lncRNA FTX对结肠癌伊立替康耐药株增殖、迁移能力的影响

Effect of lncRNA FTX on proliferation and migration of colon cancer cell line resistant to irinotecan

目的研究长链非编码RNA FTX(long noncoding RNA FTX,lncRNA FTX)对结肠癌细胞伊立替康(irinotecan,CPT-11)耐药株增殖、迁移的影响。方法对从lnCAR数据库(https://lncar.renlab.org/)中获取的80例临床样本FTX的表达情况以及70例结肠癌患者生存率进行统计学分析。采用药物浓度梯度构建人结肠癌伊立替康耐药细胞株(HCT-116/CPT-11R),通过CCK-8(cell counting kit-8)测定其耐药指数,并通过荧光实时定量PCR(real-time quantitative PCR,qPCR)检测正常结肠细胞NCM460、结肠癌细胞HCT-116以及构建的耐药细胞株的FTX表达水平。构建敲低lncRNA FTX的慢病毒载体(sh-FTX)及对照空白载体(sh-NC)转染HCT-116/CPT-11R耐药株中,经过嘌呤霉素筛选获得lncRNA FTX稳定下调的结肠癌细胞耐药株,并通过qPCR鉴定敲低效率。通过克隆形成实验及CCK-8实验分别检测lncRNA FTX对sh-NC组、sh-FTX组耐药细胞克隆形成能力及生长活力的影响。利用划痕实验检测lncRNA FTX对sh-NC组、sh-FTX组耐药株细胞迁移能力的影响。通过Western blot检测sh-NC组、sh-FTX组耐药株细胞增殖相关蛋白c-myc和迁移相关蛋白Vimentin、MTA-1表达水平。结果统计数据显示,结肠癌患者的FTX表达高于正常人(P<0.01);且生存分析结果显示FTX低表达组的生存率高于FTX高表达组(P<0.05),其中低表达组患者的5年生存率约为高表达组的5倍。结肠癌伊立替康耐药株HCT-116/CPT-11R构建成功,耐药株中lncRNA FTX的表达水平显著高于正常结肠细胞以及结肠癌细胞(P<0.01)。sh-FTX能显著降低耐药细胞株中FTX的表达水平(P<0.001)。与sh-NC组比较,sh-FTX组HCT-116/CPT-11R的增殖能力和迁移能力被显著抑制(P<0.005)。lncRNA FTX被敲低后,HCT-116/CPT-11R细胞中增殖相关蛋白c-myc以及转移相关蛋白Vimentin、MTA-1蛋白表达水平降低,HCT-116耐药株的迁移和增殖能力被显著抑制(P<0.05或P<0.005)。结论lncRNA FTX在结肠癌细胞系耐药株HCT-116/CPT-11R中高表达,体外实验发现敲低lncRNA FTX能显著抑制HCT-116/CPT-11R增殖和迁移能力。

Objective To investigate the effects of long noncoding RNA FTX(lncRNA FTX)on the proliferation and migration of irinotecan(CPT-11)resistant colon cancer cells.Methods From lnCAR database(https://lncar.renlab.org/),FTX expression in 80 cases of clinical samples and the survival rates of 70 patients with colon cancer were collected and analyzed.Human colon cancer irinotecan drug-resistant cell line(HCT-116/CPT-11R)was constructed using drug concentration gradient,and its drug resistance index was determined by CCK-8(cell counting kit-8).The expression level of FTX in normal colon cells NCM460,colon cancer cells HCT-116 and constructed drug-resistant cell lines HCT-116/CPT-11R was measured by real-time quantitative PCR(RT-qPCR).The HCT-116/CPT-11R resistant strains were transfected with lentiviral vector with knockdown of lncRNA FTX(sh-FTX)and the control blank vector(sh-NC),respectively.Then the drug-resistant strains with stable downregulation of lncRNA FTX in colon cancer cells were screened by puromycin,and the knockdown efficiency was identified by qPCR.The effects of lncRNA FTX on the clone formation ability and the growth viability of drug-resistant cells in sh-NC group and sh-FTX group were detected by clone formation assay and CCK-8 assay,respectively.The effect of lncRNA FTX on cell migration in sh-NC group and sh-FTX group was detected by scratch test.The expression levels of proliferation-related protein c-myc,migration-related protein Vimentin and MTA-1 in sh-NC group and sh-FTX group were detected by Western blot.Results The statistical data showed that the expression of FTX in colon cancer patients was higher than that in normal controls(P<0.01).The survival analysis showed that the survival rate in FTX low expression group was higher than that in FTX high expression group(P<0.05),and the five-year survival rate in low expression group was about twice that in high expression group.Colon cancer irinotecan resistant strain HCT-116/CPT-11R was successfully constructed,and the expression level of lncRNA FTX in the resistant strain was significantly higher than that in normal colon cells and colon cancer cells(P<0.01).The sh-FTX significantly reduced the expression of FTX in drug-resistant cell lines(P<0.001).Compared with sh-NC group,the proliferation of HCT-116/CPT-11R and the migration ability of HCT-116/CPT-11R were significantly inhibited in sh-FTX group(P<0.005).After FTX knockdown of lncRNA,the expression levels of c-myc,Vimentin and MTA-1 in HCT-116/CPT-11R cells were decreased,and the migration and proliferation abilities of HCT-116 drug-resistant strains were inhibited(P<0.05 or P<0.005).Conclusion The lncRNA FTX is highly expressed in colon cancer cell line HCT-116/CPT-11R,and the knockdown of lncRNA FTX can significantly inhibit the proliferation and migration of HCT-116/CPT-11R in vitro.