目的：探讨骨髓间充质干细胞（bone marrow mesenchymal stem cell，BMSC）来源的外泌体对前列腺癌细胞PC-3 的增殖、迁移和侵袭的影响及其作用机制。方法：采用qPCR检测miR-21-5p 在前列腺癌细胞系中的表达水平。采用电子显微镜观察BMSC分离出的外泌体形态，Western blotting 检测外泌体表面标志物的表达以及上皮间质转化（epithelial-mesenchymal transition，EMT）相关蛋白E-cadherin、N-cadherin 和Vimentin 的表达。采用双荧光素酶报告基因实验检测miR-21-5p 和同源血小板富亮氨酸复重蛋白磷酸酶2（PH domain leucine-rich repeat protein phosphatase 2，PHLPP2）的靶向调控关系。向PC-3 细胞培养液中加入10 μl 的BMSC外泌体悬液（Exo 组）、转染sh-PHLPP2 或antagomiR，CCK-8 和Transwell 实验检测PC-3 细胞增殖和迁移能力。结果：miR-21-5p 在前列腺癌PC-3 细胞系中高表达。成功分离BMSC培养液上清中的外泌体，透射电子显微镜下观察到外泌体典型的囊泡状结构，且表达CD9、CD63 和CD81 等特异性蛋白。Exo组中PC-3 细胞的增殖、侵袭[ （421.34±22.45）vs（200.09±14.22）个，P<0.05]、迁移能力和N-cadherin、Vimentin和miR-21-5p的表达水平均显著高于对照组（均P<0.05）。证实PHLPP2 是miR-21-5p的靶基因。与对照组相比，Exo 组和sh-PHLPP2 组PC-3 细胞中PHLPP2 的表达明显降低（0.66±0.09、0.42±0.05 vs 1.09±0.08，均P<0.01），细胞增殖、侵袭和迁移[ （87.23±12.67）%、（82.45±10.13）% vs（66.46±9.13）%]能力均显著提高（均P<0.01），E-cadherin 表达水平显著降低而N-cadherin 和Vimentin 表达水平显著升高（均P<0.05）。结论：miR-21-5p 在前列腺癌PC-3 细胞系中高表达，BMSC外泌体miR-21-5p通过靶向下调PHLPP2提高PC-3 细胞的增殖、迁移和侵袭能力。

Objective: To investigate the effects of exosome originated from bone marrow mesenchymal stem cell (BMSCs) on proliferation,migration and invasion of prostate cancer PC-3 cell and its mechanism. Methods: qPCR was used to detect the expression level of miR-21-5p in prostate cancer cell lines. The morphology of exosomes isolated from BMSCs was observed with an electron microscope.Western blotting was used to detect the expressions of exosome surface markers and the epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin and Vimentin). Dual luciferase reporter gene experiment was used to detect the targeted regulation relationship between miR-21-5p and PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2). PC-3 cells were co-cultured with 10 μl BMSCs exosomes suspension (Exo group), transfected with sh-PHLPP2 or antagomiR, then CCK-8 and Transwell experiments were used to detect changes in proliferation, migration and invasion of PC-3 cell. Results: miR-21-5p was highly expressed in prostate cancer PC-3 cell line. The exosomes in the supernatant of BMSCs culture fluid were successfully isolated, and the typical vesicle-like structures of exosomes were observed under transmission electron microscope. Exosomes expressed specific proteins such as CD9, CD63 and CD81. In the Exo group, the proliferation, invasion, migration, as well as the expressions of N-cadherin, Vimentin and miR-21-5p in PC-3 cells were significantly higher than those in the control group (all P<0.05). PHLPP2 is a target gene of miR-21-5p. Compared with the control group, the expression of PHLPP2 in PC-3 cells of Exo group and sh-PHLPP2 group was significantly reduced (0.66±0.09, 0.42±0.05 vs 1.09±0.08, all P<0.01); cell viability, invasion and migration were significantly improved (all P<0.01); and E-cadherin expression level was significantly reduced while N-cadherin and Vimentin expressions were significantly increased (both P<0.05). Conclusion: miR-21-5p is highly expressed in prostate cancer PC-3 cell line. BMSC exosome miR-21-5p can increase the proliferation, migration and invasion ability of PC-3 cells through targeted down-regulation of PHLPP2.

泸州市科技局资助项目（No. 2017LZXNYD-T09）