QCR2调控p53泛素化对SiHa细胞株的周期阻滞作用

QCR2 regulates the cycle arrest effect of p53 ubiquitination on SiHa cell line

目的研究泛醇-细胞色素C还原酶亚基2(QCR2)对宫颈癌SiHa细胞株周期阻滞的影响,并探讨相关机制。方法取对数期SiHa细胞,脂质体转染法将QCR2 siRNA、control siRNA转染至SiHa细胞,设为QCR2 siRNA组、control siRNA组,未经处理细胞设空白组。采用qRT-PCR、Western blot检测QCR2、p53 mRNA与蛋白相对表达量;碘化丙啶(PI)染色检测细胞周期。取QCR2 siRNA组细胞,以终浓度50 nmol/L泛素-蛋白酶体抑制剂PS341干预,设为QCR2 siRNA+PS341组,另取QCR2 siRNA组细胞以等量生理盐水(NS)干预,设为QCR2 siRNA+NS组。Western blot检测p53蛋白相对表达量;免疫共沉淀实验检测p53泛素化水平。结果荧光显微镜下观察,QCR2 siRNA组、control siRNA组转染效率均大于80%;与空白组、control siRNA组比较,QCR2 siRNA组QCR2 mRNA与蛋白相对表达量均降低(P<0.05),G_(0)/G_(1)占比升高(P<0.05),S、G_(2)/M占比降低(P<0.05);p53 mRNA相对表达量组间比较,差异无统计学意义;与空白组、control siRNA组比较,QCR2 siRNA组p53蛋白相对表达量升高(P<0.05);与空白组、QCR2 siRNA+PS341组比较,QCR2 siRNA组、QCR2 siRNA+NS组p53蛋白相对表达量升高(P<0.05);与空白组、control siRNA组比较,QCR2 siRNA组p53泛素化程度降低(P<0.05)。结论沉默QCR2可将SiHa细胞阻滞在G_(0)/G_(1)期,其机制可能与抑制p53泛素化,提高其蛋白表达有关。

Objective To study the effect of ubiquinol-cytochrome C reductase subunit 2(QCR2)on cell cycle arrest of cervical cancer SiHa cell line,and to explore related mechanisms.Methods The log-phase SiHa cells were taken,and QCR2 siRNA and control siRNA were transfected into SiHa cells by liposome transfection,which were set as QCR2 siRNA group and control siRNA group,and untreated cells were set as a blank group.qRT-PCR and Western blot were used to determine the relative expression of QCR2,p53 mRNA and protein.Propidium iodide(PI)staining was used to determine cell cycle.The cells in the QCR2 siRNA group were taken,and were intervened with a final concentration of 50 nmol/L ubiquitin-proteasome inhibitor PS341as the QCR2 siRNA+PS341 group.In addition,the cells in the QCR2 siRNA group were intervened with the same amount of normal saline(NS)and set as the QCR2 siRNA+NS group.Western blot was used to determine the relative expression of p53 protein.The immunoprecipitation test was used to determine the level of p53 ubiquitination.Results Observed under a fluorescence microscope,the transfection efficiency of QCR2 siRNA group and control siRNA group were both>80%.Compared with the blank group and control siRNA group,the relative expression of QCR2 mRNA and protein in the QCR2 siRNA group decreased(P<0.05),the proportion of G0/G1 increased(P<0.05),and the proportion of S,G2/M decreased(P<0.05).There was no significant difference in the relative expression of p53 mRNA between the groups.Compared with the blank group and control siRNA group,the relative expression of p53 protein in the QCR2 siRNA group increased(P<0.05).Compared with the blank group and QCR2 siRNA+PS341 group,the relative expression of p53 protein in the QCR2 siRNA group and QCR2 siRNA+NS group increased(P<0.05).Compared with the blank group and control siRNA group,the degree of p53 ubiquitination in the QCR2 siRNA group was reduced(P<0.05).Conclusion Silencing QCR2 can block SiHa cells in G_(0)/G_(1) phase.Its mechanism may be related to the inhibition of p53 ubiquitination and the increase of its protein expression.