Mechanism Of The Occurrence And Development Of Cervial Cancer Led By Human Papillomavirus16Integrated Into The Host Genome

Backgroud and Objective:As one of the most common gynecological cancer, cervical cancer(CC) is a serious threaten to women's health and survival. there are about 50 million new cervical cancer cases and 230,000 patients died of cevical cancer each year worldwide. It has been revealed that infection with high-risk human papillomaviruses (HPV) is a prerequisite for the development of cervical cancer. HPV DNA is detected in almost every (99.7%) cervical cancer sample analyzed with HPV 16 being the most common type of HPV found in cervical cancer samples. Although infection of high-risk HPV is recognized as an essential initiating event in cervical tumorigenesis, this alone is not sufficient for the progression to invasive cancer. There are About 90% of HR-HPV infections are self-limited and regress spontaneously within several months. In about 10% of the cases, however, the infection persists and may progress to a transforming HR-HPV infection that induces outgrowth of high-grade preneoplastic lesions or invasive cancers. In spite of the recent progress in molecular aspects of CC, the genetic basis of progression of HR-HPV infection to invasive cancer in the multi-step progression of CC remains poorly understood. Therefore identification of other "genetic hits" in CC is important in understanding its biology. Integration of HPV DNA into the host genome is observed in most invasive cancers but rarely in CIN and is thus likely to constitute a key step in oncogenesis. The trigger and frequent sites of the integration is unclear, but it seems to be a critical event in cervical carcinogenesis preceding the development of chromosomal abnormalities that drive the malignant progression. Important unanswered questions in HR-HPV biology concern the mechanisms by which the site of integration contributes to selection of a particular integrant. Furthermore, no integration site of chinese was report at all. This study is designed to explores the distribution rule of integration site of HPV16 in a large number of clinical samples, finds the target gene which play a key role after HPV16 integrate into the specical site in the population with same integration site and same clinical and molecular biological characteristics, and to verificate the occurence and devlopment of cervical cancer with HPV16 integrate into that specific site by constructing several cell models. The aim of this study is to provides new theoretical foundation platform for the molecular typing of cervical cancer and to realize efficacious biotherapy for cervical cancer targeting the key carcinogenic factor of HPV16 and to make a great contribution to cut down the incidence and mortality of cervical cancer.Methods:1. Screening of HPV16 infection and infection status of the samples:we select the frozen tissue of cervical cancer from patients who has received extensive hysterectomy, and extract DNA from the tissues according to the kit box. PCR analysis is used to repeatedly identify and ascertain the HPV16 infection. In order to determine the infection status of HPV16 of the samples multiple PCR amplification is being used.2. The distribution of HPV16 integration sites in cervical cancer tissues and its clinical significance:the frozen tissues with defined HPV 16 infection arc selected for the process of RNA extracting and the reverse translation. products of reverse translation are further utilized for analysis of the features of the adjacent host gene and gene locis by NEST PCR. clinical characteristics of patients according to the classification of integration sites are compared to clarify the intrinsic link between HPV integration sites and the clinical manifestations of the host.3. Screening of key target genes in the samples with HPV16 integrated into the mitochondria, which generate effect during the development of cervical cancer:as to samples with different integration sites, Real-time PCR method is used to detect the mitochondrial gene expression. results are compared according to three different groups: samples with HPV DNA integrated into mitochondria, into the host's chromosome and normal cervical tissues without HPV infection. And then the clinical characteristics of the group with HPV 16 DNA integrated into the host's mitochondrial are seriously analyzed. Four normal cervical specimens, three specimens with HPV DNA integrated into the host chromosome, and another four cases with mitochondrial integrated are selected randomly for Gene Microarray Analysis. key target genes are screened out through the chip test results of the groups with HPV DNA integrated into the mitochondria. immunohistochemistry paraffinsections are applyed at clinical samples with different levels of cervical lesions, that will give help to the validation of the Real-time PCR results targeting the appropriate tissue gene expression of RNA.what's more, different cell models simulating the occurrence of HPV 16 DNA integrate into the host's mitochondria are constructed in order to investigate the mechanism of mitochondria integration causing cervical cancer.4. Establishing primary culture of the Cervical keratinocyte model:Selecting fresh normal cervical intraepithelial samples from patients with nonmalignant tumor diseases simultaneously HPV infection and precancerous lesions of uterinecervix not being found. We cultivate cervical Angle epithelial with direct culture method and the 3T3 cell feeding method respectively. Laser confocal microscope is used for cell classification appraisal, eliminating fibroblasts pollution, we validate cells with normal diploid cells whether their chromosome form consistent after blocking its life cycle.Results1. In 192 cases of cervical cancer tissue samples, the HPV16 infection rate is 56.57%; among the 112 cases of cervical cancer samples with HPV 16 infection,73 cases are found with pure integration of HPV 16, with the rate 65.18%(73/112); 17 cases are found with the free type of HPV 16 infection (with the rate 15.18%); and the number of samples with HPV 16 free and integrated mixed infection reaches 22,with the rate 19.64%.2. among the 112 cases of cervical cancer tissue samples with HPV16 infection, transcriptional integration body have been discovered within 86 cases, totally with 98 integration sites. Especially,12 cases occur mitochondrial integration, with 8 cases located in the ND3, and 4 cases located in the RNR2 sites. Others integration sites are widely distributed in different host chromosomes except chromosome 8,9,18,22, including 26 cases occurring at common chromosomes fragile site,8 cases at rare fragile sites, and 52 cases at non-fragile sites.3.9 kinds of mitochondrial gene expression in the samples with mitochondrial integrated are significantly lower than those in normal tissues. On the contrary, in samples with sole host nuclear DNA integrated, these gene expressions are obviously elevated or slightly decreased. Seven cases of the twelve patients with mitochondrial Integration are observed with lower differentiation in pathology and with theâ…¡a period in FIGO. Another four cases are moderately differentiated squamous cell carcinoma. And among them 1 case is forâ… b period, but it was recurrence within two years. Clustering results of chip detection indicate: difference of gene expression among the three groups are obvious; great difference among hundreds of kinds of gene expression,including the mitochondrial gene expression can be observed.4. Cervical keratinocyte epithelial cells cultivated by the two different methods have been tested by Immune fluorescence detection of epithelial angle protein, and the Immune fluorescence staining of vimitin antibody. Both results indicate that the epithelial component is pure and normal, without anomalistic fiber cell's existence. Karyotype analysis implies that the number and form of chromosomes in each cell consist with that of normal diploid cell.conclusions1. HPV16 is the most common type in samples of cervical cancer. It exists on mainly integration, however, the minority samples are of mixture or free type integration2. The integration sites of HPV16 are widely distributed on host chromosomes,with the rate of 39.53% at fragile sites, mainly at common fragile sites. The integration of HPV16 in host mitochondria occurs with relatively high incidence, and the integration sites are obviously centralized. there is no affirmative relationship between the distribution of the HPV16 integration sites and it's clinical feature. But in most patients, if not all, with HPV16 integrated into mitochondrial, uaually have a higher incidence of lymph node metastasis.3. It is a special kind of cervical cancer with HPV DNA integrated into the host's mitochondrial,which owns unique clinical feature, abnormal expression of genome of mitochrodia and a unique gene fingerprint. However, the pathogenesis is remain unkown.4. The primary culture of model of cervical keratization epithelium can passage in vitro, which can provide the enough and reliable normal diploid cervical keratization cell model to research the tumorigenesis of cervical cancer.