miR-185-5p mimic阻碍黑色素瘤A375细胞的侵袭、迁移和上皮间质转化及肿瘤形成

miR-185-5p Mimic Blocks the Invasion, Migration, Epithelial-mes-enchymal Transition and Tumor Formation of Melanoma A375 Cells

目的研究miR-185-5p mimic靶向MAPK14阻碍黑色素瘤A375细胞的侵袭,迁移和上皮间质转化,抑制肿瘤生长的影响.方法采用LipofecamineTM2000分别或同时对A375细胞转染miR-185 mimic和pcDNA-MAPK14,通过RT-PCR与荧光素酶实验验证miR-185-5p与MAPK14的靶向关系;transwell小室和细胞划痕实验分析miR-185-5p过表达对A375细胞侵袭、迁移能力的影响;Western印迹分析miR-185-5p过表达对A375细胞VEGF、 E-cadherin、 N-cadherin水平的影响.构建移植瘤裸鼠模型,检测30 d时肿瘤重量, RT-PCR分析肿瘤组织中miR-185-5p和MAPK14 的表达量;免疫组化分析肿瘤组织中Ki67 及VEGF水平.结果 miR-185 mimic能够促进miR-185-5p表达,抑制MAPK14表达( P<0. 05),荧光素酶报告实验表明miR-185-5p和MAPK14之间存在靶向调控关系. miR-185-5pmimic能够抑制A375细胞的侵袭、迁移能力( P<0. 05),抑制细胞 VEGF、 N-cadherin 水平,增加 E-cadherin 水平( P <0. 05).移植瘤裸鼠模型显示, miR-185 mimic能够明显抑制移植瘤的生长,上调肿瘤组织中miR-185-5p表达,下调MAPK14表达,降低Ki67、 VEGF水平(P<0. 05).结论 miR-185-5p mimic 靶向 MAPK14 通过阻断细胞的 EMT 机制,降低Ki67、 VEGF水平,抑制黑色素瘤A375细胞的侵袭、迁移和肿瘤形成.

Objective To study the effects of miR-185-5p mimic blocking the invasion, mi-gration, epithelial-mesenchymal transition and tumor formation of melanoma A375 cells by targeting MAPK14. Methods LipofecamineTM2000 was adopted to transfect miR-185 mimic and pcDNA-MAPK14 separately or simultaneously into A375 cells. RT-PCR and luciferase assay were used to prove the targeting relationship between miR-185-5p and MAPK14. Transwell chamber and cell wound scratch assays were used to test the influence of miR-185-5p overexpression on invasion and migration of A375 cells. Western blotting was used to analyze the effects of miR-185-5p overexpres-sion on the levels of VEGF, E-cadherin and N-cadherin in A375 cells. Tumor transplant model of nude mouse was established, and the tumor weight at 30 days was detected, and RT-PCR was used to analyze the expression levels of miR-185-5p and MAPK14 in the tumor tissues, and immunohisto-chemistry to measure the expression levels of Ki67 and VEGF in the tumor tissues. Results miR- 185 mimic could promote miR-185-5p expression while inhibiting MAPK14 expression ( P<0. 05), and the luciferase reporter assay showed that there was a targeting relationship betweenmiR-185-5p and MAPK14. miR-185-5p mimic could prevent the invasion and migration of A375 cells ( P <0. 05), inhibit the levels of VEGF and N-cadherin and increase the E-cadherin level ( P<0. 05). In tumor transplant models of nude mouse, it was found that miR-185 mimic could significantly pre-vent the growth of transplant tumor, and upregulate miR-185-5p expression and downregulate MAPK14 expression in the tumor tissue, and reduce the levels of Ki67 and VEGF ( P <0. 05 ). Conclusion miR-185-5p mimic inhibits the invasion, migration and tumor formation of melanoma A375 cells by targeting MAPK14, blocking the EMT mechanism of cells and reducing the levels of Ki67 and VEGF.