lncRNA ZFAS1调控HMGA2表达对肝癌细胞增殖、侵袭、迁移的影响

Effects of lncRNA ZFAS1 on the proliferation,invasion and migration of liver cancer cells by regulating HMGA2 expression

目的探讨长链非编码RNA(lncRNA)锌指蛋白反义链1(ZFAS1)通过调控高迁移率族蛋白A2(HMGA2)表达对肝癌细胞增殖、侵袭、迁移的影响。方法利用GEPIA分析癌症基因组图谱(TCGA)数据库中肝癌组织和正常组织lncRNA ZFAS1和HMGA2 mRNA表达水平。实时定量反转录聚合酶链反应(qRT-PCR)检测在本院收集的58对肝癌组织、癌旁组织以及体外培养的人正常肝细胞(QSG-7701)和肝癌细胞系(Hep3b、SK-HEP-1、HuH-7、HepG2)中lncRNA ZFAS1和HMGA2 mRNA表达水平。利用脂质体转染试剂对HepG2细胞进行转染,并分成Blank组、sh-NC组、sh-lncRNA ZFAS1组、sh-lncRNA ZFAS1+si-NC组和sh-lncRNA ZFAS1+si-HMGA2组,48 h后,细胞计数试剂盒8(CCK8)法评估细胞增殖情况;Transwell实验和划痕实验检测细胞侵袭和迁移;免疫共沉淀实验检测lncRNA ZFAS1和HMGA2的结合关系;蛋白免疫印迹法检测HMGA2蛋白水平。结果与正常组织或癌旁组织相比,肝癌组织中lncRNA ZFAS1和HMGA2 mRNA表达均显著升高(P<0.05)。与QSG-7701相比,Hep3b、SK-HEP-1、HuH-7、HepG2细胞中lncRNA ZFAS1和HMGA2 mRNA表达明显增加(P<0.05),其中lncRNA ZFAS1和HMGA2 mRNA在HepG2细胞中表达最高,因此选择该细胞进行转染实验。转染sh-lncRNA ZFAS1后,HepG2细胞中lncRNA ZFAS1表达明显降低,24 h、48 h、72 h的OD值明显减小,侵袭和迁移细胞数目明显减少,24 h的平均划痕距离明显增加(P<0.05)。lncRNA ZFAS1和HMGA2在肝癌HepG2细胞中直接结合。同时转染sh-lncRNA ZFAS1和si-HMGA2后,HepG2细胞中HMGA2蛋白表达明显下降,24 h、48 h、72 h的OD值明显降低,侵袭和迁移细胞数显著减少,24 h的平均划痕距离明显增加(P<0.05)。结论lncRNA ZFAS1通过直接结合HMGA2促进肝癌细胞增殖、侵袭、迁移。

Objective To investigate the impacts of long non-coding RNA(lncRNA)zinc finger protein antisense strand 1(ZFAS1)on the proliferation,invasion and migration of liver cancer cells by regulating the expression of high mobility group group A2(HMGA2).Methods The expression levels of lncRNA ZFAS1 and HMGA2 mRNA in liver cancer tissues and normal tissues in The Cancer Genome Atlas(TCGA)database were analyzed by GEPIA.Real-time quantitative reverse transcription polymerase chain reaction(QRT-PCR)was performed to measure the lncRNA ZFAS1 and HMGA2 mRNA expression levels in 58 pairs of liver cancer tissues and adjacent tissues in the hospital,and human normal hepatocytes(QSG-7701)and liver cancer cell lines(Hep3b,SK-HEP-1,HuH-7,HepG2)cultured in vitro.HepG2 cells were transfected with lipofection reagent and separated into Blank group,sh-NC group,sh-lncRNA ZFAS1 group,sh-lncRNA ZFAS1 combine si-NC group and sh-lncRNA ZFAS1 combine si-HMGA2 group.After 48 h,Cell Counting Kit 8(CCK8)method was performed to assess the cell proliferation;Transwell assay and scratch assay were performed to measure cell invasion and migration.Co-immunoprecipitation assay was performed to measure the binding relationship between lncRNA ZFAS1 and HMGA2.Western blotting was performed to measure the protein level of HMGA2.Results Compared with normal tissues or adjacent tissues,the mRNA expression of lncRNA ZFAS1 and HMGA2 in liver cancer tissues was greatly increased(P<0.05).Compared with QSG-7701,the mRNA expression of lncRNA ZFAS1 and HMGA2 was greatly increased in Hep3b,SK-HEP-1,HuH-7,and HepG2 cells(P<0.05),among them,the expression of lncRNA ZFAS1 and HMGA2 mRNA was the highest in HepG2 cells,so they were selected for transfection experiments.After transfection with sh-lncRNA ZFAS1,the expression of lncRNA ZFAS1 in HepG2 cells was greatly decreased,the OD value at 24 h,48 h,and 72 h was greatly decreased,the numbers of invasive and migrating cells were greatly decreased,and the average scratch distance at 24 h was greatly increased(P<0.05).lncRNA ZFAS1 and HMGA2 were directly bound in hepatoma HepG2 cells.After simultaneous transfection of sh-lncRNA ZFAS1 and si-HMGA2,the expression of HMGA2 protein in HepG2 cells was greatly decreased,the OD value at 24 h,48 h,and 72 h was greatly decreased,the numbers of invasive and migrating cells were greatly decreased,and the average scratch distance at 24 h was greatly increased(P<0.05).Conclusion lncRNA ZFAS1 promotes the proliferation,invasion and migration of liver cancer cells by directly binding to HMGA2.