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目的构建转录因子激活蛋白-2α(transcription factor AP-2 alpha,TFAP2α)重组真核质粒及其稳转细胞株,研究过表达TFAP2α对肾透明细胞癌细胞增殖能力的影响。方法采用反转录PCR方法从293T细胞全基因组DNA中扩增TFAP2α基因编码序列,运用质粒酶切、连接及转化构建重组真核质粒p LV-EGFP(2A)Puro-TFAP2α,测序鉴定。利用慢病毒包装系统将重组质粒转染入293T细胞,制备病毒悬液转染质粒入肾透明细胞癌细胞系786-O和Caki-2中,嘌呤霉素加压筛选,形成稳定转染细胞株后扩大培养,并通过实时定量PCR法和Western Blot法检测转染细胞中TFAP2α的m RNA和蛋白的表达。通过MTS实验、平板克隆实验和细胞周期检测,观察未转染质粒组、转染空载体组和转染重组质粒组细胞增殖和周期的变化。结果成功构建重组真核质粒p LV-EGFP(2A)Puro-TFAP2α和稳定转染过表达TFAP2α的786-O和Caki-2细胞株。重组质粒组TFAP2αm RNA表达水平和蛋白表达水平均明显高于未转染组和空载体组(P<0.05)。MTS实验中,重组质粒组490 nm处光吸收值较未转染组和空载体组显著降低(P<0.05)。平板克隆实验中,重组质粒组的平板克隆率较未转染组和空载体组亦显著降低(P<0.05)。与未转染组和空载体组比较,重组质粒组G_0/G_1期细胞比例显著升高(P<0.05),S期细胞比例显著降低(P<0.05),G_2/M期细胞比例无统计学差异(P>0.970)。结论过表达TFAP2α能明显降低肾透明细胞癌细胞系786-O和Caki-2细胞增殖能力,影响细胞周期变化,停留在G_0/G_1期的细胞明显增加,而S期的细胞明显减少,使其发生G_1/S期阻滞从而抑制细胞增殖。
Objective To construct a recombinant eukaryotic plasmid of transcription factor AP-2 alpha (TFAP2α) and its stable cell line to investigate the effect of over-expressed TFAP2α on the proliferation of renal clear cell carcinoma. Methods The coding sequence of TFAP2α gene was amplified from total genomic DNA of 293T cells by reverse transcription polymerase chain reaction (RT-PCR). Purified recombinant plasmid p LV-EGFP (2A) Puro-TFAP2α was constructed by restriction enzyme digestion, ligation and transformation. Recombinant plasmids were transfected into 293T cells by lentiviral packaging system to prepare virus suspension transfection plasmids into renal clear cell carcinoma cell lines 786-O and Caki-2, and puromycin was screened by pressure to form stable transfected cell lines Then expanded and cultured. The mRNA and protein expression of TFAP2α in transfected cells were detected by real-time quantitative PCR and Western Blot. MTS assay, plate clone test and cell cycle assay were used to observe the changes of cell proliferation and cycle in untransfected plasmid group, transfected empty vector group and transfected recombinant plasmid group. Results The recombinant eukaryotic plasmid p LV-EGFP (2A) Puro-TFAP2α and 786-O and Caki-2 cell lines stably transfected with TFAP2α were successfully constructed. The expression level of TFAP2αmRNA and the protein expression level of recombinant plasmid group were significantly higher than that of the untransfected group and the empty vector group (P <0.05). In MTS experiment, the absorbance at 490 nm of the recombinant plasmid group was significantly lower than that of the untransfected and empty vector groups (P <0.05). In the plate cloning experiment, the plate cloning efficiency of the recombinant plasmid group was significantly lower than that of the untransfected group and the empty vector group (P <0.05). Compared with the untransfected and empty vector groups, the proportion of cells in G 0 / G 1 phase was significantly increased (P <0.05), the proportion of cells in S phase was significantly decreased (P <0.05), and the proportion of cells in G 2 / M phase was not statistically Difference (P> 0.970). Conclusion Overexpression of TFAP2α can significantly reduce the proliferation of renal clear cell line 786-O and Caki-2 and affect the cell cycle. The number of cells staying in G_0 / G_1 phase increased significantly while the number of cells in S phase decreased significantly G_1 / S arrest occurs to inhibit cell proliferation.