC-1 cells(F=157.7,P<0.001).Bcl-2 protein expression levels of NC,Cep63(-)and Cep63(+)groups were respectively 1.07±0.03,0.49±0.01 and 1.99±0.09,and BAX protein expression levels of three groups were respectively 0.64±0.02,1.06±0.01 and 0.21±0.03.WB showed that the expression level of Bcl-2 decreased(F=183.2,P<0.001),while the expression level of BAX was significantly up-regulated(F=283.7,P<0.001).Conclusion Cep63 may regulate the apoptotic process of TPC-1 cells through Bcl-2/BAX pathway and Cep63 may be a potential oncogene of PTC.