上调RI对小鼠黑色素瘤B16-F10细胞EMT及转移的影响

Effects of up-regulating RI on EMT and metastasis of mouse melanoma B16-F10

目的研究核糖核酸酶抑制因子基因表达对小鼠黑色素瘤B16-F10细胞EMT及转移的影响。方法构建RI真核表达质粒p IRES2-EGFP-RI,稳定转染B16-F10细胞。RT-PCR、Western blot和免疫荧光检测RI的表达;HE染色及相差显微镜观察细胞形态;FITC标记的鬼笔环肽染色,激光共聚焦观察细胞骨架;黏附实验、划痕实验和Transwell法检测细胞黏附、迁移和侵袭能力的变化;Western blot检测EMT及转移相关蛋白的表达;分别将各组B16-F10细胞眼眶静脉注射到c57/BL小鼠,建立肺转移动物模型,注射3周后处死小鼠。取肺称重,在解剖镜下计数肺转移结节数;肺组织切片HE染色观察肿瘤细胞转移;免疫组化检测肺转移瘤组织中转移及EMT相关蛋白的表达。结果 RI表达上调后,细胞由间质型向上皮型转换,细胞骨架重排;B16-F10-RI细胞组黏附、迁移和侵袭能力下降;与对照组相比,B16-F10-RI细胞中MMP2、MMP9、snail、slug、vimentin、twist和N-cadherin的表达明显降低,而E-cadherin,nm23-H1蛋白的表达明显增加(P<0.01或P<0.01)。实验组与对照组比小鼠肺的转移结节明显减少,同时EMT及转移相关蛋白在瘤组织中表达与体外细胞一致。结论上调核糖核酸酶抑制因子能够显著抑制B16-F10细胞EMT及侵袭、转移,RI可望作为治疗黑色素瘤的靶蛋白。

Objective To investigate the effects of ribonuclease inhibitor expression on EMT and metastasis of mouse melanoma B16-F10 cells. Methods RI eukaryotic expression plasmid p IRES2-EGFP-RI was constructed,then transfected into B16-F10 cells,RI expression was identified by RT-PCR,Western blot and immunofluorescence assay. Cell morphology was observed by HE staining and phase contrast microscopy. The cytoskeleton change was detected with FITC phalloidin staining with laser scanning confocal microscopy. The adhesion assay,scratch assay and Transwell assay were used to detect the cell adhesion,migration and invasion ability. The expressions of metastasis and EMT related proteins were determined by Western blot. Various B16-F10 cells were respectively injected into the vein of the eye socket of c57 / BL mice to establish pulmonary metastasis animal model. Three weeks after injection,the mice were sacrificed,lungs were removed and weighed. And then the number of metastasis nodules was counted under a dissecting microscope. The lung tissue sections were stained with HE staining,and theexpressions of metastasis and EMT related proteins were detected by immunohistochemistry. Results Up-regulation of RI inhibited migration and invasion as well as changes cell morphology,adhesion and rearranged cytoskeleton in vitro. In addition,Over-expression RI induced up-regulation of E-cadherin,accompanied with decreased expressions of proteins associated with EMT such as N-cadherin,snail,slug,vimentin and twist both in vitro and in vivo.Furthermore,RI restrained matrix metalloproteinase MMP-2 and MMP-9 secretions in B16-F10 melanoma cells. RI suppressed invasiveness and metastasis by the experimental metastasis models of melanoma with lighter lung weight,a fewer metastasis nodules and a lower incidence rate. Conclusions Up-regulating ribonuclease inhibitor may significantly inhibit EMT,invasion and metastasis of B16-F10 cell. These results suggest that RI is a potential therapeutic target protein of melanoma.