目的: 探讨外泌体分泌miR-133a-3p调控TGFBR1从而促进前列腺癌进展的分子机制。方法: 提取前列腺癌细胞中的外泌体。免疫印迹实验(Western blot, WB)实验检测外泌体相关标记蛋,如白细胞分化抗原81(CD81)和白细胞分化抗原63(CD63)的表达;miRNA矩阵测序检测miR-133a-3p的表达;细胞克隆实验、细胞增殖8(CCK-8)实验、EDU细胞增殖实验、细胞迁移实验检测过表达miR-133a-3p对前列腺癌增殖和转移能力的影响;双荧光素酶报告基因实验检测miR-133a-3p结合转化生长因子β受体1(transforming growth factor β receptor 1, TGFBR1)的情况;实时荧光定量逆转录聚合酶链反应(qRT-PCR)和WB检测调控miR-133a-3p后对TGFBR1表达的影响。结果: 相较于生理盐水(NC)组,共培养外泌体的CD81和CD63表达升高(P＜0.05)。细胞克隆实验、CCK-8实验、EDU实验、划痕、细胞迁移实验提示,相较于NC组,转染miR-133a mimic模拟物组(miR-133a-3p组)的细胞增殖、转移能力下降(P＜0.05)。双荧光素酶报告基因实验显示,miR-133a-3p结合TGFBR1序列,且过表达miR-133a-3p后,抑制了TGFBR1的表达(P＜0.05)。WB结果显示,相较于NC组,si-TGFBR1组中的WNT、GSK3β、C-MYC和β-catenin的表达下降,oe-TGFBR1组中WNT、GSK3β、C-MYC和β-catenin的表达上升(P＜0.05),而oe-TGFBR1+miR-133a-3p组中WNT、GSK3β、C-MYC和β-catenin的表达与NC组无差异。结论: 前列腺癌细胞来源外泌体miR-133a-3p靶向调控TGFBR1从而促进前列腺癌细胞恶性表型。
Objective: To investigate the molecular mechanism by which exosomal secretion of miR-133a-3p regulates TGFBR1 and promotes the progression of prostate cancer. Methods: Exosomes were extracted from prostate cancer cells, and the expression of exosom-associated markers such as leukocyte differentiation antigen 81 (CD81) and leukocyte differentiation antigen 63 (CD63) was detected by Western blot assay (WB). The expression of miR-133a-3p was detected by miRNA matrix sequencing. Cell cloning experiment, cell proliferation 8 (CCK-8) experiment, EdU cell proliferation experiment and cell migration experiment were used to detect the effects of co-cultured exosomes+miR-133a-3p on the proliferation and metastasis of prostate cancer. Dual luciferase reporter gene assay was used to detect the binding of miR-133a-3p to TGFBR1. Real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) and WB were used to detect the effect of regulation of miR-133a-3p on transforming growth factor β receptor 1 (TGFBR1) . Results: Compared with NC group, the expressions of CD81 and CD63 in co-cultured exosomes were increased (P＜0.05). Cell cloning experiment, CCK-8 experiment, EDU experiment, scratch experiment, and cell migration experiment showed that compared with NC group, the cell proliferation and metastasis ability of Mir-133a-3P group (transfected with miR-133a mimic) decreased (P＜0.05). Dual luciferase reporter gene experiment showed that miR-133a-3p binding TGFBR1 sequence and overexpression of miR-133a-3p inhibited the expression of TGFBR1 (P＜0.05). WB results showed that compared with NC group, the expressions of WNT, GSK3β, C-MYC and β-catenin in si-TGFBR1 group were decreased, while the expressions of WNT, GSK3β, C-MYC and β-catenin in oe-TGFBR1 group were increased (P＜0.05). However, the expressions of WNT, GSK3β, C-MYC and β-catenin in oe-TGFBR1+miR-133a-3p group were not different from those in NC group. Conclusion: Prostate cancer cell-derived exosome miR-133a-3p targets TGFBR1 to promote the malignant phenotype of prostate cancer cells.
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