LPA对子宫颈癌干细胞生物学特性的影响及对RhoA/Rock2通路调控作用

Effect of LPA on Biological Characteristics of Cervical Cancer Stem Cells and Regulation of RhoA/Rock2 Pathway

目的:研究和探讨溶血磷酸酯(lysophosphatidic acid,LPA)对子宫颈癌干细胞生物学特性的影响及对RhoA/Rock2通路的调控作用,进一步明确宫颈癌的发病机制,并为将来研制宫颈癌靶点药物提供理论依据。方法:以宫颈癌Siha细胞株为原代细胞,球囊培养富集宫颈癌干细胞,观察细胞的克隆形成能力,检测细胞表面标志物。成功培养子宫颈癌干细胞,分为对照组、LPA组及抑制组,对照组正常培养,LPA组分别加入不同浓度(1、5、10、15、20umoL/L)的LPA,抑制剂组以最佳促增殖浓度的LPA细胞组为基础,加入Rho激酶抑制剂Y-27632,观察细胞增殖情况并绘制细胞生长曲线图,采用PCR法检测各组细胞LPA、RhoA、Rock2的mRNA水平,western blot法检测RhoA及Rock2的表达情况。结果:经培养后有悬浮细胞球形成,细胞表型为CD44^+、CK17^+、CD34^-、CD105^-,成功获得子宫颈癌干细胞,加入LPA后,对癌细胞增殖有显著的促进作用,且存在时间与剂量依赖性关系(P<0.05),加入Y-27632后对细胞增殖有显著的抑制作用(P<0.05),LPA组的LPA、RhoA、Rock2的mRNA水平及RhoA、Rock2蛋白表达量均显著高于对照组与抑制组(P<0.05)。结论:LPA可以促进子宫颈癌干细胞的增殖,其作用机制可能是通过调控RhoA/Rock2通路来实现的。

Objective:To study and explore the effects of lysophosphatidic acid(LPA)on the biological characteristics of cervical cancer stem cells and the regulation of RhoA/Rock2 pathway,further clarify the pathogenesis of cervical cancer and develop cervical cancer targets for the future.Point drugs provide theoretical basis.Methods:Siha cell lines were used as primary cells.The cervical cancer stem cells were cultured with balloon-encapsulated cells to observe the ability of cell colony formation.Cell surface markers were detected by FACS.After the successful cultivation of cervical cancer stem cells,they were divided into control group,LPA group and inhibition group.The control group was cultured normally.In the LPA group,LPA of different concentrations(1,5,10,15,20 umol/L)was added,and the inhibitor group was The optimal proliferative concentration of LPA cells group was based on the addition of Rho kinase inhibitor Y-27632.Cell proliferation was observed and cell growth curves were plotted.The mRNA levels of LPA,RhoA and Rock2 in each group were detected by PCR.The expression of RhoA and Rock2 was detected.Results:After culture,there were suspension cell spheres with CD44^+,CK17^+,CD34^-and CD105-phenotypes.Cervical cancer stem cells were successfully obtained.After adding LPA,the proliferation of cancer cells was significantly promoted,and there was a time In a dose-dependent manner(P<0.05),addition of Y-27632 significantly inhibited cell proliferation(P<0.05).LPA,RhoA,and Rock2 mRNA levels and western blot were significantly higher in LPA group than in the control group and inhibition.Group(P<0.05).Conclusion:LPA can promote the proliferation of cervical cancer stem cells,and its mechanism of action can be achieved by regulating the RhoA/Rock2 pathway.