AFAP1-AS1对结肠癌细胞增殖、凋亡和PI3K/Akt信号通路的影响

Effects of AFAP1-AS1 on proliferation,apoptosis and PI3K/Akt signaling pathway in colon cancer cells

目的探讨长非编码RNA肌动蛋白纤维相关蛋白1-反义RNA 1(AFAP1-AS1)对结肠癌细胞增殖、凋亡和磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号通路的影响。方法采用实时定量PCR(qPCR)检测本院手术切除的30例结肠癌组织相对于癌旁组织及结肠癌细胞(SW620、HT-29、HCT116、Caco-2和SW480)相对于人正常结肠上皮细胞NCM460的AFAP1-AS1水平。脂质体介导AFAP1-AS1特异性干扰RNA(siRNA)转染SW480细胞(si-AFAP1-AS1组)并设转染无关siRNA的SW480细胞为si-NC组和未行转染的SW480细胞为对照组,qPCR、MTT比色法和Annexin V-FITC/PI双染流式细胞术检测AFAP1-AS1水平、增殖活力和凋亡率,qPCR和Western blotting检测Bcl-2、Bax、caspase-3、p-PI3K和p-Akt水平。结果结肠癌组织的AFAP1-AS1水平为2.669±0.171,高于对应癌旁组织的1.381±0.085(P<0.05)。结肠癌细胞的AFAP1-AS1水平均高于NCM460细胞(P<0.05),选取AFAP1-AS1水平最高的SW480细胞进行后续功能实验。si-AFAP1-AS1组SW480细胞的AFAP1-AS1水平和增殖活力均低于si-NC组和对照组(P<0.05)。si-AFAP1-AS1组SW480细胞的凋亡率为(19.486±0.674)%,高于对照组的(10.671±1.025)%和si-NC组的(10.224±0.683)%(P<0.05)。与对照组和si-NC组相比,si-AFAP1-AS1组的p-Akt、p-PI3K和Bcl-2水平降低,而Bax和caspase-3水平升高(P<0.05)。对照组和si-NC组上述指标的差异无统计学意义(P>0.05)。结论结肠癌中高表达的AFAP1-AS1可能通过激活PI3K/Akt通路来促进结肠癌细胞增殖并抑制凋亡,发挥促癌功效,AFAP1-AS1有可能成为结肠癌治疗的新靶点。

Objective To investigate the effects of long non-coding RNA actin filament-associated protein 1-antisense RNA 1(AFAP1-AS1)on proliferation,apoptosis and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway in colon cancer cells.Methods Real-time quantitative PCR(qPCR)was used to detect the levels of AFAP1-AS1 in 30 cases of colon cancer tissues in relative to paracancerous tissues and colon cancer cells(SW620,HT-29,HCT116,Caco-2 and SW480)in relative to human normal colon epithelial cells NCM460.SW480 cells were transfected with liposome mediated specific interfering RNA(siRNA)of AFAP1-AS1(si-AFAP1-AS1 group).SW480 cells transfected with irrelevant siRNA were set as si-NC group and SW480 cells without transfection were used as control group.QPCR,MTT colorimetry and flow cytometry via Annexin V-FITC/PI double staining were used to detect the AFAP1-AS1 level,proliferation activity and apoptotic rate.Levels of Bcl-2,Bax,caspase-3,p-PI3K and p-Akt were detected by Western blotting and qPCR.Results AFAP1-AS1 level in colon cancer tissues was 2.669±0.171,higher than 1.381±0.085 in corresponding adjacent tissues(P<0.05).The AFAP1-AS1 level in colon cancer cells was higher than that in NCM460 cells(P<0.05).SW480 cells with the highest AFAP1-AS1 level were selected for subsequent functional verification test.The AFAP1-AS1 level and proliferation activity of SW480 cells in si-AFAP1-AS1 group were lower than those of si-NC group and control group(P<0.05).The apoptotic rate of SW480 cells in si-AFAP1-AS1 group was(19.486±0.674)%,higher than(10.671±1.025)%of control group and(10.224±0.683)%of si-NC group(P<0.05).Compared with the control group and si-NC group,levels of p-Akt,p-PI3K and Bcl-2 in si-AFAP1-AS1 group were decreased,while levels of Bax and caspase-3 were increased(P<0.05).There was no significant difference between control group and si-NC group(P>0.05).Conclusion High expression of AFAP1-AS1 in colon cancer may promote the proliferation and inhibit apoptosis of colon cancer cells by activating PI3K/Akt pathway.AFAP1-AS1 may become a new target for colon cancer treatment.