circRNA_103809对结肠癌SW480细胞线粒体功能和炎症因子水平的调节作用

Regulation of circRNA_103809 on mitochondrial function and inflammatory factor levels in colon cancer SW480 cells

目的:探究环状RNA(circRNA)_103809对结肠癌SW480细胞作用。方法:qRT-PCR检测结肠癌组织及细胞、正常组织及细胞中circRNA_103809表达。将SW480细胞分为对照组(Control),过表达circRNA_103809组(LV-circRNA_103809)及其阴性对照组(LV-scramble)。除对照组外,按分组分别转染LV-circRNA_103809和LV-scramble质粒。qRT-PCR检测各转染组SW480细胞中circRNA_103809、Ki67和p21表达;CCK8检测各组细胞增殖倍数;流式细胞术检测各组细胞凋亡率;线粒体膜电位的变化;试剂盒检测各组细胞超氧化物歧化酶(SOD)和丙二醛(MDA)含量;ELISA检测各组细胞上清液中IL-1β、IL-6和IL-4炎症因子水平,免疫荧光检测p65的核转移;Western blot检测Bax/Bcl-2,活化胱天蛋白酶(cleaved cas)-9/cas-9,cleaved cas-3/cas-3相对表达量及细胞核中p65蛋白的表达。结果:结肠癌组织及细胞中circRNA_103809表达显著下调,p21的表达显著上调(P<0.05)。与对照组相比,过表达circRNA_103809组SW480细胞中circRNA_103809的表达显著上调,Ki67的表达显著下调(P<0.05),细胞增殖倍数显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),线粒体膜电位降低,Bax/Bcl-2、cleaved cas-9/cas-9和cleaved cas-3/cas-3显著升高(P<0.05),SOD含量减少,MDA含量增加(P<0.05),促炎因子IL-1β和IL-6水平显著降低,抗炎因子IL-4水平显著升高(P<0.05),细胞核中p65蛋白表达显著下调,核定位显著减少(P<0.05)。结论:circRNA_103809在结肠癌SW480细胞中发挥抑癌作用,抑制细胞增殖,通过增强氧化应激促进细胞凋亡,抑制炎症反应阻止癌细胞发展进程。

Objective:To explore effect and mechanism of circular RNA(circular RNA)_103809 on colon cancer SW480cells.Methods:qRT-PCR was used to detect expression of circRNA_103809 in colon cancer tissues and cells,normal tissues and cells.SW480 cells were divided into control group,overexpression circRNA_103809 group(LV-circRNA_103809 group)and negative control group(LV-scramble).In addition to control group,LV-circRNA_103809 and LV-scramble plasmids were transfected in groups.qRT-PCR was used to detect expressions of circRNA_103809,Ki67 and p21 in SW480 cells of each transfection group;CCK8was performed to detect cell proliferation of each group;flow cytometry was commended to detect cell apoptosis rate of each group;flow cytometry was employed to detect changes in mitochondrial membrane potential of each group of cells;kits was used to detect content of SOD and MDA in each group of cells;ELISA was performed to detect levels of IL-1β,IL-6 and IL-4 inflammatory factors in cell supernatant of each group,and immunofluorescence was performed to detect nuclear metastasis of p65;Western blot was commended to detect relative expression of Bax/Bcl-2,cleaved caspase(cas)-9/cas-9,cleaved cas-3/cas-3 and expression of p65 protein in nucleus.Results:Expression of circRNA_103809 in colon cancer tissues and cells was significantly reduced(P<0.05).Compared with control group,expression of circRNA_103809 in SW480 cells in overexpression group of circRNA_103809 was significantly up-regulated,expression of Ki67 was significantly down-regulated,expression of p21 was significantly up-regulated(P<0.05),cell proliferation ratio was significantly reduced(P<0.05),and apoptosis rate was significantly increased(P<0.05),mitochondrial membrane potential decreased,ratio of Bax/Bcl-2,cleaved cas-9/cas-9 and cleaved cas-3/cas-3 were increased significantly(P<0.05),SOD content was decreased,MDA content was increased(P<0.05),levels of pro-inflammatory factors IL-1βand IL-6 were significantly reduced,levels of anti-inflammatory factor IL-4 was significantly increased(P<0.05),expression of p65 protein in nucleus was significantly down-regulated,and nuclear localization was significantly reduced(P<0.05).Conclusion:circRNA_103809 has a significant anti-tumor effect in colon cancer SW480 cells,inhibits cell proliferation,promotes apoptosis by enhancing oxidative stress,and inhibits inflammation to prevent cancer cell development.