SIRT3通过促进肿瘤相关巨噬细胞M2极化协助结直肠癌HCT-116细胞转移

SIRT3 promotes metastasis of HCT-116 cells by M2 polarization of tumor related macrophages

目的 观察SIRT3对巨噬细胞极化的作用,并探讨其对结直肠癌HCT-116细胞增殖、凋亡、转移和上皮间质转化等生物学功能的影响。方法 构建SIRT3 RNA干扰慢病毒载体,并验证其效率。将SIRT3干扰慢病毒转染至RAW264.7巨噬细胞,免疫荧光染色和ELISA方法检测巨噬细胞极化的标志物;然后将极化的巨噬细胞和结直肠癌HCT-116细胞共培养,吸取共培养体系中的上清液,检测趋化因子CCL18、CCL22和基质金属蛋白酶MMP9表达,Transwell迁移实验检测结直肠癌转移能力。RT-qPCR检测上皮间质转化标志物Vimentin、Fibronectin、Snail2和E-Cadherin的表达。CCK8和TUNEL实验检测结直肠癌细胞增殖和凋亡。结果 抑制SIRT3可减少巨噬细胞M2极化标志物CD16/32、IL10和TGF-β表达。共培养M2极化巨噬细胞和结直肠癌HCT-116细胞后,抑制巨噬细胞SIRT3表达可降低促肿瘤转移细胞因子CCL18、CCL22和MMP9分泌;抑制巨噬细胞SIRT3可减少Vimentin、Fibronectin和Snail2表达,促进E-Cadherin表达。CCK8和TUNEL结果表明,抑制巨噬细胞SIRT3表达不影响结直肠癌增殖和凋亡。结论 SIRT3通过促进肿瘤相关巨噬细胞M2极化协助结直肠癌HCT-116细胞转移。

Objective To observe the effect of SIRT3 on the polarization of macrophages,and to investigate the effects of SIRT3 on the proliferation,apoptosis,metastasis and epithelial mesenchymal transformation of HCT-116 cells in colorectal cancer.Methods SIRT3 RNAi lentivirus was constructed and its expression efficiency was verified.SIRT3 RNAi lentivirus was transfected into RAW264.7 macrophages.Immunofluorescence and ELISA were used to detect the markers of macrophage polarization.Then the macrophages and colorectal cancer HCT-116 cells were co-cultured.The levels of CCL18,CCL22 and MMP9 in the supernatants were identified by ELISA.Transwell migration test was used to detect the metastatic ability.The expressions of Vimentin,Fibronectin,Snail2 and E-Cadherin(markers of epithelial-mesenchymal transformation)were detected by RT-qPCR.Finally,the proliferation and apoptosis of colorectal cancer cells were detected by CCK8 and TUNEL.Results Inhibiting SIRT3 reduced the expression of M2 polarization markers CD16/32,IL-10.After co-culture of RAW264.7and HCT-116,inhibiting SIRT3 of macrophages reduced the expression of CCL18,CCL22 and MMP9 in MGC-803.Inhibiting SIRT3 of macrophages decreased the expression of Vimentin,Fibronectin and Snail2,and increased the expression of E-Cadherin.CCK8 and TUNEL showed that there was no difference in the proliferation and apoptosis of colorectalcancer.Conclusion SIRT3 contributes to the metastasis of colorectal cancer by promoting the M2 polarization of tumor-associated macrophages.