miR-210通过MMP-2下调M2/M1巨噬细胞比例抑制肺癌细胞生长

miR-210 targeting MMP-2 inhibits the growth of lung cancer cells by down-regulating the M2/M1 macrophage ratio

目的研究miR-210通过MMP-2调控M2/M1巨噬细胞比例对肺癌细胞生长的影响及对相关机制进行初步探讨。方法将小鼠Lewis lung carcinoma (LLC)-1细胞皮下植入C57BL/6小鼠构建小鼠肺癌模型,荧光定量PCR检测miR-210表达。进一步取40只肺癌建模成功的小鼠,并随机分为4组:对照组小鼠尾静脉注射AAV9-Fugw腺相关病毒;AAV9-OE-miR-210组小鼠尾静脉注射AAV9-OE-miR-210腺相关病毒;AAV9-OE-MMP-2组小鼠尾静脉注射AAV9-OE-MMP-2腺相关病毒;AAV9-OE-miR-210+AAV9-OE-MMP-2组小鼠尾静脉注射AAV9-OE-miR-210和AAV9-OE-MMP-2腺相关病毒。所有小鼠注射腺相关病毒滴度为108TU,病毒注射时间为4周。检测4组小鼠肿瘤大小变化,WGA染色检测4组小鼠肺癌细胞大小变化。另外,取生长融合至80%的LLC1细胞,并随机分为2组:WT组转染包含野生型MMP-2 3’-UTR的构建物,Mut组转染突变型MMP-2 3’-UTR的构建物。细胞转染48 h后,荧光素酶实验检测miR-210和MMP-2(matrix metalloproteinases-2)的结合情况。另取生长融合至80%的LLC1细胞,并随机分为3组:对照组常规培养细胞,不给予任何特殊处理;miR-210 mimic组给予50μmol/L的miR-210 mimic药物刺激;miR-210 inhibitor组给予100μmol/L的miR-210 inhibitor药物刺激,以上细胞处理48 h后进行检测。荧光定量PCR检测小鼠肺组织中巨噬细胞M1型标志物白细胞介素IL-6和诱导型一氧化氮合酶iNOS的表达以及M2型标志物精氨酸酶Arg-1和白细胞分化抗原CD206的表达。结果荧光定量PCR实验显示,miR-210在小鼠肺癌组织中表达下调;肿瘤质量检测发现,肺癌小鼠尾静脉注射AAV9-OE-miR-210后肿瘤质量减轻,肺癌小鼠尾静脉注射AAV9-shmiR-210后肿瘤质量增加;WGA染色结果发现,肺癌小鼠尾静脉注射AAV9-OE-miR-210后肺癌细胞变小,肺癌小鼠尾静脉注射AAV9-sh-miR-210后肺癌细胞变大。荧光定量PCR结果发现,肺癌小鼠尾静脉注射AAV9-OE-miR-210后,小鼠肺组织中巨噬细胞M1型标志物白细胞介素IL-6和诱导型一氧化氮合酶iNOS表达升高,M2型标志物精氨酸酶Arg-1和白细胞分化抗原CD206表达明显降低;肺癌小鼠尾静脉注射AAV9-sh-miR-210后,小鼠肺组织中巨噬细胞M1型标志物白细胞介素IL-6和诱导型一氧化氮合酶iNOS表达降低,M2型标志物精氨酸酶Arg-1和白细胞分化抗原CD206表达明显升高。荧光素酶实验发现miR-210可以和MMP-2 3’-UTR区域结合。肿瘤质量检测和WGA染色发现,过表达miR-210可以逆转过表达MMP-2导致的肿瘤质量增加和肺癌细胞的变大。结论 miR-210通过调控M2/M1巨噬细胞比例抑制肺癌细胞生长。

To investigate the effects of miR-210 targeting MMP-2 on the growth of lung cancer cells by regulating the ratio of M2/M1 macrophages,and to preliminarily explore the relevant mechanisms.Lewis lung carcinoma(LLC)-1 cells were subcutaneously implanted into C57 BL/6 mice to establish mouse lung cancer model,and the expression of miR-210 was detected by fluorescence quantitative PCR.Further,forty lung cancer model mice were selected and randomly divided into 4 groups:control group mice were injected with AAV9-Fugw adenoassociated virus through tail vein;AAV9-OE-miR-210 group mice were injected with AAV9-OE-miR-210 adeno-associated virus through tail vein;AAV9-OE-MMP-2 group mice were injected with AAV9-OE-MMP-2 adeno-associated virus through tail vein;AAV9-OEmiR-210+AAV9-OE-MMP-2 group mice were injected with AAV9-OE-miR-210 and AAV9-OE-MMP-2 adeno-associated virus through tail vein.All mice were injected with adeno-associated virus at titers of 10^(8) TU for 4 weeks.The changes of tumor size were detected,and the changes of lung cancer cells were detected by WGA staining.In addition,LLC1 cells that grew and fused to 80% were randomly divided into two groups:WT group was transfected with constructs containing wild-type MMP-2 3 ’-UTR,and Mut group was transfected with constructs containing mutant MMP-2 3’-UTR.The binding of miR-210 and MMP-2(matrix metalloproteinases-2) was detected by luciferase assay at 48 h after the transfection.In addition,LLC1 cells grown and fused to 80% were randomly divided into three groups:the control group was conventionally cultured without any special treatment;miR-210 mimic group was given 50 μmol/L miR-210 mimic drug stimulation;miR-210 inhibitor group was given 100 μmol/L of miR-210 inhibitor drug stimulation.And the cells in the three groups were tested after 48 h treatment.Fluorescence quantitative PCR was used to detect the expression of M1 macrophage marker interleukin IL-6 and inducible nitric oxide synthase(iNOS),the expression of M2-type marker argininase Arg-1,and leukocyte differentiation antigen CD206 in mouse lung tissues.Data showed that the expression of miR-210 was downregulated in mouse lung cancer tissues;the tumor weight of lung cancer mice was reduced after the caudal vein injection of AAV9-OE-miR-210,while the tumor weight of lung cancer mice was increased after the caudal vein injection of AAV9-sh-miR-210.The size of lung cancer cells decreased after the caudal vein injection of AAV9-OE-miR-210,while the size of lung cancer cells increased after the caudal vein injection of AAV9-sh-miR-210.Furthermore,the expression of IL-6 and iNOS in lung tissue of mice were increased after the tail vein injection of AAV9-OE-miR-210,while the expression of Arg-1 and CD206 were significantly decreased.After AAV9-shmiR-210 was injected into the tail vein of lung cancer mice,the expressions of IL-6 and iNOS were decreased,while the expressions of Arg-1 and CD206 were significantly increased.Luciferase assay showed that miR-210 could bind to the 3 ’-UTR region of MMP-2.Tumor weight detection and WGA staining showed that overexpression of miR-210 reversed the increase in tumor weight and enlargement of lung cancer cells caused by overexpression of MMP-2.Taken together,miR-210 can inhibit the growth of lung cancer cells by regulating the M2/M1 macrophage ratio.