The Molecular Mechanism Of HPV16E6and HTERT In Cervical Carcinogenesis

After breast and colorectal cancer, cervical cancer is the third mostcommon cause of death from cancer in women worldwide.It is well known thatpersistent infection of high-risk human papillomavirus (HR-HPV) is a necessarycausal event in cervical carcinogenesis. HPV E6is capable of eliciting a widerange of pathological effects, including inhibition of apoptosis, disruption of celladhesion, abrogation of anti-viral immune responses, decreased genomicstability, p53inactivation, aberrant GPCR-mediated signal transduction, andreversal of epithelial differentiation.More than200HPV genotypes have beenidentified, and approximately40distinct HPV types infect the mucosalepithelium of the genital tract.HPVs are grouped according to their associationwith cervical cancer and their genomic sequence into oncogenic high-risk(16,18,45,31,33,58,52,35,59,56,51,68,39,82,73,66, and70) and non-oncogeniclow-risk (6,11,40,42,43,44,54,61,64,72,81and89).Lowrisk HPVs areprimarily found in genital warts, while high-risk are found in precancerouslesions and cervical cancer.So far,HPV16is the most common subtype whichwas detected most frequently in cervical cancer(50%).Therefore,we select themost common subtype HPV16as our point of penetration. On account of the contribution to the cervical carcinogenesis of E6and E7,and in contrast to most human somatic ells, approximately90%of immortalizedand cancer cells express telomerase activity and consequently maintain minimal,stable telomeres and indefinite proliferative potential. Therefore, telomeraseactivation is considered a critical event in the process of cell immortalization.Most tumors show the telomerase activity which may be crucial for the changeof a variety of genetic in the process of tumor development, and which isprobably the most of universal and specific tumor molecular marker. Being thecatalytic subunit, human telomerase reverse transcriptase (hTERT) is the keystructure and the major regulatory subunit of telomerase. We speculate that thethat activation of telomerase by E6is critical for cell immortalization by HPV.E6executes this increase in telomerase activity by multiple mechanisms. But themechanism underlying this effect that E6simulate telomerase activity remains tobe defined. The hTERT proteins have been investigated in several studies onsome disease such as adenomyosis,colorectal tumors,lung cancer cell,vulvarintraepithelial neoplasias (VINs), squamous cell carcinoma， oral cancer andpre-cancer,but to our knowledge, hTERT expression has not been evaluated inall tumors,including a spectrum of cervical lesions: cervicitis, various grades ofdysplasia (cervical intraepithelial neoplasia gradeⅠ～Ⅲ,CINⅠ～Ⅲ), andcancer (squamous cell carcinoma, adenocarcinoma).We identified the contributions of various human papillomavirus (HPV)genotypes in archival cervical tissue from women with different cervicallesion.And we also investigated immunohistochemical HPV16E6and hTERTexpression and its relationship with the clinicopathologic features,and identifyhigh-risk HPVE6/E7mRNA transcript in a spectrum of cervical lesions.In theend, to further confirm our preliminary findings, we observed the influence of biological behavior of HPV16E6-shRNA and hTERT-shRNA which weretransfected to Caski cells by lentiviral vector,and preliminarily explore theinteraction of hTERT and HPV16E6in the carcinogenic mechanism for cervicalcancer.Including the following three parts:Part1Distribution of human papillomavirus genotypes in differentcervical lesionsObjectiveTo identify the contributions of various HPV genotypes in archival cervicaltissue from women with different cervical lesion in Guangxi,China.MethodIn the retrospective study, a total of236formalin-fixed, paraffin-embedded(FFPE) specimens between2009and2013from women diagnosed withdifferent cervical diseases: cervicitis, various grades of dysplasia (cervicalintraepithelial neoplasia grade1-3),and cancer(squamous cell carcinoma,adenocarcinoma)were enrolled for presence and genotyping of HPV DNA usinga PCR-RDB assay.Results1. The relationship between HPV infection in defferent cervical lesionsOverall, in total of236different cervical diseases samples,65.68%ofthem were tested positive for HPV DNA,including218HPV genotypes(singleor multiple HPV infections).HPV infective rates of each cervical disease groupwere:24.39%（10/41）in cervicitis;46.67%（21/45）in CINⅠ;59.52%（25/42）in CINⅡ;95.12%（39/41）in CINⅢ;97.44%（38/39）in cervical squamous cell carcinoma (SCC)、78.57%（22/28） in cervical adenocarcinoma (ADC).Asignificant rise of HPV infective rates can be found with more advancedcervicallesions (P<0.05).2. HPV genotypes distribution in deffrent grade of cervical lesionsAmong a total of23kinds of HPV subtypes can be detected, there were18subtypes detected (including14high risk types and4low-risk types). HPV16,52,58,33,18and6were the priority HPV types.The distribution of these typeswas as below: HPV16was the most predominant type(32.20%,76/236),followed by HPV18(112.29%,29/236), HPV58(8.47%,20/236),HPV52(5.93%,14/236), HPV33(5.08%,12/236), HPV45(5.08%,12/236) and low-risk typesHPV11(2.54%,6/236),HPV6(1.70%,4/236) respectively.3. Single or multiple HPV infections in cervical lesionsAmong a total of236HPV infective cases in the study, there were115(74.19%,115/155)cases of single type infection,40(25.81%,40/155) cases ofdouble/multiple type infections.HPV16,18were the most common high-riskdouble infections.The types of multiple infections in cervicitis or≤CINⅡsamples were mainly low-risk multiple infections or co-infection with high-risktype and low-risk type, but in CINⅡ+or cervical cancer samples, high risk typemultiple infections or co-infections with high-risk type and low-risk type weremore common.Conclusion1. HPV infections were closely related with cervical lesions. High HPVprevalence was found the most frequently in the group of cervical squamouscell carcinomas, and a significant rise of HPV infective rates can be foundwith more advanced cervicallesions;2. HPV16, HPV18, HPV58, HPV52, HPV33were the most common HPV types in cervical lesions. HPV16was the predominant type, which wasclosely related to SCC, however, the second common HPV type in cervicalcancer samples was HPV18, which was closely related to ADC. Part2The expression of HPV16E6and hTERT in cervical lesions:correlations with clinicopathologic featuresObjectiveTo investigate immunohistochemical HPV16E6and hTERT expression andits relationship with the clinicopathologic features,and to identify high-riskHPVE6/E7mRNA transcript in a spectrum of cervical lesions.MethodA total of236formalin-fixed, paraffin-embedded (FFPE) specimens fromwomen with different cervical diseases: cervicitis, various grades of dysplasia(cervical intraepithelial neoplasia gradeⅠ-Ⅲ), and cancer(squamous cellcarcinoma, adenocarcinoma)were enrolled for presence of the HPV16E6andhTERT protein expression by immunohistochemical method and thetranscription of HPV E6/E7mRNA by a bDNA assay.Results1. Immunohistochemical detection of HPV16E6proteinThe positive rate of cervicitis, CINⅠ、CINⅡ、CINⅢ、squamous cellcarcinoma and adenocarcinoma was0.00%（0/34）,0%（0/35）,4.35%（1/23）,12.00%（3/25）,35.90%（14/39）,52.17%（12/23）for HPV16E6proteinrespectively and0.00%（0/41）,2.22%（1/45）,7.14%（3/42）,39.02% （16/41）,92.31%（36/39）,92.86%（26/28）for hTERT protein respectively.There was also significant difference among them(P＜0.05). The staining levelof these two protein in cervical cancer were significantly higher than incervicitis and various grades of dysplasia(P＜0.05).2.The correlations between HPV16E6/hTERT immunohistochemicalexpression and clinicopathologic features.The E6expression in cervicitis, various grades of dysplasia, or cervicalcancer was found relate to HPV infections（Z=-2.168, P=0.029）.And thehTERT expression as found relate to associated with HPV infections（Z=-2.050,P=0.041） and family history of cervical cancer(Z=-2.124, P=0.034）3. The correlations between HPV16E6and hTERT proteinThe correlations between HPV16E6and hTERT protein expression ratewas significant（reho=0.928，P＞0.05）.4. Transcription of high-risk HPV E6/E7mRNA in single or multiplehuman papillomavirus infections in cervical lesions.Presence of mRNA was detected for57.8%（126/218）of the in total218HPV infections present in the samples,and more commonly in high-gradelesions.In the <CINⅡ group and CINⅡ+group,the positive rate of HPV E6/E7mRNA transcription was35.48%（11/31） and92.74%（115/124）respectively.There was significant difference between them(P＜0.05). Presenceof mRNA was increasing with severity of lesion (P＜0.05).Presence of mRNAcould more often be detected in samples with multiple infections than in sampleswith single infections (3.48%VS.36.52%,P＜0.05).5. HPV E6/E7mRNA expression correlated-genotype which might beresponsible for cellular transformation.The genotype most prone to express mRNA in high-grade lesions was HPV45(91.7%),followed by HPV16(71.9%),HPV18(69.8%),HPV31(65.2%),andHPV52(56.0%),less prone was HPV59. Expression of mRNA wassignificantly enhanced in CINⅡ+lesions, an association also found for HPV16.Presence of mRNA could more often be detected in CINⅡ+samples than in<CINⅡ+samples with HPV16infection (81.2%VS.50%, P＜0.05).ConclusionThe expression of HPV16E6and hTERT may use the aggressiveness ofthe cervical lesions as a marker, but they are not related to clinicopathologicdata.Presence of E6/E7mRNA was more common in high-grade lesions. Thefrequent expression of E6/E7mRNA by HPV45may promote oncogenicity andcould be of clinical importance. In multiple HPV infections, E6/E7mRNAtesting may identify the genotype that causes transformation. Part3Effect of targeted silencing of HPV16E6and hTERT bylentivirus-mediated shRNA on cervical cancer cell caski in vitroObjectiveTo observe the influence of biological behavior of HPV16E6-shRNA andhTERT-shRNA which were transfected to Caski cells by lentiviral vector,andpreliminarily explore the interaction of hTERT and HPV16E6in thecarcinogenic mechanism for cervical cancer.MethodThe HPV16E6-shRNA, hTERT-shRNA and NC-shRNA were transfected into cervical HPV16-positive cell Caski with lentiviral vector. The expression ofE6and hTERT mRNA were evaluated by Real-time PCR. The E6and hTERTprotein was measured by western blotting. Then the most effective transfectantswere selected and established: E6-shRNA-19and hTERT-shRNA-1515.Contrastthe proliferation, invasion and migration ability of Caski cell between before andafter transfection.The effects of E6-shRNA and hTERT-shRNA on growth,proliferation, cell cycle, invasion, apoptosis, migration and ability of Caski cellswere measured by Cell scratch test, flow cytometric, transwell and flowcytometry.Results1. Construction of HPV16E6-shRNA and hTERT-shRNA lentiviralexpression vectorSequence of hTERT/HPV16E6-targeted shRNA was designed based on themRNA sequence of hTERT/HPV16E6which was obtained from the Genbank.They were recombined with the plasmid pGLV3/H1/GFP+Puro (BamHI/EcoRI)respectively which were identified by gene sequencing to make sure they werecorrectly connected.2. Production of lentivirus expressing shRNALentivirus was produced by co-transfecting recombinant plasmid andpackaging plasmid expression plasmid DNA into the293T cells,and then wereharvested and titered. The lentiviral stock titer was1×108TU/ml. Then thelentivirus were used to transfect the Caski cells(1:10-1:100), which transfectionefficiency was70～80%.3.Screening for the effective interference sequenceAnalysis of HPV16E6E6and hTERT mRNA levels by RT-PCR andscreening for effective interference sequence：levels of both E6and hTERT mRNA and proteins were decreased in Caski cells transduced by the72H-TERT-1515and96H-E6-19transcript-targeting LV3-shRNA comparedwith the related scramble control,bank group and NC group(P＜0.5).4. Efficiencies of E6/hTERT gene knockdown by LV-shRNA targeting theE6and the hTERTCell scratch test showed that the growth of HPV16E6-shRNA andhTERT-shRNA group Caski cells was slow-healing (P＜0.05).The averagehealing rate of the Caski cells in the B group, NC group, E6-19group andhTERT-1515group were0.927±0.01,0.783±0.07,0.637±0.04,0.58±0.11respectively. CCK8assay and flowcytometry showed that cell proliferation wassignificantly inhibited, cell cycle progression was blocked in G0/G1（P＜0.05）and apoptosis rate was higher (F=53.647,P=0.000) in HPV16E6-shRNA andhTERT-shRNA group Caski cells (4.15±0.13%,4.79±0.25%).Transwell assayshowed that invasiveness and migration of the cells in HPV16E6-shRNA andhTERT-shRNA group were significantly inhibited comparison with B group andLv-shRNA-NC group.（P＜0.05）ConlusionThe HPV16E6and hTERT gene may influences biological behavior ofhuman cervical cancer cells Caski in vitro. Silincing of these two genes wouldinhibit the proliferation, imigration and invasion of Caski cells, and induce theapoptosis. The HPV16E6and hTERT gene may interact with each other andplay a important role in the carcinogenic mechanism for cervical cancer.