RNA干扰介导的CT120A基因表达下调抑制肺腺癌细胞生长

Down-regulation of CT120A by RNA interference suppresses lung cancer cells growth

目的研究新基因CT120A的表达与肺癌细胞生长的关系。方法构建特异性靶向于CT120A基因的RNA干扰表达质粒小发夹RNA(shRNA)H,并将其转染肺腺癌细胞株SPCA1。逆转录聚合酶链反应(RTPCR)和Western印迹测定干扰后CT120A表达水平;采用BrdUTdR掺入实验和软琼脂克隆形成实验进行细胞体外增殖分析;裸鼠成瘤实验测定CT120A基因表达下调后SPCA1细胞在体内致瘤能力的改变;流式细胞仪检测细胞凋亡率。结果RNA干扰表达质粒shRNAH可以有效抑制CT120A基因的表达,使CT120AmRNA表达减少50%～70%,蛋白表达减少80%;CT120A基因表达下调抑制了SPCA1细胞在体外的增殖,降低其在软琼脂中生长形成克隆和在裸鼠体内成瘤的能力,提高细胞对紫外线诱导凋亡的敏感性。结论新基因CT120A与肺癌细胞生长有着密切的关系,CT120A基因可能可以作为药物治疗肺癌的一个新的靶点。

Objective To validate our obtained outcomes and clarify the relationship between CT120A, a novel human plasma membrane-associated gene, and proliferation of lung cancer cells. Methods A vector-based small hairpin RNA (shRNA) was transfected into the human lung adenocarcinoma SPC-A-1 cells to specifically target CT120A cDNA. RT-PCR and Western blotting were used to analyze the CT120A expression. The cell proliferation rate was analyzed by BrdU-TdR incorporation assay, the ability of cells to grow in soft agarose and the tumorigenicity in nude mice were measured. Flow cytometry was performed to analyze cell apoptosis. Results When compared with the scrambled control cell line, CT120A transcripts were reduced by 70% and 50% in two shRNA-H stable transfectants, H2 and H3 clones, respectively. The protein of CT120A was reduced by about 80% in both the H2 and H3 clones. By BrdU incorporation assay, up to the 6th day a dramatic decrease in the cell growth rate (30% to 40%) was observed in the shRNA-H2 and shRNA-H3 cell lines. The colony formation rate in soft agarose of the two cell lines was about one half that of the control cells. In addition, a remarkable reduction of tumorigenicity of the two cell lines was observed as compared with that of the control. The suppression of CT120A expression also sensitized cells to ultraviolet-induced apoptosis. Conclusion Down-regulation of CT120A by RNA interference suppresses lung cancer cell growth. The successful knockdown of CT120A expression by RNA interference implicates that CT120A may be a new candidate of drug target for treatment of lung cancers.