摘要
目的探讨小干扰RNA沉默宫颈癌细胞中PHF19基因表达后对宫颈癌细胞的增殖、凋亡以及对NF-κB信号通路的影响。方法培养人宫颈癌He La细胞,根据转染的si RNA-PHF19及其浓度和时间进行分组。MTT比色法筛选出最佳的si RNA-PHF19及其作用浓度与时间,q RT PCR和Western Blot检测加入转染试剂si RNA-PHF19后PHF19基因及蛋白的表达量,Ed U检测加入转染试剂si RNA-PHF19后He La细胞的增殖情况,TUNEL法检测加入转染试剂si RNA-PHF19后He La细胞的凋亡情况,q RT PCR检测加入转染试剂si RNA-PHF19后Bax、Bcl-2、Caspase-3、TNF、NFKBIA、p53基因的表达量。结果通过q RT PCR分析显示,对比正常组He La细胞中的PHF19的表达量,在24 h时,加入si RNA-PHF19-1-50μM、si RNAPHF19-3-10μM干扰试剂对PHF19基因的抑制极其显著(P<0.0001),加入si RNA-PHF19-2-50μM、si RNA-PHF19-3-50μM干扰试剂对PHF19基因的抑制最显著(P<0.0001)。通过Western blot检测24h的正常He La细胞以及加入转染试剂si RNAPHF19-2-50μM、si RNA-PHF19-3-50μM和Non-specific control(NC)组的He La细胞中PHF19蛋白的表达发现,对比正常组He La细胞中PHF19的表达,加入si RNA-PHF19-2-50μM和NC组干扰试剂的He La细胞中PHF19降低有显著的统计学差异(P<0.01)。Ed U检测细胞增殖,与He La组相比,加入转染试剂si RNA-PHF19-2-50μM、si RNA-PHF19-3-50μM和NC后Ed U阳性率极低,表现出抑制细胞增殖的作用。TUNEL法检测细胞凋亡,与He La组细胞相比,si RNA-PHF19-2-50μM处理的He La细胞TUNEL阳性细胞显著增多。通过q RT PCR分析显示,对比正常组He La细胞中Bax、Bcl-2和Caspase-3基因的表达量无统计学意义,对比正常组He La细胞中TNF的表达,加入si RNA-PHF19-2-50μM干扰试剂组He La细胞中TNF基因表达量上调极其显著(P<0.001),加入si RNA-PHF19-3-50μM和NC干扰试剂组He La细胞中TNF基因表达量下降有统计学意义(P<0.05)。对比正常组He La细胞中NFKBIA的表达,加入si RNA-PHF19-2-50μM、NC组干扰试剂的NFKBIA基因表达上调最显著(P<0.0001)。结论 PHF19基因在He La细胞中高表达,si RNA-PHF19干扰He La细胞后,会对He La细胞的增殖与凋亡产生影响,其主要作用是使He La细胞发生凋亡,He La细胞凋亡的产生主要是由死亡受体途径介导的,在死亡受体途径中,TNF激活下游的NF-κB通路,使NFKBIA过表达,NFKBIA可能在He La细胞中介导p53基因失活。
Objective To investigate the effect of small interfering RNA on the proliferation, apoptosis and NF-κB signaling pathway of HeLa cells by silencing of PHF19 gene expression in HeLa cells.Methods The optimal siRNA-PHF19 and its concentration and time were screened by MTT colorimetric assay.By transfected with siRNA-PHF19, the expression of PHF19 gene and protein was detected by qRT-PCR and Western Blot, the proliferation was detected by EdU method, the apoptosis was detected by TUNEL and the expression of Bax, Bcl-2, Caspase-3, TNF, NFKBIA and p53 were detected by qRT-PCR.Results By qRT-PCR and Western blot, compared with the expression of PHF19 in normal HeLa cells, the expression of PHF19 gene and protein was reduced extremely at 24 h ( P <0.0001)( P <0.01).The positive rate of EdU was extremely reduced and the TUNEL-positive cells were increased after transfection.There was no significant difference in the expression of Bax, Bcl-2 and Caspase-3 genes.The expression of TNF gene was increased significantly by added siRNA- PHF19-2-50 μM ( P <0.001).The up-regulation of NFKBIA gene expression was the most significant ( P <0.0001).Conclusion The expression of PHF19 gene is highly expressed in HeLa cells.The interference of siRNA-PHF19 on HeLa cells plays a key role in inhibiting proliferation and inducing apoptosis of HeLa cells.The apoptosis of HeLa cells is mainly caused by death receptor pathway.In the death receptor pathway, TNF activates the downstream NF-κB pathway and overexpresses NFKBIA. NFKBIA may mediate p53 gene inactivation in HeLa cells.
作者
陈梦茜
金在顺
徐春艳
刘瑞芳
杨光
董程骥
CHEN Meng-xi(Department of Pathology, Mudanjiang Medical University, Mudanjiang 157011,China)
出处
《牡丹江医学院学报》
2019年第2期16-21,共6页
Journal of Mudanjiang Medical University
基金
黑龙江省省属高校基本科研业务费项目(2018-KYYWFMY-0008)
牡丹江市科学技术计划项目(Z2017s0050)