Metabolomics of Siha Supernatant in Cervical Cancer Cells with Down-regulated HPV16 E6/E7 Expression / 昆明医科大学学报

Objective To detecte the differential metabolites and related pathways in Siha cells of cervical cancer by screening the inhibition of HPV16 E6/E7 expression based on 1H NMR metabolomics so as to identify the key metabolic markers involved in the development of high-risk HPV16 cervical cancer.Methods Siha cells were transfected with RNAi fragments to down-regulate the expression of E6/E7,which were divided into the normal control group(Siha cells),no-load group(si-NON),si-E6 group and si-E7 group,and their transfection efficiency was verified.1H NMR metabolomics was used to reveal the differential metabolites involved in interfering E6/E7 expression in Siha cells.Combined with MetaboAnalyst 5.0 online software,differential metabolites and related metabolic pathways were obtained.Results Fluorescence was observed by inverted fluorescence microscope.Western blotting results showed that compared with Siha group,the expression of E6/E7 in si-E6 group and si-E7 group was decreased(F=145.8,P<0.001).After down-regulating the expression of E6/E7,13 common differential metabolites,including Isoleucine,Leucine and valine,were detected.The results of MetaboAnalyst 5.0 online software analysis suggested that the above metabolites were mainly involved in the biochemical synthesis pathway of aminoacyl-trNA,biochemical synthesis pathway of isoleucine,Leucine and valine;There were 10 metabolic pathways of tyrosine,phenylalanine and tryptophan biochemical synthesis.Conclusion After HPV16 infection,changes of glucose and amino acid metabolism can promote the progression of cervical cancer,which provide a theoretical basis for the prevention and treatment of cervical cancer.

Value of HPV DNA,HPV E6/E7 protein and TCT in cervical intraepithelial neoplasia and cervical cancer screening / 中国现代医生

@#Objective To investigate the value of human papilloma virus(HPV)DNA,HPV E6/E7 protein and thin-prep cytology test(TCT)in cervical intraepithelial neoplasia(CIN)and cervical cancer screening.Methods A total of 190 adult women who received early cervical cancer screening in Department of Gynecology,Zhuji People's Hospital from July 2021 to June 2022 were selected as the study objects.HPV DNA,HPV E6/E7 protein and TCT were detected,respectively,and further colposcopic biopsy was performed.The diagnostic efficacy of HPV DNA,HPV E6/E7 protein and TCT in patients with different lesions were compared.Results The positive rates of HPV DNA,HPV E6/E7 protein,TCT and combined detection in CIN3 and cervical cancer patients were significantly higher than those in cervicitis patients(P<0.05).The positive rates of HPV DNA,HPV E6/E7 protein,TCT and combined detection in cervical cancer patients were significantly higher than those in CIN1 patients(P<0.05).The positive rates of HPV DNA,HPV E6/E7 protein,TCT and combined detection in CIN2+ patients were significantly higher than those in CIN1-patients.The sensitivity,specificity,positive predictive value and negative predictive value of HPV DNA,HPV E6/E7 protein and TCT in the diagnosis of high-grade lesions were 90.80%,30.10%,52.32%and 79.48%,respectively.Conclusion HPV DNA,HPV E6/E7 protein and TCT can be used as screening methods for cervical cancer and precancerous lesions,with the combination of the three being the most sensitive.

Assessment of the diagnostic value and prognosis of different detection markers in endocervical adenocarcinoma / 中华肿瘤杂志

Objective: To study the diagnostic value of different detection markers in histological categories of endocervical adenocarcinoma (ECA), and their assessment of patient prognosis. Methods: A retrospective study of 54 patients with ECA in the Cancer Hospital, Chinese Academy of Medical Sciences from 2005-2010 were performed. The cases of ECA were classified into two categories, namely human papillomavirus-associated adenocarcinoma (HPVA) and non-human papillomavirus-associated adenocarcinoma (NHPVA), based on the 2018 international endocervical adenocarcinoma criteria and classification (IECC). To detect HR-HPV DNA and HR-HPV E6/E7 mRNA in all patients, we used whole tissue section PCR (WTS-PCR) and HPV E6/E7 mRNA in situ hybridization (ISH) techniques, respectively. Additionally, we performed Laser microdissection PCR (LCM-PCR) on 15 randomly selected HR-HPV DNA-positive cases to confirm the accuracy of the above two assays in identifying ECA lesions. Receiver operating characteristic (ROC) curves were used to analyze the efficacy of markers to identify HPVA and NHPVA. Univariate and multifactorial Cox proportional risk model regression analyses were performed for factors influencing ECA patients' prognoses. Results: Of the 54 patients with ECA, 30 were HPVA and 24 were NHPVA. A total of 96.7% (29/30) of HPVA patients were positive for HR-HPV DNA and 63.3% (19/30) for HR-HPV E6/E7 mRNA, and 33.3% (8/24) of NHPVA patients were positive for HR-HPV DNA and HR-HPV E6/E7 mRNA was not detected (0/24), and the differences were statistically significant (P<0.001). LCM-PCR showed that five patients were positive for HR-HPV DNA in the area of glandular epithelial lesions and others were negative, which was in good agreement with the E6/E7 mRNA ISH assay (Kappa=0.842, P=0.001). Analysis of the ROC results showed that the AUC of HR-HPV DNA, HR-HPV E6/E7 mRNA, and p16 to identify HPVA and NHPVA were 0.817, 0.817, and 0.692, respectively, with sensitivities of 96.7%, 63.3%, and 80.0% and specificities of 66.7%, 100.0%, and 58.3%, respectively. HR-HPV DNA identified HPVA and NHPVA with higher AUC than p16 (P=0.044). The difference in survival rates between HR-HPV DNA (WTS-PCR assay) positive and negative patients was not statistically significant (P=0.156), while the difference in survival rates between HR-HPV E6/E7 mRNA positive and negative patients, and p16 positive and negative patients were statistically significant (both P<0.05). Multifactorial Cox regression analysis showed that International Federation of Obstetrics and Gynecology (FIGO) staging (HR=19.875, 95% CI: 1.526-258.833) and parametrial involvement (HR=14.032, 95% CI: 1.281-153.761) were independent factors influencing the prognosis of patients with ECA. Conclusions: HR-HPV E6/E7 mRNA is more reflective of HPV infection in ECA tissue. The efficacy of HR-HPV E6/E7 mRNA and HR-HPV DNA (WTS-PCR assay) in identifying HPVA and NHPVA is similar, with higher sensitivity of HR-HPV DNA and higher specificity of HR-HPV E6/E7 mRNA. HR-HPV DNA is more effective than p16 in identifying HPVA and NHPVA. HPV E6/E7 mRNA and p16 positive ECA patients have better survival rates than negative.

Protein-based Bioinformatics Analysis of Cervical Cancer Related Genes / 医学信息学杂志

Purpose/Significance To explore the characteristics and clinical significance of differentially expressed genes closely re-lated to HPV E6/E7 by using bioinformatics.Method/Process The cervical tissue and clinical information of cervical cancer in TCGA and GTEx of UCSC are used as the training set.The expression profile chip GSE63514 related to cervical cancer in GEO is used as the validation set.Firstly,the limma package of R software is used to screen DEGs of tumor and normal samples,and Venn map of genes re-lated to E6/E7 protein in MigDB is made.Survival analysis is performed by survival kit and verified by ROC and protein expression lev-els.Secondly,key genes are obtained by copy number variation and methylation correlation.Finally,the specific co-expression network is constructed and enrichment analysis and immune infiltration analysis are performed.Result/Conclusion There are 101 differentially expressed genes related to HPV E6/E7 protein,and three genes are found to have significance after screening,namely E2F1,MCM4 and PCNA.At the same time,it is found that the genes in the specific coexpression network are significantly enriched in the DNA replication and chromosome organization pathways.Immune correlation analysis shows that key genes are significantly associated with CD4 T cells,B cells and neutrophils.DNA replication,chromosome organization,etc.,are the molecular mechanisms and key genes significantly related to the development of cervical squamous cell carcinoma and HPV E6/E7 encoded proteins.

Correlation analysis between the expression of CD47 and the degree of cervical lesions / 安徽医科大学学报

Objective@#To investigate the correlation between CD47 and cervical lesions.@*Methods@#The expression and overall survival of CD47 in cervical cancer was analyzed using TCGA and TCGA Target GTEx databases. Based on GEO databases , the expression of CD47 was analyzed between cervical sample infected by HPV 16/18 and nonHPV. The differences of CD47 expression in the degrees of cervical lesions infected by HPV 16/18 were compared.The patholgical sections of colposcopy biopsy came from 320 patients who were admitted cervical cancer screening ,including HPV typing and E6/E7 mRNA detection. Immunohistochemistry were used to determine CD47 expression , and histochemistry score(H⁃score) was used to quantify the levels of CD47. The correlation among HPV 16/18 , pathological grades and CD47 was analyzed.@*Results@#Bioinformatics tool analysis and immunohistochemistry showed that CD47 was up⁃regulated in HSIL and CC , associated with the prognosis of CC. The expression levels of CD47 in cervical tissues increased significantly after HPV 16/18 infection ( t = 2. 494 , P < 0. 05 ) , and in HPV 16/18 infected tissues , with the increase of pathological grades. The expression levels of CD47 significantly increased(F = 4. 351 , P < 0. 05 ; rs = 0. 278 , P < 0. 001) . The expression of CD47 in the E6/E7 mRNA( + ) was higher than that in the E6/E7 mRNA( - ) ( t = 5. 710 , P < 0. 000 1) , and the copies of E6/E7 were related to the degree of cervical lesions , and were positively correlated with the expression level of CD47 ( F = 15. 557 , P <0. 000 1 ; rs = 0. 649 , P < 0. 000 1) ( rs = 0. 73 , P < 0. 000 1) .@*Conclusion@#CD47 is overexpressed in HSIL and CC after HPV 16/18 infection. The expression levels of CD47 increases with the degree of cervical lesions , and is correlated with E6/E7 mRNA. CD47 may be a potential target for cervical disease progression and therapy.

Clinical analysis on the application value of human papillomavirus E6/E7 mRNA testing for screening under 30 years old / 中国医师进修杂志

Objective:To evaluate the clinical value of human papillomavirus (HPV) E6/E7 mRNA testing in cervical cancer screening under 30 years old.Methods:The clinical data of 330 young women (less than 30 years old) with minor abnormalities of thinprep cytologic test (TCT) screening for in Dalian Women′s and Children′s Medical Center (Group) Chunliu Region from January 2020 to June 2021 were retrospectively analyzed. Among them, 165 patients underwent HPV DNA typing test (DNA group), and 165 patients underwent HPV E6/E7 mRNA typing test (mRNA group). The positive rate of cervical intraepithelial neoplasia (CIN) Ⅱ and above (CIN Ⅱ +) was compared between two groups, and the positive rates of CIN Ⅱ + in different high risk HPV types. Results:The positive rate of high risk HPV types in mRNA group was significantly lower than that in DNA group: 32.7% (54/165) vs. 47.9% (79/165), and there was statistical difference ( χ2 = 7.87, P<0.05). There was no statistical difference in the incidence of CIN Ⅱ + of patients with positive of high risk HPV types between DNA group and mRNA: 45.6% (36/79) and 59.3% (32/54), P>0.05. There was no statistical difference in the incidence of CIN Ⅱ + of patients with HPV 16, 18 or 45 positive between DNA group and mRNA group: 38.5% (10/26) and 6/10, P>0.05. The incidence of CIN Ⅱ + of patients without HPV 16, 18 or 45 positive in mRNA group was significantly higher than that in DNA group: 60.9% (28/46) vs. 41.3% (26/63), and there was statistical difference ( P<0.05). Conclusions:Without increasing the rate of missed diagnosis, HPV E6/E7 mRNA testing plays an important shunt role in women under 30 years old, and the predicted value of CIN Ⅱ + is higher for patients who are not infected with HPV16/18/45 with HPV E6/E7 mRNA testing.

Establishment and identification of human immortalized keratinocytes stably expressing human papillomavirus type 16 E6/E7 gene / 中华皮肤科杂志

Objective:To construct human immortalized keratinocytes stably expressing human papillomavirus type 16 (HPV16) E6/E7 gene, and provide a cell model for studying mechanisms underlying HPV16 E6/E7-induced cell immortalization and malignant transformation.Methods:Primary human foreskin keratinocytes (HFKs) were isolated by sequential two-step enzymatic digestion. Cultured HFKs were stably transfected with a HPV16 E6/E7 gene-overexpressing lentiviral vector LV5-HPV16 E6/E7, and consecutively cultured for more than 30 passages. Then, immortalized keratinocytes were screened out and divided into 3 groups: (1) blank control group: second-passage primary HFKs; (2) experimental group: HFKs transfected with LV5-HPV16 E6/E7 at different passages, and the second-passage primary HFKs transfected with LV5-HPV16 E6/E7 were referred to as A0 cells, thereafter, the transfected HFKs were named according to their passage number, such as A1, A2, ... A30; (3) positive control group: the HPV16-positive cervical cancer cell line SiHa. Real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were performed to determine the mRNA expression of HPV16 E6/E7 and protein expression of HPV16 E6/E7 and CK14, respectively, in the blank control group, experimental group and positive control group. Cell counting kit-8 (CCK8) assay and Transwell insert invasion assay were conducted to assess the cellular proliferative and invasive activity. In vivo tumor formation experiment in nude mice was conducted to investigate the tumorigenicity of A30 cells in the experimental group and SiHa cells in the positive control group. Results:Primary HFKs were successfully isolated. After the primary HFKs were transfected with the recombinant plasmid LV5-HPV16 E6/E7, the blank control group showed no fluorescence in the cells, but showed senescent phenotypes after serial passages, while in the experimental group, the volume and morphology of A30 cells were similar to those of the primary HFKs with the proportion of fluorescence-positive cells being 100%. Compared with the blank control group, the experimental group showed significantly increased mRNA expression levels of HPV16 E6 and E7 in A1, A10, A20 and A30 cells (HPV16 E6: t = 7.12, 8.07, 6.53, 5.66, P < 0.001, < 0.001, = 0.001, = 0.005, respectively; HPV16 E7: t = 3.20, 4.29, 3.75, 4.22; P = 0.024, 0.008, 0.013, 0.014, respectively) . The protein expression of HPV16 E6/E7 was absent in the blank control group, but was observed in A30 and SiHa cells. CCK8 assay showed that the proliferative activity of A10, A20 and A30 cells was significantly higher than that of the blank control group ( t = 6.49, 7.55, 9.43, P = 0.003, 0.002, 0.001, respectively) , while there was no significant difference in the proliferative activity between A1 cells and the blank control group ( t = 2.40, P = 0.074) . Transwell insert invasion assay showed that A30 cells could not cross the basement membrane, but SiHa cells could pass through the basement membrane and were stained blue. Two months after the inoculation with A30 cells in the nude mice, no visible tumors were found, which was confirmed by a histological study. Subcutaneous tumors were formed in the nude mice after the inoculation with SiHa cells. Conclusion:Human immortalized keratinocytes were successfully established by lentivirus-mediated transfection with HPV16 E6/E7 gene, and can serve as an ideal cell model for HPV-related research.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

Application and clinical significance of human papilloma virus E6/E7 mRNA detection in cervical atypical glandular cells / 中国医师进修杂志

Objective To investigate the application and clinical significance of human papilloma virus (HPV) E6/E7 mRNA detection in cervical atypical glandular cells (AGC). Methods Four hundred and forty-five cervical AGC patients diagnosed by thin-layer liquid-based cytology in the Maternity Affiliated Hospital of Dalian Medical University from January 2014 to March 2018 were collected. Histological follow-up data and HPV E6/E7 mRNA detection results were analyzed, and histological differences in HPV E6/E7 mRNA positive and negative patients were compared. Results The histological result of 445 patients with cervical AGC showed that negative was in 306 cases (68.76%), and clinical significant lesion was in 129 cases (28.99%). In 445 patients with cervical AGC, HPV E6/E7 mRNA result was positive in 121 cases (27.19%), among whom the positive rate of HPV 16 and 18/45 type was 54.55% (66/121); HPV E6/E7 mRNA result was negative in 324 cases (72.81% ), including 13 non-cervical lesions. The negative rate of histological results in HPV E6/E7 mRNA negative patients was significantly higher than that in HPV E6/E7 mRNA positive patients: 91.05% (295/324) vs. 9.09% (11/121), and there was statistical difference (P＜0.01); the rates of adenocarcinoma in situ (AIS), high-grade squamous intraepithelial lesion (HSIL) and cervical adenocarcinoma of histological result in HPV E6/E7 mRNA positive patients were significantly higher than those in HPV E6/E7 mRNA negative patients: 40.50% (49/121) vs. 1.23% (4/324), 44.63% (54/121) vs. 1.23% (4/324), 3.31% (4/121) vs. 0.31% (1/324), and there were statistical differences (P＜0.01). The sensitivity of HPV E6/E7 mRNA in detecting clinical significant lesion of cervical AGC patients was 82.95% (107/129), the specificity was 95.57% (302/316), positive predictive value was 88.43% (107/121), and negative predictive value was 93.21% (302/324). Conclusions The histological result of cervical AGC shows that the amount of negative patients is significantly higher than clinical significant lesion. For cervical AGC patients with HPV E6/E7 mRNA negative results, conservative follow-up can be adopted after excluding extracervical lesions and fully assessing the risk of cervical lesions. However, the cervical AGC patients with HPV E6/E7 mRNA positive results need further examination to detect lesion and choose treatment earlier.

Application of AHPV and Its Genotyping in Low-grade Squamous Intraepithelial Lesions / 实用妇产科杂志

Objective:To investigate the application of Aptima high-risk human papillomavirus (HR-HPV) E6/E7 mRNA (AHPV) and its genotyping (GT) in the risk assessment of low-grade squamous intraepithelial lesions (LSIL).Methods:The AHPV and its genotype (AHPV-GT) incervical exfoliated cells were detectedin 529 women with LSIL.The DNA-Based Hybrid Capture 2 HPV Test (HC2-HPV), colposcopy and cervical biopsy were performedsimultaneously.Results:① In 529 patients with LSIL, the positive rate of HC2-HPV in the group of ＜30 years old was significantly higher than that in the group of ≥30 years old (92.2％ vs 83.6％, P=0.026).There was no significant difference in AHPV positive rate among different age groups (82.5％ vs 77.7％, P=0.284).No significant difference of the genotyping (AHPV-GT) was detected between the two groups, either.In the 529 cases, 83 cases of HSIL+were confirmed by histology.81 cases (97.6％) were AHPV positive in the patients with HSIL+;② Compared with other 11 positive types of HR-HPV, the incidence of HSIL+in GT+ women increased significantly (P＜0.05).In the group ≥30 years old, the OR value of HSIL+exposure risk of AHPV16 positive women was the highest (141.00), which was significantly higher than that of 18/45+, GT +, AHPV + (P=0.005, 0.000, 0.000).However, in the group of ＜30 years old, the OR value of HSIL+exposure risk of AHPV16 positive women was 8.50, which showed no significant difference from that of 18/45+ and AHPV-(P=1.000, 0.070).③ In group over 30 years old, the specificity of detecting HSIL+by AHPV was higher than that by HC2-HPV (P＜0.05).There was no significant difference in detection specificity between AHPV and HC2-HPV in women under 30 years old.Conclusions:AHPV and its GT detection are reliable methods for colposcopic screening and risk stratification in women aged over 30 years old with LSIL, more attention should be focused on AHPV16 positive.Better biological markers should be explored for younger women.

Analysis of the value of HPV E6/E7 mRNA detection in screening lesions at grade CIN 2 and above / 国际检验医学杂志

Objective To explore the clinical value of HPV E6/E7 mRNA in screening lesions at grade CIN2 and above.Methods A total of 120 cases with CIN and suspected cervical cancer treated in our hospital from January 2014 to September 2016 were selected.According to the results of pathological examination,60 patients with CIN2,CIN3 and invasive cancer were selected as the research group.60 patients with normal CIN1 and CIN1 were selected as control group.The results of TCT,HPV DNA and HPV E6/E7 mRNA were detected and analyzed.Results In 120 patients,the total positive rate of TCT was 55%,the total positive rate of HPV DNA was 87.5%,the total positive rate of HPV E6/E7 mRNA 60.8% HPV E6/E7;control group mRNA positive rate of 78.3% was significantly higher than the positive rate of HPV DNA 36.7%(P<0.05),HPV in the study group were E6/E7 mRNA positive rate of 85.0% was significantly lower than the positive rate of HPV DNA(96.7%);HPV E6/E7 mRNA CIN1,quantitative CIN2,CIN3 and invasive carci-noma were(4 867.31 ± 694.84),(8 943.51 ± 986.23),(28 243.10 ± 10 963.21),(3 610.84 ± 412.64)copies/mL;HPV E6/E7 mRNA quantitative CIN3 values were significantly higher than that of CIN 2,CIN1 and infil-tration of cancer and the control group(P<0.05),E6/E7 mRNA CIN2 quantitative HPV the value of CIN1 was significantly higher than that of control group(P< 0.05).Two methods of detecting HPV DNA and HPV E6/E7 mRNA in the screening of CIN2 and above lesions,the sensitivity of 84.5% DNA 93.8% HPV sensitivity was higher than that of HPV E6/E7 mRNA(P<0.05)HPV E6/E7 21.6%;specificity the speci-ficity of mRNA was 53.6% higher than that of HPV DNA(P<0.05),HPV DNA.HPV ROC curve E6/E7 mRNA were 0.581 and 0.681,the difference was statistically significant(P<0.05).Conclusion The specific-ity and accuracy of HPV,E6/E7 and mRNA in detecting cervical lesions over CIN2 and above are higher than those of HPV and DNA.It is of certain diagnostic value for screening cervical lesions over CIN 2 and above.

The significance of combined detection of cervical E6/E7 examination and Thin-Cytologic Test in cervical cancer screening / 国际检验医学杂志

Objective To evaluate the effect of combined detection of cervical E 6/E7 and liquid based cytology(TCT)in the screening of cervical cancer.Methods A total of 206 cases of high-risk patients with cervical cancer from March 2015 to March 2017 in Yongding Hospital of Suzhou were taken the cervical E6/E7 detection and TCT two screening methods respectively,and the pathological diagnosis was taken as the gold standard,the efficiency difference among TCT,cervical E6/E7 detection,and the com-bined detection of cervical cancer was compared.Results The sensitivity and negative predictive value of E6/E7 were significantly higher than those of TCT(P<0.05).The specificity of TCT was significantly higher than that of cervical E 6/E7(P<0.05).The accuracy of combined diagnosis was significantly higher than that of cervical E6/E7 and TCT alone.The difference was statistically significant(P<0.05).Compared with the cervical E6/E7,the specificity and positive predictive value of the combined diagnosis were higher,and the difference was statistically significant(P<0.05).The sensitivity and negative predictive value of the combined diagnosis were significantly higher than those of TCT,and the difference was statistically significant(P<0.05).The receiver oper-ating characteristic curve(ROC curve)showed that the area under the combined diagnostic curve(AUC)was significantly higher than that of the cervical E6/E7 and TCT alone,and the difference was statistically significant(P<0.05).Conclusion Cervical E6/E7 detection has a low specificity,and sensitivity of TCT is low;the combined detection of cervical E6/E7 detection and TCT has a high screening accuracy,and can improve the sensitivity and specificity of individual diagnosis.

O papel da autofagia no estresse oncogênico promovido por HRASG12V em queratinócitos humanos imortalizados por E6E7 / The role of autophagy in the face of the oncogenic stress triggered by HRASG12V in human keratinocytes immortalized by E6E7

RAS é a oncoproteína mutada mais encontrada em tumores sólidos, o que mostra seu grande potencial transformador. Não obstante, células que carregam essa mutação apresentam estresse oncogênico gerado por excessiva sinalização mitogênica, o que direciona preferencialmente as células portadoras para a morte em detrimento da transformação maligna. Basicamente a transformação direcionadapela proteína RAS mutada age sinergicamente com deficiência em supressores de tumor para evitar o destino celular preferencial frente ao estresse oncogênico. Este é o caso da interação observada entre HRASG12V e E6E7 de HPV, sendo que a infecção pelo vírus aparentemente é condição necessária em cânceres cervicais e muito presente em cânceres de cabeça e pescoço. A sinergia entre HRASG12V e queratinócitos imortalizados por E6E7 desequilibra o balanço homeostático entre subsistemas pró-morte, devido ao estresse oncogênico, ou pró-sobrevivência, que garante viabilidade por meio de novos padrões de robustez celular. Em ambos os casos, o processo que gera uma célula transformada, ou as elimina pelo caminho, apresenta pistas de vulnerabilidades às quais os queratinócitos são expostos uma vez que carreguem tal combinação de fatores. Apresentamos nesse trabalho os principais atores que compõem o estresse oncogênico deletério desencadeado pela atividade de HRASG12V: estresse mitogênico, replicativo e oxidativo; todos eles são responsáveis por provocar dano no DNA, que por sua vez promove parada no ciclo celular até que as células não possam mais suportar tamanha injúria, o que acaba levando-as maciçamente a morte. Mostramos que a alta intensidade mitogênica gerada pela atividade de HRASG12V provoca um desequilíbrio metabólico que leva ao aumento de espécies oxidantes e ao estresse replicativo. Todavia, um tratamento exógeno com o antioxidante NAC restaurou parcialmente a proliferação celular assim como a sobrevivência, agindo como um amenizador do dano no DNA gerado pelas espécies oxidantes. Já uma suplementação com nucleosídeos exógenos restaurou fortemente a sobrevivência celular, sugerindo que o desequilíbrio metabólico pode estar agindo no pool de nucleotídeos, o que poderia ser uma das causas do estresse replicativo. Como mecanismo intrínseco de sobrevivência, a autofagia se intensifica em resposta ao desequilíbrio sistêmico desencadeado pela atividade de HRASG12V. Por meio de sublinhagens defectivas para autofagia, mostramos que o processo retarda o aparecimento de espécies oxidantes, além de evitar sua elevação a níveis ainda mais drásticos, o que consequentemente ameniza o dano no DNA observado. Além disso, hipotetizamos que o processo poderia também estar contribuindo fortemente para a reciclagem de substratos básicos tais como nucleotídeos, assim acarretando em menor estresse replicativo. Na literatura atual, debate-se a noção de que, dependendo do contexto celular, a autofagia poderia promover tanto morte celular como transformação maligna. Entretanto, nesta tese mostramos que, na interação entre queratinócitos, E6E7 e HRASG12V, a autofagia é um mecanismo pró-sobrevivência: se por um lado a demanda autofágica é recrutada além de sua capacidade de processamento, fazendo com que seu fluxo seja bloqueado, por outro a eliminação do sistema se torna demasiadamente deletério, direcionando as células expostas ao estresse oncogênico causado pela atividade de HRASG12V necessariamente à morte.

Mutated RAS is the oncoprotein most found in solid tumors, which shows its huge tumorogenic potential. Despite of that, mutated RAS triggers a strong oncogenic stress, which very often drives cells to death instead of malignant transformation. Basically, the success of the Ras malignant transformation driving activity depends on a synergy between that mutated protein and inhibition of tumor suppression genes. This is the case of the interaction between HRASG12V and E6E7 proteins of HPV: It seems that HPV infection is an initial necessary condition for cervical cancer development and is also very frequent in head and neck carcinomas. The synergy between HRASG12V and E6E7, in pre-malignant keratinocytes, imbalances the homeostasis between pro-death subsystems, due oncogenic stress, and pro-survival subsystems that ensure new patterns of cellular robustness. In both cases, the process responsible for generating a malignant transformed cell or, more frequently, eliminating those cells carrying the combined characteristics, exposes the keratinocytes vulnerabilities. We showed in this work that the main actors of deleterious oncogenic stress triggered by HRASG12V activity are: Mitogenic, replicative and oxidative stresses; all of them induce DNA damage, hence cell cycle arrests until the cell cannot resist such injury any further, which leads to massive cell death. The intense mitogenic activity triggered by HRASG12Vcauses metabolic imbalance, which is responsible for an increase of oxidative species and replicative stress levels; the exogenous treatment with antioxidant NAC partially restored cell growth and cell survival, acting as a softener of the DNA damage caused by oxidative species. On the other hand, nucleoside supplementation strongly restored cell survival, suggesting that the aforementioned metabolic imbalance might be acting in the pool of nucleotides, hence it might be a possible cause of replicative stress. As an intrinsic survival mechanism, the autophagy is intensified in response to systemic imbalance triggered by HRASG12V activity. Through the autophagy defective subline, we showed that that mechanism both delays the increase of oxidative species and avoids their elevation to catastrophic highlevels. Furthermore, autophagy could strongly contribute to the recycling of basic substrates such as nucleotides, which might be mitigating the replicative stress. Nowadays, there is a debate on the role of autophagy promoting either malignant transformation or cell death depending on metabolic context. In this work, we showed an instance of the interaction between E6E7 and HRASG12V triggering autophagy pro-survival mechanisms and hence increasing general cell viability

Clinical significance of HPV E6/E7 mRNA test combining TCT test to detect early cervical dysplasia / 实用医学杂志

Objective To investigate the value of HPV E6/E7 mRNA test combining liquid-based cytology test in cervical cancer screening. Methods A total of 377 samples from Wenzhou People's Hospital from June 2014 to September 2015 were collected and screened by HPV E6/E7 mRNA test combining with liquid-based cytology test , and the results was compared with the findings from the gold criteria of histology and pathology. Results The combination of HPV E6/E7 mRNA test and liquid-based cytology test can enhance the testing sensitivity and specificity. The sensitivity of the combination of HPV E6/E7 mRNA test and liquid-based cytology test for the diagnosis of LSIL was 94.41%, and that for the diagnosis of HSIL was 96.36%. Based on the gold criteria of histology and pathology , the sensitivity , specificity , positive-predictive value and negative predictive value of HPV E6/E7 mRNA test for the diagnosis of HSIL was 90%, 60.67%, 48.53% and 92.49%respectively. The sensitivity, specificity, positive-predictive value and negative- predictive value of liquid-based cytology test for the diagnosis of HSIL was 72.73%, 75.28%, 54.79% and 87.01% respectively. Conclusions The sensitivity of HPV E6/E7 mRNA test is superior to that of liquid-based cytology test , while the specificity of HPV E6/E7 mRNA test is inferior to that of liquid-based cytology test. The negative predictive value of HPV E6/E7 mRNA test is more meaningful than that of liquid-based cytology test. The combination of HPV E6/E7 mRNA test and liquid-based cytology test can enhance the testing sensitivity , but it does not increase the specificity.

Progress in human papilloma virus E6/E7 in cervical cancer / 肿瘤研究与临床

The occurrence of cervical cancer is the result of long-term synergy from a variety of carcinogenic factors.Human papilloma virus (HPV),the main biological cause of cervical cancer,is closely related to the occurrence of cervical cancer,while HPV E6/E7 protein plays an important role in the malignant transformation of cervical cancer.This article summarizes the carcinogenic mechanism of HPV E6/E7 and application in cervical cancer screening,and reviews the targeting therapy aiming at HPV E6/E7.

The clinical value of HPV E6/E7 mRNA and HPV DNA in early screening of cervical cancer / 天津医药

Objective To analyze the differences of positive detection rate and copy number of human papillomavirus (HPV) DNA and E6/E7 mRNA between different grades of cervical lesions, and evaluate their clinical values in early screen?ing of cervical cancer. Methods The cervical exfoliated cell samples from 154 women undergoing biopsy examination and 32 objects undergoing hysterectomy (control group) were collected in Tianjin Central Hospital of Gynecology Obstetrics in 2014. According to the pathological results of cervical biopsy, 154 samples were divided into low-grade squamous intraepi?thelial lesion group (LSIL, n=51), high-grade squamous intraepithelial lesion group (HSIL, n=71), and squamous cell carci?noma group (SCC, n=32). HPV DNA was tested with hybrid capture technology, and E6/E7 mRNA was detected with fluores?cence quantitative hybridization. Immunohistochemistry was performed by detecting E6/E7 protein in all patients after sur?gery or cervical biopsy. Results Combined results of HPV DNA and E6/E7 mRNA demonstrated that the positive detection rate was significantly lower in control group than that of all levels of lesion groups (P 10 000 E6/E7 were significantly increased in high-grade squamous intraepithelial lesion group. Immunohistochemical results showed that the positive detection rate of E6/E7 was significantly lower in control group than that of all levels of lesion groups (P<0.05). The positive rate of E6/E7 was significantly higher in the high-grade squa?mous intraepithelial lesion group than that of low-grade group (P<0.05). Conclusion HPV infection is closely related to cervical abnormalities, which is one of effective measures for early screening of cervical cancer. The negative result of HPV DNA is very helpful to exclude the cervical abnormality, whereas the positive detection of mRNA has great value in predict?ing the disease. Combined results of positive detection and copy number make a comprehensive evaluation for the risk of cer?vical lesions.

Interaction between folate and the expression of human papillomavirus 16 E6/E7 mRNA in the progression of cervix carcinogenesis / 中华流行病学杂志

Objective To explore the interaction between folate and the expression of HPV16 E6/E7 mRNA in the progression of cervix carcinogenesis.Methods Subjects were selected from the participants who were diagnosed pathologically,including 64 patients with cervical squamous cell carcinoma (SCC),55 patients with low-grade cervical intraepithelial neoplasm (CIN1),55 patients with high-grade cervical intraepithelial neoplasm (CIN2 +) and 80 with normal cervix (NC).The levels of serum folate and RBC folate were detected by microbiological assay,and the expression levels of HPV16 E6/E7 mRNA were measured,using the real-time polymerase chain reaction (real-time PCR).Data was analyzed by methods as chi-square test,analysis of variance (ANOVA),Welch test,Kruskal-Wallis H test and ordinal logistic regression.Spearman correlation was tested using the SPSS statistical software (version 16.0) while the interaction effects were evaluated by additive model.Results There was a positive correlation seen between the serum folate and RBC folate (r=0.41,P＜0.001).The levels of serum folate and RBC folate decreased gradually along with the severity of cervical lesions (x2=32.71,P＜0.001;x2=16.32,P＜0.001).The expression levels of HPV 16 E6/E7 mRNA increased gradually with the severity of cervical lesions (x2 =30.11,P＜ 0.001;x2 =38.99,P＜0.001).There was a negative correlation between the levels of RBC folate,expression levels of HPV16 E6 (E6:r=-0.14,P=0.009) and HPV16 E7 mRNA (E7:r=-0.21,P=0.001),respectively.Both RBC folate deficiency and HPV16 E6/E7 mRNA high expression showed additive interaction in CIN 1,CIN2 + and SCC.Conclusion Folate deficiency and high expression of HPV16 E6/E7 mRNA might increase the risk of cervical cancer and cervix precancerous lesions,and having a synergistic action in the progression of cervix carcinogenesis.

Interaction between folate and the expression of human papillomavirus 16 E6/E7 mRNA in the progression of cervix carcinogenesis / 中华流行病学杂志

Objective To explore the interaction between folate and the expression of HPV16 E6/E7 mRNA in the progression of cervix carcinogenesis.Methods Subjects were selected from the participants who were diagnosed pathologically,including 64 patients with cervical squamous cell carcinoma (SCC),55 patients with low-grade cervical intraepithelial neoplasm (CIN1),55 patients with high-grade cervical intraepithelial neoplasm (CIN2 +) and 80 with normal cervix (NC).The levels of serum folate and RBC folate were detected by microbiological assay,and the expression levels of HPV16 E6/E7 mRNA were measured,using the real-time polymerase chain reaction (real-time PCR).Data was analyzed by methods as chi-square test,analysis of variance (ANOVA),Welch test,Kruskal-Wallis H test and ordinal logistic regression.Spearman correlation was tested using the SPSS statistical software (version 16.0) while the interaction effects were evaluated by additive model.Results There was a positive correlation seen between the serum folate and RBC folate (r=0.41,P＜0.001).The levels of serum folate and RBC folate decreased gradually along with the severity of cervical lesions (x2=32.71,P＜0.001;x2=16.32,P＜0.001).The expression levels of HPV 16 E6/E7 mRNA increased gradually with the severity of cervical lesions (x2 =30.11,P＜ 0.001;x2 =38.99,P＜0.001).There was a negative correlation between the levels of RBC folate,expression levels of HPV16 E6 (E6:r=-0.14,P=0.009) and HPV16 E7 mRNA (E7:r=-0.21,P=0.001),respectively.Both RBC folate deficiency and HPV16 E6/E7 mRNA high expression showed additive interaction in CIN 1,CIN2 + and SCC.Conclusion Folate deficiency and high expression of HPV16 E6/E7 mRNA might increase the risk of cervical cancer and cervix precancerous lesions,and having a synergistic action in the progression of cervix carcinogenesis.

Clinical Efficacy of Human Interferon Alpha-2b in the Patients with HPV Infection / 中国药师

Objective:To observe the efficacy of human interferon alpha-2b in the patients with HPV infection. Methods:Totally 128 patients with HPV infection were divided into two groups by the random number table with 64 cases in each. The patients in the observation group were treated with human interferon alpha-2b and those in the control group were treated with Baofukang suppositories. The course of treatment was 30 days for two groups. The expression level of HPV E6 / E7 mRNA,p16 and p53 before and after the treatment and the severe adverse reactions in each group were studied. Results:The effective rate of the observation group was 93. 75% ,and that of the control group was 84. 38%(P < 0. 05),the effictive rate of the observation group was better than the control group. After the treatment,the median(M)and the positive expression rate of E6 / E7mRNA,p16 and p53 in the two groups were significantly changed when compared with those before the treatment(P < 0. 05),and the difference between the two groups was also significant(P < 0. 05). After treatment the expression and the positive rate of E6 / E7 mRNA P16 and P53 for different types of diseases in two groups were significanst difference(P < 0. 05),comparisons the parameters between groups were significant difference(P < 0. 05). Conclusion:The clinical efficacy of recombinant human interferon alpha-2b for HPV infection is better and the adverse drug reactions are mild,which can reduce the expression and positive rate of E6 / E7mRNA,p16 and p53 in the patients with HPV infection.