摘要
目的长链非编码RNA肿瘤易感性候选基因9(long non-coding RNA tumor susceptibility candidate gene 9,lncRNACASC9)有助于肿瘤的发生和发展,但它是否影响着肝细胞癌(hepatocellular carcinoma,HCC)细胞的干性特征罕见报道。本研究旨在探讨LncRNA CASC9对HCC细胞干性特征的影响及作用机制。方法培养人HCC细胞系SNU-475、HepG2、SNU-398、Huh-7和人永生化肝上皮细胞L02,实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)法检测各细胞中LncRNA CASC9表达水平。将Huh-7细胞分为空白对照组(Control)、siRNA阴性对照组(siRNA-NC)和CASC9 siRNA干扰组(siRNA-CASC9),并将CASC9 siRNA干扰质粒及其阴性对照质粒转染至Huh-7细胞中,采用qRT-PCR检测转染后细胞中lncRNACASC9表达水平及干性相关基因OCT-4、SOX2、Bmi1和Nanog等mRNA水平;克隆形成实验和细胞成球实验检测转染后细胞的干性细胞特征,流式细胞仪检测干细胞(EpCAM^+AFP^+)亚群的比例,蛋白质印迹法检测转染后细胞中OCT-4、SOX2、Bmi1、Nanog、p-STAT3、STAT3、KLF4和c-Myc等蛋白表达水平。结果 L02、SNU-475、HepG2、SNU-398和Huh-7细胞系培养48h后,细胞中lncRNACASC9表达量分别为1.03±0.07、2.06±0.19、1.77±0.20、2.88±0.14和3.63±0.37,差异有统计学意义,F=64.115,P<0.001。其中SNU-475(P<0.001)、HepG2(P=0.002)、SNU-398(P<0.001)和Huh-7(P<0.001)细胞中LncRNACASC9表达量均低于L02细胞。CASC9干扰48h后,siRNA-CASC9组细胞中lncRNA CASC9表达量为0.41±0.05,低于Control组(1.03±0.07)和siRNA-NC组(1.01±0.03),F=144.970,P<0.001;siRNA-CASC9组细胞克隆形成数为(45.00±10.81)个,低于Control组(277.33±7.77)个和siRNA-NC组(264.67±9.71)个,F=565.358,P<0.001;siRNA-CASC9组细胞成球率为(12.74±2.56)%,低于Control组的(25.93±3.89)%和siRNA-NC组的(24.61±4.72)%,F=10.810,P=0.010;siRNA-CASC9组EpCAM^+AFP^+细胞亚群比例为(4.68±0.33)%,低于Control组的(8.64±0.84)%和siRNA-NC组的(7.91±1.08)%,F=20.289,P=0.002;siRNA-CASC9组细胞中OCT-4、Nanog、SOX2和Bmi1等mRNA和蛋白相对表达水平分别低于Control组和siRNA-NC组,差异有统计学意义,P<0.05;siRNA-CASC9组细胞中p-STAT3/STAT3、KLF4和c-Myc等蛋白相对表达水平均低于Control组和siRNA-NC组,F值分别为13.525、25.122和35.038,均P<0.05。结论 LncRNACASC9在HCC细胞系中的表达水平高于正常肝细胞,干扰lncRNACASC9表达能降低HCC细胞的干性特征,其机制可能与STAT 3信号通路的抑制有关。
OBJECTIVE Long non-coding RNA tumor susceptibility candidate gene 9(lncRNACASC9)contributes to the occurrence and development of tumor,but whether it affects the stemness characteristics of liver cancer cells has not been reported.This study aimed to explore the effect of lncRNACASC9 on the stemness characteristics of liver cancer cells and the mechanism.METHODS Cultured hepatocellular carcinoma cell line SNU-475,HepG2,SNU-398,Huh-7 and normal hepatic epithelial cell line L02.The expression of lncRNACASC9 in each group was detected by qRT-PCR.Human hepatocyte cancer cell lines SNU-475,HepG2,SNU-398,Huh-7 and human immortalized hepatocyte epithelial cells L02 were cultured,and lncRNACASC9 expression levels in each cell were detected by qRT-PCR.Huh-7 cells were divided into blank group(Control),siRNA negative control group(siRNA-NC)and CASC9 siRNA interference group(siRNA-CASC9),and CASC9 siRNA interference plasmid and its negative control plasmid were transfected into huh-7 cells.qRT-PCR was used to detect the expression of lncRNA CASC9 and the mRNA levels of stemness related genes such as OCT-4,SOX2,Bmi1 and Nanogin the transfected cells,as well as clone formation and sphere formation were used to observe the stemness characteristics of Huh-7 cells.The proportion of EpCAM^+AFP^+stem cell subpopulations was detected by flow cytometry.Western blot was used to detect the expression levels of OCT-4,SOX2,Bmi1,Nanog,p-STAT3,STAT3,KLF4,c-Myc and other proteins in transfected cells.RESULTS The expression levels of LncRNA CASC9 in L02,SNU-475,HepG2,SNU-398,Huh-7 cell lines after 48 hof culture were 1.03±0.07,2.06±0.19,1.77±0.20,2.88±0.14 and 3.63±0.37,respectively,the differences were statistically significant,F=64.115,P<0.001,and the expression levels of lncRNA CASC9 in SNU-475,HepG2,SNU-398 and Huh-7 cells were lower than those in L02 cells(P<0.001,0.002,<0.001 and<0.001).After CASC9 interference for 48 h,lncRNACASC9 expression in siRNA-CASC9 cells was 0.41±0.05,lower than that in Control group(1.03±0.07)and siRNA-NC group(1.01±0.03),F=144.970,P<0.001.The number of cell clones in LncRNACASC9 group was 45.00±10.81,lower than that in Control group(277.33±7.77)and siRNA-NC group(264.67±9.71),F=565.358,P<0.001.The spherification rate of siRNA-CASC9 group was(12.74±2.56)%,lower than that of Control group(25.93±3.89)%and siRNA-NC group(24.61±4.72)%,F=10.810,P=0.010.The proportion of EpCAM^+AFP^+stem cell subsets was(4.68±0.33)%,lower than that in Control group(8.64±0.84)%and siRNA-NC group(7.91±1.08)%,F=20.289,P=0.002.The relative mRNA and protein expression levels of OCT-4,SOX2,Bmi1 and Nanog in the siRNA-CASC9 group were lower than those in the Control group and siRNA-NC group,with statistically significant differences(P<0.05).The relative expression levels of p-STAT3/STAT3,KLF4 and c-Myc in siRNA-CASC9 cells were all lower than those in Control group and siRNA-NC group,with Fvalues of 13.525,25.122 and 35.038,all P<0.05.CONCLUSIONS The expression level of lncRNACASC9 in liver cancer cell lines was significantly higher than that in normal liver cells.LncRNACASC9 can reduce the stemness of HCC cells by inhibiting the activation of the STAT 3 signaling pathway.
作者
徐志广
陈诗怡
张朴花
XU Zhi-guang;CHEN Shi-yi;ZHANG Pu-hua(Affiliated Nanhua Hospital,University of South China,Hengyang 421002,P.R.China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2020年第13期1049-1055,共7页
Chinese Journal of Cancer Prevention and Treatment
