RACK1通过调控脂质合成促进子宫颈癌ME180细胞侵袭、迁移及增殖

RACK1 promotes the invasion,migration and proliferation of ME180 cells by regulating lipid synthesis

目的探讨下调RACK1表达对子宫颈癌ME180细胞的脂质合成及迁移、侵袭和增殖的影响。方法qRT-PCR法检测子宫颈癌组织和细胞以及正常子宫颈组织和细胞中RACK1的mRNA表达水平,随后利用Western blot法检测RACK1蛋白表达;BODIPY染色观察H8、ME180和SiHa细胞中脂滴含量。采用慢病毒转染技术构建RACK1干扰稳转细胞株,利用qRT-PCR法及Western blot法验证敲降效果。qRT-PCR法检测组织和细胞中脂肪酸合成关键基因FASN和ACC1的表达,Western blot法检测FASN和ACC1的蛋白表达;BODIPY染色和电镜观察脂滴含量变化;采用柠檬酸、脂肪酸试剂盒检测子宫颈癌ME180细胞中柠檬酸及脂肪酸含量;将shRACK1-ME180组细胞进行油酸(OA)处理后,Transwell观察OA处理前后细胞的侵袭及转移能力;平板克隆实验检测细胞增殖情况。结果子宫颈癌组织中RACK1 mRNA表达(2.38±1.78)高于正常子宫颈组织(0.43±0.47)(P<0.001);子宫颈癌细胞ME180与SiHa中RACK1的mRNA(1.67±0.05、1.39±0.08)及蛋白(1.58±0.08、1.32±0.08)表达均高于H8细胞(1.00±0.03、0.41±0.08)(P均<0.05),且ME180细胞中的表达最高;ME180细胞内脂滴含量(1.10±0.15)较SiHa细胞(0.36±0.21)丰富,且2株子宫颈癌细胞内脂质含量均高于H8细胞(0.15±0.01)(P<0.05);子宫颈癌组织中FASN(2.98±1.53)和ACC1(2.41±1.25)mRNA表达量显著高于正常子宫颈组织(0.85±0.37、0.69±0.28)(P<0.05),且RACK1 mRNA表达与FASN和ACC1 mRNA表达呈正相关(r=0.693,P<0.001;r=0.752,P<0.001);shRACK1-ME180组FASN(0.28±0.02 vs 1.00±0.01)和ACC1表达(0.24±0.03 vs 1.00±0.01)及细胞内脂质(0.62±0.09 vs 1.09±0.16)、脂肪酸(0.94±0.07 vs 2.13±0.27)及柠檬酸(3.46±0.77 vs 5.89±0.47)含量均低于shNON-ME180组(P均<0.05);且shRACK1-ME180组细胞侵袭、迁移及增殖能力显著下降(P<0.001)。经OA处理后,shRACK1-ME180组细胞侵袭、迁移及增殖能力恢复,且与shNON-ME180组相近。结论下调RACK1表达干扰子宫颈癌ME180细胞脂质合成,抑制细胞侵袭、迁移及增殖能力,为探究子宫颈癌的发生、发展提供理论依据。

Purpose To investigate the effects of down-regulated RACK1 expression on lipid synthesis,migration,invasion and proliferation of ME180 cell lines.Methods The transcription level of RACK1 in cervical cancer tissues/cells and normal cervical tissues/cells was detected by qRT-PCR,and the protein level of RACK1 of cell lines was detected by Western blot.The content of lipid droplets in H8,ME180 and SiHa cells was observed by BODIPY staining.Lentivirus transfection technology was used to construct stable shRACK1 cell lines,and the effect was verified by qRT-PCR and Western blot.qRT-PCR was used to detect the expression of FASN and ACC1 in tissues and cells which are the key genes for fatty acid synthesis,and Western blot was used to detect the protein expression of FASN and ACC1 of cell lines.The changes of lipid droplet content were observed by BODIPY staining and electron microscopy.The contents of citric acid and fatty acid in cervical cells were detected by citric acid/fatty acid determination kit.shRACK1-ME180 cells were treated with oleic acid(OA),and the invasion and migration of shRACK1-ME180 cells before and after OA were observed by Transwell assay.Cell proliferation was detected by plate cloning experiment.Results The mRNA expression of RACK1 in cervical cancer tissue(2.38±1.78)was higher than that in normal cervical tissue(0.43±0.47,P<0.001).The mRNA(1.67±0.05,1.39±0.08)and protein(1.58±0.08,1.32±0.08)levels of RACK1 in ME180 and SiHa cells were higher than those in H8 cells(1.00±0.03,0.41±0.08)(P<0.05),and the mRNA and protein expression of RACK1 in ME180 cells was the highest.The content of lipids in ME180 cells(1.10±0.15)was more abundant than that in SiHa cells(0.36±0.21),and the content of lipids in ME180 and SiHa cervical cancer cells was higher than that in H8 cells(0.15±0.01,P<0.05).The mRNA expression of FASN(2.98±1.53)and ACC1(2.41±1.25)gene in cervical cancer tissues was significantly higher than those in normal cervical tissues(0.85±0.37,P<0.05,0.69±0.28,P<0.05),and the mRNA expression of RACK1 was positively correlated with the mRNA expression of FASN and ACC1 gene(r=0.693,P<0.001,r=0.752,P<0.001).The expression of FASN(0.28±0.02 vs 1.00±0.01,P<0.05)and ACC1(0.24±0.03 vs 1.00±0.01,P<0.05)and intracellular lipid content(0.62±0.09 vs 1.09±0.16,P<0.05),fatty acid(0.94±0.07 vs 2.13±0.27,P<0.05)and citric acid(3.46±0.77 vs 5.89±0.47,P<0.05)in shRACK1-ME180 were lower than those in shNON-ME180 cell.The invasion,migration and proliferation of shRACK1-ME180 were significantly decreased(P<0.001).After oleic acid(OA)treatment,the invasion,migration and proliferation of shRACK1-ME180 cells were restored,which similar to that of shNON-ME180 cells.Conclusion Knockdown of RACK1 influence lipid synthesis of ME180 cells and inhibits the invasion,migration and proliferation of ME180 cells,providing theoretical reference for the study of the occurrence and development of cervical cancer.