CDCA7基因在食管鳞癌细胞系中的表达及其对细胞增殖凋亡的影响

Expression of CDCA7 gene in esophageal squamous cell carcinoma cell lines and its effect on cell proliferation and apoptosis

目的明确CDCA7基因在食管鳞癌各种细胞系中的表达量及其对细胞表型的影响。方法 (1)实时聚合酶链反应(real-time PCR)和蛋白质印迹法检测食管鳞癌细胞系及永生化食管上皮细胞系中的CDCA7基因的表达水平;(2)构建CDCA7基因的敲除载体pLKO.1-purosh RNA-CDCA7;(3)将pLKO.1-puro-sh RNA-CDCA7和对照空载体pLKO.1-puro转染到内源性高表达CDCA7基因的食管鳞癌细胞系ECA109中;(4)用real-time PCR和蛋白质印迹法验证筛选出的单克隆细胞;(5)四甲基偶氮唑盐(MTT)试验、克隆形成试验、细胞凋亡试验验证CDCA7基因敲除后对细胞表型的影响。结果 (1)在食管鳞癌的各种细胞系中,ECA109是内源性高表达CDCA7基因的细胞系之一;(2)成功构建了pLKO.1-puro-sh RNA-CDCA7的敲除质粒,并筛选出稳定敲除CDCA7基因的单克隆细胞株;(3)MTT、克隆形成试验结果显示,敲除CDCA7基因可以降低食管鳞癌细胞的增殖能力和克隆形成能力;(4)细胞凋亡试验结果显示,敲除CDCA7基因能够加速食管鳞癌细胞的凋亡。结论 CDCA7基因的敲除能够对食管鳞癌细胞系的细胞表型产生影响,会抑制癌细胞的增殖能力和克隆形成能力,加速癌细胞的凋亡。

Objective To determine the expression of CDCA7 gene in various cell lines of esophageal squa-mous cell carcinoma （ESCC） and its effect on the cell phenotype. Methods ①Real-time PCR and Western blotting were used to determine the expression level of CDCA7 gene in ESCC cell lines and immortalized esophageal epithelial cell lines.②The vector pLKO.1-puroshRNA-CDCA7 was constructed for knocking down CDCA7 gene. ③The pLKO1-puro-shRNA-CDCA7 and control empty vector pLKO.1-puro were transfected into ESCC cell line, ECA109 with en-dogenous high expression of CDCA7 gene. ④Real-time PCR and Western blotting were used to validate the screened monoclonal cells. ⑤MTT assay, clone formation assay, and apoptosis assay were used to verify the effect of CDCA7 gene knockout on cell phenotype. Results ①In various cell lines of ESCC, ECA109 was one of the cell lines with endogenous high expression of CDCA7 gene. ②The knockout plasmid of pLKO1-puro-shRNA-CDCA7 was successful-ly constructed, and the monoclonal cell line of CDCA7 gene was screened. ③MTT and clone formation assay showed that the knockout of CDCA7 gene could decrease the proliferation and clone formation of ESCC cells. ④The findings of apoptosis assay showed that the knockout of CDCA7 gene could accelerate the apoptosis of ESCC cells. Conclusion The knockout of CDCA7 gene can affect the cell phenotype of ESCC cell lines, inhibit the proliferation and clone for-mation of cancer cells, and accelerate the apoptosis of cancer cells.