摘要
目的探讨外泌体miR-940在前列腺癌(PCa)骨转移中的作用。方法采集2017年6月在温州医科大学附属第一医院就诊的3例正常老年男性及3例PCa骨转移患者的血清标本,采用RT-PCR法检测血清外泌体中微小RNA(miRNA)表达并绘制miRNA谱。培养PCa细胞株PC-3及正常前列腺上皮细胞株RWPE-1,提取并鉴定细胞外泌体;采用RT-PCR法检测细胞外泌体中miR-940表达。PC-3细胞经转染后与人骨髓间充质干细胞(hMSC)共培养48 h,采用RT-PCR法检测成骨细胞分化相关基因表达,Von Kossa染色法检测矿化面积占比。结果与正常老年男性比较,PCa骨转移患者血清外泌体中多个miRNA表达明显升高,其中miR-940尤为明显。在电镜下,PC-3细胞外泌体呈40~100 nm的盘状囊泡,外泌体标志性蛋白VDAC、CD63、Hsp70、CD81表达均明显上调。PC-3细胞外泌体中miR-940表达明显高于RWPE-1细胞外泌体(P<0.05)。在PCa-hMSC共培养体系中,miR-940过表达组ALP、骨钙素(BGLAP)基因表达及矿化面积占比较miR-940敲减组、空白对照组均明显降低(均P<0.05)。结论外泌体miR-940可以有效抑制PCa-hMSC成骨分化,这为转移性PCa个体化治疗提供了新的靶点。
Objective To investigate the role of exosomal miR-940 in prostate cancer(PCa)bone metastasis.Methods Serum samples were collected from 3 normal elderly men and 3 PCa patients with bone metastasis in the First Affiliated Hospital of Wenzhou Medical University in June 2017.RT-PCR was used to detect the expression of microRNA(miRNA)in serum exosomes.Human PCa cells PC-3 and prostate epithelial cells RWPE-1 were cultured,and exosomes were extracted and identified,RT-PCR was used to detect the expression of miR-940 in exosomes.PC-3 cells were transfected with siRNA,then co-cultured with human bone marrow mesenchymal stem cells(hMSC)for 48 h,RT-PCR was used to detect the expression of genes related to osteoblast differentiation,and Von Kossa staining was used to detect the proportion of mineralized area.Results Compared with normal elderly men,the expression of multiple miRNAs in serum exosomes of PCa patients with bone metastasis was significantly increased,especially miR-940.Electron microscopy showed that the exosomes of PC-3 cells were 40-100 nm disc-shaped vesicles,and the expressions of exosomal marker proteins VDAC,CD63,Hsp70,and CD81 were all significantly up-regulated.The expression of miR-940 in PC-3 exosomes was significantly higher than that in RWPE-1 exosomes(P<0.05).In the PCa-hMSC co-culture system,the expression of alkaline ALP and osteocalcin(BGLAP)genes and the area of mineralization in the miR-940 over-expressing PC-3 cells were significantly lower than those in the miR-940-knockdown PC-3 cells and the blank control PC-3 cells(all P<0.05).Conclusion The exosomal miR-940 can effectively inhibit the osteogenic differentiation of PCa-hMSC,which may provide a new target for treatment of metastatic prostate cancer.
作者
黄航
李萍
叶雪挺
陈伟
张方毅
HUANG Hang;LI Ping;YE Xueting;CHEN Wei;ZHANG Fangyi(Department of Urology,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000,China)
出处
《浙江医学》
CAS
2020年第24期2645-2648,2652,I0015,共6页
Zhejiang Medical Journal
基金
浙江省自然科学基金资助项目(LQ20H050001、LY20H160013)
温州市基础性科研项目(Y20190066)。
