乙型肝炎病毒整合在原发性肝细胞癌中的作用再评价
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摘要
背景和目的：原发性肝细胞癌(HCC)是我国最常见的恶性肿瘤之一,病死率极高。在中国,大约80%的HCC与乙型肝炎病毒(HBV)感染相关。已知HBV感染的致癌作用机制包括慢性感染所致肝脏的持续炎症反应,肝细胞破坏和肝细胞代偿性增生；病毒HBx蛋白和突变的前S蛋白的直接致癌作用；以及HBVDNA整合致肝细胞基因组。虽然病毒DNA整合被认为在慢性乙型肝炎病毒肝炎感染相关的肝细胞癌的发生中有着重要作用,但现有证据主要来自对少量肝细胞癌组织的研究,且缺少大样本配对癌旁组织的研究结果。因此,开展系统性的大样本配对癌与癌旁组织HBV的整合研究十分必要。我们对60例HBV阳性肝癌患者配对的肝细胞癌与癌旁组织进行系统性的比较研究,对HBV整合在肝细胞癌中的作用进行了重新评价。
材料和方法：
入选病人信息：60位HCC患者来自河南省肿瘤医院(年龄30-70岁；男性：女性=42：18)。所有病例均为乙型肝炎表面抗原(HBsAg)阳性；其中57标本为HBeAg阴性,3例为HBeAg阳性；除2例为肝纤维化外,其余患者均为肝硬化背景；所有患者血清抗-HCV均为阴性；HBV C基因型。
DNA提取：使用蛋白酶K消化后用酚/氯仿法提取60对配对癌组织和癌旁组织基因组DNA。QIAGEN DNA提取试剂盒提取25例患者配对的癌与癌旁组织基因组DNA,用于基于芯片技术的比较基因组杂交(array-based Comparative Genomic Hybridization, aCGH)分析。
整合序列获得：使用连接接头介导的PCR (LM-PCR)和Alu-PCR方法特异性扩增HBV整合序列。LM-PCR方法使用接头特异引物和HBV特异性引物；Alu-PCR方法使用人Alu序列和HBV特异引物。HBV特异性引物包括HBV X基因区上游的引物和HBV核心基因特异性引物。对PCR产物进行直接测序,测序失败的进行克隆测序。同时采用杂交捕获结合高通量测序方法对其中28对配对的癌与癌旁组织的整合位点进行验证分析。整合序列通过NCBI Blast工具和UCSChg19数据库进行分析整合序列中的病毒和宿主基因序列。设计跨HBV X到C基因的PCR上下游引物扩增肝癌及癌旁组织中的HBV基因组。整合序列中的X基因来自于己测序证实的病毒-宿主整合序列。使用荧光定量PCR方法检测了10例整合附近的基因mRNA水平。
比较基因组杂交技术(aCGH)分析染色体不稳定性。在aCGH实验中,每个癌组织标本均使用自身癌旁组织作为参照。
肿瘤组织TP53基因突变分析：分4个区段PCR反应扩增全部60例患者癌及癌旁组织TP53基因的外显子2到外显子11(外显子1不能翻译氨基酸)序列。同时对扩增区域内的SNP位点进行分析,选择了9个SNP位点(rs1642785. rs17878362、rs17883323, rs1042522、rs77624624、s2909430、rs12947788.rs12951053和rs6503048)。计算肿瘤组织中杂合子基因座两等位基因峰高比值与相配对的癌旁组织对应基因座两等位基因峰高比值的比。如有2个以上的SNP位点出现峰高比值的比为<0.5或>2.0,则作为判断肿瘤组织TP53基因LOH的判定标准。
使用SAS软件对实验结果进行统计学分析：统计描述采用频数表示,对完全随机下两组频数分布进行x2检验。对全基因组拷贝数变异的总数目(通过aCGH芯片得到结果)与整合的数目进行两个变量之间的相关分析。由于样本资料不符合正态分布,不适合用直线相关描述两个变量之间的相关关系,考虑用秩相关进行分析。P<0.05有统计学意义。
结果：
1.在癌组织和配对的癌旁组织中,整合发生的频率和在染色体分布未见差异。
使用PCR方法,88%(53/60)的患者可检测到整合。68%(41/60)的肿瘤组织和72%(43/60)的癌旁组织可检测到整合。约50%(22/41)的肿瘤组织和91%(39/43)的癌旁组织可检测到多个位点的整合序列。在获得的287个整合序列中,233个位点可以精确定位到人染色体DNA上,其中81个整合序列来自癌组织,152个整合序列来自癌旁组织。其它54个序列为不能定位的序列和高度重复序列。通过杂交捕获结合高通量测序方法,在>8reads数目支持的整合序列中,癌组织共发现201个整合断点,癌旁组织共发现91个整合断点。
整合的序列分布在除chr22外所有的常染色体和性染色体上。基于PCR技术的和基于杂交捕获结合高通量测序方法的整合分析均显示染色体的长度越长,整合序列数目越多。后者还发现在部分标本整合发生在线粒体DNA上。
对整合位点的染色体进行细化分析,与癌旁组织相比,HBV整合位点在肝癌组织中更容易发生在脆性位点附近。在癌组织可定位的81个整合位点中,有31个位点发生在脆性位点附近；而在癌旁组织可定位的151个整合位点中,有33个位点发生在脆性位点附近。两组之间的差异具有统计学差异(x2=7.1132,P=0.0077)。下面的脆性位点被发现整合次数大于1次：5例整合发生于脆性位点FRA19B(19p13);在脆性位点FRA1A(1p36)、FRA5A(5p13)和FRA7J (7q11)位置分别发现有4个整合位点；在脆性位点FRA7B (7p22)和FRA1L (1p31)各发现有3个整合位点；在脆性位点FRA1F(1q21)、FRA12A(12q13.1)、FRA3D (3q25)、FRA6D (6q13)和FRA19A (19q13)各发现有2个整合位点。
2.HBV整合易发生在富含基因的区域
.在基于PCR方法得到的233个可定位整合序列中,47%(110个)的整合位点插入至细胞基因的内含子,其分布在癌组织中和癌旁组织无差异[癌组织43%(35/81)vs.癌旁组织49%(75/152)]。整合发生于基因的外显子区的有10例(4例在癌组织和6例在癌旁组织)。另外,有26个整合位点10kb之内有人基因编码序列,这其中11例发生在癌组织,15例发生在癌旁组织。基于杂交捕获结合高通量测序方法的研究发现：对>8reads数目支持的整合序列进行定位分析,28例癌组织中有28.86%(58/201)的病毒序列插入在内含子和外显子区域(其中7个序列在外显子,51个序列在内含子上),28例配对的癌旁组织中有34.07%(31/91)的病毒插入在内含子和外显子内(其中4个序列在外显子,27个序列在内含子)。其分布在癌组织和癌旁组织无差异。
在60个HCC病人中,有3个癌组织在hTERT基因检测到IHBV DNA整合,而癌旁组织无一检测到此位点整合。此外,共有12个病毒插入在FN1基因附近,其中有10例发生在癌旁组织,2例发生在癌组织。
3.整合序列中的病毒断点分布在配对的癌与癌旁组织未见差异
对基于PCR；方法获得的287整合序列进行分析,其中163个序列含有X基因、121个序列含有病毒前C／C区,还有3个序列为前S/S基因区(既不含有X基因也不含有病毒前C/C区域)。与既往报道一致,大部分断点分布在病毒直接重复序列1(DRl)附近。本研究首次发现如此多的整合序列含有病毒前C/C区。
HBV核苷酸序歹nt1601-nt1834为断裂热点,大约75%的病毒断点分布在,其中24%(68/287)的断点位于5'-CTTTTT-3'拓扑异构酶I结构域(ntl820-1825)和DRl区(nt1824-nt1834)。断点分布在DR2(nt1590-1600)的整合病毒远少于断点在DR1者。
在癌组织和癌旁组织中,我们未发现病毒断点分布在两组间存在差异。
4.整合序列中病毒突变在配对的癌与癌旁组织未见差异
来自整合的病毒序列中C1653T、T1753V和A1762T/G1764A突变率与既往报导的的慢乙肝患者血清中HBV突变率相似,但低于肝癌和肝硬化患者；而游离HBV的突变率则与肝癌、肝硬化患者血清中的突变率相似。然而,无论是整合还是游离的HBV,C1653T、T1753V和A1762T/G1764A突变频率在配对的癌与癌旁组织中分布相近,未发现存在统计学差异。
比较整合的病毒序列和游离HBV发现,C1653T和A1762T/G1764A突变率在游离组要高于整合组[C1653T,整合组vs.游离组=9%(7/75)vs.22%(15/68),x2=4.4366,P=0.0352:T1762/A1764,整合组vs.游离组=54%(31/57)vs.78%(47/60),x2=7.5434,P=0.0060].对整合中X基因序列进行分析,我们发现134个含有X基因的整合序列均为3’端缺失的x基因。在癌组织和癌旁组织未见差异。
5.肝癌与癌旁组织中的整合序列附近的基因组重排
整合序列中含有的前C/C区较短,不能分析整合序列中的病毒重排。我们仅对含有X基因序列的整合进行了分析。在癌组织中,25.86%(15/58)的病毒序列进行了重排,高于癌旁组织中12.26%(15/58)的病毒序列重排(x2=4.8958,p=0.0269).HBV基因的重排包括缺失、插入、扩增和倒置等多种形式,以缺失最为常见。
在杂交捕获结合高通量测序方法对28对HCC标本检测结果中,癌组织和癌旁组织均发现HBV整合可多次发生在染色体某个位点几个kb左右区域,并且Reads支持数很接近,此结果提示HBV整合可能在染色体局部有一定选择性。
6.未发现HBV整合与全基因组基因拷贝数异常相关。
aCGH可得到全基因组水平拷贝数目的改变。对60例中的25例HCC患者进行aCGH分析(10例为女性,15例为男性),通过Spearman相关分析做整合频率与宿主全基因组水平拷贝数改变问的相关性分析,结果显示HBV整合存在与否及数目与对应肿瘤组织全基因组拷贝数变异无关。
我们进一步分析了60例患者中的TP53状态。共在20个肿瘤组织中发现了21个TP53点突变。其中1个肿瘤组织含有2个点突变位点(191eu和24lys)。其中20个点突变为错义突变,导致氨基酸编码改变,1个点突变未导致氨基酸编码改变。在所有的点突变中,有10个点突变为TP53249密码子点突变(AGG突变成AGT)。从所在外显子分布看,62%(13/21)的点突变位于外显子7。外显子2、4和5分别含有2个点突,外显子8有1个点突变。而在配对的癌旁组织中,未发现有任何标本含有TP53突变。
在60个HCC患者有51个可进行TP53的杂合性丢失分析,剩余9个患者的癌与癌旁组织均不含有有效的SNP信息,所以不能进行LOH分析。41%(22/51)的患者含有TP53杂合性丢失。综合考虑TP53点突变和/或杂合性丢失,我们共在48%的肝癌组织中发现有TP53异常。结合60例患者临床标本资料分析显示,未发现TP53基因状态与肝细胞癌患者性别、年龄、临床分期、AFP水平相关。
我们发现抑癌基因的异常数目(包括RBI、CDKN2A、TP53、BRCA1、BRCA2和TP53BP2)与癌组织全基因组基因拷贝数变异的关系具有统计学意义(r=0.6625,P=0.0003)。
7.整合附近基因表达水平
选取整合位点附近明确存在人基因组编码基因(hTERT和另外9个基因)的10个配对肝癌及癌旁组织,分析病毒DNA整合对整合位点周围宿主基因表达的潜在影响。使用CTBP1作为内参基因,与配对的癌旁组织相比,这些基因在癌组织的mRNA水平改变结果如下：在肿瘤中整合附近的DNLZ、SNAPCA4和ATP8B2基因上调2倍以上,而其他6个基因的mRNA水平无改变。同时发现,在两例整合在hTERT基因启动子区的癌组织标本中,我们发现其mRNA水平上调非常明显；而在另外一例HBV整合在hTERT基因转录起始位点上游55kb处,并且病毒序列和人基因序列方向相反,该肿瘤组织中的hTERT基因mRNA水平未发生改变。
结论：
通过对整合序列中的病毒突变分析,我们推测HBV整合发生在疾病进程的早期。人全基因组拷贝数变异可能与抑癌基因的状态相关,而整合可能与其无关。在脆性位点附近的整合可能与肝细胞癌的发生相关。PCR方法和HBV杂交捕获结合高通量测序方法得到的整合数据显示：除脆性位点外,在染色体的分布上,整合在癌组织和癌旁组织未见明显差异；人肝癌中常见的基因组水平上的染色体不稳定性未见与HBV整合相关。约27%到47%的HBV插入在人内含子区；还有约7%的HBV插入在人外显子区域。但这些在癌与癌旁组织两组的分布未见差异。在肝细胞癌中,约48%的患者存在TP53基因异常。基于芯片技术的比较基因组杂交结果提示抑癌基因的拷贝数异常与全基因组基因拷贝数目异常相关。我们目前的数据提示在肝癌中发现的多数HBV整合与肝细胞癌的发生没有明显相关性。
Background and aims:Primary hepatocellular carcinoma (HCC) is the most common cause of cancer death in China. It is estimated that more than80%of HCC is etiologically associated with HBV in China. Chronic HBV infection results in persistent inflammatory hepatocytes damage and compensatory regeneration. Oncogenic viral proteins such as HBx and mutant large surface protein were also considered playing direct pathogenic roles. In addition, it has been proposed more than three decades ago that HBV DNA integration into the hepatocytes cellular genome played a causative role in hepatocarcinogenesis. However, this speculation was mostly based on small scale observational studies using only tumor tissue and lacked the comparative control of the non-tumor tissue counterpart. Therefore, analysis of adjacent non-tumor tissue and consequently a systematic investigation of this hypothesis are still required. Through this systematic investigation, carcinogenesis significance of HBV integration in hepatocarcinogenesis was re-evaluated in this study.
Materials and method:Sixty HCC patients underwent surgical operation were recruited from He'nan Cancer Hospital from2008to2009(ranging age from30years to70years, mean age=50.7±8.46years; male:female=42:18;3of them were HBeAg-positive and57were HBeAg-negative);58of them were accompanied with liver cirrhosis.
DNAs were extracted from60paired frozen HCC tissues and corresponding adjacent non-tumor liver tissues using proteinase K followed by the standard pheol/chloroform extraction and ethanol precipitation method. For aCGH study, the genomic DNAs were extracted using the Genomic DNA purification Kit (Qiagen, USA).
LM-PCR was employed using cassette primers and primers specific to HBV sequences to amplify viral-host junctions. Alu-PCR was employed using specific primers to human Alu sequences and to HBV sequences to efficiently amplify viral-host junctions. The HBV specific primers were HBV X gene forward primers and HBV core gene reverse primers. The PCR products were subjected to agarose gel electrophoresis, and the bands were cut out of the gels for subsequent sequencing. We subcloned the PCR products into TA cloning vector when direct sequencing failed. HBV-captured deep DNA sequencing was performed in28paired tumor and non-tumor tissues. The viral-host sequences were analyzed by using the NCBI Blast tool and UCSC database hg19to identify viral genome sequences, and to map the integration sites in human genome separately.
The sequences for the 'free' HBx region were detected by PCR using primers acrossed from X gene to C gene. The about1100bp PCR products was directly sequenced. The integrated HBV X region sequences were acquired from the confirmed viral-host junction sequences.
A total of25paired DNA samples derived from tumor tissues and the corresponding tissues were prepared. Chromosome aberration was comprehensively analyzed via aCGH. In the assay, each corresponding paired adjacent non-tumor tissue DNA was used as reference DNA.
The exons2to11of TP53were amplified by4independent PCR reactions. The PCR products were directly sequenced to identify mutation. Meanwhile,9SNP sites (rs1642785, rs17878362, rs17883323, rs1042522, rs77624624, rs2909430, rs12947788, rs12951053and rs6503048) in this region were also analyzed for any potential loss of heterozygosity (LOH), in comparison with the corresponding non-tumor tissues.
Through comparing the electrophography peak values of the heterozygote SNP sites, LOH was defined according to the following formula:LOH index:L=(T2/T1)/(N2:N1)(T is tumor tissue, N is the adjacent non-tumor tissue;1and2are the intensities of smaller and larger alleles.). If the LOH index was less than0.5or more than2.0, then define the case as a potential LOH site. TP53LOH was defined when there were two or more than two potential LOH sites in each tissue. This method had been validated using the25aCGH analyzed tumor and non-tumor tissues.
The relative expression of genes surrounding viral insertion sites were quantitatively analyzed by qRT-PCR. The cccDNA level was also analyzed in some of the paired samples. All statistical analyses were performed using the SPSS14.0for windows.
Results:
HBV DNA integration showed no difference either in frequency or chromosome distribution between tumor derived and the corresponding non-tumor derived samples.
A total of287different inserted sequences were identified amongst the88% (53/60) integration positive patients. Among them,84viral-host junctions being identified in68%(41/60) tumor derived samples and148in72%(43/60) non-tumor derived samples. Multiple integration events were found in50%(22/41) of the HCCs and91%(39/43) of the non-tumor derived samples. Amongst the viral-host junctions identified,233could be precisely mapped to chromosomes, of which81were from tumor derived tissues and152from non-tumor samples. The remaining54virus-host junctions could not be uniquely mapped due to repetitive or unidentified sequences. Using HBV captured deep sequencing method,292different viral-host junctions were found (such junctions were more than8reads supported). Among them,201were in tumor tissues while91in non-tumor tissues.
As expected, larger chromosomes harbored more integration. However, when normalized to the number of integrations per108base pairs, no obvious preference of chromosome was observed in either tumor derived or in non-tumor samples. Amongst the233precisely mapped viral insertion sites,64were found to lie within a known fragile site. Amongst the233precisely mapped viral insertion sites,64were found to lie within a known fragile site. The following fragile sites were found being hit more than once:FRA19B in5cases, FRA1A, FRA5A, and FRA7J each in4cases, FRA7B and FRAIL in3cases, FRA1F, FRA12A, FRA3D, FRA6D and FRA19A in2cases. Interestingly, a significant disparity in the frequency with which fragile sites were mapped occurred between the tumor derived and non-tumor samples was observed (31/81sites in tumor vs.33/152sites in non-tumor, P=0.0077).
Cellular gene containing areas in the human genome were the favored target site for HBV integration.
Alignment analysis using the UCSC blat revealed that47%(110/233) of the viral insertion sites mapped were in introns (35/81in tumor vs.75/152in non-tumor) and4%(10/233) fell in exons (4/81in tumor vs.6/152in non-tumor). The remaining48% (113/233) were mapped to non-coding regions of the human genome (43/81in tumor vs.70/152in non-tumor). In addition,11of82integration sites mapped from tumor derived samples fell within+5kb of transcription start sites whilst the same was true for only15of the152integration sites mapped for non-tumor derived samples. These data indicate that, promoter, exon and intron areas in the genome are the favored target sites for HBV integration. Of course, it is possible that direct inserted into a gene area could affect the function of the targeted gene. In the HBV captured deep sequencing technique group,27%(78/292) of the viral insertion sites mapped were in introns (58/201in tumor vs.31/91in non-tumor) and4%(11/292) fell in exons (7/201in tumor vs.4/91in non-tumor). The remaining48%(113/233) were mapped to non-coding regions of the human genome (43/81in tumor vs.70/152in non-tumor). However, no significant differences of integration preference were observed between the tumor derived and the non-tumor derived samples.
In the present study, insertion in or around the hTERT gene was found in3tumor derived samples. This observation provides additional evidence that hTERT is frequently hit by HBV integration. HBV insertion directly into the FN1gene was also found in12cases. However, only2of these were found in the tumor derived samples.
No difference in the viral break point pattern was found between tumor derived and non-tumor derived samples.
Sequence analysis of the287inserted viral fragments showed that163of them harbored partial X gene sequences and121harbored preC/C sequences, leaving3that contained neither the X gene nor preC/C sequences. As previously reported, most of the break points occurred around the DR1site. This is the first study in which large numbers of inserted preC/C sequences have been found in HCC patients.
About75%of the break-points mapped between nt1601and nt1834of the viral genome, with24%(68/287) of them being located in the5'CTTTTT-3' topoisomerase motif (1820to1825nt) and the DR1region (1824-1834nt). The DR2(1590-1600nt) region was rarely found as the break point. Therefore, overall the data indicated that the topoisomerase I motif and the DR1region of the viral genome were the preferred HBV genome break-points in the mapped integration sites, but failed to reveal any difference between those from tumor derived and non tumor derived samples.
Comparative analysis of mutations in the inserted viral DNA failed to reveal any difference between tumor derived and non-tumor derived samples.
Meta-analysis of our own previously published data and that of others has shown that the number of mutations of the HBV genome (C1653T, T1753V and A1762T/G1764A) gradually increased with disease progression and correlated with hepatocarcinogenesis.
Direct sequencing of PCR amplicons generated from both tumor derived and non-tumor derived integrated viral sequences (integrated group) revealed that they were carrying fewer point mutations than were found in 'free' non integrated HBV genomes. The frequencies of C1653T, T1753V and A1762T/G1764A mutations found were at the same level as that seen in the serum derived samples of the CHB group, and were significantly lower than that in the LC and HCC group. A1.1kb amplicon (from nt1264-2362) from 'free' non integrated HBV DNA was successfully amplified and sequenced from a total of30tumor derived and34non-tumor derived samples (Table2). As expected, the frequencies of C1653T, T1753V, A1762T/G1764A mutations in these 'free' HBV DNAs from both tumor derived and non-tumor derived samples were at the same level as that for the serum derived samples of the LC group and HCC group. It is worthy to note that the frequency of either C1653T or A1762T/G1764A mutations seen was significantly higher than that found for the integrated group (C1653T,9%vs.22%, P=0.0110; T1762/A1764,78% vs.54%, P=0.0060). This significant difference remained when the frequencies of the T1762or A1764point mutation (P<0.05) were separately compared.
None of134confirmed X region insertions sequenced carried a whole X gene, with all of the inserts having a31terminal truncation. No significant difference was found for these or the other mutations described above between integrated viral genomes in tumor derived and non-tumor derived samples.
Rearrangements of host DNA surrounding integration sites was a rare event in both tumor derived and non-tumor derived samples.
The position of the sequencing primer on the X gene side of the break-point in the viral genome has a sufficient distance to the viral-host junction, so that viral genome rearrangements including deletions, inversions and duplications of viral sequence could be observed in the integrants. These rearrangements were found in15of58insertions in tumor and13of106insertions in non-tumor respectively (x2=4.8958, P=0.0269)(Figure3). In contrast the position of the preC/C sequencing primer was much closer to the viral-host junction which meant that the integrated viral sequences were too short for a similar analysis for viral genome rearrangements to be carried out Sequencing of the host cell genome close to the viral-host junction revealed only a few micro-deletions, micro-insertions, point mutations and translocations, with no significant difference being found between tumor derived and non-tumor derived samples.
We found that HBV inserted in a specific area more than two times in one sample by using the HBV captured deep sequencing method. However, it showed no difference between tumor group and non-tumor group.
The number of chromosome aberrations found in tumor derived samples did not correlated with HBV integration.
A comprehensive aCGH assay was used to analyze host cell chromosomal abnormalities in25individuals from the60recruited patient cohorts, selected to allow any effect of the number of HBV insertion events on chromosomal aberration to be examined. In each assay, material obtained from corresponding adjacent non-tumor tissue was used as the reference control. The number of HBV integration events detected in across25tumor samples analyzed ranged from0to11and the total number of genome aberrations (gain and loss) detected in the assay ranged from11to537. No correlation was found between the number of genome aberrations identified and the number of HBV integration in the tumor samples analyzed (P=0.6520).
The TP53status in all60patients was then analyzed. Twenty-one TP53point mutations were found in20(33%) of the tumor derived tissues, including18single nucleotide missense mutations and1single nucleotide synonymous mutation. One sample contained2point mutations at TP53191eu and241ys. Amongst the point mutations found10of them were AGG to AGT transversion at codon249of TP53. The majority of the point mutations were located in exon7(62%,13/21), with2mutations in exon2,2in exon4,2in exon5, and1in exon8, respectively. In contrast, no mutational changes in TP53were found in the paired adjacent non-tumor derived samples.
LOH of TP53was successfully assayed in51of the60samples. The remaining9samples had no informatic SNP data and therefore could not be assayed for LOH. Twenty-two of the51(41%) patients successfully assayed showed LOH for TP53. TP53point mutation and LOH concurrence was present in20%(12/60) of the patients. Overall mutational change (point mutation and/or LOH) causing loss of TP53function was detected in48%(29/60) of the recruited patient cohort. No correlation was found between mutational change in TP53and the number of HBV integration events.
Interestingly, among the25patients whose samples were subjected to aCGH analysis9of the11tumor derived samples harboring TP53mutations were found to have higher numbers of chromosomal aberrations. In contrast,7of the14tumor derived samples that had no mutations in TP53was found lie amongst those with a higher number of chromosomal aberrations.
The mutational status of various tumor suppressor genes such as retinoblastoma1(RBI), TP73, cyclin-dependent kinase inhibitor2A (CDKN2A), breast cancer1, early onset (BRCA1), BRCA2, TP53and TP53BP2(tumor protein p53binding protein,2) in the patient samples subjected to aCGH analysis was also examined (Table3). As expected, a positive correlation was found between the number of specific tumor suppressor gene mutations identified and the number of whole chromosomal aberrations observed (r=0.6625, P=0.0003).
The relative expression levels of genes successive to HBV integration
Except hTERT gene,9other genes successive to the integration sites were randomly selected to determine if their expression was affected by HBV DNA insertion. Using C-terminal binding protein1(CTBP1) gene as an internal control, in comparison with the adjacent non-tumor tissue, the mRNA levels of DNLZ, SNAPCA4, and ATP8B2in tumor tissues with HBV integration nearby were detected with more than2fold up-regulation. The remained6genes showed no changes at the mRNA levels.
The hTERT expression level was associated with the distance between viral insertion and the start site of gene. The hTERT up-regulation was found in2tumor tissues in which the HBV sequence inserted into the hTERT promoter regions. No difference of hTERT expression was observed in the third tumor tissues when compared with its paired non-tumor tissue, in which the HBV inserted into55Kb downstream of the hTERT with opposite orientation.
Conclusion
HBV integration events occurred at the early stage of disease progression. Instead of HBV integration, the status of several tumor suppressor genes might play a crucial role in the number of chromosome aberration. With exception of a significant high frequency of fragile sites hit in tumor group, all other properties of HBV insertion were highly similar between tumor and non-tumor. Our data failed to demonstrate a strong co-relationship between HBV integration and hepatocarcinogenesis.
材料和方法：
入选病人信息：60位HCC患者来自河南省肿瘤医院(年龄30-70岁；男性：女性=42：18)。所有病例均为乙型肝炎表面抗原(HBsAg)阳性；其中57标本为HBeAg阴性,3例为HBeAg阳性；除2例为肝纤维化外,其余患者均为肝硬化背景；所有患者血清抗-HCV均为阴性；HBV C基因型。
DNA提取：使用蛋白酶K消化后用酚/氯仿法提取60对配对癌组织和癌旁组织基因组DNA。QIAGEN DNA提取试剂盒提取25例患者配对的癌与癌旁组织基因组DNA,用于基于芯片技术的比较基因组杂交(array-based Comparative Genomic Hybridization, aCGH)分析。
整合序列获得：使用连接接头介导的PCR (LM-PCR)和Alu-PCR方法特异性扩增HBV整合序列。LM-PCR方法使用接头特异引物和HBV特异性引物；Alu-PCR方法使用人Alu序列和HBV特异引物。HBV特异性引物包括HBV X基因区上游的引物和HBV核心基因特异性引物。对PCR产物进行直接测序,测序失败的进行克隆测序。同时采用杂交捕获结合高通量测序方法对其中28对配对的癌与癌旁组织的整合位点进行验证分析。整合序列通过NCBI Blast工具和UCSChg19数据库进行分析整合序列中的病毒和宿主基因序列。设计跨HBV X到C基因的PCR上下游引物扩增肝癌及癌旁组织中的HBV基因组。整合序列中的X基因来自于己测序证实的病毒-宿主整合序列。使用荧光定量PCR方法检测了10例整合附近的基因mRNA水平。
比较基因组杂交技术(aCGH)分析染色体不稳定性。在aCGH实验中,每个癌组织标本均使用自身癌旁组织作为参照。
肿瘤组织TP53基因突变分析：分4个区段PCR反应扩增全部60例患者癌及癌旁组织TP53基因的外显子2到外显子11(外显子1不能翻译氨基酸)序列。同时对扩增区域内的SNP位点进行分析,选择了9个SNP位点(rs1642785. rs17878362、rs17883323, rs1042522、rs77624624、s2909430、rs12947788.rs12951053和rs6503048)。计算肿瘤组织中杂合子基因座两等位基因峰高比值与相配对的癌旁组织对应基因座两等位基因峰高比值的比。如有2个以上的SNP位点出现峰高比值的比为<0.5或>2.0,则作为判断肿瘤组织TP53基因LOH的判定标准。
使用SAS软件对实验结果进行统计学分析：统计描述采用频数表示,对完全随机下两组频数分布进行x2检验。对全基因组拷贝数变异的总数目(通过aCGH芯片得到结果)与整合的数目进行两个变量之间的相关分析。由于样本资料不符合正态分布,不适合用直线相关描述两个变量之间的相关关系,考虑用秩相关进行分析。P<0.05有统计学意义。
结果：
1.在癌组织和配对的癌旁组织中,整合发生的频率和在染色体分布未见差异。
使用PCR方法,88%(53/60)的患者可检测到整合。68%(41/60)的肿瘤组织和72%(43/60)的癌旁组织可检测到整合。约50%(22/41)的肿瘤组织和91%(39/43)的癌旁组织可检测到多个位点的整合序列。在获得的287个整合序列中,233个位点可以精确定位到人染色体DNA上,其中81个整合序列来自癌组织,152个整合序列来自癌旁组织。其它54个序列为不能定位的序列和高度重复序列。通过杂交捕获结合高通量测序方法,在>8reads数目支持的整合序列中,癌组织共发现201个整合断点,癌旁组织共发现91个整合断点。
整合的序列分布在除chr22外所有的常染色体和性染色体上。基于PCR技术的和基于杂交捕获结合高通量测序方法的整合分析均显示染色体的长度越长,整合序列数目越多。后者还发现在部分标本整合发生在线粒体DNA上。
对整合位点的染色体进行细化分析,与癌旁组织相比,HBV整合位点在肝癌组织中更容易发生在脆性位点附近。在癌组织可定位的81个整合位点中,有31个位点发生在脆性位点附近；而在癌旁组织可定位的151个整合位点中,有33个位点发生在脆性位点附近。两组之间的差异具有统计学差异(x2=7.1132,P=0.0077)。下面的脆性位点被发现整合次数大于1次：5例整合发生于脆性位点FRA19B(19p13);在脆性位点FRA1A(1p36)、FRA5A(5p13)和FRA7J (7q11)位置分别发现有4个整合位点；在脆性位点FRA7B (7p22)和FRA1L (1p31)各发现有3个整合位点；在脆性位点FRA1F(1q21)、FRA12A(12q13.1)、FRA3D (3q25)、FRA6D (6q13)和FRA19A (19q13)各发现有2个整合位点。
2.HBV整合易发生在富含基因的区域
.在基于PCR方法得到的233个可定位整合序列中,47%(110个)的整合位点插入至细胞基因的内含子,其分布在癌组织中和癌旁组织无差异[癌组织43%(35/81)vs.癌旁组织49%(75/152)]。整合发生于基因的外显子区的有10例(4例在癌组织和6例在癌旁组织)。另外,有26个整合位点10kb之内有人基因编码序列,这其中11例发生在癌组织,15例发生在癌旁组织。基于杂交捕获结合高通量测序方法的研究发现：对>8reads数目支持的整合序列进行定位分析,28例癌组织中有28.86%(58/201)的病毒序列插入在内含子和外显子区域(其中7个序列在外显子,51个序列在内含子上),28例配对的癌旁组织中有34.07%(31/91)的病毒插入在内含子和外显子内(其中4个序列在外显子,27个序列在内含子)。其分布在癌组织和癌旁组织无差异。
在60个HCC病人中,有3个癌组织在hTERT基因检测到IHBV DNA整合,而癌旁组织无一检测到此位点整合。此外,共有12个病毒插入在FN1基因附近,其中有10例发生在癌旁组织,2例发生在癌组织。
3.整合序列中的病毒断点分布在配对的癌与癌旁组织未见差异
对基于PCR；方法获得的287整合序列进行分析,其中163个序列含有X基因、121个序列含有病毒前C／C区,还有3个序列为前S/S基因区(既不含有X基因也不含有病毒前C/C区域)。与既往报道一致,大部分断点分布在病毒直接重复序列1(DRl)附近。本研究首次发现如此多的整合序列含有病毒前C/C区。
HBV核苷酸序歹nt1601-nt1834为断裂热点,大约75%的病毒断点分布在,其中24%(68/287)的断点位于5'-CTTTTT-3'拓扑异构酶I结构域(ntl820-1825)和DRl区(nt1824-nt1834)。断点分布在DR2(nt1590-1600)的整合病毒远少于断点在DR1者。
在癌组织和癌旁组织中,我们未发现病毒断点分布在两组间存在差异。
4.整合序列中病毒突变在配对的癌与癌旁组织未见差异
来自整合的病毒序列中C1653T、T1753V和A1762T/G1764A突变率与既往报导的的慢乙肝患者血清中HBV突变率相似,但低于肝癌和肝硬化患者；而游离HBV的突变率则与肝癌、肝硬化患者血清中的突变率相似。然而,无论是整合还是游离的HBV,C1653T、T1753V和A1762T/G1764A突变频率在配对的癌与癌旁组织中分布相近,未发现存在统计学差异。
比较整合的病毒序列和游离HBV发现,C1653T和A1762T/G1764A突变率在游离组要高于整合组[C1653T,整合组vs.游离组=9%(7/75)vs.22%(15/68),x2=4.4366,P=0.0352:T1762/A1764,整合组vs.游离组=54%(31/57)vs.78%(47/60),x2=7.5434,P=0.0060].对整合中X基因序列进行分析,我们发现134个含有X基因的整合序列均为3’端缺失的x基因。在癌组织和癌旁组织未见差异。
5.肝癌与癌旁组织中的整合序列附近的基因组重排
整合序列中含有的前C/C区较短,不能分析整合序列中的病毒重排。我们仅对含有X基因序列的整合进行了分析。在癌组织中,25.86%(15/58)的病毒序列进行了重排,高于癌旁组织中12.26%(15/58)的病毒序列重排(x2=4.8958,p=0.0269).HBV基因的重排包括缺失、插入、扩增和倒置等多种形式,以缺失最为常见。
在杂交捕获结合高通量测序方法对28对HCC标本检测结果中,癌组织和癌旁组织均发现HBV整合可多次发生在染色体某个位点几个kb左右区域,并且Reads支持数很接近,此结果提示HBV整合可能在染色体局部有一定选择性。
6.未发现HBV整合与全基因组基因拷贝数异常相关。
aCGH可得到全基因组水平拷贝数目的改变。对60例中的25例HCC患者进行aCGH分析(10例为女性,15例为男性),通过Spearman相关分析做整合频率与宿主全基因组水平拷贝数改变问的相关性分析,结果显示HBV整合存在与否及数目与对应肿瘤组织全基因组拷贝数变异无关。
我们进一步分析了60例患者中的TP53状态。共在20个肿瘤组织中发现了21个TP53点突变。其中1个肿瘤组织含有2个点突变位点(191eu和24lys)。其中20个点突变为错义突变,导致氨基酸编码改变,1个点突变未导致氨基酸编码改变。在所有的点突变中,有10个点突变为TP53249密码子点突变(AGG突变成AGT)。从所在外显子分布看,62%(13/21)的点突变位于外显子7。外显子2、4和5分别含有2个点突,外显子8有1个点突变。而在配对的癌旁组织中,未发现有任何标本含有TP53突变。
在60个HCC患者有51个可进行TP53的杂合性丢失分析,剩余9个患者的癌与癌旁组织均不含有有效的SNP信息,所以不能进行LOH分析。41%(22/51)的患者含有TP53杂合性丢失。综合考虑TP53点突变和/或杂合性丢失,我们共在48%的肝癌组织中发现有TP53异常。结合60例患者临床标本资料分析显示,未发现TP53基因状态与肝细胞癌患者性别、年龄、临床分期、AFP水平相关。
我们发现抑癌基因的异常数目(包括RBI、CDKN2A、TP53、BRCA1、BRCA2和TP53BP2)与癌组织全基因组基因拷贝数变异的关系具有统计学意义(r=0.6625,P=0.0003)。
7.整合附近基因表达水平
选取整合位点附近明确存在人基因组编码基因(hTERT和另外9个基因)的10个配对肝癌及癌旁组织,分析病毒DNA整合对整合位点周围宿主基因表达的潜在影响。使用CTBP1作为内参基因,与配对的癌旁组织相比,这些基因在癌组织的mRNA水平改变结果如下：在肿瘤中整合附近的DNLZ、SNAPCA4和ATP8B2基因上调2倍以上,而其他6个基因的mRNA水平无改变。同时发现,在两例整合在hTERT基因启动子区的癌组织标本中,我们发现其mRNA水平上调非常明显；而在另外一例HBV整合在hTERT基因转录起始位点上游55kb处,并且病毒序列和人基因序列方向相反,该肿瘤组织中的hTERT基因mRNA水平未发生改变。
结论：
通过对整合序列中的病毒突变分析,我们推测HBV整合发生在疾病进程的早期。人全基因组拷贝数变异可能与抑癌基因的状态相关,而整合可能与其无关。在脆性位点附近的整合可能与肝细胞癌的发生相关。PCR方法和HBV杂交捕获结合高通量测序方法得到的整合数据显示：除脆性位点外,在染色体的分布上,整合在癌组织和癌旁组织未见明显差异；人肝癌中常见的基因组水平上的染色体不稳定性未见与HBV整合相关。约27%到47%的HBV插入在人内含子区；还有约7%的HBV插入在人外显子区域。但这些在癌与癌旁组织两组的分布未见差异。在肝细胞癌中,约48%的患者存在TP53基因异常。基于芯片技术的比较基因组杂交结果提示抑癌基因的拷贝数异常与全基因组基因拷贝数目异常相关。我们目前的数据提示在肝癌中发现的多数HBV整合与肝细胞癌的发生没有明显相关性。
Background and aims:Primary hepatocellular carcinoma (HCC) is the most common cause of cancer death in China. It is estimated that more than80%of HCC is etiologically associated with HBV in China. Chronic HBV infection results in persistent inflammatory hepatocytes damage and compensatory regeneration. Oncogenic viral proteins such as HBx and mutant large surface protein were also considered playing direct pathogenic roles. In addition, it has been proposed more than three decades ago that HBV DNA integration into the hepatocytes cellular genome played a causative role in hepatocarcinogenesis. However, this speculation was mostly based on small scale observational studies using only tumor tissue and lacked the comparative control of the non-tumor tissue counterpart. Therefore, analysis of adjacent non-tumor tissue and consequently a systematic investigation of this hypothesis are still required. Through this systematic investigation, carcinogenesis significance of HBV integration in hepatocarcinogenesis was re-evaluated in this study.
Materials and method:Sixty HCC patients underwent surgical operation were recruited from He'nan Cancer Hospital from2008to2009(ranging age from30years to70years, mean age=50.7±8.46years; male:female=42:18;3of them were HBeAg-positive and57were HBeAg-negative);58of them were accompanied with liver cirrhosis.
DNAs were extracted from60paired frozen HCC tissues and corresponding adjacent non-tumor liver tissues using proteinase K followed by the standard pheol/chloroform extraction and ethanol precipitation method. For aCGH study, the genomic DNAs were extracted using the Genomic DNA purification Kit (Qiagen, USA).
LM-PCR was employed using cassette primers and primers specific to HBV sequences to amplify viral-host junctions. Alu-PCR was employed using specific primers to human Alu sequences and to HBV sequences to efficiently amplify viral-host junctions. The HBV specific primers were HBV X gene forward primers and HBV core gene reverse primers. The PCR products were subjected to agarose gel electrophoresis, and the bands were cut out of the gels for subsequent sequencing. We subcloned the PCR products into TA cloning vector when direct sequencing failed. HBV-captured deep DNA sequencing was performed in28paired tumor and non-tumor tissues. The viral-host sequences were analyzed by using the NCBI Blast tool and UCSC database hg19to identify viral genome sequences, and to map the integration sites in human genome separately.
The sequences for the 'free' HBx region were detected by PCR using primers acrossed from X gene to C gene. The about1100bp PCR products was directly sequenced. The integrated HBV X region sequences were acquired from the confirmed viral-host junction sequences.
A total of25paired DNA samples derived from tumor tissues and the corresponding tissues were prepared. Chromosome aberration was comprehensively analyzed via aCGH. In the assay, each corresponding paired adjacent non-tumor tissue DNA was used as reference DNA.
The exons2to11of TP53were amplified by4independent PCR reactions. The PCR products were directly sequenced to identify mutation. Meanwhile,9SNP sites (rs1642785, rs17878362, rs17883323, rs1042522, rs77624624, rs2909430, rs12947788, rs12951053and rs6503048) in this region were also analyzed for any potential loss of heterozygosity (LOH), in comparison with the corresponding non-tumor tissues.
Through comparing the electrophography peak values of the heterozygote SNP sites, LOH was defined according to the following formula:LOH index:L=(T2/T1)/(N2:N1)(T is tumor tissue, N is the adjacent non-tumor tissue;1and2are the intensities of smaller and larger alleles.). If the LOH index was less than0.5or more than2.0, then define the case as a potential LOH site. TP53LOH was defined when there were two or more than two potential LOH sites in each tissue. This method had been validated using the25aCGH analyzed tumor and non-tumor tissues.
The relative expression of genes surrounding viral insertion sites were quantitatively analyzed by qRT-PCR. The cccDNA level was also analyzed in some of the paired samples. All statistical analyses were performed using the SPSS14.0for windows.
Results:
HBV DNA integration showed no difference either in frequency or chromosome distribution between tumor derived and the corresponding non-tumor derived samples.
A total of287different inserted sequences were identified amongst the88% (53/60) integration positive patients. Among them,84viral-host junctions being identified in68%(41/60) tumor derived samples and148in72%(43/60) non-tumor derived samples. Multiple integration events were found in50%(22/41) of the HCCs and91%(39/43) of the non-tumor derived samples. Amongst the viral-host junctions identified,233could be precisely mapped to chromosomes, of which81were from tumor derived tissues and152from non-tumor samples. The remaining54virus-host junctions could not be uniquely mapped due to repetitive or unidentified sequences. Using HBV captured deep sequencing method,292different viral-host junctions were found (such junctions were more than8reads supported). Among them,201were in tumor tissues while91in non-tumor tissues.
As expected, larger chromosomes harbored more integration. However, when normalized to the number of integrations per108base pairs, no obvious preference of chromosome was observed in either tumor derived or in non-tumor samples. Amongst the233precisely mapped viral insertion sites,64were found to lie within a known fragile site. Amongst the233precisely mapped viral insertion sites,64were found to lie within a known fragile site. The following fragile sites were found being hit more than once:FRA19B in5cases, FRA1A, FRA5A, and FRA7J each in4cases, FRA7B and FRAIL in3cases, FRA1F, FRA12A, FRA3D, FRA6D and FRA19A in2cases. Interestingly, a significant disparity in the frequency with which fragile sites were mapped occurred between the tumor derived and non-tumor samples was observed (31/81sites in tumor vs.33/152sites in non-tumor, P=0.0077).
Cellular gene containing areas in the human genome were the favored target site for HBV integration.
Alignment analysis using the UCSC blat revealed that47%(110/233) of the viral insertion sites mapped were in introns (35/81in tumor vs.75/152in non-tumor) and4%(10/233) fell in exons (4/81in tumor vs.6/152in non-tumor). The remaining48% (113/233) were mapped to non-coding regions of the human genome (43/81in tumor vs.70/152in non-tumor). In addition,11of82integration sites mapped from tumor derived samples fell within+5kb of transcription start sites whilst the same was true for only15of the152integration sites mapped for non-tumor derived samples. These data indicate that, promoter, exon and intron areas in the genome are the favored target sites for HBV integration. Of course, it is possible that direct inserted into a gene area could affect the function of the targeted gene. In the HBV captured deep sequencing technique group,27%(78/292) of the viral insertion sites mapped were in introns (58/201in tumor vs.31/91in non-tumor) and4%(11/292) fell in exons (7/201in tumor vs.4/91in non-tumor). The remaining48%(113/233) were mapped to non-coding regions of the human genome (43/81in tumor vs.70/152in non-tumor). However, no significant differences of integration preference were observed between the tumor derived and the non-tumor derived samples.
In the present study, insertion in or around the hTERT gene was found in3tumor derived samples. This observation provides additional evidence that hTERT is frequently hit by HBV integration. HBV insertion directly into the FN1gene was also found in12cases. However, only2of these were found in the tumor derived samples.
No difference in the viral break point pattern was found between tumor derived and non-tumor derived samples.
Sequence analysis of the287inserted viral fragments showed that163of them harbored partial X gene sequences and121harbored preC/C sequences, leaving3that contained neither the X gene nor preC/C sequences. As previously reported, most of the break points occurred around the DR1site. This is the first study in which large numbers of inserted preC/C sequences have been found in HCC patients.
About75%of the break-points mapped between nt1601and nt1834of the viral genome, with24%(68/287) of them being located in the5'CTTTTT-3' topoisomerase motif (1820to1825nt) and the DR1region (1824-1834nt). The DR2(1590-1600nt) region was rarely found as the break point. Therefore, overall the data indicated that the topoisomerase I motif and the DR1region of the viral genome were the preferred HBV genome break-points in the mapped integration sites, but failed to reveal any difference between those from tumor derived and non tumor derived samples.
Comparative analysis of mutations in the inserted viral DNA failed to reveal any difference between tumor derived and non-tumor derived samples.
Meta-analysis of our own previously published data and that of others has shown that the number of mutations of the HBV genome (C1653T, T1753V and A1762T/G1764A) gradually increased with disease progression and correlated with hepatocarcinogenesis.
Direct sequencing of PCR amplicons generated from both tumor derived and non-tumor derived integrated viral sequences (integrated group) revealed that they were carrying fewer point mutations than were found in 'free' non integrated HBV genomes. The frequencies of C1653T, T1753V and A1762T/G1764A mutations found were at the same level as that seen in the serum derived samples of the CHB group, and were significantly lower than that in the LC and HCC group. A1.1kb amplicon (from nt1264-2362) from 'free' non integrated HBV DNA was successfully amplified and sequenced from a total of30tumor derived and34non-tumor derived samples (Table2). As expected, the frequencies of C1653T, T1753V, A1762T/G1764A mutations in these 'free' HBV DNAs from both tumor derived and non-tumor derived samples were at the same level as that for the serum derived samples of the LC group and HCC group. It is worthy to note that the frequency of either C1653T or A1762T/G1764A mutations seen was significantly higher than that found for the integrated group (C1653T,9%vs.22%, P=0.0110; T1762/A1764,78% vs.54%, P=0.0060). This significant difference remained when the frequencies of the T1762or A1764point mutation (P<0.05) were separately compared.
None of134confirmed X region insertions sequenced carried a whole X gene, with all of the inserts having a31terminal truncation. No significant difference was found for these or the other mutations described above between integrated viral genomes in tumor derived and non-tumor derived samples.
Rearrangements of host DNA surrounding integration sites was a rare event in both tumor derived and non-tumor derived samples.
The position of the sequencing primer on the X gene side of the break-point in the viral genome has a sufficient distance to the viral-host junction, so that viral genome rearrangements including deletions, inversions and duplications of viral sequence could be observed in the integrants. These rearrangements were found in15of58insertions in tumor and13of106insertions in non-tumor respectively (x2=4.8958, P=0.0269)(Figure3). In contrast the position of the preC/C sequencing primer was much closer to the viral-host junction which meant that the integrated viral sequences were too short for a similar analysis for viral genome rearrangements to be carried out Sequencing of the host cell genome close to the viral-host junction revealed only a few micro-deletions, micro-insertions, point mutations and translocations, with no significant difference being found between tumor derived and non-tumor derived samples.
We found that HBV inserted in a specific area more than two times in one sample by using the HBV captured deep sequencing method. However, it showed no difference between tumor group and non-tumor group.
The number of chromosome aberrations found in tumor derived samples did not correlated with HBV integration.
A comprehensive aCGH assay was used to analyze host cell chromosomal abnormalities in25individuals from the60recruited patient cohorts, selected to allow any effect of the number of HBV insertion events on chromosomal aberration to be examined. In each assay, material obtained from corresponding adjacent non-tumor tissue was used as the reference control. The number of HBV integration events detected in across25tumor samples analyzed ranged from0to11and the total number of genome aberrations (gain and loss) detected in the assay ranged from11to537. No correlation was found between the number of genome aberrations identified and the number of HBV integration in the tumor samples analyzed (P=0.6520).
The TP53status in all60patients was then analyzed. Twenty-one TP53point mutations were found in20(33%) of the tumor derived tissues, including18single nucleotide missense mutations and1single nucleotide synonymous mutation. One sample contained2point mutations at TP53191eu and241ys. Amongst the point mutations found10of them were AGG to AGT transversion at codon249of TP53. The majority of the point mutations were located in exon7(62%,13/21), with2mutations in exon2,2in exon4,2in exon5, and1in exon8, respectively. In contrast, no mutational changes in TP53were found in the paired adjacent non-tumor derived samples.
LOH of TP53was successfully assayed in51of the60samples. The remaining9samples had no informatic SNP data and therefore could not be assayed for LOH. Twenty-two of the51(41%) patients successfully assayed showed LOH for TP53. TP53point mutation and LOH concurrence was present in20%(12/60) of the patients. Overall mutational change (point mutation and/or LOH) causing loss of TP53function was detected in48%(29/60) of the recruited patient cohort. No correlation was found between mutational change in TP53and the number of HBV integration events.
Interestingly, among the25patients whose samples were subjected to aCGH analysis9of the11tumor derived samples harboring TP53mutations were found to have higher numbers of chromosomal aberrations. In contrast,7of the14tumor derived samples that had no mutations in TP53was found lie amongst those with a higher number of chromosomal aberrations.
The mutational status of various tumor suppressor genes such as retinoblastoma1(RBI), TP73, cyclin-dependent kinase inhibitor2A (CDKN2A), breast cancer1, early onset (BRCA1), BRCA2, TP53and TP53BP2(tumor protein p53binding protein,2) in the patient samples subjected to aCGH analysis was also examined (Table3). As expected, a positive correlation was found between the number of specific tumor suppressor gene mutations identified and the number of whole chromosomal aberrations observed (r=0.6625, P=0.0003).
The relative expression levels of genes successive to HBV integration
Except hTERT gene,9other genes successive to the integration sites were randomly selected to determine if their expression was affected by HBV DNA insertion. Using C-terminal binding protein1(CTBP1) gene as an internal control, in comparison with the adjacent non-tumor tissue, the mRNA levels of DNLZ, SNAPCA4, and ATP8B2in tumor tissues with HBV integration nearby were detected with more than2fold up-regulation. The remained6genes showed no changes at the mRNA levels.
The hTERT expression level was associated with the distance between viral insertion and the start site of gene. The hTERT up-regulation was found in2tumor tissues in which the HBV sequence inserted into the hTERT promoter regions. No difference of hTERT expression was observed in the third tumor tissues when compared with its paired non-tumor tissue, in which the HBV inserted into55Kb downstream of the hTERT with opposite orientation.
Conclusion
HBV integration events occurred at the early stage of disease progression. Instead of HBV integration, the status of several tumor suppressor genes might play a crucial role in the number of chromosome aberration. With exception of a significant high frequency of fragile sites hit in tumor group, all other properties of HBV insertion were highly similar between tumor and non-tumor. Our data failed to demonstrate a strong co-relationship between HBV integration and hepatocarcinogenesis.
引文
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29. Ma NF, Lau SH, Hu L, Xie D, Wu J, et al. (2008) COOH-terminal truncated HBV X protein plays key role in hepatocarcinogenesis. Clin Cancer Res 14:5061-5068.
30. Aoki N, Robinson WS (1989) State of hepatitis B viral genomes in cirrhotic and hepatocellular carcinoma nodules. Mol Biol Med 6:395-408.
31. Wang Y, Lau SH, Sham JS, Wu MC, Wang T, et al. (2004) Characterization of HBV integrants in 14 hepatocellular carcinomas:association of truncated X gene and hepatocellular carcinogenesis. Oncogene 23:142-148.
32. Bonilla GR, Roberts LR (2005) The role of hepatitis B virus integrations in the pathogenesis of human hepatocellular carcinoma. J Hepatol 42:760-777.
33. Minami M, Daimon Y, Mori K, Takashima H, Nakajima T, et al. (2005) Hepatitis B virus-related insertional mutagenesis in chronic hepatitis B patients as an early drastic genetic change leading to hepatocarcinogenesis. Oncogene 24:4340-4348.
34. Murakami Y, Saigo K, Takashima H, Minami M, Okanoue T, et al. (2005) Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut 54: 1162-1168.
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36. Dillon LW, Burrow AA, Wang YH (2010) DNA instability at chromosomal fragile sites in cancer. Curr Genomics 11:326-337.
37. Liu S, Zhang H, Gu C, Yin J, He Y, et al. (2009) Associations between hepatitis B virus mutations and the risk of hepatocellular carcinoma:a meta-analysis. J Natl Cancer Inst 101:1066-1082.
38. Subirana JA, Anokian E (2011) Visualization of sequence and structural features of genomes and chromosome fragments. Application to CpG islands, Alu sequences and whole genomes. Gene 473:76-81.
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2. Nikolova PV, Wong KB, DeDecker B, Henckel J, Fersht AR (2000) Mechanism of rescue of common p53 cancer mutations by second-site suppressor mutations. EMBO J 19:370-378.
3. Suzuki K, Matsubara H (2011) Recent advances in p53 research and cancer treatment. J Biomed Biotechnol 2011:978312.
4. Nakajima T, Moriguchi M, Mitsumoto Y, Sekoguchi S, Nishikawa T, et al. (2004) Centrosome aberration accompanied with p53 mutation can induce genetic instability in hepatocellular carcinoma. Mod Pathol 17:722-727.
5. Suad O, Rozenberg H, Brosh R, Diskin-Posner Y, Kessler N, et al. (2009) Structural basis of restoring sequence-specific DNA binding and transactivation to mutant p53 by suppressor mutations. J Mol Biol 385:249-265.
6. Selivanova G, Wiman KG (2007) Reactivation of mutant p53:molecular mechanisms and therapeutic potential. Oncogene 26:2243-2254.
7. Ezzikouri S, Essaid El Feydi A, Afifi R, Benazzouz M, Hassar M, et al. (2011) Impact of TP53 codon 72 and MDM2 promoter 309 allelic dosage in a Moroccan population with hepatocellular carcinoma. Int J Biol Markers 26:229-233.
8. Friedler A, DeDecker BS, Freund SM, Blair C, Rudiger S, et al. (2004) Structural distortion of p53 by the mutation R249S and its rescue by a designed peptide:implications for "mutant conformation". J Mol Biol 336:187-196.
9. Besaratinia A, Kim SI, Hainaut P, Pfeifer GP (2009) In vitro recapitulating of TP53 mutagenesis in hepatocellular carcinoma associated with dietary aflatoxin B1 exposure. Gastroenterology 137: 1127-1137,1137 el 121-1125.
10. Zhao X, He M, Wan D, Ye Y, He Y, et al. (2003) The minimum LOH region defined on chromosome 17p13.3 in human hepatocellular carcinoma with gene content analysis. Cancer Lett 190:221-232.
11. Zhu GN, Zuo L, Zhou Q, Zhang SM, Zhu HQ, et al. (2004) Loss of heterozygosity on chromosome 10q22-10q23 and 22ql 1.2-22q12.1 and p53 gene in primary hepatocellular carcinoma. World J Gastroenterol 10:1975-1978.
12. Wada I, Horiuchi H, Mori M, Ishikawa Y, Fukumoto M, et al. (1999) High rate of small TP53 mutations and infrequent loss of heterozygosity in malignant liver tumors associated with thorotrast:implications for alpha-particle carcinogenesis. Radiat Res 152:S125-127.
13. Navas MC, Suarez I, Carreno A, Uribe D, Rios WA, et al. (2011) Hepatitis B and Hepatitis C Infection Biomarkers and TP53 Mutations in Hepatocellular Carcinomas from Colombia. Hepat Res Treat 2011:582945.
14. Abedi-Ardekani B, Gouas D, Villar S, Sotoudeh M, Hainaut P (2011) TP53 Mutations and HBX Status Analysis in Hepatocellular Carcinomas from Iran:Evidence for Lack of Association between HBV Genotype D and TP53 R249S Mutations. Hepat Res Treat 2011:475965.
15. Villar S, Le Roux-Goglin E, Gouas DA, Plymoth A, Ferro G, et al. (2011) Seasonal variation in TP53 R249S-mutated serum DNA with aflatoxin exposure and hepatitis B virus infection. Environ Health Perspect 119:1635-1640.
16. Nogueira JA, Ono-Nita SK, Nita ME, de Souza MM, do Carmo EP, et al. (2009) 249 TP53 mutation has high prevalence and is correlated with larger and poorly differentiated HCC in Brazilian patients. BMC Cancer 9:204.
17. Zhong Y, Han J, Zou Z, Liu S, Tang B, et al. (2011) Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR. Clin Chim Acta 412:1905-1911.
18. Nassal M (2008) Hepatitis B viruses:reverse transcription a different way. Virus Res 134:235-249.
19. Levrero M, Pollicino T, Petersen J, Belloni L, Raimondo G, et al. (2009) Control of cccDNA function in hepatitis B virus infection. J Hepatol 51:581-592.
20. Caruntu FA, Molagic V (2005) CccDNA persistence during natural evolution of chronic VHB infection. Rom J Gastroenterol 14:373-377.
21. Reaiche GY, Le Mire MF, Mason WS, Jilbert AR (2010) The persistence in the liver of residual duck hepatitis B virus covalently closed circular DNA is not dependent upon new viral DNA synthesis. Virology 406:286-292.
22. Zoulim F (2005) New insight on hepatitis B virus persistence from the study of intrahepatic viral cccDNA. J Hepatol 42:302-308.
23. Kock J, Rosler C, Zhang JJ, Blum HE, Nassal M, et al. (2010) Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner. PLoS Pathog 6:e1001082.
24. Lentz TB, Loeb DD (2011) Roles of the envelope proteins in the amplification of covalently closed circular DNA and completion of synthesis of the plus-strand DNA in hepatitis B virus. J Virol 85:11916-11927.
25. Xie Q, Zhang S, Wang W, Li YM, Du T, et al. (2012) Inhibition of hepatitis B virus gene expression by small interfering RNAs targeting cccDNA and X antigen. Acta Virol 56:49-55.
26. Edman JC, Gray P, Valenzuela P, Rall LB, Rutter WJ (1980) Integration of hepatitis B virus sequences and their expression in a human hepatoma cell. Nature 286:535-538.
27. Wang HC, Huang W, Lai MD, Su IJ (2006) Hepatitis B virns pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. Cancer Sci 97:683-688.
28. Feitelson MA, Lee J (2007) Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis. Cancer Lett 252:157-170.
29. Ma NF, Lau SH, Hu L, Xie D, Wu J, et al. (2008) COOH-terminal truncated HBV X protein plays key role in hepatocarcinogenesis. Clin Cancer Res 14:5061-5068.
30. Aoki N, Robinson WS (1989) State of hepatitis B viral genomes in cirrhotic and hepatocellular carcinoma nodules. Mol Biol Med 6:395-408.
31. Wang Y, Lau SH, Sham JS, Wu MC, Wang T, et al. (2004) Characterization of HBV integrants in 14 hepatocellular carcinomas:association of truncated X gene and hepatocellular carcinogenesis. Oncogene 23:142-148.
32. Bonilla GR, Roberts LR (2005) The role of hepatitis B virus integrations in the pathogenesis of human hepatocellular carcinoma. J Hepatol 42:760-777.
33. Minami M, Daimon Y, Mori K, Takashima H, Nakajima T, et al. (2005) Hepatitis B virus-related insertional mutagenesis in chronic hepatitis B patients as an early drastic genetic change leading to hepatocarcinogenesis. Oncogene 24:4340-4348.
34. Murakami Y, Saigo K, Takashima H, Minami M, Okanoue T, et al. (2005) Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut 54: 1162-1168.
35. Jin H, Wang J, Yan L, Nie JJ, Li J, et al. (2008) [Establishment of a nested PCR to identify hepatitis B virus genotypes A-D and subgenotypes Bl, B2, Cl and C2]. Zhonghua Liu Xing Bing Xue Za Zhi 29:1235-1239.
36. Dillon LW, Burrow AA, Wang YH (2010) DNA instability at chromosomal fragile sites in cancer. Curr Genomics 11:326-337.
37. Liu S, Zhang H, Gu C, Yin J, He Y, et al. (2009) Associations between hepatitis B virus mutations and the risk of hepatocellular carcinoma:a meta-analysis. J Natl Cancer Inst 101:1066-1082.
38. Subirana JA, Anokian E (2011) Visualization of sequence and structural features of genomes and chromosome fragments. Application to CpG islands, Alu sequences and whole genomes. Gene 473:76-81.
39. Chen LL, Carmichael GG (2008) Gene regulation by SINES and inosines:biological consequences of A-to-I editing of Alu element inverted repeats. Cell Cycle 7:3294-3301.
40. Hancks DC, Kazazian HH, Jr. (2010) SVA retrotransposons:Evolution and genetic instability. Semin Cancer Biol 20:234-245.
41. McVean G (2010) What drives recombination hotspots to repeat DNA in humans? Philos Trans R Soc Lond B Biol Sci 365:1213-1218.
42. Karon M, Henry P, Weissman S, Meyer C (1966) The effect of 1-beta-D-arabinofuranosylcytosine on macromolecular synthesis in KB spinner cultures. Cancer Res 26:166-171.
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