c-Myc调控PIK3R3抑制黑色素瘤B16-F10细胞的迁移和侵袭

c-Myc promoted migration and invasion of malignant melanoma B16-F10 cells through regulating PIK3R3

目的:探讨c-Myc调控PIK3R3对黑色素瘤B16-F10细胞的迁移和侵袭的影响。方法:运用免疫组化检测PIK3R3在正常皮肤组织和黑色素瘤组织的表达;检测慢病毒(LV3-c-Myc和LV3-PIK3R3)对c-Myc和PIK3R3基因的沉默效率;使用Ed U增殖实验检测沉默c-Myc和PIK3R3后黑色素瘤细胞株B16-F10的增殖情况;Transwell侵袭实验检测c-Myc和PIK3R3的表达对黑色素瘤细胞B16-F10的侵袭能力的影响;划痕实验检测c-Myc和PIK3R3的表达对黑色素瘤细胞B16-F10迁移能力的影响;q PCR检测沉默c-Myc和PIK3R3后miR-199a的表达;q PCR检测转染miR-199a mimics和miR-199a inhibitor后c-Myc和PIK3R3的表达;双荧光素酶报告基因系统检测miR-199a对c-Myc和PIK3R3转录活性的影响。结果:和正常皮肤组织比较,PIK3R3在黑色素瘤组织中表达明显增加;沉默c-Myc和PIK3R3基因后,黑色素瘤细胞株B16-F10的增殖、侵袭和转移能力明显降低;沉默c-Myc和PIK3R3基因后,miR-199a表达上调;转染miR-199a mimics后,c-Myc和PIK3R3表达降低;转染miR-199a inhibitor后,c-Myc和PIK3R3表达上调;双荧光素酶报告基因系统检测结果显示,miR-199a可以直接调控c-Myc和PIK3R3的转录活性。结论:c-Myc可以通过miR-199a调控PIK3R3的表达,从而促进黑色素瘤细胞的增殖、侵袭和迁移能力。

Objective： To investigate the effect and the related mechanism of c-Myc on the proliferation,invasion and migration ability of malignant melanoma B16-F10 cells. Methods： We detected the expression of PIK3R3 in malignant melanoma and normal tissues. Efficiency of gene silencing of c-Myc and PIK3R3 was determined by q PCR and Western blot. We detected the proliferate ability of B16-F10 cells after silencing c-Myc and PIK3R3 using Ed U assay. We detected the migration and invasion ability of B16-F10 cells after silencing c-Myc and PIK3R3 using wound healing assays and Transwell matrigel invasion assays. The expression of miR-199 a after silencing c-Myc and PIK3R3 using q PCR. The expression of c-Myc and PIK3R3 was detected by q PCR after transfecting miR-199 a mimics or miR-199 a inhibitor. Dual-luciferase reporter assay system was used to detect miR-199 a regulating c-Myc and PIK3R3. Results： Compared normal skin tissue,expression of PIK3R3 was significantly increased in malignant melanoma tissue; after silencing c-Myc and PIK3R3 gene,the proliferation,invasion and metastasis of melanoma cell line B16-F10 were significantly reduced;expression of miR-199 a upregulated after silencing c-Myc and PIK3R3 genes,expression of c-Myc and PIK3R3 decreased after transfecting miR-199 a mimics,expression of c-Myc and PIK3R3 upregulated after transfecting miR-199 a inhibitor,dual luciferase reporter system test results revealed miR-199 a can directly regulate c-Myc and PIK3R3 transcription activity. Conclusion： miR199 a regulated the expression of c-Myc,then promote proliferation,migration and invasion in malignant melanoma cells.