Diagnostic value of serum miRNA-9-5p, miRNA-21-5p and miRNA-3923 in cervical cancer / 肿瘤研究与临床

Objective:To investigate the diagnostic value of serum miRNA-9-5p (miR-9-5p), miRNA-21-5p (miR-21-5p) and miRNA-3923 (miR-3923) in patients with cervical cancer.Methods:The data of 100 cervical cancer patients in Shanxi Provincial Cancer Hospital from July 2016 to June 2018 (the experimental group) and 100 healthy subjects (the healthy control group) during the same period were collected. The real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) combined with probe hybridization was used to detect the expression levels of human papillomavirus (HPV) E6/E7 mRNA in paraffin-embedded tissues of patients with cervical cancer and in cervical exfoliated cells of the healthy control people. Ct value ≤ 40 cycles was considered as HPV E6/E7 positive. Serum samples from 3 patients with cervical cancer and 3 healthy people were taken out; microRNA (miRNA) Array was used to detect the expression level of 384 miRNA; the differential expression of miRNA was screened out and cluster analysis was performed, and then the screened miRNAs was verified by using qRT-PCR. Finally, the receiver operation characteristics (ROC) curves of screened miRNAs alone and HPV E6/E7 alone or the combination of three miRNAs and HPV E6/E7 in the diagnosis of cervical cancer were drawn to make comparison of diagnostic efficacy.Results:Among the paraffin-embedded tissues from 100 patients with cervical cancer, there were 65 cases (65%) HPV E6/E7 positive; in the healthy control group, HPV E6/E7 in cervical exfoliated cells of 100 people was negative. A total of 248 differentially expressed miRNAs were detected from the serum samples in 3 patients with cervical cancer and 3 healthy people. The cluster analysis finally identified 16 abnormally regulated miRNAs. qRT-PCR verification confirmed that differences in the expression levels of miR-9-5p, miR-21-5p and miR-3923 in the healthy control group and cervical cancer group were statistically significant (all P < 0.01), and then the three were selected to make diagnosis of cervical cancer. The expression levels of miR-9-5p, miR-21-5p and miR-3923 in HPV E6/E7 positive cervical cancer group were higher than those in the healthy control group (all P < 0.05), expression levels of miR-21-5p and miR-3923 in HPV E6/E7 negative cervical cancer group were increased ( P = 0.008, P = 0.038); expression levels of the three miRNAs in HPV E6/E7 positive group were higher than those in HPV E6/E7 negative group (all P < 0.05). ROC curve analysis showed that the area under the curve (AUC) of miR-3923 was the biggest (0.843), the specificity was the highest (82%, the cut-off value was 2.88) and the sensitivity of miR-21-5p was the highest (85%, the cut-off value was 4.08) when miR-9-5p, miR-21-5p and miR-3923 were respectively applied to diagnose the cervical cancer; AUC (0.924), the sensitivity and the specificity (85%, 94%; the cut-off value was 4.04) of the combination of the three indicators were higher than those of the single indicator in the diagnosis of cervical cancer. AUC was 0.766 when HPV E6/E7 was kused alone to diagnose. The diagnostic efficacy of HPV E6/E7 combined with miR-9-5p, miR-21-5p, miR-3923 respectively was further improved, the corresponding AUC was 0.914, 0.848, 0.932, respectively; the diagnostic efficacy of the combination of the four indicators was the highest (AUC was 0.942). Conclusion:miR-9-5p, miR-21-5p and miR-3923 may be helpful in the diagnosis of cervical cancer.

Presença do papilomavirus humano tipo 16 e expressão gênica da proteína p16ink4a e oncoproteína e7 no carcinoma colorretal / Hpv16 and expression of protein p16ink4a and e7 oncoprotein in colorectal carcinoma

RESUMO - INTRODUÇÃO: O papilomavírus humano (HPV) é agente das doenças sexualmente transmissíveis de maior prevalência no mundo que estão associadas ao câncer do colo do útero e canal anal. A ação do HPV na carcinogênese colorretal não está ainda estabelecida. OBJETIVO: Estudar a eventual correlação entre a presença do HPV tipo 16 e a expressão gênica da proteína p16INK4a e da oncoproteína E7 de HPV e de seus níveis no tecido do carcinoma colorretal. METODOS: Estudo retrospectivo caso-controle de 79 doentes com carcinoma colorretal divididos em dois grupos: HPV presente e HPV ausente. Foi realizada reação em cadeia da polimerase (PCR), além da hibridização do tipo dot blot para o HPV 16 e o HPV 18 Amostras do tecido colorretal também foram submetidas ao estudo imuno-histoquimico para avaliar o nível tecidual das proteínas E7 e p16INK4a. RESULTADOS: O HPV foi identificado em 36 (45,6%) casos. Não houve diferença significante entre os grupos quanto ao sexo (p=0,056), idade (p=0,1), localização cólica e/ou retal (0,098) e presença do HPV. A expressão gênica da oncoproteína E7 de HPV estava presente em 3,12% dos casos (p=0,9) e a expressão da proteína p16INK4a foi observada em 46,3% (p=0,27) dos indivíduos com detecção do HPV. CONCLUSÃO: A expressão gênica e os níveis teciduais da oncoproteína E7 e da proteína p16INK4a encontrados nos pacientes positivos para o HPV sugerem a ausência de atividade oncogênica do HPV tipo 16 no carcinoma colorretal.

ABSTRACT - BACKGROUND: Human papillomavirus (HPV) is the agent of the most prevalent sexually transmitted diseases in the world associated with cervix and anal canal cancer. The action of HPV on colorectal carcinogenesis is not yet established. OBJECTIVE: This research aimed to study the possible correlation between the presence of HPV16 and the gene expression of p16INK4a protein and HPV E7 oncoprotein and their levels in colorectal carcinoma tissue. METHODS: A retrospective case-control study of 79 patients with colorectal carcinoma was divided into two groups: HPV-positive and HPV-negative. The polymerase chain reaction was performed, in addition to dot-blot hybridization for HPV16 and HPV18. Colorectal tissue samples were also subjected to immunohistochemical study to assess the tissue level of E7 and p16INK4a proteins. RESULTS: HPV was identified in 36 (45.6%) cases. There was no significant difference between groups regarding gender (p=0.056), age (p=0.1), colic and/or rectal location (0.098), and presence of HPV. Gene expression of HPV E7 oncoprotein was present in 3.12% of cases (p=0.9), and p16INK4a protein expression was observed in 46.3% (p=0.27) of those selected with HPV detection. CONCLUSION: Gene expression and tissue levels of E7 oncoprotein and p16INK4a protein found in HPV-positive patients suggest the absence of HPV16 oncogenic activity in colorectal carcinoma.

Diagnostic value of flow cytometry in detecting human papillomavirus E6／E7 mRNA in cervical lesions / 中国综合临床

Objective To explore the diagnostic value of flow cytometry in detecting HPV E6／E7 mRNA of human papilloma virus (HPV) in the diagnosis of cervical lesions. Methods From January 2017 to September 2018,119 women with suspected cervical lesions in the department of gynecology and obstetrics of Shanxi Maternal and Child Health Hospital were selected. Flow cytometry was used to detect HPV E6 ／ E7 mRNA in cervical exfoliated cells of women,and the DNA of HPV was detected by the method of hybrid capture 2 (HC2). Results 31. 09%(37／119) HPV E6／E7 mRNA and 57. 14%(68 ／ 119) HPV DNA were positive in 119 cases. The positive rate of HPV E6／E7 mRNA in cervical intraepithelial neoplasia ( CIN)2+ group was 77. 78%(28／36),which was statistically significant compared with 20. 00%(4／20) in CIN1 group (χ2＝15. 246,P＜0. 01),and was statistically significant compared with 7. 94%(5／63) in nilm group (χ2＝50. 286,P＜0. 01) . In nilm group,HPV E6 ／ E7 mRNA positive rate was 7. 94%(5／63) and HPV DNA positive rate was 30. 16%(19 ／ 63),which was statistically significant (χ2＝10. 088,P＝0. 001) . In cin1 group,HPV E6／ E7 mRNA positive rate was 20. 00%(4 ／ 20) and HPV DNA positive rate was

A phase 1/2a, dose-escalation, safety and preliminary efficacy study of oral therapeutic vaccine in subjects with cervical intraepithelial neoplasia 3 / 부인종양

OBJECTIVE: Persistent infection of HPV increases the chance of carcinoma in situ of cervix through stages of cervical intraepithelial neoplasia (CIN) 1, 2, and 3, and finally progresses into cervical cancer. We aimed to explore the safety and efficacy of BLS-M07 which is orally administered agent expressing human papillomavirus (HPV) 16 E7 antigen on the surface of Lactobacillus casei in patients with CIN 3. METHODS: Patients with CIN 3 were recruited in our clinical trial. Reid Colposcopic Index (RCI) grading and serum HPV16 E7 specific antibody production were used to evaluate efficacy of BLS-M07. In phase 1, BLS-M07 was administered orally, 5 times a week, on weeks 1, 2, 4, and 8 with dosages of 500 mg, 1,000 mg, and 1,500 mg. In phase 2a, patients were treated with 1,000 mg. The primary endpoints were the safety and the pathologic regression on colposcopic biopsy. RESULTS: Nineteen patients were enrolled in the CIN 3 cohort. In phase 1, no patients experienced dose limiting toxicity. No grade 3 or 4 treatment-related adverse events or deaths were observed. At 16 weeks after treatment, RCI grading was improved and serum HPV16 E7 specific antibody production increased (p<0.05). Six of 8 (75%) patients with CIN 3 were cured in phase 2a. CONCLUSIONS: Oral immunization with BLS-M07 increases production of serum HPV16 E7 specific antibody which induces protective humoral immunity. The safety of this oral vaccine was proved and could be a competitive non-surgical therapeutic agent of CIN 3. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02195089

Effects of lentivirus-delivered short hairpin RNA targeting human papillomavirus 16 E7 gene on the expression of DNA methyltransferases in SiHa cells / 中华皮肤科杂志

Objective To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) targeting human papillomavirus 16 (HPV16) E7 gene on the expression of 4 kinds of DNA methyltransferases (DNMTs),including DNMT1,DNMT3A,DNMT3B and DNMT3L,in HPV16-positive cervical cancer cell line SiHa.Methods The recombinant plasmid containing HPV16 E7 gene-targeting shRNA was constructed firstly.Then,the BLOCK-iTTM lentiviral RNAi expression system kit was used to package the lentiviral vector,which was transfected into 293T cells.The lentivirus-containing supernatants were collected at 48 and 72 hours after transfection.The SiHa cells were divided into 3 groups to be cultured with lentiviral supernatant containing HPV16 E7 gene-targeting shRNA recombinant plasmids mixed with complete medium at a ratio of 1:1 (shRNA group),lentiviral supernatant containing empty plasmids mixed with complete medium at a ratio of 1:1 (negative control group),and complete medium alone (blank control group),respectively.Real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to measure mRNA expression of HPV16 E7 and 4 kinds of DNMTs in the above 3 groups at 0,48,96 hours after infection,and Western blot analysis to determine protein expression of the 4 DNMTs at 48,96 hours after infection.Results There were no significant differences in the mRNA expression of HPV16 E7 and the 4 DNMTs among the shRNA group,negative control group and blank control group at 0 hour after infection (all P ＞ 0.05).At 48,96 hours after infection,the mRNA expression of HPV16 E7 and the 4 DNMTs decreased significantly in the shRNA group compared with the negative control group and blank control group (all P ＜ 0.05),but did not differ between the negative control group and blank control group (all P ＞ 0.05).Additionally,E7,DNMT1,DNMT3A,DNMT3B and DNMT3L gene-silencing efficiencies in the shRNA group were 71.13％,50.53％,13.72％,46.27％ and 17.92％ at 48 hours,and 83.50％,74.2％,47.8％,64.7％ and 48.9％ at 96 hours after infection,respectively.Western blot analysis showed that the protein expression of the 4 DNMTs significantly decreased in the shRNA group compared with the negative control group and blank control group at 48,96 hours after infection (all P ＜ 0.01).Moreover,the protein expression of DNMT1,DNMT3A,DNMT3B and DNMT3L in the shRNA group gradually decreased over time,and was inhibited by 84％,37.2％,59.8％ and 49.3％ at 48 hours respectively,and by 73.1％,68.7％,55.5％ and 65.5％ at 96 hours after infection respectively.Conclusion Targeted silencing of E7 gene in HPV16-positive SiHa cells can interfere with the mRNA and protein expression of DNMT1,DNMT3A,DNMT3B and DNMT3L.

Effects of a small interfering RNA targeting HPV16E7 on proliferation and apoptosis of SiHa cells and expressions of six tumor suppressor genes / 中华皮肤科杂志

Objective To evaluate effects of human papilloma virus(HPV)16E7 on expressions of six tumor suppressor genes(including MT1G, NMES1, RRAD, SFRP1, SPARC and TFPI2)in a cell line SiHa, as well as on its proliferation and apoptosis. Methods SiHa cells were divided into two groups to be transfected with a small interfering RNA targeting HPV16E7(E7SiRNA, experimental group)and an empty vehicle(negative control group) respectively, with SiHa cells receiving no treatment serving as the blank control group. After 48 hours, qPCR was performed to measure the mRNA expressions of E7 and six tumor suppressor genes, CCK?8 assay to evaluate cellular proliferative activity, and flow cytometry to assess apoptosis of SiHa cells. Results At 48 hours after the transfection, the experimental group showed significantly decreased E7 mRNA expression(0.25 ± 0.036, P0.05). Conclusion E7 may participate in HPV16?induced cellular malignant transformation by suppressing the mRNA expressions of 6 tumor suppressor genes, including MT1G, NMES1, RRAD, SFRP1, SPAR and TFPI2.

Application of HPV E6/E7 mRNA detection in screening of moderately severe cervical intraepithelial neoplasia / 中华检验医学杂志

Objective This study aimed to explore the clinical value of human papilloma virus ( HPV) E6/E7 mRNA tests in identifying precancerous lesions of the uterine cervix-cervical intraepithelial neoplasia 2 or more CIN2+( CINⅡand CINⅢ).Methods This study is a cross-sectional survey design , between December 2011 to December 2013.The specimens were collected from the First People′s Hospital of Huizhou and the Third People′s Hospital of Huizhou in Department of Obstetrics and Gynecology outpatient and inpatient of cervical disease suspected patients , with thin-prep cytologic test ( TCT ) and histopathological results as reference , detected 345 patients of exfoliated cervical epithelial cells by using the branched DNA (b-DNA) technology to evaluate the application value of high risk HPV E 6 /E7mRNA in the clinical diagnosis of CIN.Using spss 19.0 software for data analysis.Results (1)Compared with TCT, the positive rate of E6/E7 mRNA in 325 samples were grading by cytology as follows: no intraepithelial lesion cells (NILM) 21.1%(40/190), atypical squamous cells (ASC) 38.5%(15/39 ), low-grade squamous intraepithelial lesions ( LSIL ) 76.9% ( 30/39 ) , atypical squamous cells can not exclude high-grade intraepithelial lesions (ASC-H) (8/10), high-grade squamous intraepithelial lesions (HSIL) 72.3%(34/47), TCT grades and HPV E6/E7 mRNA positive rate showed linear association (χ2 =67.654,P<0.01;r=0.497, P<0.01 ); and with HPV E6/E7 mRNA copy number was also relevant ( r =0.511, P <0.01).(2) Compared with pathological results , the positive rate of E6/E7 mRNA in 164 women samples were grading by pathology as follows:with NILM was 27.8%(10/36), with CIN Ⅰwas 65.9%(29/44), with CINⅡwas 80.6%(54/67), and with CINⅢwas 82.4%(14/17), pathological grades and HPV E6/E7 mRNA positive rate showed a linear correlation (χ2 =26.426, P<0.01; r=0.438, P<0.01); and the number of copies correlated with the increase of pathological grades too (r=0.543, P<0.01).(3) Screening effectiveness analysis results showed , the sensitivity of HPV E6 /E7mRNA was 84.6% while TCT was 47.7%.The sensitivity and specificity were 40.0% and 91.1% respectively when HPV E6/E7 mRNA and TCT processed as sequential detection test.The CIN2 +( CINⅡand CIN Ⅲ) best diagnostic critical point of 890.26 copies/ml,was established using receiver operating characteristic ( ROC) curve.The sensitivity and specificity were 58.5% and 93.7%, respectively.Conclusions The sensitivity of HPV E6/E7 mRNA test is better than TCT, the specificity is high in HPV E6/E7 mRNA and TCT processed as sequential detection test.Using the optimal cut-off value of ROC curve to detect CIN 2+has high sensitivity and specificity, so the detection of HPV E6/E7 mRNA may have some clinical value in screening and risk assessment of precancerous lesions of the uterine cervix.

Study on phenotype and function of antigen-specific CD8+ cytotoxic T cells induced by HPV 16 E7 peptide in vitro / 中华皮肤科杂志

Objective To investigate the phenotype and function of antigen-specific CD8+ cytotoxic T cells (CTL) from HLA-A2+ healthy human donors induced by high-carcinogenic HPV 16 E7 peptide in vitro.Methods Peripheral blood T cells from 26 HLA-A2+ healthy human donors were incubated with HPV 16E711-20 peptide (HLA-A*0201/YMLDLQPETT), recombinant human interleukin 7 (IL-7) and IL-2 for 7 days to yield antigen-specific CD8+ CTL. Then, four-color flow cytometric analysis was performed to detect the percentage of antigen-specific CD8+ CTL expressing different surface markers including CD45 and CD27. The expression of three intracellular cytokines including perforin, granzyme-B and FasL in the antigen-specific CD8+ CTL was also measured by intracellular flow cytometry. A wrong peptide, HBVcoxe18-27 (FLPSDFFPSV),was used as an isotype control. Results A significant increase was observed in the percentage of antigen-specific CD8+ CTL in peripheral T cells stimulated with HPV 16 E7-peptide compared with the non-stimulated T cells (0.73% ± 0.33% vs 0.02% ± 0.03%, P ＜ 0.01). The percentage of CD45RA+CD27- effector T cells,CD45RA-CD27- effector memory T (TEM) cells, CD45RA-CD27+ central memory T (TCM) cells, and CD45RA+CD27+ naive T cells in antigen-specific CD8+ CTL was 26.07% ± 13.46%, 7.97% ± 7.11%, 33.25% ± 19.68%and 32.73% ± 13.89%, respectively in HPV 16 E7-stimulated group, significantly higher than that in nonstimulated group (0.02% ± 0.03%, 0.02% ± 0.03%, 0.02% ± 0.03% and 0.02% ± 0.05%, all P ＜ 0.01 ). Elevated proportions of perforin-, granzyme-B- and FasL-expressing antigen specific CTL were observed in HPV 16 E7-stimulated group compared with non-stimulated group (47.01% ± 18.69% vs 0.38% ± 0.55%, 80.53% ±13.32% vs 0.34% ± 0.22%, 26.48% ± 7.81% vs 0.16% ± 0.16%, all P ＜ 0.01 ). Conclusions HPV 16 E7peptide could induce the clonal proliferation of CD8+ T cells, generation of antigen-specific CD8+ CTL and secretion of toxic cytokines, finally lead to a highly efficient and specific killing of virus-infected target cells through different mechanisms, hence, it might play a crucial role in antiviral immune responses.

Role of human interferon α-2b as an immunological adjuvant for human papillomavirus (HPV) 11-E7 DNA vaccine / 中华皮肤科杂志

ngthen cellular immunity, especially Th1-type immune response to HPV11-E7 DNA vaccine in mice.

Effects of hyperthermia on the expression of E6 and E7 genes of human papillomavirus type 6 and 11 / 中华皮肤科杂志

Objective To investigate the effect of hyperthermia on the expression of E6 and E7 genes of human papillomavirus (HPV) type 6 and 11 in HPV-infected human skin. Methods Tissue samples were obtained from the lesions of condyloma accuminatum (CA) in 6 patients after informed consent. Each sample was divided into 4 parts: one was embedded and directly stored at -80 ℃; the other 3 parts were placed in culture medium and the surface of the samples was irradiated for 30 minutes with a thermotherapy apparatus at 37℃, 42 ℃, 45 ℃, respectively, then the samples were taken out and stored at -80 ℃. RNA was extracted from the specimens, real time quantitative PCR (qPCR) was performed to detect the expression of E6 and E7 genes of HPV-6 and -11. Results Of the 6 patients, 2 were infected with HPV-6 and -11 respectively, 4 with both HPV-6 and HPV-11. The expression of E6 and E7 mRNA decreased with the increase in irradiation temperature. The relative mRNA expression levels at 37 ℃, 42 ℃ and 45 ℃ were 1.00 ± 0.00, 0.61 ± 0.17, 0.27 ± 0.15, respectively, for HPV-6 E6 gene, 1.00 ± 0.00, 0.56 ± 0.21, 0.16 ± 0.11 respectively, for HPV-6 E7 gene, 1.00 ± 0.00, 0.60 ± 0.22, 0.16 ± 0.08, respectively, for HPV-I1 E6 gene, 1.00 ± 0.00, 0.55 ± 0.15, 0.24 ± 0.06, respectively, for HPV-11 E7 gene; statistical difference was noted among them between the specimens irradiated at different temperature (all P < 0.01). Conclusion Hyperthermia can remarkably suppress the expression of HPV-6/I 1 E6 and E7 genes, which may be a possible mechanism under the regression of warts induced by local hyperthermia.

Immunocytochemical detection of HPV16 E7 in cervical smear

Cervical cancer is characterized by a long period of preclinical dysplasia or carcinoma in situ progressing into invasive cancer. Although Papanicolaou (Pap) smear test has contributed significantly to the early detection of precursor lesions, the cytological screening has inherent problems that produce considerable false negative/positive results. Since the infection of high-risk type of human papillomavirus (HPV) is strongly associated with cervical cancer, we investigated the feasibility of an immunostaining test to detect cells infected by HPV in cervical smear. We produced monoclonal antibodies against HPV16 E7 in mice by repeated injections with the recombinant HPV16 E7. Western blot analysis and immunocytochemical assay demonstrated that the selected monoclonal antibody, mAb (130-9-7), reacts specifically with cultured cervical cancer cell lines infected by HPV16. Specific staining was observable with the HPV16-positive smear specimens obtained from the cervical cancer patients, whereas no staining was detected with the HPV-negative smear specimens. To achieve the desired sensitivity, specificity and reproducibility, we modified and optimized the conventional immunocytochemical procedure for cervical smear specimens. Our results suggest that this immunostaining method for detecting high-risk HPV in cervical smear may be used as a strategy to distinguish a high-risk group, especially those patients with low grade cytological abnormality.