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Abstract：
OBJECTIVE: To reveal the mechanisms by which miR-513b-5p inhibits metastasis of colon cancer stem cells (CCSCs) through IL-6/STAT3 in HCT116 cells.
METHODS: Sphere formation media and magnetic cell sorting were used to enrich and screen CCSCs. We used a colony formation assay, cell proliferation and viability assays, and a nude mouse transplantation tumor assay to identify CCSCs. ELISA was performed to identify IL-6 in the cell culture medium, and the growth, viability, wound healing, and transwell migration of distinct cell groups were compared to differentiate them. Dual-luciferase reporter assay, RT-PCR, and/or Western Blot analysis were conducted to determine the correlation between them.
RESULTS: CD133+CD44+ HCT116 cells were shown to have higher cloning efficiency, greater proliferation ability and viability, and stronger tumorigenicity. A dual-luciferase reporter assay revealed that miR-513b-5p negatively affected STAT3 expression. RT-PCR and/or Western Blot analysis suggested that miR-513b-5p negatively affected STAT3 and Vimentin, while positively affecting E-cadherin expression. The STAT3 overexpression vector + miR-513b-5p inhibitor cell group had the highest efficiency, greatest proliferation ability and viability, and the highest IL-6 level in the experiments.
CONCLUSIONS: Mir-513b-5p inhibited the epithelial-mesenchymal transition (EMT) of CCSCs through IL-6/STAT3. This potential mechanism may provide a new therapeutic target for colon cancer.
METHODS: Sphere formation media and magnetic cell sorting were used to enrich and screen CCSCs. We used a colony formation assay, cell proliferation and viability assays, and a nude mouse transplantation tumor assay to identify CCSCs. ELISA was performed to identify IL-6 in the cell culture medium, and the growth, viability, wound healing, and transwell migration of distinct cell groups were compared to differentiate them. Dual-luciferase reporter assay, RT-PCR, and/or Western Blot analysis were conducted to determine the correlation between them.
RESULTS: CD133+CD44+ HCT116 cells were shown to have higher cloning efficiency, greater proliferation ability and viability, and stronger tumorigenicity. A dual-luciferase reporter assay revealed that miR-513b-5p negatively affected STAT3 expression. RT-PCR and/or Western Blot analysis suggested that miR-513b-5p negatively affected STAT3 and Vimentin, while positively affecting E-cadherin expression. The STAT3 overexpression vector + miR-513b-5p inhibitor cell group had the highest efficiency, greatest proliferation ability and viability, and the highest IL-6 level in the experiments.
CONCLUSIONS: Mir-513b-5p inhibited the epithelial-mesenchymal transition (EMT) of CCSCs through IL-6/STAT3. This potential mechanism may provide a new therapeutic target for colon cancer.
摘要：
目的：探讨miR-513b-5p通过IL-6/STAT3抑制结肠癌干细胞(CCSCs)在HCT116细胞中转移的机制。
方法：球体形成培养基和磁性细胞分选用于富集和筛选CCSCs。我们用了集落形成试验,细胞增殖和活力测定，和裸鼠移植瘤试验以鉴定CCSCs。进行ELISA以鉴定细胞培养基中的IL-6，和增长，生存能力，伤口愈合，并比较不同细胞群的transwell迁移以区分它们。双荧光素酶报告基因测定,RT-PCR,进行和/或WesternBlot分析以确定它们之间的相关性。
结果：CD133+CD44+HCT116细胞具有更高的克隆效率，更大的增殖能力和生存能力，和更强的致瘤性。双荧光素酶报告基因分析显示miR-513b-5p对STAT3表达有负面影响。RT-PCR和/或WesternBlot分析提示miR-513b-5p对STAT3和波形蛋白有负面影响，同时积极影响E-cadherin表达。STAT3过表达载体+miR-513b-5p抑制剂细胞组有效率最高,最大的增殖能力和生存能力，和实验中最高的IL-6水平。
结论：Mir-513b-5p通过IL-6/STAT3抑制CCSCs的上皮-间质转化(EMT)。这种潜在的机制可能为结肠癌提供新的治疗靶点。
方法：球体形成培养基和磁性细胞分选用于富集和筛选CCSCs。我们用了集落形成试验,细胞增殖和活力测定，和裸鼠移植瘤试验以鉴定CCSCs。进行ELISA以鉴定细胞培养基中的IL-6，和增长，生存能力，伤口愈合，并比较不同细胞群的transwell迁移以区分它们。双荧光素酶报告基因测定,RT-PCR,进行和/或WesternBlot分析以确定它们之间的相关性。
结果：CD133+CD44+HCT116细胞具有更高的克隆效率，更大的增殖能力和生存能力，和更强的致瘤性。双荧光素酶报告基因分析显示miR-513b-5p对STAT3表达有负面影响。RT-PCR和/或WesternBlot分析提示miR-513b-5p对STAT3和波形蛋白有负面影响，同时积极影响E-cadherin表达。STAT3过表达载体+miR-513b-5p抑制剂细胞组有效率最高,最大的增殖能力和生存能力，和实验中最高的IL-6水平。
结论：Mir-513b-5p通过IL-6/STAT3抑制CCSCs的上皮-间质转化(EMT)。这种潜在的机制可能为结肠癌提供新的治疗靶点。
