Effects of HPV Pseudotype Virus in Cutting E6 Gene Selectively in SiHa Cells / 华中科技大学学报（医学）（英德文版）

The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer.After designing specific gRNA sequences targeting HPV 16 E6,generating hCas9-EGFP and E6-gRNA-RFP plasmids,and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids,we determined the titer of the pseudotype virus using the TCID50 method.We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells.Experimental subjects were divided into control group,empty virus group,E6-gRNA transfected group,Cas9 transfected group and Cas9+E6-gRNA transfected group.The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis,and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time.RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups;the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups.Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6.The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells,and there were two cutting zones in the Cas9+E6-gRNA transfected group.However,the empty virus group,E6-gRNA transfected group and Cas9 transfected group had no corresponding zone.Compared with those in the control group,the empty virus group,E6-gRNA transfected group and Cas9 transfected group,the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9+E6-gRNA transfected group (P＜0.01).In addition,the proliferation and migration abilities of SiHa cells were significantly inhibited (P＜0.01).There were no significant differences among the other groups.In contrast to the control group,the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model (P＜0.01).The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells,which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors.Taken together,these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease,particularly in HPV-related tumors.

Effects of HPV Pseudotype Virus in Cutting E6 Gene Selectively in SiHa Cells / 华中科技大学学报（医学）（英德文版）

The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer.After designing specific gRNA sequences targeting HPV 16 E6,generating hCas9-EGFP and E6-gRNA-RFP plasmids,and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids,we determined the titer of the pseudotype virus using the TCID50 method.We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells.Experimental subjects were divided into control group,empty virus group,E6-gRNA transfected group,Cas9 transfected group and Cas9+E6-gRNA transfected group.The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis,and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time.RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups;the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups.Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6.The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells,and there were two cutting zones in the Cas9+E6-gRNA transfected group.However,the empty virus group,E6-gRNA transfected group and Cas9 transfected group had no corresponding zone.Compared with those in the control group,the empty virus group,E6-gRNA transfected group and Cas9 transfected group,the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9+E6-gRNA transfected group (P＜0.01).In addition,the proliferation and migration abilities of SiHa cells were significantly inhibited (P＜0.01).There were no significant differences among the other groups.In contrast to the control group,the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model (P＜0.01).The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells,which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors.Taken together,these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease,particularly in HPV-related tumors.

Effect of human papillomavirus 16 E6 gene on the expression of E-cadherin in human cervical cancer cell line SiHa / 肿瘤研究与临床

Objective To investigate the effect of human papillomavirus (HPV) 16 E6 gene on the expression of E-cadherin (E-cad) in cervical cancer cell line SiHa. Methods HPV16 E6 expression system was established in the cervical cancer cell line SiHa by using transient transfection system, MTT method was used to detect SiHa cell proliferation activity, and reverse transcription-polymerase chain reaction (RT-PCR) and western blot method was used respectively to detect E-cad mRNA and protein expression level in cells after HPV16 E6 transfection. Results Compared with the blank control group (non-transfected plasmid) and the vector group (the addition of pcDNA3.1), the cell viability rate of the E6 group (pcDNA3-1-HPV16 E6) was significantly increased (P<0 .05), while there was no significant difference between the vector group and the blank control group (P>0.05). The relative expressions of E-cad mRNA in the E6 group, the vector group and the blank control group were 0.26±0.12, 0.82±0.14, 0.83±0.21 respectively, then the protein relative expressions in the three groups were 0.62±0.02, 1.33±0.04, 1.31±0.05 respectively. The expressions level of E-cad mRNA and protein in E6 group were significantly lower than those in the other two groups (all P<0.05). However, there was no significant difference between the vector group and the blank control group (both P>0.05). Conclusion The instantaneous transfection of HPV16 E6 gene can reduce the expression of E-cad in cervical cancer cells, which is closely related to the occurrence and development of cervical cancer.

Effect of human papillomavirus 16 E6 gene on the expression of E-cadherin in human cervical cancer cell line SiHa / 肿瘤研究与临床

Objective To investigate the effect of human papillomavirus (HPV) 16 E6 gene on the expression of E-cadherin (E-cad) in cervical cancer cell line SiHa. Methods HPV16 E6 expression system was established in the cervical cancer cell line SiHa by using transient transfection system, MTT method was used to detect SiHa cell proliferation activity, and reverse transcription-polymerase chain reaction (RT-PCR) and western blot method was used respectively to detect E-cad mRNA and protein expression level in cells after HPV16 E6 transfection. Results Compared with the blank control group (non-transfected plasmid) and the vector group (the addition of pcDNA3.1), the cell viability rate of the E6 group (pcDNA3-1-HPV16 E6) was significantly increased (P<0 .05), while there was no significant difference between the vector group and the blank control group (P>0.05). The relative expressions of E-cad mRNA in the E6 group, the vector group and the blank control group were 0.26±0.12, 0.82±0.14, 0.83±0.21 respectively, then the protein relative expressions in the three groups were 0.62±0.02, 1.33±0.04, 1.31±0.05 respectively. The expressions level of E-cad mRNA and protein in E6 group were significantly lower than those in the other two groups (all P<0.05). However, there was no significant difference between the vector group and the blank control group (both P>0.05). Conclusion The instantaneous transfection of HPV16 E6 gene can reduce the expression of E-cad in cervical cancer cells, which is closely related to the occurrence and development of cervical cancer.

Molecular detection of HPV 16/18 E6 genes from cervical cells.

Background: Epidemiological, clinical and molecular studies have established the link between genital infection with high risk human papillomavirus (HPV) and cervical cancer but there is great challenge in establishing early infection by both clinicians and the laboratories. The virus cannot be grown in conventional cell cultures and serology cannot different between active and past infection. Molecular studies remain the goal standard as it detects viral nucleic acid or cellular antigens indicative of oncogenic potential in cytology or biopsy specimen. The study was aimed to molecularly determine the presence of HPV 16/18 and expression of E6 gene in squamous intraepithelial lesions. Methods: Cervical cells were collected from 18 women with positive cytology test results and 32 controls in gynaecology clinic of Murtala Muhammad Specialist Hospital, Kano Nigeria and HPV 16/18 were detected with E6 gene specific PCR primers. Results: Overall, HPV E6 gene was found in 76% of the women, 88.3% of positive cytology specimens and 71.2% controls. Conclusions: There is very high prevalence of HPV infection. The presence of HPV 16/18 E6 genes in cervical intraepithelial lesions may serve as a useful predictor of diagnosis and possible clinical outcome of the disease.

Diseño de un sistema de PCR para la detección del virus del papiloma humano mediante el uso de oligonucleótidos degenerados de la región E6 / Desing of PCR system for detection of human papilloma virus by using degenerate oligonucleotides of the E6 region

Diseñar y estandarizar un sistema de PCR para detectar un amplio espectro de tipos de VPH en una sola reacción utilizando como blanco la región E6 del genoma viral. Utilizando secuencias del gen E6 de distintos tipos de VPH del NCBI y las herramientas informáticas Mult Alin versión 5.4.1. y Oligo Analyzer 1.1.2 se diseñaron oligonucleótidos degenerados que permitían la amplificación de un fragmento de aproximadamente 214 pb. Las muestras seleccionadas incluyen: 25 muestras de ADN, cada una positiva para un solo tipo de VPH tipificados por el sistema PCR-RFLP MY11/MY09 del gen L1. Se obtuvieron amplificados de aproximadamente 214 pb de 25 tipos distintos de VPH, se detectaron amplificados de E6 en muestras negativas para el sistema MY09/MY11. Se pudo determinar que el diseño de los oligonucleótidos degenerados fue altamente eficiente para la amplificación de por lo menos 25 tipos distintos de VPH lo que facilitaría la detección en muestras clínicas

Design and standardize a PCR system to detect a broad spectrum of HPV types in a single reaction using target the E6 region of the viral genome. Using E6 gene sequences of different HPV types and NCBI tools Mult Alin Oligo Analyzer version 1.1.2 5.4.1. Degenerate oligonucleotides were designed that allowed amplification of a fragment of approximately 214 bp. Selected samples include: 25 DNA samples, each positive for a single HPV type typified by PCR-RFLP system MY11/MY09 L1 gene. We obtained approximately 214 bp amplified from 25 different HPV types were detected in samples amplified from E6 negative MY09/MY11 system. It was determined that the design of degenerate oligonucleotides was highly efficient for amplification of at least 25 different HPV types which would facilitate detection in clinical samples

Induction of SiHa Cells Cells Apoptosis by Nanometer Realgar Suspension and Its Mechanism / 华中科技大学学报（医学）（英德文版）

The effects of nanometer realgar suspension on proliferation and apoptosis of human uterine cervix cancer cell line SiHa cells and oncogenic genes HPV16E6/E7 were investigated. A "micro-jet efflux" strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations (6.25,12.5,25 and 50mg/L) for different durations (12,24,48 and 72h). The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MIT method. Special morphological changes of apoptosis were observed by transmission electron microscopy (TEM) and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry (FCM). The expression of HPV16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25-50mg/L nanometer realgar suspension for 48h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group (P<0.01), and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50mg/L for 48h.RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

Inhibitory effects of chemically synthetic siRNA on the expression of E6 gene in the cervical cancer cell line SiHa / 中国癌症杂志

Background and purpose:The genesis and progression of cervical cancer are closly related to E6 and E7 oncogenes of HPV.Special ribozyme and antisense oligonucleotide could inhibit the expression of E6 or E7.but both of them were not the best methods.The aim of this study was to investigate the inhibitive effect of chemically synthetic siRNA on E6 gene in the cervical cancer cell line SiHa and the following biological changes of SiHa cells.Methods:Three specific siRNAs targeting to HPV16 E6 were synthesized and transfected into SiHa cells by lipofectamine.The level of E6 mRNA was tested by RT-PCR,proliferation activities,the cell cycle distribution,expression of p53 protein and the ultramicrostructure changes of SiHa cells were detected by MTT,FCM,immunochemistry and TEM respectively.Results:All the 3 siRNAs could inhibit the level of E6 mRNA singificantly,among which siRNA1 was most effective.Decreased cellular proliferation activity was observed by MTT,especially when the cells were transfected with siRNA1 at 50 nmol/L concentration.Flow cytometry revealed obvious G1 arrest in cell cycle.The expression of p53 protein in transfected cells(0.75?0.06)was increased compared to the control group(0.43?0.03),the difference was singificant.Cell-substance concentration and vacuolus were found in endochylema by TEM.Conclusions:Chemically synthetic siRNA can interfere in the expression of E6 mRNA in SiHa cells specifically and induce the biological changes of the target cells.

siRNA inhibits HPV18 E6 gene and its effect on the apoptosis of HeLa cells / 基础医学与临床

Objective To study the effect of small interfering RNA(siRNA) of human papillomavirus(HPV) 18(E6) gene on apoptosis of HPV-related cervical HeLa cell line.Methods siRNA targeting HPV18 E6 mRNA was designed and generated by PCR amplification.The PCR products containing U6 promoter and the siRNA sequence were then transfected into HeLa cells via Lipofectamine()~(TM)2000.Cell viability was determined by MTT assay.Apoptosis was detected by morphological observation and flow cytometry analysis.The expression level of HPV18 E6 mRNA was assayed by RT-PCR.Results The cell growth and viability of(siRNA) transfected group were significantly inhibited(P

Construction of HPV16 E6 siRNA expressing recombinants and identification of transfection efficiency in vitro / 重庆医科大学学报

Objective:To silence the expression of HPV16 E6 gene in Ca Ski cell line with RNA interference technique.In order to understand if the HPV16 E6 siRNA could inhibit the expression of E6 gene specifically,recombinant eukaryotic vectors were constructed and transfected into Ca Ski cell line.The transfection efficiency was observed.Methods: According to the sequence of HPV16 E6 gene,the E6 siRNA was designed and cloned into the EGFP reporter plasmid pGenesil-1,and then transfected into CaSki cell line.The fluorescence expression was observed and the transfection efficiency was identified.Results: Two siRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into Ca Ski cell successfully,and the transfection efficiency achieved about 50%.Conclusion: The construction of recombinant E6 siRNA eukaryotic vectors and transfection of them into Ca Ski cell line successfully established a favourable foundation for further study.

Telomerase Activity in Gynecologic Cancers and correlation of HPV E6 gene expression in cervical cancer tissues / 대한산부인과학회잡지

OBJECT: In this study, to evaluate the putative role of telomerase in gynecologic malignancies (cervical ca, ovarian ca, endometrial ca), we measured telomerase activity in malignant gynecologic tumor tissues and normal tussues, and compared it with prognostic factors in cervical cancer. To evaluate the correlation of telomerase activity and human papillomavirus (HPV) infection in cervical cancer, the analysis of HPV E6 gene was performed. METHOD: Specimens were obtained from 51 women who underwent gynecologic radical operation and 13 normal tissues (from December 1995 to December 1996) in the Department of Obstetrics and Gynecology, Kyung-Hee Univ. Medical Center. With Telomerase PCR ELISA (Boehring Mannheim), modified TRAP (Telomere Repeat Amplication Protocol), we examined telomerase activity of 32 cervical carcinomas, 11 ovarian carcinomas, 8 endometrial carcinomas, 5 normal cervical tissues, 4 normal ovarian tissues and 4 normal endometrial tissues. The analysis of HPV E6 gene was performed by PCR amplication. We compared the abnormally high telomerase activity with prognostic factors, also compared the telomerase activity with the expression of HPV E6 gene in cervical cancer tissues. RESULT: We detected the abnormally high telomerase activity in all cervical carcinomas, 10 of 11 (90.9%) ovarian carcinomas, 6 of 8 (75.0%) endometrial carcinomas, but couldn't detect in each normal tissues. There was statistically no significant difference of telomerase activity levels according to age, clinical stage, pathology, differentiation, LN involvement, depth of invasion and tumor size except lymphovascular space invasion in cervical carcinomas (p<0.05). According to the analysis of HPV E6 gene, 29 of 32 (90.6%) in 32 cervical cancer tissues showed HPV E6 positivity. So it was considered that telomerase activation was closely related with the expression of HPV E6 gene. CONCLUSION: Telomerase activation is associated with immortalization or malignant transformation of gynecologic cancers. The expression of HPV E6 gene is considered to activate telomerase in cervical cancer.

Human Papilloma Virus Type 16 E7 Genes Protect Astrocytes against Apoptotic and Necrotic Death Induced by Hydrogen Peroxide

Hydrogen peroxide is considered to be a dose- and time-dependent mediator in apoptotic and necrotic death. In this study, we examined the signaling of the E6 and E7 proteins with respect to apoptosis or necrosis after H2O2 injury using an in vitro model with overexpressed E6 or E7 genes. For this purpose, the E6 and E7 gene expressing astrocytes were exposed to 0.01 mM and 0.2 mM H2 O2 solutions. Twenty- four hours after treatment with the lower dosage(0.01 mM H2O2), control, E6-expressing cells suffered about 45% injury and LXSN-expressi ng cells decreased by 67% as assessed by LDH release. However, E7-expressing cells showed less injury, resulting in 20-30% of LDH release. Astrocytes expressing E6, E7, LXSN and mock-infected cells showed a typical apoptotic death patter n on the DNA gel after treatment with a low-dose of H2O2 (0.01 mM), however the y died from necrotic death after a high-dose (0.2 mM) H2O2. Overexpression of HPV-E7 genes protected the cells from apoptotic death after a low-dose of H2O2 and from necrotic death after a high-dose of H2O2, while the overexpression of E 6 genes from the necrotic death. E7 expressing astrocytes showed higher catalas e activity and the levels of E2F protein surged more than 100-folds compared with the control astrocytes. We believe that the activity of E7 protein to protect astrocytes from H2O2 injury was at least partly due to increased catalase, a scavenger protein.

Vaccine preparation of dendritic cell transfected with HPV16E6 antigen gene and its biological characteristics / 中国免疫学杂志

Objective:To prepare the vaccine of DC derived from human peripheral blood and transfected with HPV16E6 antigen gene, and to detect its morphological character,surface marker and immunological effect.Methods:DC-enriched populations were prepared from human peripheral blood mononuclear cell(PBMC) with the combination of rhGM-CSF,rhIL-4 and rhTNF-?. The plasmid containing HPV16E6 gene was transfected into DC with lipofectamine. The morphology of DC was observed dynamically, and the expression of surface markers of DC vaccine could be detected using immuno-cytochemical staining and flow cytometry. MTT assay was applied to detect the activity of CTL in vitro.Results:The transfected DC had typical morphologic and phenotypic characteristics, and expressed E6 protein 47.3%, CD80 82.5%, CD86 79.8% and CD83 85.7%. The killing activities of CTL to Caski cells induced by transfected DC were higher evidently than that of control groups(P

Polymorphisms in E6 Gene of Human Papillomavirus Type 16 Found in Cervical Tissues from Korean Women / 대한암학회지

PURPOSE: To examine the distribution of HPV 16 E6 polymorphisms and analyse the possible association between the polymorphisms and cervical cancer development in Korean women. MATERIALS AND METHODS: Fifty-four cases of uterine cervical tissues containing HPV 16 DNA confirmed by polymerase chain reaction (PCR) from Korean women were subjected to investigate the E6 gene mutations. PCR-amplified products were sequenced by the fluorescent dideoxy ter mination method and the results obtained from sequencing were analysed. And newly designed PASA method was tried to develop rapid test for identification of the most commonly detected variation. RESULTS: Among the 27 cervical cancer cases, only two (7.4%) was found as a prototype. Among 11 kind of variants identified in total, 4 variants (5 nucleotide sites) which were never reported before has been found, registered firstly to GenBank. The most frequently found variation was D25E, absolutely different from the previous reports from the western country. There was no statistically significant trend for the D25E variation to be more frequently detected in cancerous lesions than in noncancerous lesions. All of the DNA sequencing results observed could be confirmed by PASA method. CONCLUSION: These results suggest that Korean-specific genetic factors might operate during the cervical carcinogenesis.

Inhibition effect of silencing HPV16 E6 gene on human cervical cancer CaSki cell carcinoma xenograft in nude mice / 第三军医大学学报

Objective To learn the effect on carcinoma xenograft in nude mice by inhibiting human papillomavirus 16(HPV16) E6 gene expression in CaSki cell. Methods The recombinant plasmids expressing HPV16 E6 small interference RNA (siRNA) were transfected into CaSki cell. The cells expressing recombinant plasmid was screened out with G418. The expression of E6 mRNA was determined by RT-PCR. The cells were inoculated into BALB/c nude mice subcutaneouly and the growth of the xenograft carcinoma was observed. After the pGensil-CH2 recombinant was injected into the carcinoma, the growth of carcinoma and pathological changes of carcinoma were observed. Results The CaSki cell expressing E6 siRNA was obtained, and HPV16 E6 mRNA expression in CaSki cell was down-regulated. The oncogenicity of the CaSki cell expressing E6 siRNA was degraded, the inhibition rate was up to 71.4% as compared with that of control group. The growth of tumor in nude mice was inhibited after the E6 siRNA plasmids were injected into the nude mice. The volume and weight of the tumor treated by siRNA were smaller than that of control group significantly. More necrotic area and less cell division phase were observed under light microscope in the E6 siRNA treated tumor. Conclusion The oncogenicity of the CaSki cell was degraded after silencing HPV16 E6 gene in CaSki cell by E6 siRNA.