知母皂苷AⅢ对肝癌细胞Akirin2基因表达及细胞增殖的抑制作用

The inhibitory effect of saponin AⅢfrom Anemarrhena asphodeloides on Akirin2 gene expression and cell proliferation in liver cancer cells

目的:研究Akirin2在人肝癌组织中的表达及知母皂苷AⅢ(TA-Ⅲ)抑制人肝癌细胞(HepG2)增殖和促进凋亡的潜在机制。方法:应用RT-qPCR法和免疫组织化学法检测103例肝癌组织中Akirin2的表达。应用MTT法分别检测Akirin2过表达组、Akirin2 inhibitor组以及不同浓度TA-Ⅲ(0、5、10、20、40 mol/L)、紫杉醇组(80 g/ml)干预后对HepG2细胞增殖活力的影响;平板克隆实验和流式细胞术分别检测Akirin2过表达、Akirin2抑制以及TA-Ⅲ干预后对HepG2细胞克隆、凋亡的影响;Akirin2和凋亡蛋白采用WB法检测。TA-Ⅲ(40 mol/L)处理HepG2细胞的同时转染Akirin2 mimics(TA-Ⅲ+Akirin2过表达组),比较过表达Akirin2对TA-Ⅲ干预细胞增殖、凋亡的影响。结果:肝癌患者肝癌组织中Akirin2呈显著高表达(P<0.05),且Akirin2的高表达与患者的不良预后独立相关(P<0.05)。Akirin2过表达组的细胞增殖能力、克隆能力显著高于对照组(P<0.05),凋亡相关蛋白表达和细胞凋亡率低于对照组(P<0.05)。Akirin2 inhibitor组的细胞增殖能力、克隆能力显著低于对照组(P<0.05),凋亡相关蛋白表达和细胞凋亡率高于对照组(P<0.05)。TA-Ⅲ处理组的HepG2细胞增殖活力显著抑制,细胞增殖活性抑制与干预浓度、时间成正比。TA-Ⅲ不同浓度处理HepG2细胞增殖活力、细胞克隆能力、Akirin2蛋白表达显著低于对照组(P<0.05),细胞凋亡率和凋亡蛋白显著高于对照组(P<0.05)。Akirin2过表达后,TA-Ⅲ对HepG2细胞的细胞增殖能力细胞凋亡率和凋亡相关蛋白的影响作用被部分逆转。结论:Akirin2在肝癌中呈显著高表达,TA-Ⅲ可通过抑制Akirin2表达抑制癌细胞增殖,促进凋亡。

Objective:To investigate the expression of Akirin2 in human liver cancer tissue and the potential mechanism of anemone saponin AⅢ(TA-Ⅲ)inhibiting the proliferation and promoting apoptosis of human liver cancer cells(HepG2).Methods:The RT-qPCR and immunohistochemistry was used to detect the expression of Akirin2 in 103 liver cancer tissues.in vitro experiment.The MTT method were used to detect the effects of Akirin2 overexpression group,Akirin2 inhibitor group,as well as different concentrations of TA-Ⅲ(0,5,10,20,40 mol/L)and paclitaxel group(80 g/ml)on the proliferation activity of HepG2 cells after intervention,plate cloning experiment.The flow cytometry was used to detect the effects of Akirin2 overexpression,Akirin2 inhibition,and TA-Ⅲintervention on HepG2 cell cloning and apoptosis,respectively;Akirin2 and apoptotic protein were detected using the WB method.Treatment of HepG2 cells with TA-Ⅲ(40 mol/L)and simultaneous transfection of Akirin2 mimics(TA-Ⅲ+Akirin2 overexpression group)were performed to compare the effects of overexpression of Akirin2 on TA-Ⅲintervention in cell proliferation and apoptosis.Results:Akirin2 was significantly overexpressed in the tissues of liver cancer patients(P<0.05),and the overexpression of Akirin2 was independently associated with poor prognosis in patients(P<0.05).The cell proliferation and cloning ability of the Akirin2 overexpression group were significantly higher than those of the control group(P<0.05),while the expression of apoptosis related proteins and cell apoptosis rate were lower than those of the control group(P<0.05).The cell proliferation and cloning ability of the Akirin2 inhibitor group were significantly lower than those of the control group(P<0.05),while the expression of apoptosis related proteins and cell apoptosis rate were higher than those of the control group(P<0.05).The proliferation activity of HepG2 cells in the TA-Ⅲtreatment group was significantly inhibited,and the inhibition of cell proliferation activity was proportional to the intervention concentration and time.The proliferation activity,cell cloning ability,and Akirin2 protein expression of HepG2 cells treated with TA-Ⅲat different concentrations were significantly lower than those of the control group(P<0.05),and the cell apoptosis rate and apoptotic protein were significantly higher than those of the control group(P<0.05).After overexpression of Akirin2,the effects of TA-Ⅲon the cell proliferation ability,apoptosis rate,and apoptosis related proteins of HepG2 cells were partially reversed.Conclusion:Akirin2 is significantly overexpressed in liver cancer,and TA-Ⅲcan inhibit cancer cell proliferation and promote apoptosis by inhibiting Akirin2 expression.