叉头转录因子O亚族6对结直肠癌细胞增殖和侵袭能力的作用

Effects of FoxO6 on proliferation and invasion of colorectal cancer cells

目的探讨叉头转录因子O亚族6(FoxO6)对结肠癌细胞增殖和侵袭能力的影响及其分子机制。方法将FoxO6 siRNA转染结直肠癌细胞HCT116和SW480,干扰FoxO6表达。构建过表达载体pcDNA.3.1-c-Myc,转染FoxO6沉默的HCT116和SW480细胞,过表达c-Myc。实时荧光定量聚合酶链反应(RT-qPCR)和Western blot法检测HCT116和SW480细胞中FoxO6、c-Myc、p21的mRNA和蛋白表达量,溴脱氧尿苷(BrdU)法检测细胞增殖能力,Transwell侵袭实验检测细胞侵袭能力。将FoxO6 shRNA慢病毒(LV-FoxO6)转染的SW480细胞注射至BAL b/c裸鼠右侧腋窝皮下构建荷瘤模型,注射后第10、13、16、19、22、25天测量瘤体体积。结果正常结肠细胞FHC、结肠癌细胞系HCT116和SW480中FoxO6的mRNA表达水平分别为0.91±0.04、1.72±0.07和2.03±0.06,蛋白表达水平分别为0.7±0.04、1.35±0.08和1.56±0.07,结肠癌细胞系HCT116和SW480中FoxO6的mRNA和蛋白表达水平均高于FHC细胞(均P<0.05)。转染FoxO6 siRNA后,HCT116和SW480细胞中FoxO6 mRNA和蛋白表达均降低(均P<0.05),细胞增殖能力降低(吸光度分别为0.26±0.07和0.27±0.06,均P<0.05),侵袭能力下降[侵袭细胞数分别为(42.3±3.3)个和(45.7±4.1)个,均P<0.05],c-Myc的mRNA和蛋白表达量降低(均P<0.01),p21的mRNA和蛋白表达量升高(均P<0.01)。转染pcDNA.3.1-c-Myc到FoxO6沉默的HCT116和SW480细胞后,p21表达量减少,细胞增殖能力增强(吸光度分别为0.54±0.09和0.58±0.07,均P<0.01),侵袭能力增强[侵袭细胞数分别为(79.2±5.9)个和(80.5±6.4)个,均P<0.01]。裸鼠接种细胞后第25天,LV-FoxO6-SW480组的肿瘤体积为(190.6±36.2)mm^3,明显低于SW480组[(437.8.6±69.2)mm3,P<0.05]。结论FoxO6可通过c-Myc调节p21的表达,进而调控结直肠癌细胞的增殖和侵袭能力。

Objective To investigate the effects and the mechanism of FoxO6 on the proliferation and invasion of colorectal cancer cells.Methods FoxO6 siRNA was transfected into colorectal cancer cell HCT116 and SW480.The overexpression vector pcDNA.3.1-c-Myc was constructed and co-transfected into HCT116 and SW480 cells with FoxO6 siRNA.Real-time fluorescent quantitative PCR(RT-qPCR)and western blot were used to detect the mRNA and protein expressions of FoxO6,c-Myc,and p21 in HCT116 and SW480 cells.Bromodeoxyuridine(BrdU)was used to detect cell proliferation and Transwell assay was performed to detect the invasion ability of these cells.SW480 cells transfected with FoxO6 shRNA lentivirus(LV-FoxO6)and were injected into the right armpit of BAL b/c nude mice to construct a tumor-bearing mode and the tumor volumes were measured on the days of 10,13,16,19,22,and 25 after injection.Results The FoxO6 mRNA were 0.91±0.04,1.72±0.07,and 2.03±0.06,and protein expression were 0.70±0.04,1.35±0.08,and 1.56±0.07 in normal colon cell FHC,colorectal cancer cells HT116 and SW480,respectively.The protein and mRNA levels of FoxO6 in HCT116 and SW480 were significantly higher than those in FHC(both P<0.05).Knockdown of FoxO6 in HCT116 and SW480 cells decreased the mRNA and protein expressions of FoxO6(both P<0.05),the cell proliferation ability(absorbances were 0.26±0.07 and 0.27±0.06,both P<0.05),cell invasion ability(the invaded cell numbers were 42.3±3.3 and 45.7±4.1,both P<0.05),and the mRNA and protein expressions of c-Myc,while increased the mRNA and protein expressions of p21(both P<0.01).Overexpression of Myc in FoxO6 silenced HCT116 and SW480 cells decreased the expression of p21,while increased the cell proliferation ability(absorbances were 0.54±0.09 and 0.58±0.07,both P<0.01)and invasion ability(the invaded cell numbers were 79.2±5.9 and 80.5±6.4,both P<0.01).On the 25th day after cell inoculation in nude mice,the tumor volume of LV-FoxO6 group was(190.6±36.2)mm^3,significantly lower than(437.8.6±69.2)mm3 of LV-NC group(P<0.05).Conclusion FoxO6 promotes the proliferation and invasion of colorectal cancer cells through facilitating c-Myc mediated p21 expression inhibition.