Sphk1抑制剂PF543对宫颈癌SiHa细胞凋亡和增殖的影响及其机制

Effect of Sphk1 inhibitor PF543 on the apoptosis and proliferation of cervical cancer SiHa cells and its mechanism

目的:研究鞘氨醇激酶1(Sphk1)抑制剂PF543对宫颈癌SiHa细胞凋亡和增殖的影响。方法:体外培养SiHa细胞,并分为SiHa、SiHa+20μmol/L PF543和SiHa+40μmol/L PF543三组,分别采用流式细胞实验检测细胞凋亡和细胞周期情况,CCK-8实验和Edu实验检测细胞增殖能力,RT-PCR和Western Blot实验检测各组Sphk1、Caspase3、Bax、Bcl-2、Ki67和Cyclin D1的表达情况。结果:与SiHa组相比,20和40μmol/L PF543组均可增加Bax和cleaved-Caspase3表达,抑制Sphk1、Bcl-2、Cyclin D1和Ki67的表达,具有剂量依赖性(P<0.05),而总Caspase3蛋白表达无明显变化;同时,20和40μmol/L PF543组细胞活率逐渐下降,显微镜下红色荧光占比逐渐减少(P<0.05)。流式细胞实验中,随着PF543浓度的增加,细胞凋亡率逐渐升高,G1期细胞占比逐渐增加,S期占比逐渐减少(P<0.05)。结论:PF543下调Sphk1后促进SiHa细胞凋亡、阻滞细胞于G1期和抑制细胞增殖。这可能与增加促凋亡基因cleaved-Caspase3和Bax表达,抑制抗凋亡基因Bcl-2和促增殖基因Ki67、Cyclin D1表达有关。

Objective:To study the effect of sphingosine kinase 1(Sphk1) inhibitor PF543 on the apoptosis and proliferation of cervical cancer SiHa cells.Methods:SiHa cells were cultured in vitro and divided into three groups:SiHa,SiHa+20 μmol/L PF543,and SiHa+40 μmol/L PF543 group.Flow cytometry assay was used to detect cell apoptosis and cycle status,CCK-8 assay and Edu assay were used to detect the cell proliferation,and real-time quantitative PCR and Western Blot assay were used to detect the expression of Sphk1,Caspase3,Bax,Bcl-2,Ki67,and Cyclin D1 in each group.Results:Compared with SiHa group,both 20 and 40 μmol/L PF543 treatment increased Bax and cleaved-Caspase3 expression,inhibited Sphk1,Bcl-2,Cyclin D1,and Ki67 expression in a dose-dependent manner(P<0.05),while there was no significant change in total Caspase3 protein expression.Meanwhile,cell survival rate gradually decreased in the 20 and 40 μmol/L PF543 groups,and the percentage of red fluorescence under the microscope decreased gradually(P<0.05).In the flow cytometry assay,the apoptosis rate gradually increased with the increase of PF543 concentration,the percentage of G1 phase cells gradually increased and the percentage of S phase decreased gradually too(P<0.05).Conclusion:PF543 could promote SiHa cell apoptosis,block cells in G1 phase,and inhibit SiHa cell proliferation by downregulation of Sphk1.This may be related to increasing the expression of pro-apoptotic genes cleaved-Caspase3 and Bax,and inhibiting the expression of anti-apoptotic gene Bcl-2 and pro-proliferative genes Ki67 and CyclinD1.