| Background:Prostate Cancer(PCa)is the most common malignant tumor of the male genitourinary system.Its incidence ranks second among male malignancies.In 2020,there were approximately 1.4 million new cases and 370,000 deaths worldwide.The inadequateness of specific symptoms in the early stage of PCa has caused most patients to have metastases at the time of diagnosis in my country.The bone is the most important metastasis site of PCa,and the survival time of patients with bone metastasis is significantly reduced.Bone metastasis of PCa is currently diagnosed mainly through clinical manifestations and imaging examinations.However,due to the short of specific clinical manifestations,imaging changes and biochemical indicators in the early stage of bone metastasis,the best time for diagnosis is often delayed.Therefore,looking for non-invasive biomarkers for early diagnosis and prediction of PCa bone metastasis have become a hot topic.As a kind of tiny membrane vesicles,exosomes have a special double-layer membrane structure to protect RNA from nuclease degradation.A large number of literatures have reported that exosomal miRNAs are differentially expressed in many tumors.The high stability of exosomal miRNA and the difference in tumor expression mean that exosomal miRNA may become a valuable non-invasive biomarker for disease diagnosis and prognosis monitoring.Therefore,this study mainly screens differential miRNAs molecules from peripheral venous blood exosomes in patients with localized PCa and bone metastases,and finds new biomarkers for PCa bone metastasis.Objective:By analyzing the changes in plasma exosomal miRNAs levels in patients with localized PCa and prostate cancer patients with bone metastases,the differentially expressed miRNAs are screened and evaluated for their diagnostic value for PCa bone metastases.Methods:A total of 46 patients with PCa were selected by prostate puncture in our hospital,33 patients with bone metastases were diagnosed by bone scan and magnetic resonance examination,and 13 patients with localized PCa without metastasis.We draw 10ml of fasting blood,extracting plasma exosomes by using high-speed centrifugation and kit methods,and utilized transmission electron mcroscopy,nanoparticle tracking analysis technology and flow cytometry to identify exosomes.With high-throughput sequencing technology,miRNA sequencing was performed on the plasma exosomes of 3 patients with localized PCa and 3 patients with PCa bone metastasis.With this method,the miRAN molecules with differences were screened.Through pre-experimental results,exosomal miRNA database expression,literature review and other methods,the most diagnostic miRNAs molecules were screened out.The real-time quantitative PCR technology was used to verify 13 patients with localized prostate cancer and 33 patients with bone metastasis.The diagnostic efficiency of expression was analyzed by ROC curve.Based on the GEO gene database,the expression of miRNA molecules in prostate cancer tissue and plasma were further verified by bioinformatics methods,and the molecular mechanism of miRNA involvement in tumors were explored by combining target gene enrichment analysis and pathway analysis.Results:Under the electron microscope and nanoparticle tracking analysis,it can be seen that the exosomes are approximately round double-layer vesicles.The diameter of the vesicles is in the range of 20nm-200nm,with typical exosomes size and characteristics.It is found that the two specific proteins CD63 and CD81 on the surface of exosomes have positive signals under the detect of Flow cytometry.High-throughput sequencing of exosomes shows that the proportion of miRNA content is greater than 50%,and the distribution of miRNA expression density is basically the same.In the high-throughput results,a total of 61 miRNAs molecules in patients with bone metastases have different expressions compared with localized PCa,including 30 up-regulateds and 31 down-regulateds.According to the results of preliminary experiments,expression of exosomal miRNA database and literature review,hsa-miR-130b-3p and hsa-miR-654-3p are selected for verification.RT-qPCR reveals that Plasma exosomal hsa-miR-130b-3p is up-regulated in patients with bone metastasis(1.00±0.07 VS 1.94±0.26 P=0.0069),and hsa-miR-654-3p is down-regulated in bone metastases(1.37±0.23 VS 0.76±0.04 P=0.026),the differences were statistically significant.ROC shows that the area under the curve(AUC)of plasma exosomes hsa-miR-130b-3p and hsa-miR-654-3p for the diagnosis of bone metastasis are 0.76(95%CI=0.61-0.90)and 0.72(95%CI=0.51-0.92).Through GEO database verification,it is found that hsa-miR-130b-3p is up-regulated in metastatic PCa(mPCa)tissue and plasma chips,and hsa-miR-654-3p is down-regulated in mPCa.,which are consistent with the experimental results,and the difference is statistically significant.Functional enrichment analysis has showed that hsa-miR-130b-3p and hsa-miR-654-3p target genes are mainly enriched in protein binding,RNA polymerase Ⅱ promoter transcription negative regulation,cell proliferation regulation,transcription regulation and transcription regulation regions DNA binding,which are closely related to tumor proliferation and metastasis.Conclusion:1 Exosomes with a complete structure and a diameter of 20nm-200nm can be extracted from the plasma of PCa patients,to express CD63 and CD81 specific surface markers.2 The content of miRNA in Plasma exosomal RNA is greater than 50%,and there exists differences in the expression of plasma exosomes in the localized PCa group and the bone metastasis group.3 Plasma exosomal hsa-miR-130b-3p and exosomal hsa-miR-654-3p have potential value as biological indicators for the diagnosis of bone metastasis of PCa.4 Gene function enrichment of exosomal hsa-miR-130b-3p and hsa-miR-654-3p show that they are closely related to tumor cell proliferation and invasion. |
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