摘要
目的探究氧调节蛋白150(ORP150)对宫颈癌细胞凋亡与自噬的影响及其作用机制。方法采用RT-qPCR和Western blot检测正常宫颈上皮细胞系(HUCEC)、宫颈癌细胞系(C33a、CaSki、HeLa、SiHa)中ORP150表达;将SiHa细胞分为对照组、shNC组、shORP150组、shNC+4-苯丁酸(4-PBA)组、shORP150+4-PBA组,Western blot检测葡萄糖调节蛋白78(GRP78)、内质网调节激酶(PERK)、真核起始因子2α(eIF2α)、C/EBP同源蛋白(CHOP)蛋白表达,CCK-8法检测细胞生长抑制率,流式细胞术检测细胞凋亡率,细胞免疫荧光染色检测微管相关蛋白1轻链3B(LC3B)表达,Western blot检测LC3Ⅱ/LC3Ⅰ比值和Beclin1蛋白表达。结果4种宫颈癌细胞系(C33a、CaSki、HeLa、SiHa)中的ORP150 mRNA和蛋白表达均高于正常宫颈上皮细胞系HUCEC(P<0.05);shORP150组SiHa细胞中ORP150 mRNA和蛋白相对表达量低于对照组、shNC组(P<0.05);与对照组、shNC组比较,shORP150组SiHa细胞中GRP78、PERK、eIF2α、CHOP的蛋白表达上调,细胞生长抑制率和细胞凋亡率增加,LC3B荧光染色强度增加,LC3Ⅱ/LC3Ⅰ比值和Beclin1蛋白表达水平上调(P<0.05)。与shORP150组比较,shORP150+4-PBA组SiHa细胞中GRP78、PERK、eIF2α、CHOP的蛋白表达下调,细胞生长抑制率和细胞凋亡率减少,LC3B荧光染色强度降低,LC3Ⅱ/LC3Ⅰ比值和Beclin1蛋白表达水平也下调(P<0.05)。结论ORP150在宫颈癌细胞系中上调表达,敲减其表达能够通过触发内质网应激诱导宫颈癌细胞发生凋亡与自噬,进而发挥抗宫颈癌的作用。
Objective To investigate the effect of oxygen-regulated protein 150(ORP150)on apoptosis and autophagy in cervical cancer cells and its mechanism.Methods RT-qPCR and Western blot were used to detect the expression of ORP150 in normal cervical epithelial cell lines(HUCEC)and cervical cancer cell lines(C33a,CaSki,HeLa,SiHa).SiHa cells were divided into control group,shNC group,shORP150 group,shNC+4-phenylbutyric acid(4-PBA)group,shORP150+4-PBA group,Western blot was used to detect the expression of glucose-regulated protein 78(GRP78),endoplasmic reticulum-regulated kinase(PERK),eukaryotic initiation factor 2α(eIF2α),and C/EBP homologous protein(CHOP),CCK-8 method was used to detect the cell growth inhibition rate.Flow cytometry was used to detect the cell apoptosis rate.Cell immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3B(LC3B).Western blot was used to detect the ratio of LC3Ⅱ/LC3Ⅰ and Beclinl protein expression.Results The mRNA and protein expressions of ORP150 in 4 cervical cancer cell lines(C33a,CaSki,HeLa,SiHa)were higher than those in normal cervical epithelial cell lines HUCEC(P<0.05).The relative expression levels of ORP150 mRNA and protein in SiHa cells in shORP150 group were lower than those in control group and shNC group(P<0.05).Compared with the control group and shNC group,the protein expressions of GRP78,PERK,eIF2α and CHOP in SiHa cells in shORP150 group were up-regulated,the cell growth inhibition rate and apoptosis rate were increased,and the fluorescent staining intensity of LC3B was increased,LC3Ⅱ/LC3Ⅰ ratio and Beclinl protein expression level were up-regulated(P<0.05).Compared with shORP150 group,the protein expressions of GRP78,PERK,eIF2α and CHOP in SiHa cells in shORP150+4-PBA group were down-regulated,while the cell growth inhibition rate and apoptosis rate were decreased,the fluorescence staining intensity of LC3B was decreased,the ratio of LC3Ⅱ/LC3Ⅰ and the expression level of Beclinl protein were decreased(P<0.05).Conclusion The expression of ORP150 is up-regulated in cervical cancer cell lines,and the inhibition of ORP150 expression can induce apoptosis and autophagy of cervical cancer cells by triggering endoplasmic reticulum stress,so as to play an anti-cervical cancer role.
作者
朱静
陆月梅
王秀美
丁其培
ZHU Jing;LU Yuemei;WANG Xiumei;DING Qipei(Gynecology Department,Hai'an People's Hospital,Hai'an,Jiangsu 226600,China)
出处
《中国优生与遗传杂志》
2025年第9期1889-1895,共7页
Chinese Journal of Birth Health & Heredity
基金
南通市基础科学研究和社会民生科技计划项目(MSZ2022131)
南通大学临床医学专项科研基金项目立项(2024LZ024)。
关键词
宫颈癌
氧调节蛋白150
凋亡
自噬
内质网应激
cervical cancer
oxygen regulatory protein 150
apoptosis
autophagy
endoplasmic reticulum stress
