SKA3蛋白在胆管癌组织中的表达及其体外对胆管癌SSP-25细胞功能和PI3K-AKT信号通路的影响

Expression of SKA3 protein in cholangiocarcinoma tissues and its effect on the function of cholangiocarcinoma SSP-25 cells in vitro and PI3K-AKT signaling pathway

目的探讨胆管癌组织中SKA3蛋白的表达和临床意义,以及体外干扰SKA3表达对胆管癌SSP-25细胞增殖、侵袭和迁移的影响和可能机制。方法回顾性收集2015年1月至2020年12月首都医科大学附属北京友谊医院172例远端胆管癌患者临床病理资料及癌组织、癌旁组织标本。采用免疫组织化学法检测癌组织和癌旁组织中SKA3蛋白的表达水平。向胆管癌SSP-25细胞转染SKA3小干扰RNA(siRNA)(si-SKA3组),以未经处理的SSP-25细胞为对照组。采用细胞免疫荧光染色和蛋白质印迹法检测转染效果;采用CCK-8法和细胞集落形成实验观察细胞增殖变化;采用细胞划痕实验检测细胞侵袭能力;采用蛋白质印迹法检测PI3K-AKT信号通路相关蛋白的表达情况。结果172例胆管癌患者中,男性116例,女性56例;年龄<60岁54例,≥60岁118例。胆管癌组织SKA3蛋白阳性率高于癌旁组织[78.49%(135/172)比13.95%(24/172)],差异有统计学意义(χ2=42.78,P<0.01);有神经侵犯[84.35%(124/147)比44.00%(11/25)]、淋巴结转移[88.78%(87/98)比64.86%(48/74)]患者癌组织SKA3蛋白阳性率均高于无神经侵犯、淋巴结转移患者,差异均有统计学意义(均P<0.05),年龄、性别、TNM分期、肿瘤分化分层患者的癌组织SKA3蛋白阳性率差异均无统计学意义(均P>0.05)。CCK-8法检测示,培养72 h si-SKA3组SSP-25细胞增殖能力(以450 nm处吸光度值表示)低于对照组(0.56±0.05比0.83±0.06),差异有统计学意义(t=3.06,P=0.006)。集落形成实验显示,培养2周后si-SKA3组SSP-25细胞集落形成数少于对照组。培养24 h,si-SKA3组和对照组SSP-25细胞划痕愈合率分别为(31±6)%、(72±5)%,差异有统计学意义(t=5.63,P=0.013)。蛋白质印迹法检测显示,PI3K-AKT信号通路中p-PI3K、p-AKT蛋白相对表达量均低于对照组,差异均有统计学意义(均P<0.05)。结论胆管癌组织中SKA3蛋白高表达,与神经侵犯、淋巴结转移可能相关。干扰SKA3表达可抑制胆管癌SSP-25细胞的增殖和侵袭,其机制可能与抑制PI3K-AKT信号通路有关。

Objective To investigate the expression and clinical significance of SKA3 protein in cholangiocarcinoma,and the effect of interfering SKA3 expression in vitro on the proliferation,invasion,and migration of cholangiocarcinoma SSP-25 cells,as well as its possible mechanism.Methods The clinicopathological data,cancer tissues,and paracancerous tissues from 172 patients with distal cholangiocarcinoma in Beijing Friendship Hospital,Capital Medical University between January 2015 and December 2020 were retrospectively collected.Immunohistochemical method was used to detect the expression level of SKA3 protein in cancer tissues and paracancerous tissues.Transfection of SKA3 small interfering RNA(siRNA)into cholangiocarcinoma SSP-25 cells was used as si-SKA3 group,and the untreated SSP-25 cells were used as the control group.Cell immunofluorescence staining and Western blot were used to detect the transfection effect;CCK-8 method and cell colony formation experiment were used to observe changes in cell proliferation;cell scratch assay was used to monitor cell invasion;Western blot was used to detect the expression of PI3K-AKT signaling pathway related proteins.Results Among 172 patients with cholangiocarcinoma,there were 116 males and 56 females;the age of 54 cases was under 60 years,and age of 118 cases was equal to or more than 60 years.The positive rate of SKA3 protein in cholangiocarcinoma tissues was higher than that in paracancerous tissues[78.49%(135/172)vs.13.95%(24/172)],and the difference was statistically significant(χ2=42.78,P<0.01).The positive rate of SKA3 protein in cancer tissues of cholangiocarcinoma patients with nerve invasion[84.35%(124/147)vs.44.00%(11/25)]and lymph node metastasis[88.78%(87/98)vs.64.86%(48/74)]was higher than that of patients without nerve invasion and without lymph node metastasis,and the differences were statistically significant(all P<0.05).There were no statistically significant differences in the positive rate of SKA3 protein in cancer tissues of patients stratified by age,gender,tumor diameter,TNM stage,and tumor differentiation(all P>0.05).The CCK-8 method showed that after 72 h of cultivation,the proliferation ability of SSP-25 cells in the si-SKA3 group(expressed as absorbance value at 450 nm)was lower than that in the control group(0.56±0.05 vs.0.83±0.06),and the difference was statistically significant(t=3.06,P=0.06).After 2 weeks of cultivation,the colony formation experiment showed that the number of colony formation of SSP-25 cells in the si-SKA3 group was lower than that in the control group.After 24 h of cultivation,the scratch healing rates of SSP-25 cells in the si-SKA3 group and the control group were(31±6)%and(72±5)%,respectively,and the difference was statistically significant(t=5.63,P=0.013).Western blot analysis showed that the relative expression levels of p-PI3K and p-AKT proteins in the PI3K-AKT signaling pathway were lower than those in the control group,and the difference was statistically significant(all P<0.05).Conclusions SKA3 protein is highly expressed in cholangiocarcinoma tissues,and may related to nerve invasion and lymph node metastasis.Interfering SKA3 expression can inhibit the proliferation and invasion of cholangiocarcinoma SSP-25 cells,and its mechanism may be related to the inhibition of the PI3K-AKT signaling pathway.