hnRNP C 및 CNOT7 상호 작용을 통한 Bcl-XL mRNA 안정성 하향 조절로 인한 BTG2의 암세포 사멸 촉진
BTG2-enhanced cancer cell death is mediated by downregulating mRNA stability of Bcl-XL via interaction with hnRNP C and CNOT7
초록/요약
BTG2, as a founding member of anti-proliferative gene family, has been reported to render cancer cells more sensitive to chemotherapy and radiotherapy. Recent study suggested that BTG2 can activate general mRNA deadenylation and degradation as a binding partner of mRNA deadenylase, CNOT7. However, molecular mechanism of cell death regulation has not yet been fully elucidated in terms of mRNA stability regulation of BTG2. Therefore, the mechanism of enhanced cell death by BTG2 and its clinical significance were investigated. Among anti-apoptotic genes including Bcl-2, Bcl-XL, and Mcl-1, mRNA stability of Bcl-XL was reduced by BTG2. By protein chip array, HnRNP C, mRNA binding protein, was discovered as new interacting protein of BTG2. In vivo binding of BTG2 and HnRNP C was validated by immunoprecipitation. The binding of HnRNP C to 3' UTR of Bcl-XL mRNA was also confirmed by RNA immunoprecipitation and pull down assay of biotin labeled RNA. The decreased mRNA stability of Bcl-XL and enhanced cell death after cisplatin treatment by BTG2 were not observed in BTG2 mutant defective in CNOT7 binding. There results suggested that BTG2-CNOT7 complex can bind to 3' UTR of Bcl-XL mRNA by hnRNP C and decrease Bcl-XL mRNA stability with enhancing cell death. Similar to the results of cell culture experiments, response of platinum-based chemotherapy and progression-free and overall survival were better in advanced lung squamous cell carcinoma patients with high BTG2 expression. In conclusion, these results indicated that BTG2 can augment chemotherapy-induced cancer cell death by regulation of Bcl-XL mRNA stability via mediating interaction of hnRNP C-3' UTR of Bcl-XL and mRNA deadenlyase, CNOT7.
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I. INTRODUCTION 1
1.1. Discovery and tissues expression of BTG2 1
1.2 Regulation of BTG2 1
1.3. Biological functions of BTG2 response 2
1.4. Molecular functions of BTG2 4
1.5. Aim of the study 5
II. MATERIALS AND METHOD 6
2.1. Cells and reagents. 6
2.2. Preparation and transduction of adenoviruses 6
2.3. Reverse transcriptional and real-time PCR analysis 6
2.4. Western blot and immunoprecipitation (IP) analyses 6
2.5. Knockdown of BTG2 using siRNA 6
2.6. Construction of BTG2 and CNOT7 overexpressing plasmids and site-directed mutagenesis 7
2.7. Immunoprecipitation of RNP complexes and RT-PCR. 7
2.8. Synthesis of Biotinylated Transcripts and Biotin pull-down assays 7
2.9. Measurement of cell viability 8
2.10. Immunohistochemical staining for BTG2 8
2.11. Patients and clinical review 9
2.12. Public database analysis 9
2.13. Statistical analysis 9
2.14. BTG2 interaction proteins screening with protein chip array 10
III. RESULTS 11
3.1. BTG2 regulates mRNA and protein level of anti-apoptotic protein, Bcl-XL 11
3.2. BTG2 regulates mRNA stability of Bcl-XL 16
3.3. BTG2 interacts with CNOT7 and hnRNP C 20
3.4. HnRNP C interacts with 3’-UTR of Bcl-XL mRNA 25
3.5. mRNA downregulation of Bcl-XL is dependent on interaction between BTG2 and CNOT7 28
3.6. BTG2 enhances cell death through the regulation of Bcl-XL 31
3.7. High BTG2 expression was associated with favorable platinum-based chemotherapy response and prognosis in advanced squamous carcinoma of lung 37
IV. DISCUSSION 48
REFERENCE 52
국문요약 59
