沉默EZH2基因表达对宫颈癌细胞增殖能力影响的分析

The Effect Analysis of Silencing EZH2 Gene Expression on the Proliferation of Cervical Cancer Cells

本实验旨在探讨EZH2基因在宫颈癌中表达情况、对宫颈癌细胞增殖能力的影响及其机制。通过免疫组织化学法检测20例正常宫颈、20例CIN及60例宫颈鳞癌组织中EZH2蛋白表达;采用RT-PCR法检测4个宫颈癌细胞株中EZH2 m RNA的表达。通过Lipofectamin2000介导EZH2 si RNA瞬时转染C33A细胞株,Western blotting法检测si RNA的干扰效率。MTT、流式细胞仪检测EZH2沉默后细胞增殖及细胞周期变化;Western blotting检测p21表达变化。结果显示,正常宫颈组织、CIN、宫颈鳞癌中EZH2阳性表达率分别为15%、30%、76.7%,呈逐级升高趋势,后者与前两者见差异有统计学意义(p<0.01);EZH2的表达同宫颈鳞癌细胞分化程度、间质浸润深度及淋巴结转移显著相关(p<0.05)。EZH2 m RNA在宫颈癌细胞株He La、Si Ha、C33A及Caski中均表达,其中C33A细胞中表达最高。si RNA沉默EZH2基因明显抑制宫颈癌C33A细胞增殖,阻滞细胞周期于G1期;Western blotting检测p21表达上调。由此得出结论,EZH2基因在宫颈癌中高表达;沉默EZH2基因可通过上调p21蛋白的表达阻滞细胞周期于G1期、抑制宫颈癌细胞的增殖。

The purpose of this research was to investigate the expression of EZH2 gene in cervical carcinoma and its effect on the multiplication capacity of cervical cancer cells as well as the mechanism. We detected the expression of EZH2 protein in tissues of normal cervix （20 cases）, CIN （20 cases） and cervical squamous carcinoma （60 cases） by immunohistochemistry; and we detected the expression ofEZH2 mRNA in 4 cell lines of cervical cancer by RT-PCR. The small interfering RNA （siRNA） for EZH2 was transiently transfected into C33A cell line with Lipofectamin2000, and Western blotting was used to detect the validity of siRNA. The cellular proliferation and cell cycle changes were detected by MTT assay and flow cytometry （FCM）, respectively, and the expression variation of p21 was detected by Western blotting. The results showed that the positive expression rates of EZH2 in normal cervical tissue, CIN and cervical squamous carcinoma were 15%, 30% and 76.7%, respectively, which it showed a gradual growing trend; the difference between the latter and the former had the statistically significance （p 〈0.01）. The expression of EZH2 was associated with differentiation, the depth of invasion andlymph node metastasis ofcvical squamous cells （p〈0.05）. EZH2 mRNA was expressed in HeLa, SiHa, C33A and Caski, and it was expressed the highest in C33A cell line. Silencing EZH2 by siRNA obviously inhibited the cell proliferation of C33A cervical cancer, and retarded cell cycle in G1 phase. Up-regulation of p21 expression was detected by Western blotting. Therefore, it could be concluded that gene the expression of EZH2 was highly expressed in cervical squamous cell carcinoma; silencing EZH2 could retard cell cycle at G1 phase, and inhibit proliferation of cervical cancer cells by up-regulating p21 expression.