摘要
目的探究高尔基体磷蛋白3(GOLPH3)通过PI3K/AKT/GSK3β信号调控子宫内膜癌(EC)细胞增殖与凋亡的机制。方法将人子宫内膜腺癌HEC-1-B细胞分为对照组、GOLPH3组和GOLPH3+GDC-0941组。通过转染GOLPH3 pcDNA质粒过表达GOLPH3,PI3K抑制剂GDC-0941用于阻断PI3K/AKT/GSK3β通路。分别通过CCK-8法、克隆形成实验和流式细胞术检测细胞活力、细胞增殖能力和细胞凋亡。通过Western blotting检测PI3K/AKT/GSK3β通路中蛋白磷酸化水平。结果转染实验成功,且PI3K抑制剂GDC-0941不会影响GOLPH3 mRNA和蛋白的表达。对照组、GOLPH3组、GOLPH3+GDC-0941组的相对细胞活力分别为(100.00±4.63)%、(131.56±7.85)%、(97.85±7.36)%,3组间差异有统计学意义(F=7.437,P<0.001);GOLPH3组高于对照组(P<0.001),GOLPH3+GDC-0941组低于GOLPH3组(P<0.001)。3组的细胞克隆形成数目分别为(46.86±3.56)个、(89.32±4.78)个、(46.48±4.05)个,差异有统计学意义(F=20.437,P<0.001);GOLPH3组多于对照组(P<0.001),GOLPH3+GDC-0941组少于GOLPH3组(P<0.001)。3组的细胞凋亡率分别为(5.17±0.61)%、(2.34±0.56)%、(6.85±0.53)%,差异有统计学意义(F=11.643,P<0.001);GOLPH3组低于对照组(P<0.001),GOLPH3+GDC-0941组高于GOLPH3组(P<0.001)。3组磷酸化PI3K/PI3K水平分别为1.00±0.09、3.32±0.19、0.93±0.06,磷酸化AKT/AKT水平分别为1.00±0.11、4.63±0.63、1.15±0.16,磷酸化GSK3β/GSK3β水平分别为1.00±0.08、4.06±0.57、1.04±0.14,差异均有统计学意义(F=12.532,P<0.001;F=16.792,P<0.001;F=15.311,P<0.001);各蛋白磷酸化水平GOLPH3组均高于对照组(均P<0.001),GOLPH3+GDC-0941组均低于GOLPH3组(均P<0.001)。结论在EC细胞中,过表达GOLPH3可通过激活PI3K/AKT/GSK3β通路促进细胞增殖并抑制细胞凋亡,提示GOLPH3参与EC的发生和发展。
Objective To explore the mechanism of Golgi phosphoprotein 3(GOLPH3)regulating the proliferation and apoptosis of endometrial cancer(EC)cells through PI3K/AKT/GSK3βsignal.Methods Human endometrial adenocarcinoma HEC-1-B cells were divided into control group,GOLPH3 group and GOLPH3+GDC-0941 group.GOLPH3 was over-expressed by transfection of the GOLPH3 pcDNA.The PI3K inhibitor GDC-0941 was used to block the PI3K/AKT/GSK3βpathway.Cell viability,cell proliferation and apoptosis were measured by CCK-8 method,clone formation experiment and flow cytometry,respectively.The protein phosphorylation level in PI3K/AKT/GSK3βpathway was detected by Western blotting.Results Transfection experiments were successful,and the PI3K inhibitor GDC-0941 did not affect GOLPH3 mRNA and protein expression.The relative cell viability of the control group,the GOLPH3 group and the GOLPH3+GDC-0941 group were(100.00±4.63)%,(131.56±7.85)%and(97.85±7.36)%,and the difference among the three groups were significant(F=7.437,P<0.001).The cell viability of the GOLPH3 group was higher than that of the control group(P<0.001).The cell viability of the GOLPH3+GDC-0941 group was lower than that of the GOLPH3 group(P<0.001).The numbers of cell clones in the three groups were 46.86±3.56,89.32±4.78 and 46.48±4.05,and the difference was significant(F=20.437,P<0.001).GOLPH3 group had more clones than control group(P<0.001).The number of clones formed in the GOLPH3+GDC-0941 group was less than that in the GOLPH3 group(P<0.001).The apoptosis rates of the three groups were(5.17±0.61)%,(2.34±0.56)%and(6.85±0.53)%,and the difference was significant(F=11.643,P<0.001).The apoptosis rate of the GOLPH3 group was lower than that of the control group(P<0.001),and the apoptosis rate of the GOLPH3+GDC-0941 group was higher than that of the GOLPH3 group(P<0.001).The phosphorylated PI3K/PI3K levels of the three groups were 1.00±0.09,3.32±0.19 and 0.93±0.06,respectively;phosphorylated AKT/AKT levels were 1.00±0.11,4.63±0.63 and 1.15±0.16,respectively;phosphorylated GSK3β/GSK3βlevels were 1.00±0.08,4.06±0.57 and 1.04±0.14,respectively.The differences were statistically significant(F=12.532,P<0.001;F=16.792,P<0.001;F=15.311,P<0.001).The phosphorylation levels of each protein in the GOLPH3 group were higher than those in the control group(all P<0.001),and the GOLPH3+GDC-0941 group were lower than the GOLPH3 group(all P<0.001).Conclusion In EC cells,over-expression of GOLPH3 can promote cell proliferation and inhibit apoptosis by activating the PI3K/AKT/GSK3βpathway,suggesting that GOLPH3 is involved in the occurrence and development of EC.
作者
罗春翠
袁超燕
陈庆芬
Luo Chuncui;Yuan Chaoyan;Chen Qingfen(Department of Gynecology,Minda Hospital Affiliated to Hubei Minzu University,Enshi 445000,China;Obstetrics and Gynecology of the Fourth People′s Hospital of Shaanxi Province,Xi′an 710043,China)
出处
《国际肿瘤学杂志》
CAS
2020年第2期65-69,共5页
Journal of International Oncology
