| The incidence of prostate cancer(PCa)is increasing year by year,and has become one of the important diseases threatening men’s health.Castration-resistant prostate cancer(CRPC)is a highly malignant advanced prostate cancer,which is resistant to androgen deprivation therapy(ADT)and most deaths caused by prostate cancer also occur in this stage.One of the important mechanisms for the progression of CRPC is the continuous abnormal regulation of its androgen receptor(AR)or androgen-receptor splicing variants(AR-Vs).Therefore,find the way to reverse or inhibit the abnormal status of AR/AR-Vs in CRPC become important.Currently,drugs targeting the AR signaling pathway,such as Bicalutamide,Enzalutamide and Abiraterone,have been shown to be effective in prolonging the overall survival(OS)and disease-free survival(DFS).ADT based on these drugs can effectively reduce patients’ PSA and inhibit disease progression,but the effective period is often only 18-24 months.After that,tumors usually show resistance to these drugs.Therefore,finding new therapeutic targets becomes particularly important.Single inhibition has limited effect in the treatment of prostate cancer,it is urgent to find a treatment that can inhibit both AR and AR-V7.This study aims to explore the mechanism by which the newly discovered long non-coding RNA KDM4A-AS1 in recent years promotes the progression of CRPC by inhibiting the AR and its splicing variants AR-V7/AR-V567 es,and explore the role of KDM4A-AS1 in CRPC enzalutamide resistance,and evaluate its value as a therapeutic target,which may provide theoretical support for the treatment of castration-resistant prostate cancer.Objective: 1: Explore the mechanism of KDM4A-AS1 in CRPC in vivo and in vitro.2: Explore the association between KDM4A-AS1 and enzalutamide resistance,and verify the effect of targeting KDM4A-AS1 in the treatment of prostate cancer in vivo and in vitro.Methods: 1: Determine the expression of KDM4A-AS1 in prostate cancer patients and prostate cancer cell lines at different stages by RNAScope,high-throughput microarray chip and q PCR,and analyze its characteristics.2: KDM4A-AS1 was knocked down by in vitro lentivirus,and functional experiments such as Transwell,clone formation and MTT were used to determine the effect of knocking down KDM4A-AS1 on prostate cancer.3: Verify the existence of KDM4A-AS1-USP14-AR/AR-Vs complex through bioinformatics,ITRAQ mass spectrometry,RIP,RNA-Pulldown,immunohistochemistry,Western-Blot,and determine the role of KDM4A-AS1 in AR/AR-Vs regulation;4: Construct enzalutamide-resistant cell lines and the corresponding nude mouse subcutaneous xenograft model,measure the mouse tumor volume and growth rate,and use RNAScope,q PCR,Western-Blot and immunohistochemistry to confirm the effect of KDM4A-AS1 in drug resistant.Results: 1: KDM4A-AS1 was significantly up-regulated in CRPC cell lines LNCa P-AI,C4-2,and CRPC patients;2: The depletion of KDM4A-AS1 significantly reduced LNCa P-AI and C4-2 cell viability and cell proliferation.Cell migration ability in vitro and tumor growth in vivo;3: We found that by binding to the N-terminal transcriptional activation region of the androgen receptor protein(AR-NTD),KDM4A-AS1 significantly enhanced the stability of USP14-AR/AR-Vs complex,and promote the deubiquitination of AR/AR-Vs,thereby protecting it from MDM2 mediated degradation of the ubiquitin-proteasome pathway;4: Successfully constructed drug-resistant cell lines and nude mouse models,and found that simply targeting inhibition of KDM4A-AS1 can significantly inhibit tumor growth.Using short hairpin RNA targeting KDM4A-AS1 and enzalutamide can significantly restore the sensitivity of tumors to enzalutamide and kill tumor cells.Conclusion: 1: KDM4A-AS1 is significantly up-regulated in CRPC.2: KDM4A-AS1 promotes the progression of CRPC and resistance to enzalutamide by stabilizing the USP14-AR/AR-Vs complex.Targeting KDM4A-AS1 can significantly restore drug-resistant in vivo and in vitro.The sensitivity of enzalutamide has great potential for improving the prognosis of clinical prostate cancer patients. |
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