The Effect And Mechanism Of MiR-127-3p In Bone Metastasis Of Prostate Cancer

[Object]Prostatic carcinoma(PCa)is one of the most common cancers in men,ranking first among the three leading causes of cancer-related deaths in men worldwide.The high mortality rate of PCa is related to its tumor metastasis to distant organs,especially for osseous metastasis.The incidence of bone metastases in advanced PCa is as high as 90%.The treatment of PCa bone metastasis is limited,which can only relieve the symptoms and cannot be radically cured the disease.Therefore,it is very urgent to understand the underlying molecular mechanisms that lead to the migration of PCa cells to the bone,which may help to develop new therapeutic strategies for PCa bone metastasis.MicroRNA(miRNA)plays an important role in the regulation of gene transcription and affects tumor metastasis.A variety of miRNAs are dysregulated in malignant tumors,but the specific mechanism has not been fully understood.This study focuses on the role of miR-127-3p in the development of prostate cancer,and preliminary exploration of its related molecular mechanisms.[Methods]1.Screening of differentially expressed miRNAs in normal and malignant prostate tissues using data from the Cancer Genome Atlas(TCGA)and GSE21036 data sets.qRT-PCR was used to detect the expression of miR-127-3p and compared the difference between normal and cancerous prostate tissues.Analyzed the clinical samples and data to find the relationship between the expression of miR-127-3p and Gleason as well as TNM,and performed survival analysis.2.Through plasmid transfection,miR-127-3p was overexpressed in PCa cells,and cell migration and invasion experiments were conducted to explore the effect of mir-127-3p on invasion and migration of PCa cells.The bone metastasis model of mice was established by intracardial injection to explore the effect of mir-127-3p level on bone metastasis.3.Found target mRNAs of miR-127-3p through targetscan and screen them further by experiment.The interaction between miR-127-3p and PSMB5 was detected in real-time by conducting dual luciferase and RNA binding protein immunoprecipitation(RIP).The effect of miR-127-3p on PSMB5 protein expression was analyzed by western blot.PSMB5 was up-regulated for Transwell analysis to find its effect on migration and invasion of PCa cells.4.Looking for the molecular mechanism of down-regulation of miR-127-3p in PCa,identifying and screening the transcription factors involved in this process through bioinformatics analysis and qRT-PCR.ChIP-qPCR was used to analyze the relationship between the interaction between PSMB5 and miR-127-3p.Western blot and RT-PCR were used to analyze the relationship between the changes in CTCF expression and PSMB5 expression,and through miR-127-3p intervention for comprehensive analysis.5.Explore the clinical significance of CTCF/miR-127-3p/PSMB5 axis.The relationship between miR-127-3p level and PSMB5 mRNA and CTCF mRNA in PCa patients was analyzed by RT-PCR.The relationship between CTCF,miR-127-3p and PSMB5 was analyzed by immunohistochemistry[Results]1.miR-127-3p expression was reduced in bone metastatic prostate cancer.In clinical samples,its expression level was demonstrated as follows:bone metastasis tissue<carcinoma in situ tissue<normal tissue adjacent to cancer,PCa cells<normal prostate epithelial cells.At the same time,survival analysis showed that PCa patients with low miR-127-3p expression had shorter boneless metastasis survival time.Multivariate Cox regression analysis showed that the down-regulation of miR-127-3p was an independent prognostic factor for survival without bone metastasis2.The up-regulation of miR-127-3p inhibits PCa cell migration,invasion,and bone metastasis.Gene enrichment analysis determined that miR-127-3p plays a role in PCa bone metastasis,transwell analysis demonstrated its promotion to migration and invasion.In vivo experiments,a PCa bone metastasis model was established,compared with the carrier group,the up-regulation of miR-127-3p inhibited the bone metastasis ability of PC3 cells in vivo which was demonstrated by X-ray imaging.Besides,when there was no bone metastasis,mice with over-expressed miR-127-3p had shorter survival times.3.PSMB5 is the target of miR-127-3p.RIP showed a selective interaction between the two.Luciferase gene report unfolded that overexpression of miR-127-3p reduced the luciferase activity of PSMB5 3’-UTR.Western blot analysis showed that the upregulation of miR-127-3p can reduce the protein expression of PSMB5.Transwell showed that upregulation of PSMB5 promotes the migration and invasion of PCa cells,and can reverse the effect of miR-127-3p upregulation.4.CTCF transcriptionally inhibits miR-127-3p in PCa cells.Luciferase assay showed that knocking down CTCF enhanced the luciferase activity of miR-127-3p promoter.ChIP-qPCR showed that CTCF was enriched in the miR-127 promoter.qRT-PCR analysis of clinical samples showed that CTCF significantly increased in bone metastasis PCa tissue compared with non-bone metastasis tissue.Western blot and qRT-PCR analysis showed that the level of CTCF was positively correlated with PSMB5 protein and mRNA expression,and miR-127-3p could reverse CTCF-mediated PSMB5 regulation.5.The miR-127-3p level in PCa patients was inversely related to the mRNA and protein levels of PSMB5 and CTCF.[Conclusion]Our study revealed that the late bone metastasis is significantly associated with the down-regulation of miR-127-3p,and CTCF related down-regulation of miR-127-3p is achieved by targeting PSMB5 in PCa patients.In conclusion,our study provides novel insights into the CTCF/miR-127-3p/PSMB5 axis in PCa bone metastasis,which indicates that miR-127-3p can be used as a therapeutic target for PCa bone metastasis.