Objective: To investigate the role of allograft inflammatory factor-1 (AIF-1) in colorectal cancer (CRC) progression and explore the possible mechanism.
Methods: The expression levels of AIF-1 in 70 CRC tissues and paired adjacent tissues were detected using immunohistochemistry and Western blotting, and the correlation of AIF-1 expression with the clinicopathological features of the patients was analyzed. In the CRC cell line SW480, the functional role of AIF-1 in regulating tumor progression was investigated by transfecting the cells with an AIF-1-overexpressing plasmid (AIF-1) and a negative control plasmid (NC). EdU proliferation assay and flow cytometry were used to assess the cell proliferation and cell cycle changes; Transwell migration assay and Annexin V-APC/7-AAD apoptosis assay kit were used to analyze the cell migration and apoptosis. The changes in the biological behaviors of the cells were observed after application of SB203580 to block the p38 MAPK pathway. The expression levels of CDK4, cyclin D1, P21, P27, MMP2, MMP9, Bax, Bcl2, Bcl-xl, p38 and p-p38 were detected using Western blotting.
Results: AIF-1 was down-regulated in CRC tissues compared with the adjacent normal tissues, and its expression level was positively correlated with lymph node metastasis (P=0.008), TNM stage (P=0.003) and tumor size (P=0.023). Overexpression of AIF-1 in SW480 cells significantly reduced EdU-positive cells and caused obvious cell cycle arrest in G1 phase (P<0.05). AIF-1 overexpression resulted in significantly lowered protein expressions of CDK4 and cyclin D1, enhanced expressions of P21 and P27, attenuated cell migration ability (P<0.001), and decreased protein levels of MMP2 and MMP9. AIF-1 overexpression also induced obvious apoptosis of SW480 cells (P<0.01), significantly increased the protein levels of Bax and p-p38, and decreased the protein levels of Bcl-2 and Bcl-xl; SB203580 significantly attenuated the apoptosis-inducing effect of AIF-1 overexpression.
Conclusion: AIF-1 plays the role of a tumor suppressor in CRC by inhibiting cell proliferation, suppressing cell migration and inducing cell apoptosis. AIF-1 overexpression promotes the apoptosis of CRC cells by activating the p38 MAPK pathway.
目的: 探讨同种异体移植物炎症因子-1(AIF-1)在结直肠癌(CRC)进展中的作用以及可能的作用机制。
方法: 采用免疫组织化学染色和Western blot的方法检测70例CRC患者癌组织及癌旁正常组织中AIF-1的表达水平，并分析AIF-1的表达与临床病理特征的相关性。然后将AIF-1过表达质粒(AIF-1)和阴性对照质粒(NC)转染至CRC细胞株SW480，探讨AIF-1在体外细胞中的功能。EdU增殖实验和流式细胞术用来评估细胞增殖和细胞周期；Transwell迁移实验和Annexin V-APC/7-AAD凋亡检测试剂盒用来分析细胞迁移和凋亡；SB203580用来阻断p38 MAPK通路。最后用western blot的方法检测CDK4，Cyclin D1，P21，P27，MMP2，MMP9，Bax，Bcl-2，Bcl-xl，p38和p-p38的表达水平。
结果: AIF-1在结直肠癌组织中的表达明显低于癌旁正常组织，其表达水平与淋巴结转移(P=0.008)，TNM分期(P=0.003)和肿瘤大小(P=0.023)密切相关。体外增殖和周期实验结果显示，过表达AIF-1于SW480细胞后能降低EdU细胞阳性率以及阻滞细胞的G1期(P < 0.05)；且Western blot结果显示过表达AIF-1于SW480细胞后能抑制CDK4、Cyclin D1的蛋白表达，增强P21、P27的蛋白表达。Transwell迁移结果显示过表达AIF-1能抑制SW480细胞的迁移(P < 0.001)，伴随着MMP2和MMP9的蛋白水平的下调。此外，AIF-1过表达能诱导SW480细胞凋亡(P < 0.01)，增强Bax和p-p38的蛋白水平，减弱Bcl-2和Bcl-xl的蛋白水平，而利用SB203580可以明显改善AIF-1过表达引起的凋亡。
结论: AIF-1通过抑制细胞增殖和细胞迁移以及诱导细胞凋亡在结直肠癌中发挥肿瘤抑制作用，且AIF-1通过激活p38 MAPK途径促进细胞凋亡。