摘要
目的:探讨TGF-β1诱导骨髓间充质干细胞（hBMSCs）成骨分化来源的外泌体对前列腺癌（PCa）细胞PC-3的增殖、迁移和侵袭的影响。方法:通过ELISA检测碱性磷酸酶（ALP）活性和Western blot检测TGF-β1诱导hBMSCs成骨分化相关蛋白；采用超速离心法从细胞培养液中分离提取外泌体，使用投射电子显微镜（TEM）、纳米粒径追踪技术（NTA）以及Western blot对分离得到的外泌体进行鉴定；通过深度RNA测序技术鉴定TGF-β1诱导后hBMSCs外泌体中miRNA的表达谱和qPCR检测miRNA在外泌体中的表达水平；CCK8法检测细胞活力，Wound-healing检测细胞迁移能力，Trans-well试验检测细胞侵袭能力。结果:与对照组相比，TGF-β1能显著提高hBMSCs的ALP活性，以及成骨相关因子BMP-2、OCN和RUNX2蛋白表达水平（P<0.001）。与hBMSCs组相比，TGF-β1_hBMSCs组的细胞增殖、迁移和侵袭能力显著提高（P<0.001）。接着成功分离hBMSCs（hBMSCs_Exo组）和TGF-β1诱导后hBMSCs（TGF-β1_hBMSCs_Exo组）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等外泌体特异性蛋白，其中TGF-β1_hBMSCs_Exo组浓度高于hBMSCs_Exo组浓度。基于miRNA测序显示TGF-β1_hBMSCs_Exo中95个miRNA分子表达升高，选择前5个miRNA进行qPCR验证，相比较于hBMSCs_Exo组，miR-424-3p在TGF-β1_hBMSCs_Exo组中显著升高（P<0.001）。这和miRNA测序结果相一致。与miRNA NC组相比，miR-424-3p mimic组和TGF-β1_hBMSCs_Exo中PC3细胞的增殖、迁移和侵袭能力均显著升高（P<0.001）；miR-424-3p mimic组和TGF-β1-Exo组细胞增殖、迁移和侵袭能力差异无统计学意义（P>0.05）。结论:TGF-β1诱导hBMSCs外泌体miR-424-3p能显著提高PC3细胞的增殖、迁移和侵袭能力，可能为出现骨转移性PCa个体化治疗提供新的靶点。
Abstract
Objective:To investigate the effect of exosomes derived from TGF-β1-induced osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) on the proliferation, migration, and invasion of prostate cancer (PCa) PC-3 cells.Methods:The impact of TGF-β1-induced osteogenic differentiation of hBMSCs was assessed using ALP ELISA and Western blot. Exosomes were isolated from cell culture supernatants via ultracentrifugation and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. The expression profile of miRNAs in hBMSCs exosomes following TGF-β1 induction was determined using deep RNA sequencing technology, and the expression level of miRNAs in exosomes was measured using qPCR. Cell proliferation was evaluated using the CCK8 assay, cell migration ability was assessed using the Wound-healing assay, and cell invasion ability was determined using the Trans-well assay.Results:Compared to the control group, TGF-β1 treatment significantly increased the alkaline phosphatase (ALP) activity on hBMSCs and resulted in a significant up regulation of osteogenic-related factors BMP-2, OCN, and RUNX2 at the protein level, with significant differences (P<0.001). Additionally, it was found that compared to the PC3 cells in hBMSCs group, the proliferation, migration, and invasion abilities of PC3 cells in the TGF-β1-treated hBMSCs group were significantly elevated (P<0.001). Subsequently, exosomes were successfully isolated from the culture supernatants of hBMSCs (hBMSCs_Exo group) and BMSCs following TGF-β1 induction (TGF-β1_hBMSCs_Exo group). Under transmission electron microscopy, typical vesicular structures of exosomes were observed, and exosome-specific proteins such as CD9, CD63, and CD81 were expressed. Notably, the concentration of TGF-β1_hBMSCs_Exo was higher than that of hBMSCs_Exo. Based on miRNA sequencing results, 95 miRNAs were found to be upregulated in TGF-β1_hBMSCs_Exo. The top 5 miRNAs were selected for qPCR validation. The results showed that compared to hBMSCs_Exo, miR-424-3p expression was significantly increased in TGF-β1_hBMSCs_Exo (P<0.001), which was consistent with the miRNA sequencing results. Compared to the miRNA NC group, the proliferation, migration, and invasion abilities of PC3 cells in the miR-424-3p mimic group and TGF-β1_hBMSCs_Exo group were all significantly elevated (P<0.001). Furthermore, it was found that there was no significant difference between the proliferation, migration, and invasion abilities of the miR-424-3p mimic group and TGF-β1_hBMSCs_Exo group.Conclusion:The miR-424-3p present in hBMSCs exosomes induced by TGF-β1 can significantly enhance the proliferation, migration and invasion abilities of PC3 cells. These findings may provide a new target for individualized treatment of bone metastatic PCa.
基金项目
广州市科技计划项目(202102080624)
广州市医学重点学科建设项目(2021-2023年)()
万方数据