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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。
Objective: To investigate the effects of CXCR4-PI3KⅢ-autophagic axis on the migration and invasion of colorectal cancer LoVo cells. Methods: The experiment was divided into normal LoVo cell group, CXCR4-RNAi lentivirus transfected LoVo cell group (CXCR4-RNAi group) and nonsense lentivirus transfected LoVo cell group (NC group). The expression of PI3KⅢmRNA was detected by Q-PCR, the expression of PI3KⅢ, LC3-Ⅱ and Beclin 1 were detected by Western blotting. The green fluorescent particles were observed by laser confocal microscopy, the autophagy lysosome was observed by transmission electron microscopy, and the migration and invasion ability of LoVo cells were evaluated by Transwell chamber . Results: The expression level of PI3KⅢmRNA was 1.09 ± 0.11 in LoVo cell group, 1.07 ± 0.25 in NC group and 0.86 ± 0.06 in CXCR4-RNAi group. The relative expression level of PI3KⅢ in CXCR4-RNAi group was 0.227 ± 0.023, lower than that in normal LoVo cells group (0.607 ± 0.012) and 0.667 ± 0.040 (P <0.001) in NC group Statistical significance, P = 0.310. The relative expression level of LC3-Ⅱ in CXCR4-RNAi group was 0.083 ± 0.012, which was lower than that in normal LoVo cell group (0.233 ± 0.015, P <0.001) and NC group (0.253 ± 0.020, P <0.001) The difference was not statistically significant, P = 0.184. Beclin 1 relative expression level was 0.207 ± 0.012 in CXCR4-RNAi group, which was lower than 0.440 ± 0.053 (P = 0.134) in normal LoVo cell group and 0.650 ± 0.252 in NC group (P = 0.011). The number of green fluorescent particles and the number of autophagy lysosomes was significantly higher in LoVo cell group and NC group than in CXCR4-RNAi group. CXCR4-RNAi group migration assay The cell count (87.7 ± 9.1) was lower than that of the normal LoVo cell group by 126.3 ± 4.9 (P = 0.009) and NC group by 115.0 ± 18.7 (P = 0.035). The cell count of invasion assay in CXCR4-RNAi group (88.7 ± 10.5) was lower than 109.7 ± 4.7 (P = 0.019) in normal LoVo cell group and 107.7 ± 8.1 in NC group (P = 0.029). Conclusion: The CXCR4-PI3KⅢ-autophagic axis can regulate the autophagy status of colon cancer LoVo cells and affect tumor cell migration and invasion, which may be one of the mechanisms of invasion and metastasis of malignant tumor.