肿瘤相关成纤维细胞自噬调节对共培养大肠癌细胞侵袭、迁移能力的影响

Influences of autophagic regulations of carcinoma associated fibroblasts on the migration and invasion of cocultured colorectal cancer

目的通过调控肿瘤相关成纤维细胞（CAFs）的自噬状态，观察对共培养大肠癌细胞的侵袭、迁移能力的影响。方法取大肠癌患者原代培养的CAFs，微管相关蛋白1轻链3（LC3）免疫荧光法检测CAFs的自噬状态；筛选自噬上调剂雷帕霉素与自噬抑制剂巴弗洛霉素的最佳作用时间与剂量，分别对CAFs进行自噬调控；通过细胞侵袭实验和细胞划痕实验观察不同CAFs自噬状态对大肠癌侵袭与迁移能力的影响；通过对肿瘤细胞抑癌基因p53、B细胞淋巴瘤/白血病-2（bcl-2）、bcl-2相关x蛋白（bax）、天冬氨酸特异性半胱氨酸蛋白酶-3（Caspase-3）、核因子（NF）-κB等蛋白的检测，探讨自噬调控CAFs影响大肠癌细胞侵袭、迁移能力的可能机制。结果未自噬干预的CAFs培养12、24及48 h后，噻唑蓝（MTT）检测吸光度（A）值最大时间为48 h；400 nmol/L雷帕霉素可进一步增加CAFs自噬，Western blot检测结果显示损伤调节自噬调控因子（DRAM）、自噬相关基因Atg5及Beclin-1、LC3等自噬相关基因表达上调，而400 nmol/L巴弗洛霉素可抑制CAFs自噬水平，DRAM、Atg5与Beclin-1基因表达下调；各组细胞于Transwell中进行共培养48 h后，发现穿过小室肿瘤细胞以雷帕霉素预处理CAFs＋CCL244组最多[（172.0±9.3）个]，巴弗洛霉素预处理CAFs＋CCL244最少[（52.0±5.0）个]；细胞迁移实验结果显示雷帕霉素预处理组CCL244细胞向划痕处迁移程度最明显[（15.34±0.49） mm]，明显高于巴弗洛霉素预处理组[（10.32±1.08） mm]；自噬上调组细胞的bcl-2、NF-κB基因表达上调，而bax、p53及Caspase-3表达下调，自噬下调组相反。结论上调CAFs自噬水平，可增强共培养大肠癌细胞侵袭、迁移能力；而抑制CAFs自噬水平，可降低共培养大肠癌细胞侵袭、迁移能力。

Objective To explore the influences of autophagic regulations of carcinoma associated fibroblasts （CAFs） on the migration and invasion of cocultured colorectal cancer.Methods CAFs were attempted by primary cultured tissues from colorectal cancer. Autophagic regulation of CAFs was performed using rapamycin and bafilomycin, and autophagic effects were observed with anti-microtubule-associated protein 1 light 3 （LC3）. The influences of autophagic regulation of CAFs on the migration and invasion abilities of CCL244 were observed by transwell invasion assay and scratch assay. Western blotting analysis of tumor protein 53 （p53）, B-cell lymphoma/leukemia-2 （bcl-2）, bcl-2 assaciated X protein （bax）, nuclear factor-kappa B （NF-κB） and cysteine-containing aspartate-specific proteases-3 （Caspase-3） were performed determined to investigate the potential mechanisms.Results 400 nmol/L rapamycin increased autophagy and the expression of autophagy associated proteins damage-regulated autophagy modulator （DRAM）, autophagy associated gene 5 （Atg5）, Beclin-1 and LC3 was up-regulated.400 nmol/L bafilomycin inhibited autophagy of CAFs and the above proteins were down-regulated. We find that rapamycin pretreatment CAFs＋ CCL244 group has most tumor cells to across the well （172.0±9.3） and bafilomycin pretreatment CAFs＋ CCL244 group has least tumor cells to across the well （52.0±5.0） after each group cells were cultured in transwell in 48 h. The migration rate of CCL-244 cells pretreated in Rapamycin [（15.34±0.49） mm] was especially higher than the bafilomycin pretreatment group, There was no significant difference between the control group and bafilomycin pretreatment group [（10.32±1.08） mm]. Induction of autophagy in CAFs upregulated the expression of bcl-2 and NF-κB and downregulated the expression of bax, p53 and Caspase-3 in irradiated CCL244. Inhibition of autophagy in CAFs showed the opposite results in CCL244.Conclusion Up regulation of autophagy in CAFs can enhance the ability of invasion and migration of cocultured cancer cells, while inhibition of autophagy in CAFs can reduce the invasion and migration ability of cocultured cancer cells.