전립선암에서 METTL3-m⁶A-Reader 축의 후성유전학적 조절을 통한 생성 및 AR-v7 생성 및 안정성과 치료저항성 억제 기전 연구 = Epitranscriptomic Modulation of the METTL3-m⁶A-Reader Axis Suppresses AR-V7 Formation and Stability in Prostate Cancer

다국어 초록 (Multilingual Abstract)

Prostate cancer is one of the most prevalent malignancies in men, and progression to castration-resistant prostate cancer (CRPC) following androgen-deprivation therapy remains a major cause of mortality. The androgen receptor splice variant AR-V7 exhibits ligand-independent transcriptional activity and is acritical driver of resistance to second-generation anti-androgens such as enzalutamide. However, the post-transcriptional regulatory mechanisms governing AR-V7 expression during CRPC progression remain incompletely understood.

In this study, we investigated the role of m⁶A-dependent epitranscriptomic regulation in AR-V7 expression using LNCaP-LN3–derived prostate cancer models, including a hormone-sensitive control (CONT), an androgen-deprived long-termcultured model (ADL), and an enzalutamide-resistant model (ER). Western blotand quantitative PCR analyses revealed marked upregulation of AR-V7 in ADL and ER cells, accompanied by increased expression of the m⁶A writer METTL3 and the m⁶A reader HNRNPA2B1. These changes were associated with mesenchymal and survival-related signaling alterations, as indicated by increased N-cadherin, and Snail expression.

m⁶A RNA immunoprecipitation demonstrated the presence of m⁶A modifications on AR-V7 mRNA, while RNA immunoprecipitation confirmed direct binding of HNRNPA2B1 to AR-V7 transcripts. Consistently, analysis of public genomic datasets, including TCGA prostate cancer cohorts, CCLE cell line data, and metastatic CRPC patient datasets, showed elevated expression of METTL3 and HNRNPA2B1 in CRPC and AR-V7–high samples.

Collectively, these findings indicate that METTL3-mediated m⁶A methylation contributes to AR-V7 upregulation, and that the m⁶A reader HNRNPA2B1 acts as a key post-transcriptional regulator of AR-V7 in CRPC. This study highlights an epitranscriptomic mechanism underlying AR-V7–driven therapeutic resistance and suggests m⁶A-dependent RNA regulation as a potential targetable vulnerability in advanced prostate cancer.

목차 (Table of Contents)

• - ABSTRACT I
• - CONTENTS II
• - LIST OF TABLE IV
• - LIST OF FIGURE V
• -----------------
• - INTRODUCTION 1
• - MATERIAL AND METHOD 4
• 1. Establishment of Modeling Cell Lines and Cell Culture
• 2. Chemicals
• 3. RNA Extraction, cDNA Synthesis, and Quantitative PCR
• 4. Primers and Antibodies
• 5. Western Blotting
• 6. Cell Viability Assay
• 7. MeRIP (m⁶A RNA Immunoprecipitation) Assay
• 8. RIP(RNA Immunnoprecipitation) Assay
• 9. Actinomycin D Chase Assay
• 10. Bioinformatic Analysis
• 11. Statistical Analysis
• - RESULT 10
• 1. Establishment of androgen-deprivation long-term(ADL) and enzalutamide-resistant (ER) prostate cancer models 10
• 2. METTL3 is upregulated in resistant prostate cancer models and correlates with increased AR-V7 expression 11
• 3 .METTL3 inhibition decreases m⁶A methylation, destabilizes AR-V7 mRNA, and restores enzalutamide sensitivity 12
• 4. Identification of m⁶A reader proteins associated with AR-V7 abundance in resistant prostate cancer 13
• 5. Pharmacological inhibition of METTL3 reduces m⁶A modification and destabilizes AR-V7 expression in resistant prostate cancer cells 14
• 6. Increased association of the m⁶A reader HNRNPA2B1 with AR-V7 mRNA in resistant cells and its modulation by METTL3 inhibition 15
• - DISCUSSION 23
• - CONCLUSION 25
• - REFERENCE 26
• - 국문요약 31