生物钟基因PER1调控口腔鳞癌细胞中BAX、BCL2和PCNA表达的昼夜节律

Biological clock gene PER1 regulates circadian rhythms of BAX,BCL2 and PCNA expression in oral squamous carcinoma cells

目的探讨生物钟基因PER1对口腔鳞癌细胞(oral squamous cell carcinoma,OSCC)的增殖和凋亡及细胞内BAX、BCL2和PCNA表达昼夜节律的调控作用。方法建立稳转过表达PER1的口腔鳞癌SCC15细胞(OE-PER1-SCC15)和不含PER1基因序列慢病毒感染的阴性对照SCC15细胞(NC-SCC15);建立稳转沉默PER1的口腔鳞癌TSCCA细胞(RNAi-PER1-TSCCA)和不含PER1片段的scramble质粒慢病毒感染的阴性对照TSCCA细胞(Scramble-TSCCA)。通过流式细胞术、RT-qPCR和Western blot检测各组细胞中细胞增殖指数、凋亡指数及细胞内PER1、BAX、BCL2和PCNA在24 h内6个不同时间点的表达。对数据进行方差分析和余弦分析,以中值、振幅和峰值相位为指标分析各基因表达的昼夜节律特征。结果在OE-PER1-SCC15和NC-SCC15中,细胞增殖指数和凋亡指数及细胞内PER1、BAX、BCL2和PCNA mRNA和蛋白的表达具有昼夜节律性(P<0.05)。与NC-SCC15细胞相比,OE-PER1-SCC15细胞凋亡指数、PER1和BAX mRNA和蛋白表达的中值显著升高,而细胞增殖指数、BCL2和PCNA mRNA和蛋白表达的中值显著降低(P<0.05);细胞凋亡指数和BAX mRNA表达的振幅显著升高,而BCL2和PCNA mRNA表达的振幅显著降低(P<0.05);细胞凋亡指数、PER1和BAX mRNA和蛋白表达的峰值相位前移,而细胞增殖指数、BCL2和PCNA mRNA和蛋白表达的峰值相位后移。与Scramble-TSCCA细胞相比,RNAi-PER1-TSCCA细胞中BAX蛋白的表达发生昼夜节律紊乱,但细胞增殖指数、凋亡指数及细胞内PER1、BCL2和PCNA mRNA和蛋白以及BAX mRNA的表达具有昼夜节律(P<0.05),其昼夜节律特征与OE-PER1-SCC15细胞相反。结论PER1表达改变后导致其自身的昼夜节律特征发生改变,同时调控口腔鳞癌细胞增殖和凋亡及BAX、BCL2和PCNA表达的昼夜节律特征发生改变。

Objective To explore the regulative role of biological clock gene PER1 in the circadian rhythms of proliferation,apoptosis and expression of intracellular BAX,BCL2 and PCNA in oral squamous carcinoma cells(OSCC).Methods The SCC15 cells stably overexpressing PER1 gene(OE-PER1-SCC15)and negative control SCC15 cells(NC-SCC15)infected with lentiviral virus without PER1 sequence were established.The TSCCA cells with stable PER1 knockdown(RNAi-PER1-TSCCA)and the negative control TSCCA cells(Scramble-TSCCA)of scramble plasmid lentivirus infection without PER1 fragment were also constructed.The cell proliferation index,apoptosis index and the expression of intracellular PER1,BAX,BCL2 and PCNA at 6 different time points within 24 h were detected in each group by flow cytometry,RT-qPCR and Western blotting,respectively.The data were subjected to ANOVA and cosine analysis,and the mesor,amplitude and acrophase were used as indicators to analyze the circadian rhythm characteristics of each gene expression.Results In OE-PER1-SCC15 and NC-SCC15 cells,the cell proliferation index,apoptosis index,and the mRNA and protein levels of PER1,BAX,BCL2 and PCNA showed circadian rhythms(P<0.05).As compared with the NC-SCC15 cells,the OE-PER1-SCC15 cells had significantly increased mesors of apoptosis index and mRNA and protein levels of PER1 and BAX,while decreased mesors of proliferation index and mRNA and protein levels of BCL2 and PCNA(P<0.05).The amplitudes of apoptosis index and BAX mRNA level were remarkably elevated,while those of BCL2 and PCNA mRNA levels were reduced in OE-PER1-SCC15 cells(P<0.05).In addition,the acrophases of apoptosis index,and PER1 and BAX mRNA and protein levels were notably advanced,which were delayed in proliferation index,mRNA and protein levels of BCL2 and PCNA.When compared with the Scramble-TSCCA cells,the circadian rhythm of BAX protein expression was disturbed in the RNAi-PER1-TSCCA cells,but the cell proliferation index,apoptosis index and the mRNA and protein levels of PER1,BCL2 and PCNA,as well as mRNA level of BAX presented circadian rhythms(P<0.05),whose characteristics were opposite to those of OE-PER1-SCC15 cells.Conclusion The changes in PER1 expression lead to the alteration in both its own circadian rhythm characteristics,and the circadian rhythm characteristics of proliferation,apoptosis,and BAX,BCL2,and PCNA expression in OSCC cells.