【摘要】 目的:探讨调控CXCR4-PI3KⅢ-自噬轴对结直肠癌LoVo细胞迁移和侵袭能力的影响。方法:实验分为正常LoVo细胞组、介导CXCR4-RNAi慢病毒转染LoVo细胞组(CXCR4-RNAi组)和无意义慢病毒转染LoVo细胞组(NC组)。采用Q-PCR检测PI3KⅢmRNA表达,蛋白质印迹法检测PI3KⅢ、LC3-Ⅱ及Beclin 1的表达,激光共聚焦显微镜观察绿色荧光颗粒,透射电镜观察自噬溶酶体,Transwell小室评价LoVo细胞迁移与侵袭能力。结果:PI3KⅢmRNA表达量正常LoVo细胞组为1.09±0.11,NC组为1.07±0.25,CXCR4-RNAi组为0.86±0.06。PI3KⅢ相对表达量CXCR4-RNAi组为0.227±0.023,低于正常LoVo细胞组的0.607±0.012(P<0.001)和NC组的0.667±0.040(P<0.001),正常LoVo细胞组和NC组差异无统计学意义,P=0.310。LC3-Ⅱ相对表达量CXCR4-RNAi组为0.083±0.012,低于正常LoVo细胞组的0.233±0.015(P<0.001)和NC组的0.253±0.020(P<0.001),正常LoVo细胞组和NC组差异无统计学意义,P=0.184。Beclin 1相对表达量CXCR4-RNAi组为0.207±0.012,低于正常LoVo细胞组的0.440±0.053(P=0.134)和NC组的0.650±0.252,P=0.011。绿色荧光颗粒和自噬溶酶体数量正常LoVo细胞组和NC组明显多于CXCR4-RNAi组。CXCR4-RNAi组迁移实验细胞计数(87.7±9.1)低于正常LoVo细胞组的126.3±4.9(P=0.009)和NC组的115.0±18.7(P=0.035)。CXCR4-RNAi组侵袭实验细胞计数(88.7±10.5)低于正常LoVo细胞组的109.7±4.7(P=0.019)和NC组的107.7±8.1,P=0.029。结论:CXCR4-PI3KⅢ-自噬轴可调控结肠癌LoVo细胞自噬状态,影响肿瘤细胞迁移和浸润,可能是恶性肿瘤浸润转移的机制之一。更多还原

【Abstract】 OBJECTIVE:To investigate the influence of CXCR4-PI3KⅢ-autophagy to migration and invasion of LoVo cell.METHODS:The LoVo cells were grouped to normal LoVo cell group,CXCR4-RNAi(mediated with lentivirus)group,and nonsence control group(NC).PI3K Ⅲ mRNA expression level was measured with Q-PCR.Western blot was employed to measure the expression of PI3K Ⅲ,LC3-Ⅱ,and Beclin 1protein.Laser canning confocal microscope was used to identify the green fluorescence particles.Transmission electron microscope was employed to observe autolysosome.Transwell chamber was applied to evaluate the migration and invasion of LoVo cell.RESULTS:The PI3K Ⅲ mRNA expression level was 1.09±0.11in normal LoVo cell group,1.07±0.25in NC group,and 0.86±0.06in CXCR4-RNAi group.The relative expression level of PI3K Ⅲ was significantly lower in CXCR4-RNAi group(0.227±0.023)than that in normal LoVo cell group(0.607±0.012,P<0.001)and in NC group(0.667±0.040,P<0.001),and there was no significant difference between normal LoVo cell group and NC group(P=0.310).The relative expression level of LC3-Ⅱ was significantly lower in CXCR4-RNAi group(0.083±0.012)than that in normal LoVo cell group(0.233±0.015,P<0.001)and in NC group(0.253±0.020,P<0.001),there was no significant difference between normal LoVo cell group and NC group(P=0.184).The relative expression level of Beclin 1was 0.207±0.012in CXCR4-RNAi group,0.440±0.053(P=0.134)in normal LoVo cell group,and 0.650±0.252(P=0.011)in NC group.The relative expression level of PI3K Ⅲ,LC3-Ⅱ,and Beclin 1was significantly lower in CXCR4-RNAi group than that in normal LoVo cell group and in NC group.There was more green fluorescence particles and autolysosomes in normal LoVo cell group and in NC group than that in CXCR4-RNAi group.The migration experiment showed the mean number of LoVo cell in CXCR4-RNAi group(87.7±9.1)was lower than that in normal LoVo cell group(126.3±4.9,P=0.009)and in NC group(115.0±18.7,P=0.035).The invasion experiment found the mean number of LoVo cell in CXCR4-RNAi group(88.7±10.5)was lower than that in normal LoVo cell group(109.7±4.7,P=0.019)and in NC group(107.7±8.1,P=0.029).CONCLUSION:CXCR4-PI3KⅢ-autophagy axis can regulate the cell autophagy level,influence the migration and invasion power of LoVo cell,and it may be one of the mechanisms of invasion and metastasis of malignant tumors.更多还原