LincRNA-p21调控PI3K/AKT信号通路逆转结肠癌细胞伊立替康耐药性

LincRNA-p21 reverses irinotecan resistance in colon cancer cells via the PI3K/AKT signaling pathway

背景伊立替康(camptothecin-11,CPT-11)为晚期结肠癌的一线化疗用药,但CPT-11耐药性限制了CPT-11的化疗效果.研究结肠癌CPT-11耐药的机制,并恢复结肠癌细胞对CPT-11的敏感性,对延长结肠癌患者的生命时间具有十分重要的临床价值.目的探讨长链基因间非编码RNA-p21(long intergenic noncoding RNA-p21,lincRNA-p21)对结肠癌细胞CPT-11耐药的作用和机制.方法采用结肠癌HCT-8细胞和SW480细胞利用CPT-11持续接触浓度递增法分别构建伊立替康耐药HCT-8/CPT-11细胞和SW480/CPT-11细胞,并利用实时定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测细胞内lincRNA-p21表达.HCT-8/CPT-11细胞和SW480/CPT-11细胞分别转染pcDNA-lincRNA-p21或si-lincRNA-p21后,细胞计数试剂盒-8(cell counting kit-8,CCK-8)实验检测CPT-11对细胞活性的影响;蛋白质免疫印迹(Western blot)初步分析了lincRNA-p21对磷脂酰肌醇3-激酶/蛋白激酶B(phosphoinositide 3-kinase/protein kinase B,PI3K/AKT)通路是否有调节作用.采用PI3K/AKT通路抑制剂LY294002预处理已转染si-lincRNA-p21的HCT-8/CPT-11细胞和SW480/CPT-11细胞或采用PI3K/AKT通路激动剂Recilisib预处理已转染pcDNAlincRNA-p21的HCT-8/CPT-11细胞和SW480/CPT-11细胞,CCK-8实验检测CPT-11对细胞活性的影响.结果与亲本细胞相比,CPT-11耐药细胞中lincRNA-p21表达明显降低.过表达lincRNA-p21抑制HCT-8/CPT-11细胞和SW480/CPT-11细胞对CPT-11耐药性,而敲低lincRNA-p21增强HCT-8/CPT-11细胞和SW480/CPT-11细胞对CPT-11耐药性.Western blot结果显示,lincRNA-p21过表达抑制PI3K/AKT通路活性;而敲低lincRNA-p21增强PI3K/AKT通路活性.LY294002能抑制lincRNA-p21敲低对CPT-11耐药的促进作用;Recilisib能抑制lincRNA-p21过表达对CPT-11耐药的抑制作用.结论上调lincRNA-p21能抑制结肠癌细胞CPT-11耐药性,而下调lincRNA-p21能增强结肠癌细胞CPT-11耐药性,这种调节作用可能与lincRNA-p21调控PI3K/AKT信号通路活性相关.

BACKGROUND Irinotecan(camptothecin-11,CPT-11)is a first-line chemotherapy drug for advanced colon cancer,but CPT-11 resistance limits its efficacy.Studying the mechanism of CPT-11 resistance in colon cancer and restoring the sensitivity of colon cancer cells to CPT-11 are of great clinical value in prolonging the life time of colon cancer patients.AIM To investigate the effect and mechanism of long intergenic non-coding RNA-p21(lincRNA-p21)on CPT-11 resistance in colon cancer cells.METHODS HCT-8 and SW480 cells were used to construct irinotecanresistant HCT-8/CPT-11 and SW480/CPT-11 cell lines by continuously exposing them to increasing concentrations of CPT-11,and the expression of lincRNA-p21 in the cells was detected by real-time quantitative polymerase chain reaction(RT-qPCR).After transfection with pcDNAlincRNA-p21 or si-lincRNA-p21,the effect of CPT-11 on the viability of HCT-8/CPT-11 cells or SW480/CPT-11 cells was measured by cell counting kit-8(CCK-8)assay.The regulatory effect of lincRNA-p21 on the phosphoinositide 3-kinase/protein kinase B(PI3K/AKT)pathway was preliminarily analyzed by Western blot.After pretreatment with PI3K/AKT pathway inhibitor LY294002 prior to transfection with si-lincRNA-p21,or pretreatment with PI3K/AKT pathway agonist Recilisib prior to transfection with pcDNA-lincRNA-p21,the effect of CPT-11 on cell viability in HCT-8/CPT-11 cells or SW480/CPT-11 cells was measured by CCK-8 assay.RESULTS LincRNA-p21 expression in CPT-11 resistant cells was significantly lower than that in parental cells.Overexpression of lincRNA-p21 inhibited the resistance of HCT-8/CPT-11 cells and SW480/CPT-11 cells to CPT-11,while knockdown of lincRNA-p21 enhanced the resistance of HCT-8/CPT-11 cells and SW480/CPT-11 cells to CPT-11.Western blot results showed that overexpression of lincRNA-p21 inhibited the activity of the PI3K/AKT pathway,while knockdown of lincRNA-p21 enhanced the activity of the PI3K/AKT pathway.LY294002 inhibited the promotive effect of lincRNA-p21 knockdown on CPT-11 resistance,while Recilisib inhibited the inhibitive effect of lincRNA-p21 overexpression on CPT-11 resistance.CONCLUSION Up-regulation of lincRNA-p21 can inhibit the CPT-11 resistance of colorectal cancer cells,while down-regulation of lincRNA-p21 can promote their CPT-11 resistance,which may be related to the regulation of the PI3K/AKT signaling activity by lincRNA-p21.