miR-31协同5-Fu发挥抗肿瘤作用的体内研究

摘要

目的：探讨miR-31在胃癌细胞对化疗药物5-氟尿嘧啶（5-Fu）敏感性方面的影响，以及在小鼠体内miR-31联合5-Fu对胃癌生长的影响和机制。方法实时定量PCR的方法检测临床耐药胃癌患者癌组织和非胃癌患者胃组织中以及PBMC中的miR-31的表达，野生型MFC细胞和耐5-Fu 的MFC-R细胞中miR-31的表达，同时，流式细胞术检测耐药胃癌患者癌组织和非胃癌患者外周血中Treg细胞的比例，分析miR-31和FOXP3的相关性。 MTS检测miR-31对MFC细胞增殖的影响。 miR-31和5-Fu联合用药后对小鼠体内肿瘤生长的影响， LDH释放实验检测CD8＋T细胞对MFC的杀伤影响。结果临床耐药胃癌患者癌组织和PBMC中的miR-31水平显著低于非胃癌患者（P＜0唵．05），而外周CD3＋CD4＋CD25＋FOXP3＋T细胞比例显著高于非癌症患者（ P ＜0．05），且二者呈负相关（ P ＜0．05）。而在耐5-Fu的MFC细胞中，miR-31的水平降低。当转染miR-31到MFC细胞中后，miR-31的高表达增加了细胞对5-Fu的敏感性（ P ＜0．05）。小鼠体内实验证实，miR-31协同5-Fu可以显著抑制MFC细胞在小鼠体内生长，并且miR-31可以降低荷瘤小鼠肿瘤浸润以及外周的Treg的比例，增强CD8＋T细胞的CTL功能（ P ＜0．05）。结论miR-31能够抑制胃癌MFC细胞的生长可能是通过直接增加癌细胞对化疗药物的敏感性，并且通过抑制Treg细胞的功能来实现的。%Objective To explore the effects of miR-31 on the sensitivity of 5-fluorouracil (5-Fu) in gastric cancer cells in vitro,and to investigate the effects of miR-31 combined with 5-Fu on mice in vivo.Methods The expression levels of miR-31 in peripheral blood mononuclear cell ( PMBC) and the cancer tissues of patients with gastric cancer and with clinical resistance and the expression levels of miR-31 in PMBC and gastric tissues of patients without gastric cancer were detected by Real-time PCR,moreover, which in wild type MFC cells and MFC cells with chemoresistance to 5-Fu ( MFC-R cells) were also detected by Real-time PCR in vitro.Meanwhile the proportion of Treg cells ( CD3+CD4+CD2+5 FOXP3+T cells) in gastric cancer tissues of patients with gastric cancer and in peripheral blood of patients without gastric cancer were detected by flow cytometry,moreover, the correlation between miR-31and FOXP3 was analyzed.The effects of miR-31on proliferation of MFC cells were detected by MTS,moreover, the effects of miR-31 combined with 5-Fu on tumor growth of mice in vivo were detected by MTS.Besides the effects of CD8+T cells on MFC were detected by LDH releasing assay.Results The expression levels of miR-31 in cancer tissues and PMBC of patients with gastric cancer as well as clinical resistance were significantly lower than those of patients without gastric cancer ( P <0.05), however, the proportion of CD3+CD4+CD2+5FOXP3+T cells in peripheral blood of patients with gastric cancer was significantly higher than that of patients without gastric cancer ( P <0.05), furthermore, there was an negative correlation between them ( P <0.05).However in MFC cells with chemoresistance to 5-Fu,the expression levels of miR-31were decreased.After miR-31was transfected into MFC cells, the high-expression of miR-31increased the sensitivity to 5-Fu ( P <0.05) .The experimental results in mice in vivo showed that miR-31combined with 5-Fu could obviously inhibit the growth of MFC cells in mice in vivo,moreover, miR-31 could decrease tumor infiltration of mice with tumor and could decrease the proportion of Treg cells in peripheral blood of mice and enhance CTL function of CD8+T cells ( P <0.05).Conclusion miR-31can inhibit the growth of gastric cancer MFC cells by increasing the sensitivity of cancer cells to chemotherapeutic drugs directly and by inhibiting the function of Treg cells.