[논문]B형 간염 바이러스의 ntC1731T 및 G1806A의 core 프로모터 돌연변이에 의한 HBV 유전자 발현 증가 분석

조자영; 이이조; 성미소; 정재훈

doi:10.5352/jls.2022.32.2.94

B형 간염 바이러스의 ntC1731T 및 G1806A의 core 프로모터 돌연변이에 의한 HBV 유전자 발현 증가 분석
Core Promoter Mutation of ntC1731T and G1806A of Hepatitis B Virus Increases HBV Gene Expression 원문보기

생명과학회지 = Journal of life science, v.32 no.2, 2022년, pp.94 - 100

조자영 (국립수산과학원 생명공학과) , 이이조 (미얀마 의학연구국) , 성미소 (부산대학교 분자생물학과) , 정재훈 (부산대학교 분자생물학과)

초록
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B형간염바이러스(HBV)의 만성 감염은 간경화와 간세포암 발생 빈도를 현저히 높인다. HBV 감염의 임상적 결과는 숙주 유전적 요인과 바이러스의 유전자 변이, 그리고 환경적 요인 등에 결정된다. HBV 복제를 위한 HBV의 pre-genomic RNA 전사는 바이러스의 core promoter 활성화에 의해 조절된다. Core promoter 돌연변이는 급성간 질환과 간세포암 발생에 연관되어 있다. 본 연구팀은 미얀마의 HBV 감염 환자들로부터 바이러스 유전자를 획득하여 core promoter 부위의 유전자 변이들을 파악하였다. Core promoter의 상대적 유전자 활성 차이를 분석하기 위해서 core promoter를 luciferase reporter에 재조합한 시스템을 제작하였다. 분석한 core promoter의 유전자 변이들 중에서 C1731T와 G1806A 돌연변이가 HBV core promoter의 전사 활성화를 증가에 관여하였다. 돌연변이 부위를 중심으로 전사 인자들의 가능한 결합 부위 변화를 컴퓨터 프로그램 분석을 통해 조사한 결과, C/EBPβ와 XBP1 반응 부위가 새롭게 생성되었음을 도출하였다. C/EBPβ의 세포 내 발현은 C1713T 돌연변이를 가진 core promoter의 전사 활성을 증가시켰으며, XBP1 발현은 G1806A 돌연변이를 함유한 M95 promoter를 활성을 증가시켰다. HBV 감염의 치료는 약제 내성과 백신 회피 돌연변이 발생으로 문제점을 가지고 있는 상황에서, 이 연구 결과는 HBV core promoter 돌연변이의 분자생물학적 그리고 임상학적 중요성을 제공한다.

Abstract ▼ AI-Helper

Chronic infection by hepatitis B virus (HBV) greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). The outcome of HBV infection is shaped by the complex interplay of the mode of transmission, host genetic factors, viral genotype, adaptive mutations, and environmental factors. The pregenomic RNA transcription of HBV for their replication is regulated by the core promoter activation. Core promoter mutations have been the reason for acute liver failure and are associated with HCC development. We obtained HBV genes from a patient in Myanmar who was infected with HBV and identified gene variations in the core promoter region. For measuring the relative transactivation activity of the core promoter, we prepared the core-promoter reporter construct. Among the gene variations of the core promoter, the mutations of C1731T and G1806A were associated with increase in the transactivation of the HBV core promoter. Through computer analysis for searching for a tentative transcription factor binding site, we showed that the mutations of C1713T and G1806A newly created C/EBPβ and XBP1-responsive elements of the core promoter, respectively. The ectopic expression of C/EBPβ largely increased the HBV core promoter containing the C1713T mutation and that of XBP1 activated the M95 promoter containing the G1806A mutation. Our efforts to treat and prevent HBV infections are hampered by the emergence of drug-resistant mutations and vaccine-escape mutations. Our results provide the biological properties and clinical significance of specific HBV core promoter mutations.

주제어

표/그림 (4)

그림 Fig. 1. Identification of DNA sequence of HBV core promoter from M50. After obtaining HBV gene from M50 patient, the core promoter region (nt1595-nt1880) was sequenced. The M50 core promoter sequence was used as a wild type and control for comparing with other the mutated core promoter sequences.
그림 Fig. 2. HBV core promoter mutation induces HBV gene expression. (A) The HBV core promoter site from the patients was sequenced and the sequences were aligned to enable the detection of mutations. (B) Structure of the HBV core promoter reporter plasmid. HBV core promoter region from the serum of Myanmar patients M50, M82, M83, and M95 was amplified by PCR and was cloned using a PGL3B vector. (C) The effect of mutations on HBV core promoter activity. Chang liver cell were transfected with HBV core promoter M50, M82, M83, and M95 reporter construct for 24 hr. The cell lysates were determined for luciferase activity. Cloned M50 served as the control. The bar represents the mean value from at least three independent experiments. *p<0.05, **p<0.01 indicate significance when compared to C/EBPβ transfectant.
그림 Fig. 3. C/EBPβ is responsible to increase HBV core promoter activity of C1713T mutation. (A) Creation of C/EBPβ binding site on the C1713T core promoter of M82, M83, and M95. The transcription factor binding sites to the HBV core promoter mutation sites at nt1713 was searched through PROMO (http://alggenlsi.upc.es/cgibin/promo_ V3/promo/Promoinit.cgi?-dirDB=TF_8.3). (B) The effect of C/EBPβ binding on HBV core promoter activity. Chang live cells were transfected with HBV core promoter M50, M82, M83, and M95 construct for 24 hr in the presence or absence of C/EBPβ transfection. The cell lysates were applied for luciferase activity. Cloned M50 served as the control. The bar represents the mean value from at least three independent experiments. *p<0.05, **p<0.01 indicate significance when compared to C/EBPβ transfectant.
그림 Fig. 4. XBP1 is responsible to increase HBV core promoter activity of G1806A mutation. (A) Creation of XBP1 binding site on the G1806A core promoter of M82, M83, and M95. The transcription factor binding sites to the HBV core promoter mutation sites at nt1806 was searched through PROMO (http://alggenlsi.upc.es/cgibin/promo_V3/pro mo/Promoinit.cgi?-dirDB=TF_8.3). (B) The effect of XBP1 binding on HBV core promoter activity. Chang live cells were transfected with HBV core promoter M50, M82, M83, and M95 construct for 24 hr in the presence or absence of XBP1 transfection. The cell lysates were applied for luciferase activity. Cloned M50 served as the control. The bar represents the mean value from at least three independent experiments. *p<0.05, **p<0.01 indicate significance when compared to XBP1 transfectant.

AI 본문요약
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문제 정의

Hepatitis B is one of the most common and serious disease in Myanmar. Therefore, this research aims to further the development of more effective treatments that are specific to mutations and genotype. HBV can be classified into ten genotypes, A through J, based on a sequence divergence of > 8%.
It is unclear whether these mutations directly induce inflammation of the liver [3, 20]. This study indicates that the sporadic core promoter mutations of HBV increases HBV gene expression, resulting from new creation of liver-specific transcription factor binding sites for HBV DNA replication.

제안 방법

To construct HBV core promoter M50, M82, M83 and M95, the promoter fragments from serum of HBV infected patients were amplified by PCR using cloning primers contacting restriction enzyme site, KpnI and HindIII: forward, 5’- GCC GGT ACC ACG CCC ACC AAA TAT - 3’ and reverse, 5’-CGC AAG CTT TAC AAG AGA TGA TTA-3’

대상 데이터

This study used serum samples of 96 HBsAg positive patients that were received for routine HBV DNA detection from Department Medical Research (DMR) in Yangon My- anmar. The serum samples were stored at -80℃.

데이터처리

All experiments are presented as mean±standard deviation (SD) and analyzed by a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test

성능/효과

Therefore, C/EBPβ binding to mutation sites in the HBV core promoter results in the induction of the inflammatory response in the liver, leading to development of chronic hepatitis.

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저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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