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中华腔镜泌尿外科杂志(电子版) ›› 2024, Vol. 18 ›› Issue (01) : 82 -89. doi: 10.3877/cma.j.issn.1674-3253.2024.01.015

实验研究

TGF-β1诱导骨髓间充质干细胞外泌体分泌miR-424-3p促进前列腺癌细胞增殖及转移
邓瑞锋, 程璐, 周宇林, 刘远灵, 江文聪, 江敏耀, 江福能, 习明()   
  1. 510800 广州,南方医科大学第三临床医学院广州市花都区人民医院
    广州市花都区人民医院检验科
    广州市花都区人民医院泌尿外科
    510180 广州市第一人民医院,广东省临床分子医学及分子诊断重点实验室
  • 收稿日期:2023-10-10 出版日期:2024-02-01
  • 通信作者: 习明
  • 基金资助:
    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)

TGF-β1 induced hBMSCs exosomes to secrete miR-424-3p to promote the proliferation and migration of prostate cancer cells

Ruifeng Deng, Lu Cheng, Yulin Zhou, Yuanling Liu, Wencong Jiang, Minyao Jiang, Funeng Jiang, Ming Xi()   

  1. The Third School of Clinical Medicine, Southern Medical University, Huadu District People's Hospital of Guangzhou
    Department of Laboratory, Huadu District People's Hospital, Guangzhou 510800, China
    Department of Urology, Huadu District People's Hospital, Guangzhou 510800, China
    Guangzhou First People's Hospital, Guangdong key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou 510180, China
  • Received:2023-10-10 Published:2024-02-01
  • Corresponding author: Ming Xi
引用本文:

邓瑞锋, 程璐, 周宇林, 刘远灵, 江文聪, 江敏耀, 江福能, 习明. TGF-β1诱导骨髓间充质干细胞外泌体分泌miR-424-3p促进前列腺癌细胞增殖及转移[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(01): 82-89.

Ruifeng Deng, Lu Cheng, Yulin Zhou, Yuanling Liu, Wencong Jiang, Minyao Jiang, Funeng Jiang, Ming Xi. TGF-β1 induced hBMSCs exosomes to secrete miR-424-3p to promote the proliferation and migration of prostate cancer cells[J/OL]. Chinese Journal of Endourology(Electronic Edition), 2024, 18(01): 82-89.

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。

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.

图1 TGF-β1增加hBMSCs中的碱性磷酸酶活性(a)和成骨相关蛋白表达水平(b)注:***P<0.001
图2 TGF-β1处理的hBMSCs培养上清液对PC3细胞增殖实验(a)、侵袭实验(b~c)和划痕实验(d~e)注:TGF-β1能显著提高PC3细胞增殖、迁移和侵袭能力;与PC3组相比,***P<0.001;与hBMSCs+PC3组相比,###P<0.001
图3 透射电镜观察外泌体形态(a),外泌体粒径以及浓度检测(b)和外泌体特异性标志性蛋白检测(c)注:hBMSCs_Exo为从hBMSCs获得的外泌体,TGF-β1_hBMSCs_Exo为经TGF-β1处理hBMSCs获得的外泌体
图4 miRNA测序中hBMSCs细胞来源外泌体火山图(a),前5个miRNA分子热图(b)以及qPCR验证前5个miRNA分子在外泌体中的表达差异(c)注:***P<0.001,nsP>0.05
图5 转染miR-424-3p质粒以及不同来源的外泌体对PC3细胞增殖实验(a)、侵袭实验(b~c)和划痕实验(d~e)注:与PC3+miRNA NC组相比,***P<0.001
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