SIRT2在结肠癌微环境CD4^(+)T细胞向Th17细胞分化中的作用及对HIF-1α的影响

Effect of SIRT 2 on differentiation of CD4^(+)T cells into Th 17 cells in colon cancer microenvironment and its influence on HIF-1α

目的:探讨沉默信息调节因子2(SIRT2)过表达在结肠癌微环境CD4^(+)T细胞向Th17细胞分化中的作用及对缺氧诱导因子-1α(HIF-1α)的影响。方法:小鼠结肠癌细胞CT26过表达SIRT2,构建稳定SIRT2过表达的CT26细胞株(CT26/SIRT2),qRT-PCR、Western blot检测SIRT2、HIF-1α水平;CCK-8验证SIRT2过表达对CT26细胞增殖能力的影响;24只健康C57BL/6小鼠随机分为CT26组、CT26/SIRT2组,分别接种CT26细胞、CT26/SIRT2细胞,28 d后处死小鼠,Ki67染色、TUNEL染色分别观察小鼠肿瘤组织细胞增殖与凋亡,并测定组织匀浆上清液中IL-17、IL-6水平;利用流式细胞术分离Th17细胞,qRT-PCR、Western blot检测Th17细胞中SIRT2、HIF-1α水平。结果:与CT26细胞相比,CT26/SIRT2细胞SIRT2 mRNA、蛋白水平升高,HIF-1αmRNA与蛋白水平降低(P<0.05);CT26、CT26/SIRT2细胞的增殖抑制率差异无统计学意义(P>0.05);与CT26小鼠相比,CT26/SIRT2小鼠剥离瘤体体积与重量、Ki67阳性表达,小鼠肿瘤组织Th17细胞比例、组织匀浆上清液中IL-17、IL-6水平、Th17细胞HIF-1αmRNA与蛋白水平均降低(P<0.05);肿瘤组织细胞凋亡率、Th17细胞SIRT2 mRNA和蛋白水平升高(P<0.05)。结论:SIRT2可抑制结肠癌微环境CD4^(+)T细胞向Th17细胞分化,并下调HIF-1α表达。

Objective:To investigate the effect of silent information regulator 2(SIRT2)overexpression on the differentiation of CD4^(+)T cells into Th17 cells in the colon cancer microenvironment and its influence on hypoxia-inducible factor-1α(HIF-1α).Methods:CT26 overexpressed SIRT2 in mouse colon cancer cells,and a stable SIRT2 overexpressed CT26 cell line was constructed(CT26/SIRT2).The levels of SIRT2 and HIF-1αwere detected by qRT-PCR and Western blot;and the effect of SIRT2 overexpression on the proliferation ability of CT26 cells was verified by CCK-8 method.Twenty-four healthy C57BL/6 mice were randomly separated into CT26 group and CT26/SIRT2 group,inoculated with CT26 cells and CT26/SIRT2 cells,respectively.Mice were sacrificed after 28 days,the proliferation and apoptosis of mouse tumor tissue cells were observed by Ki67 staining and TUNEL staining,and the levels of IL-17 and IL-6 in the supernatant of tissue homogenate were determined;Th17 cells were isolated by flow cytometry,and the levels of SIRT2 and HIF-1αin Th17 cells were detected by qRT-PCR and Western blot.Results:Compared with CT26 cells,CT26/SIRT2 cells had higher SIRT2 mRNA and protein levels,and lower HIF-1αmRNA and protein levels(P<0.05);there was no significant difference in the proliferation inhibition rates of CT26 and CT26/SIRT2 cells(P>0.05);compared with CT26 mice,CT26/SIRT2 mice had decreased tumor volume and weight,positive expression of Ki67,proportion of Th17 cells in mouse tumor tissues,levels of IL-17 and IL-6 in tissue homogenate supernatant,mRNA and protein levels of HIF-1αin Th17 cells(P<0.05);and increased apoptotic rate of tumor cells,the levels of SIRT2 mRNA and protein in Th17 cells(P<0.05).Conclusion:SIRT2 can inhibit the differentiation of CD4^(+)T cells into Th17 cells in the colon cancer microenvironment and down-regulate the expression of HIF-1α.