인체 간암세포 염색체 DNA에 삽입된 HBV DNA의 검출과 삽입부위 구조의 결정

Abstract

[한글]

만성 B형 간염 바이러스(HBV) 감염과 간세포암 발생과는 밀접한 상관 관계가 있으며HBV에 감염된 간세포암 환자에서 HBV DNA가 암조직 염색체 DNA에 비교적 높은 확률로 삽입되어 있다는 사실이 여러 연구자들에 의해 보고되었다. 그리고, HBV DNA의 암조직 염색체 D

NA 삽입 부위에 대한 몇몇 연구가 보고되었으나 삽입 부위가 일정치 않고 특히HBV가 삽입된 염색체 ONA의 위치와 삽입된 HBV DNA가 간세포암 발생에 어떻게 영향을 미치는지에 대한 증거는 아직 미흡한 실정이다.

본 연구에서는 H8V에 감염된 간세포암 환자의 암조직 염색체 ONA에 삽입된 HBV 유전자의 확인과 삽입 부위를 확인하기 위하여, 11명의 HBV에 감염된 간세포암 환자(10명에서HBsAg 양성이고 1명에서 HBcAb 양성)의 암조직으로부터 염색체 DNA를 분리한 다음Southern

blot hybridization, 중합효소 연쇄반응 및 삽입부위의 염기서열 결정을 시행하였다.

(37)**P 로 표지한 H8V DNA probe를 이용한 Southern blot hybridization을 실시하여 7명의환자 모두에서 HBV DNA가 간세포암 조직의 염색체 DNA내에 삽입되었음이 확인되었다HBV의 특정 유전자 삽입을 확인하기 위하여 특정 유전자 부위를 중합효소 연쇄반응(polymerase chain reaction, PCR)으로 증폭한 결과 HBV X 유전자 부위 삽입은 11명중 9명의 환자에서 검출되었고, pre S와 5 유전자를 포함한 부위는 각각 1예와 8예에서, 그리고 P유전자의 일부 서열은 6예에서 검출되었으며, C 유전자 부위는 검출되지 않았다.

HBV DNA가 삽입된 주위 염색체 DNA 염기 서열을 결정하기 위하여 Pre S 유전자에 상보적인 primer와 oligo dG primer를 사용하여 중합효소 연쇄반응을 시행한 결과 약 400염기의 H8V DNA가 삽입된 염색체 DNA가 증폭되었으며 이를 pGEM4Z에 연합하여 subcloning하고

염기 서열을 결정한 결과 HBV DNA(42번 염기)가 염색체 DNA 염기 서열인 ‥‥TTG GG‥‥(HBV)에서 연합하고 있음을 확인하였다.

이상과 같은 실험 결과로부터 다음과 같은 결론을 얻었다. HBV에 감염된 간세포암 환자 암조직의 염색체 DNA에는 대부분에서 HBV DNA가 삽입되어 있었으며 특히 HBV의 X 유전자 및 5 유전자 부위가 주로 삽입되어 있었다. 또한 HBV DNA의 single strand 부위가 염색

체 DNA에 주로 삽입되고, HBV의 direct repeat sequences(ORI, DR7)가 존재하는 X 유전자 부위의 염기 서열이 HBV DNA가 염색체 DNA내로 삽입되는데 관여할 것으로 생각된다.

Detection of HBV DNA inserted into the chromosomal ONA of hepatocellular carcinoma

and determination of DNA sequence elf the inserted region

Seong-Gyu Hwang

Department of Medical Science, The graduate School, Yonsei University

(Directed by Professor Yoon Soo Kim)

A close relationship between the chronic infection of human hepatitis B

virus(HBV) and the incidence of hepatocellular carcinoma(HCC) has been reported,

and the insertion of HBV DNA into the chromosomal DNA of HCC tissues of H8V

infected patients was observed with considerable frequency. Although several

reports on the insertional site of HBV DNA into chromosomal DNA of HCC tissues are

available, it is still uncertain how HBV insertion affect on the

hepatocarcinogenesis.

In order to identify the insertion of HBV gene and the site of insertion into

chromosomal DNA of the HCC tissues of HBC infected, DNAs of HCC tissues from 11 HBV

infected patients were isolated and subjected to Southern blot hybridization,

polymerase chain reaction(PCR)and sequence analysis after subcloning of the

inserted DNA were performed.

By Southern blot hybridization using a (32)**P -labeled HBV probe, the insertion

of HBV DNA into the chromosomal DNA of HCC tissues was identified in all 7 patients

tested.

For the identification of the insertion of the specific gene locus of HBV, the

amplifications of specific genes of HBV were performed by PCR. The insertion of X

gene of HBV into the chromosomal DNA of HCC tissue was detected in 9 out of 11

patients, and the insertion of pre Sand S gene loci were detected in 1 and 8 out of

11 patients, respectively. The insertion of P gene locus of HBV into the

chromosomal DNA was detected in 6 of 11 patients, but the insertion of C gene was

not detected in any of them.

In order to determine the site of the junction of HBV DNA and the chromosomal

DNA, PCR was carried out using an oligonucleotide(20mer) complementary to pre S

gene locus and nonspecific oligo dG as primers and the chromosomal DNA as template.

About 400bp of the amplified DNA was obtained and was ligated to pGEM4Z plasmid for

the subcloning. It was identified that HBV DNA(No.42 base) was linked to

chromosomal DNA TTG GG (HBV).

From these results it is concluded that HBV DNA was inesrted into the chromosomal

DNA of most HCC tissues of HBV infected patients and that the insertion of X and S

gene loci of HBV were predominant.

The present results implicate that the single stranded sites of HBV genome were

more frequently inserted into chromosomal DNA and that X gene containing direct

repeat sequences(DRI, DR2) might involve in the HBV DNA insertion into the

chromosomal DNA of HCC tissue.

[영문]

A close relationship between the chronic infection of human hepatitis B virus(HBV) and the incidence of hepatocellular carcinoma(HCC) has been reported, and the insertion of HBV DNA into the chromosomal DNA of HCC tissues of H8V infected patients was observed with considerable frequency. Although several

reports on the insertional site of HBV DNA into chromosomal DNA of HCC tissues are available, it is still uncertain how HBV insertion affect on the hepatocarcinogenesis.

In order to identify the insertion of HBV gene and the site of insertion into chromosomal DNA of the HCC tissues of HBC infected, DNAs of HCC tissues from 11 HBV infected patients were isolated and subjected to Southern blot hybridization,

polymerase chain reaction(PCR)and sequence analysis after subcloning of the inserted DNA were performed.

By Southern blot hybridization using a (32)**P -labeled HBV probe, the insertion of HBV DNA into the chromosomal DNA of HCC tissues was identified in all 7 patients tested.

For the identification of the insertion of the specific gene locus of HBV, the amplifications of specific genes of HBV were performed by PCR. The insertion of X gene of HBV into the chromosomal DNA of HCC tissue was detected in 9 out of 11

patients, and the insertion of pre Sand S gene loci were detected in 1 and 8 out of 11 patients, respectively. The insertion of P gene locus of HBV into the chromosomal DNA was detected in 6 of 11 patients, but the insertion of C gene was not detected in any of them.

In order to determine the site of the junction of HBV DNA and the chromosomal DNA, PCR was carried out using an oligonucleotide(20mer) complementary to pre S gene locus and nonspecific oligo dG as primers and the chromosomal DNA as template.

About 400bp of the amplified DNA was obtained and was ligated to pGEM4Z plasmid for the subcloning. It was identified that HBV DNA(No.42 base) was linked to chromosomal DNA TTG GG (HBV).

From these results it is concluded that HBV DNA was inesrted into the chromosomal DNA of most HCC tissues of HBV infected patients and that the insertion of X and S gene loci of HBV were predominant.

The present results implicate that the single stranded sites of HBV genome were more frequently inserted into chromosomal DNA and that X gene containing direct repeat sequences(DRI, DR2) might involve in the HBV DNA insertion into the chromosomal DNA of HCC tissue.