骨髓间充质干细胞来源的miR-199a-5p通过抑制ZIK1/MAP3K11调节肾细胞癌细胞的增殖和侵袭

miR-199a-5p derived from bone marrow mesenchymal stem cells regulates the proliferation and invasion of renal cell carcinoma by inhibiting ZIK1/MAP3K11

目的:研究骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)来源的外泌体(exsome)中miR-199a-5p对肾细胞癌(renal cell carcinoma,RCC)细胞增殖和侵袭的影响。方法:分离骨髓间充质干细胞来源的外泌体(BM-MSCs-Exo),电子显微镜观察外泌体形态,Western blot方法检测外泌体标志蛋白CD9和CD63。CCK-8法检测RCC细胞在不同条件下的增殖速度,Transwell法检测RCC细胞的侵袭能力,q-PCR法检测miR-199a-5p以及ZIK1和MAP3K11的mRNA表达,双荧光素酶实验验证miR-199a-5p以及ZIK1和MAP3K11基因的3’UTR区域靶向结合。结果:BM-MSCs-Exo电镜下呈圆形囊泡状,直径约100 nm,且高表达CD9和CD63,RCC细胞摄取BM-MSCs-Exo后增殖和侵袭能力均被抑制,过表达miR-199a-5p的BM-MSCs-Exo能显著抑制RCC细胞的增殖和侵袭能力,抑制BM-MSCs-Exo中的miR-199a-5p后能缓解BM-MSCs-Exo对RCC的抑制作用,且发现过表达miR-199a-5p的BM-MSCs-Exo能显著降低ZIK1和MAP3K11的mRNA表达,而在BM-MSCs-Exo中抑制miR-199a-5p有相反作用。结论:BM-MSCs-Exo通过向RCC细胞转运miR-199a-5p抑制ZIK1和MAP3K11的表达,进而抑制RCC细胞的增殖和侵袭。

Objective:To investigate the effects of miR-199a-5p in exosomes derived from bone marrow mesenchymal stem cells(BM-MSCs)on proliferation and invasion of renal cell carcinoma(RCC)cells.Methods:The exosomes derived from bone marrow mesenchymal stem cells(BM-MSCs-Exo)were isolated.The morphology of the exosomes was observed by electron microscopy,and the exosomal marker proteins CD9 and CD63 were detected by Western blot.The proliferation rate of RCC cells under different conditions was detected by CCK-8 method,the invasion ability of RCC cells was detected by Transwell method,and the mRNA expression of miR-199a-5p,ZIK1 and MAP3K11 was detected by q-PCR method.Dual luciferase assay verified the targeted binding of miR-199a-5p and the 3'UTR region of ZIK1 and MAP3K11 genes.Results:Under electron microscope,BM-MSCs-Exo was a circular bubble with a diameter of about 100 nm and high expression of CD9 and CD63.The proliferation and invasion of RCC cells were inhibited after uptake of BM-MSCs-Exo.BM-MSCs-Exo with overexpression of miR-199a-5p could significantly inhibit the proliferation and invasion of RCC cells.Inhibition of miR-199a-5p in BM-MSCs-Exo can alleviate the inhibitory effect of BM-MSCs-Exo on RCC,and it was found that BM-MSCs-Exo with overexpression of miR-199a-5p can significantly reduce the mRNA expression of ZIK1 and MAP3K11.However,inhibition of miR-199a-5p in BM-MSCs-Exo had the opposite effect.Conclusion:BM-MSCs-Exo inhibits the expression of ZIK1 and MAP3K11 by transporting miR-199a-5p to RCC cells,and then inhibits the proliferation and invasion of RCC cells.