MicroRNAs (miRNAs) are short, conserved segments of non-coding RNA which play a significant role in prostate cancer development and progression. To identify miRNAs associated with castration resistance, we performed miRNA microarray analysis comparing castration resistant prostate cancer (CRPC) with androgen dependent prostate cancer (ADPC). We identified common underexpression of miR-4638-5p in CRPC compared to ADPC samples, which were further confirmed by quantitative PCR analysis. The role of miR-4638-5p in prostate cancer androgen-independent growth has been demonstrated both in vitro and in vivo. We also identified Kidins220 as a target gene directly regulated by miR-4638-5p and shRNA-mediated knockdown of Kidins220 phenocopied miR-4638-5p restoration. Subsequently, we revealed that Kidins220 activates PI3K/AKT pathway, which plays a key role in CRPC. Loss of miR- 4638-5p may lead to CRPC through the activity of Kidins220 and PI3K/AKT pathway. Furthermore, we found that miR-4638-5p, through regulating Kidins220 and the downstream activity of VEGF and PI3K/AKT pathway, influences prostate cancer progression via angiogenesis. The identification of miR-4638-5p down-regulation in CRPC and the understanding of the functional role of miR-4638-5p and its downstream genes/pathways have the potential to develop biomarkers for CRPC onset and to identify novel targets for novel forms of treatments of this lethal form of PCa.

Objective To investigate the expression of miR-363-3p in human prostate cancer cells and normal prostate epithelial cells, and explore its impact on the biological behaviors of prostate cancer cells. Methods Total RNA in human prostate cancer cells DU145, PC3 and normal prostate epithelial cells RWPE-1 were extracted respectively. The expressions of miR-363-3p in prostate cancer cells and normal prostate epithelial cells were detected by SYBR Green real-time PCR(RT-PCR). DU145 cells and PC3 cells were infected with the synthesized PCDH, miR-363-3p and miR-363-3p sponge, respectively. CCK 8 and Colony assay were utilized to evaluate cell proliferation, transwell assay was applied to detect migration ability, and microtubule assay to measure tube formation ability. Results The expressions of miR-363-3p in the DU-145 and PC3 cells were all significantly lower than that in the RWPE-1 cells (P<0.05). Compared with the control, overexpression of miR-363-3p can significantly enhance cell proliferation, migration and invasion, and microtubule formation of DU-145 and PC3 cells (P<0.05). In contrast, knockdown of miR-363-3p significantly reversed these changes in biological phenotypes mentioned above (P<0.05). Conclusion miR-363-3p was upregulated in prostate cancer cells. Overexpression of miR-363-3p can promote cell proliferation, invasion and migration and microtubule formation. miR-363-3p may play an important role in the development of prostate cancer, which may become a new target for treatment of prostate cancer.

目的 构建含有miR-363-3p的慢病毒表达载体,获得能够稳定表达miR-363-3p的DU145细胞株并初步进行细胞形态学实验.方法 利用PCR扩增miR-363-3p片段,插入慢病毒载体pCDH-CMV-MCS-EF1-tRFP中构建重组慢病毒质粒pCDH-miR-363-3p.将慢病毒包装三质粒表达系统的重组慢病毒质粒pCDH-miR-363-3p,包装质粒psPAX以及包膜质粒pMD2.G共转染人胚肾上皮细胞(HEK293T)细胞中,同时以慢病毒空载体pCDH-vector作为阴性对照.收集含有病毒颗粒细胞的病毒上清液,测定Lentivirus-miR-363-3p和Lentivirus-vector的病毒滴度.再用重组慢病毒Lentivirus-miR-363-3p及阴性对照Lentivirus-vector以相同感染复数(MOI)的病毒量分别感染DU145细胞,72 h后,在荧光显微镜下通过观察红色荧光蛋白(RFP)的表达情况观察被感染细胞数.采用反转录实时定量PCR (RT-qPCR)方法检测两组细胞系中miR-363-3p表达情况.将获得的两组细胞进行平板划痕实验,初步观察其细胞生长行为的差异.结果 PCR成功扩增出miR-363-3p片段,插入慢病毒载体pCDH-CMV-MCS-EF1-tRFP后,利用限制性内切酶酶切鉴定和核酸序列测定证实重组慢病毒质粒pCDH-miR-363-3p构建成功.通过慢病毒包装三质粒表达系统获得表达miR-363-3p的重组慢病毒,测得滴度约为1.5×107 efu/ml.以相同MOI的重组慢病毒Lentivirus-miR-363-3p以及Lentivirus-vector分别感染DU145细胞72 h后,在荧光显微镜下能够明显观察到红色荧光,且RT-qPCR检测结果显示,感染miR-363-3p DU145细胞中miR-363-3p表达水平显著高于感染miR-vector DU145细胞,差异有统计学意义(P＜0.01).平板划痕实验结果显示划痕后24 h时两组细胞生长具有明显差异(P＜O.01),但48 h时两组生长的细胞均已完全覆盖划痕.结论 成功构建了含miR-363-3p的慢病毒表达载体,获得的重组慢病毒能够有效地感染DU145细胞,为下一步实验奠定了坚实的基础.初步的细胞形态学实验-平板划痕显示划痕后24h两组细胞生长存在差异.