摘要
目的:探讨微小RNA-15b(miR-15b)与程序性死亡蛋白-1(PD-1)在细胞因子诱导的杀伤细胞(CIK)抑制宫颈癌细胞增殖过程中的调控作用,并初步分析其中miR-15b与PD-1的可能作用机制。方法:采用传统方法培养CIK细胞,采用流式细胞仪检测CIK细胞免疫表型,CCK-8法检测CIK细胞对宫颈癌细胞(Si Ha)增殖的影响。实时定量聚合酶链反应(qRT-PCR)及蛋白印迹(WB)法检测CIK细胞共培养前后Si Ha细胞中miR-15b及PD-1表达水平。实验将CIK细胞共培养Si Ha细胞分为3组:空白对照组(未进行任何处理),NC组(加入miR-15b阴性对照质粒),si-miR-15b组(加入miR-15b siRNA)。采用qRTPCR与WB分别检测各组miR-15b及PD-1表达水平;CCK-8法检测各组Si Ha细胞增殖情况。采用WB法检测细胞增殖相关蛋白增殖细胞核抗原(PCNA)及Ki67表达。结果:CIK细胞共培养组Si Ha细胞增殖抑制率显著高于单纯培养Si Ha细胞组(P <0. 05);CIK细胞共培养后Si Ha细胞中miR-15b表达水平显著高于单纯培养Si Ha细胞组(P <0. 05),而PD-1蛋白表达水平显著降低(P <0. 05);si-miR-15b组miR-15b表达水平、Si Ha细胞增殖抑制率均显著低于空白对照组、NC组(P <0. 05),而PD-1、PCNA、Ki67蛋白表达均显著升高(P <0. 05)。结论:miR-15b可能通过下调CIK细胞上PD-1表达进而抑制宫颈癌细胞增殖。
Objective:To investigate the regulatory role of microRNA-15 b(miR-15 b) and programmed death protein-1(PD-1) in cytokine-induced killer cells(CIK) inhibiting the proliferation of cervical cancer cells,and to preliminarily analyze the possible mechanism of miR-15 b and PD-1.Methods:CIK cells were cultured by traditional methods.The immunophenotype of CIK cells was detected by flow cytometry.The effect of CIK cells on the proliferation of cervical cancer cells(SiHa) was detected by CCK-8 method.Real-time quantitative polymerase chain reaction(qRT-PCR) and Western blot(WB) were used to detect the expressions of miR-15 b and PD-1 in SiHa cells before and after co-culture of CIK cells.SiHa cells co-cultured with CIK cells were divided into three groups:Blank control group(without any treatment),NC group(with negative control plasmid of miR-15 b) and si-miR-15 b group(with miR-15 b siRNA).The expression levels of miR-15 b and PD-1 in each group were detected by qRT-PCR and WB,respectively.CCK-8 method was used to detect the proliferation of SiHa cells in each group.WB method was used to detect the expressions of proliferating cell nuclear antigen(PCNA) and Ki67.Results:The proliferation inhibition rate of SiHa cells in CIK cells co-culture group was significantly higher than that in SiHa cells alone-culture group(P<0.05).After co-culture of CIK cells,the expression of miR-15 b in SiHa cells was significantly higher than that in SiHa cells alone-culture group(P<0.05).While the expression of PD-1 protein was significantly lower(P<0.05).The expression level of miR-15 b and the inhibition rate of proliferation of SiHa cells in si-miR-15 b group were significantly lower than those in blank control group and NC group(P<0.05),while the expressions of PD-1,PCNA,and Ki67 protein were significantly increased(P<0.05).Conclusion:miR-15 b may inhibit the proliferation of cervical cancer cells by down-regulating the expression of PD-1 on CIK cells.
作者
牛志军
赵书君
杨立
Niu Zhijun;Zhao Shujun;Yang Li(Department of Obstetrics and Gynecology,the Third Affiliated Hospital of Zhengzhou University,Henan Zhengzhou 450000,China)
出处
《现代肿瘤医学》
CAS
2019年第20期3587-3592,共6页
Journal of Modern Oncology
基金
河南省高等学校重点科研项目(编号:19A320052)
