MACC1通过PI3K/AKT信号通路促进结直肠癌细胞的肿瘤干细胞样特性

MACC1 facilitate cancer stem celllike properties of colorectal cancer cells through the PI3K/AKT signaling pathway

目的探究结肠癌转移相关基因-1(MACC1)对结直肠癌(CRC)肿瘤干细胞(CSCs)样特性生物学行为的影响及机制。方法选择人结肠癌细胞HCT116,将MACC1基因过表达克隆慢病毒颗粒(LV-MACC1)和空载体对照慢病毒颗粒(LV-Ctrl)转染HCT116,荧光显微镜观察转染效果;分别应用平板克隆实验、Transwell侵袭实验、肿瘤球形成实验检测细胞单克隆能力、侵袭能力及体外肿瘤形成能力;采用蛋白质印迹实验检测MACC1、CD133、蛋白激酶B(AKT)和磷酸化AKT(p-AKT)蛋白水平的表变化,使用740 Y-P验证PI3K/AKT在MACC1诱导的干细胞样特性中的作用。结果感染48 h,90%以上的细胞均有绿色荧光。LV-MACC1的MACC1蛋白相对表达量为(1.03±0.04),高于LV-Ctrl的(0.14±0.03),差异具有统计学意义(P<0.05)。在平板克隆形成实验中,LV-MACC1的菌落数为(118.70±6.12)个,多于LV-Ctrl的(27.00±4.16)个,差异具有统计学意义(P<0.05)。LV-MACC1细胞侵袭穿孔数为(66.80±3.85)个,多于LV-Ctrl的(27.80±1.99)个,差异具有统计学意义(P<0.05)。LV-Ctrl和LV-MACC1的肿瘤球数量分别是(43.8±2.1)个和(63.8±2.5)个,统计显示LV-MACC1的肿瘤球数显著高于LV-Ctrl(P<0.05)。LV-MACC1的CD44、CD133和p-AKT蛋白的相对表达量均高于LV-Ctrl(P<0.05),LV-Ctrl和LV-MACC1的总AKT蛋白表达没有差异(P>0.05)。使用740 Y-P激活PI3K/AKT通路活性后,CD44和CD133相对表达量比未使用740 Y-P显著增加,差异具有统计学意义(P<0.05)。结论MACC1通过PI3K/AKT信号通路促进CRC细胞出现肿瘤干细胞样特性和恶性生物学行为。

Objective To determine the effects and mechanism of metastasisassociated colon cancer-1(MACC1)on the biological behavior of cancer stem cell(CSCs)-like properties of colorectal cancer(CRC).Methods HCT116 cells were transfected with MACC1 overexpression vector(LV-MACC1)and MACC1 empty vector(LV-Ctrl).The transfection effect was observed under fluorescence microscope.Colony-forming assay,Transwell invasion experiment,tumor sphere formation assay were used to measure cell colony formation,invasion abilities and tumor formation capacity of cancer cells,respectively.Western blot test was used to detect the expression difference of protein level of MACC1,CD4,CD133,protein kinase B(AKT)and phosphorylated AKT(p-AKT).740 Y-P was used to determine the possible roles of PI3K/AKT in MACC1induced stem cell properties.Results 48 h after infection,more than 90%of the cells showed green fluorescence.The relative expression of MACC1 protein of LV-MACC1 was(1.03±0.04),which was higher than(0.14±0.03)of LV-Ctrl,and the difference was statistically significant(P<0.05).In the colony-forming assay,the number of colonies of LV-MACC1 was(118.70±6.12),which was more than(27.00±4.16 of LV-Ctrl),and the difference was statistically significant(P<0.05).The number of LV-MACC1 cells infested with perforations was(66.80±3.85),which was more than(27.80±1.99)of LV-Ctrl,and the difference was statistically significant(P<0.05).The numbers of tumor spheres in LV-Ctrl and LV-MACC1 were(43.8±2.1)and(63.8±2.5),and the statistics showed that the number of tumor spheres in LV-MACC1 was significantly higher than that in LV-Ctrl(P<0.05).The relative expression of CD44,CD133 and p-AKT proteins in LV-MACC1 was higher than that in LV-Ctrl(P<0.05),and there was no difference in total AKT protein expression between LV-Ctrl and LV-MACC1(P>0.05).After activation of PI3K/AKT pathway activity with 740 Y-P,the relative expression of CD44 and CD133 was significantly increased compared with that without 740 Y-P,and the difference was statistically significant(P<0.05).Conclusion MACC1 facilitates cancer stem celllike properties and malignant biological behavior in CRC cells through the PI3K/AKT signaling pathway.