沉默PRR14基因抑制结肠癌HCT116细胞增殖

PRR14 Silencing Inhibits Proliferation of Colon Cancer HCT116 Cells

已有报道显示,富脯氨酸蛋白14(proline-rich protein 14,PRR14)促进肿瘤的发生发展,但具体作用机制仍不清楚。本文以结肠癌细胞为模型,探索其对细胞增殖和细胞周期进程的影响。qPCR和Western印迹检测发现,PRR14在4个结肠癌细胞系中呈现高水平表达。合成特异靶向PRR14基因的siRNA,转染结肠癌HCT116细胞。检测发现,PRR14基因表达下调约70%。CCK8结果显示,沉默PRR14后各时间点细胞增殖能力均显著降低,克隆形成实验细胞克隆数减少约40%;流式细胞仪结果显示,沉默PRR14后,G1期细胞比例升高约10%,S期细胞比例降低约14%;BrdU标记免疫荧光检测结果显示,BrdU阳性细胞比例减少约50%,表明细胞DNA合成速率显著降低。机制分析表明:促G1/S期转换基因周期蛋白依赖性激酶2(cyclin dependent kinase 2,CDK2)mRNA水平降低约85%,对应的蛋白质水平也明显降低,G1/S期转换抑制因子周期蛋白依赖性激酶抑制因子1A(cyclin dependent kinase inhibitor 1A,CDKN1A/P21)和周期蛋白依赖性激酶抑制因子1B(cyclin dependent kinase inhibitor 1B,CDKN1B/P27)mRNA水平分别升高约1.8倍和5倍,对应的蛋白质水平也明显升高。沉默PRR14表达,G1/S期相关基因表达紊乱,导致细胞G1期阻滞并抑制细胞增殖。结肠癌细胞中PRR14高表达可促进癌细胞恶性增殖。

It has been reported that proline-rich protein 14(PRR14)regulates cell proliferation and promotes the occurrence and development of tumors,but the specific mechanism is still unclear.In this paper,colon cancer HCT116 cells were used as a model to explore its effects on cell proliferation and cell cycle progression.qPCR and Western blotting revealed high expression of PRR14 in four colon cancer cell lines.SiRNA specifically targeting PRR14 was synthesized and transfected into colon cancer HCT116 cells to construct a PRR14 silencing cell model.CCK8 assays showed that the proliferation rate was significantly reduced at each time point after PRR14 silencing,and the clone number of colony formation assays was reduced by about 40%.Flow cytometry results showed that the proportion of G1 phase cells increased by about 10%,while that of S phase cells decreased by about 14%after PRR14 silencing.The BrdU labeling was detected by immunofluorescence,results showed that the proportion of BrdU positive cells decreased by about 50%,indicating that the DNA synthesis rate was significantly reduced.The mRNA level of cyclin dependent kinase 2(CDK2)was reduced by about 85%,and the corresponding protein level was significantly reduced.The mRNA levels of cyclin dependent kinase inhibitor 1A(CDKN1A/P21)and cyclin dependent kinase inhibitor 1B(CDKN1B/P27)were increased by about 1.8 and 5 times,respectively,and the corresponding protein levels were significantly increased.Silencing PRR14 disrupts G1/S phase-related genes expression,leads to G1 phase arrest and inhibits cell proliferation.In sum,high expression of PRR14 in colon cancer cells promotes malignant proliferation of cancer cells.