| Background and objectivesHuman papilloma virus (Human papilloma virus, HPV) can cause a series of epithelial lesions. Some types of HPV associated with precancerous lesions, referred to as a high-risk type HPV. And some people infected with these high-risk HPV types can occur canceration. In1995, the International Agency for Research on Cancer (IARC) recognized that HPV-16and HPV-18were carcinogenic in human. The correlations between cervical cancer and high risk HPV types have been widely recognized. Epidemiology, molecular and clinical evidence suggested that the high-risk HPV infection was strongly associated with cervical cancer. Also, parts of the head and neck squamous cell carcinoma (Head and neck Squamous cell carcinoma, HNSCC) were closely related with high-risk HPV.At present, most of the HNSCC of HPV related research is oropharyngeal squamous cell carcinoma (Oropharyngeal squamous cell carcinoma, OSCC). Clinical evidences suggested that HPV-related OSCC usually did not have such typical history as heavy consumption of alcohol and/or tobacco use, which different from the classic OSCC in biological behavior. HPV infection or not has a significant influence on the prognosis of OSCC patients, therapeutic effect and survival rate. HPV positive patients with OSCC were superior to HPV negative OSCC patients. Laryngeal squamous cell carcinoma and hypopharyngeal squamous cell carcinomas are two common malignancies of HNSCC, which occurs mainly in middle-aged men. Heavy consumption of alcohol and/or tobacco use was recognized as carcinogenic factors. However, some patients do not have the history of heavy consumption of alcohol and/or tobacco use. For this part of the patients, the researchers turned to the HPV infection. Many research institutions detected the overall rate of HPV infection in laryngeal squamous cell carcinoma and hypopharynx squamous cell carcinoma. While these previous studies were always retrospective study, the detection of cancerous tissue from the pathology archive specimens and the patients clinical data obtained subject to many limitations. In addition, anatomical position of the larynx and hypopharynx are adjacent. Some advanced cancers alone with pathological specimens were not easy to identify the primary position. Given the incidence of laryngeal squamous cell carcinoma of were significantly higher than the hypopharyngeal squamous cell carcinoma, most researchers put hypopharyngeal squamous cell carcinoma classified as laryngeal squamous cell carcinoma. Therefore, lots of the literatures reported laryngeal squamous cell carcinoma of HPV infection, but rarely hypopharyngeal squamous cell carcinoma HPV infection. HPV infection rates and common infection in hypopharyngeal squamous cell type is not clear.The clinical manifestations of hypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma are similar, but primary in two different anatomical sites. The degree of differentiation, regional lymph node metastasis rate and5-year survival rate were significantly different. Hypopharyngeal squamous cell carcinoma prognosis is worse. Moreover, studies suggest that HPV infection is mainly associated with HNSCC prognosis. Thus, when carried out the related research of HPV infection, it is necessary to distinguish with hypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma. Hypopharyngeal squamous cell carcinoma will be as an independent disease-related research. In this study, we will test and genotype fresh frozen tissue samples which collected from28cases of hypopharyngeal squamous cell carcinoma HPV DNA, understand human HPV infection cases in laryngeal pharyngeal squamous carcinoma tissues and the common type of HPV. E6and E7genes were the early region of the HPV genome. Encoding E6and E7protein. The E6and E7protein of HPV-16play a carcinogenic role in encoded oncoprotein. Lentiviral vector (Lentivirus vector) can be high efficiency and stable transfected phases of the cell. It is a good means of delivery in integration of the gene into cells, and has the advantages of easy operation, high efficiency of infection, security. This project intends to use the lentiviral vector genome synthesis HPV-16E6-E7gene to transfect hypopharyngeal squamous cell carcinoma FaDu cells, and observe HPV-16E6-E7genes impact the FaDu cell biology behavior.MicroRNA are involved in almost all the body biological processes, including cell proliferation, differentiation, apoptosis, invasion, and migration. Abnormal expression of miRNAs associated with the occurrence and development of many tumors. However, the current understanding of HPV related miRNAs expressions in HNSCC are limited. This subject intends to detect the miRNAs expression levels in hypopharyngeal squamous cell carcinoma tissue which only infected with HPV-16and HPV-negative.Part one HPV detection and genotyping of fresh frozen tissue in the human hypopharyngeal squamous cell carcinomaMethods1. Collected fresh tissues of hypopharyngeal squamous cell carcinoma from28cases. Saved in-80℃low temperature refrigerator after frozen in liquid nitrogen.2. Applied a small amount of genomic DNA kit to extracted frozen hypopharyngeal squamous cell carcinoma tissue genomic DNA.3. PCR-reverse dot blot hybridization specimens and genotyping of HPV DNA.4. Combinded the state of HPV infection with the patient’s clinical data and made correlation Analysis.Results1.28cases of hypopharyngeal squamous cell carcinoma of HPV infection were as follows:HPV positive specimens were7cases, total positive rate was25.0% (7/28). Five cases for a single type. HPV-16in three cases, HPV-18type in1case, HPV-52type in1case.2cases were two types of HPV infection at the same time, HPV-16, HPV-33in type1case, HPV-16,52in type1case.2. Compared with HPV-positive and HPV-negative patients, less HPV-positive patients had heavy consumption of alcohol and/or tobacco use history (P<0.05), and there were no significant differences between age, gender, pathological type and tumor T stage (P>0.05).Part two HPV-16E6-E7gene integration affect FaDu cells biological behaviorMethods1. Preparation of recombinant HPV-16E6-E7lentiviral:Gene synthesis HPV-16E6-E7genes and addition HindⅢ and KpnI restriction endonuclease enzyme sites at both ends respectively. Then, connected with lentiviral vectors pLV-EGFP-C, packaging the reorganization of HPV-16E6-E7lentiviral, determining the lentiviral titer.2. Packaging of Recombinant HPV-16E6-E7lentiviralPrepared the packaging plasmid DNA:The lentivirus packaging vectors PHI, PH2, and recombinant lentivirus vectors HPV-16E6-E7in accordance with a certain ratio, mixed in500μL serum-free DMEM medium.Prepared transfection reagent dilutions:A certain volume of transfection solution Polyfect-V was mixed with500μL serum-free DMEM medium.Transfection reagent dilution was added into the packaging plasmid DNA solution, and immediately mixes thoroughly, this was transfection mixture. Incubated transfection mixture for15minutes, digestion HEK293T cells, per1ml transfection mixture was added10ml of cell suspension, and gently blowing and suction cell.The cell suspension was added to a10cm petri dish, and cultured for24hours in37℃. Removed the culture medium containing the transfection reagent, the medium was changed with10ml of virus medium. After48hours transfection, the virus particles were collected from the culture supernatan and virus titer was measured, stored at-80℃.3. Recombinant HPV-16E6-E7lentiviral infection laryngeal pharyngeal squamous carcinoma FaDu cells:recoved FaDu cells, and infected with recombinant virus HPV-16E6-E7lentiviral particles liquid. Fluorescence expression was observed after48hours. DMEM high glucose medium containing0.5μg/mL puromycin was used for screening culture, and stably transfected cells were obtained after3weeks. PCR detected the HPV-16E6-E7DNA in FaDu cells, RT-PCR detected E6, E7mRNA, Western-blot detected E6-E7protein.4. Cells were divided into three groups:the experimental group (the stable infection FaDu cells with recombinant HPV-16E6-E7lentiviral), lentiviral empty vector (stable FaDu cells infected by the vector control group) and blank control group (not infected with lentivirus FaDu cells).5. CCK-8kit was used to detect the cell proliferation. It measured the absorbance values hole in the experimental group, vector control group and blank control group cells (A), draw the growth curve of the cells in each group.6. Collected each group cells. Flow cytometer were used to determine the variation of cell cycles and apoptosis through PI and FITC-Annexin-V dying. The datas were analyzed by CellQuest acquistion and analysis program.7. Experimental group, vector control group and blank control group were cllected, then, the enzyme-linked immunosorbent assay was used to detect cell Caspase-3, Caspase-9activity.8. Transwell invasion assay was used to detect changes in vitro invasion capacity of the cells in each group. Matrigel glue was spread evenly on the surface of the polycarbonate membrane.100μL cell suspensions with three groups were added into the upper chamber.500μL DMEM complete medium was added into the lower chamber containing a chemokine. Incubated at37℃incubator for48h, and calculated invasion inhibition rate.Results1. The recombinant HPV-16E6-E7lentiviral sequencs were conformed to the design.2. Lentiviral titer measurement results:reorganization of the HPV-16E6-E7 lentiviral titer was1.15x108MOI, and empty vector control lentiviral titer was2.09x108MOI.3. The hypopharyngeal squamous cell carcinoma FaDu cells which recombinant HPV-16E6-E7lentiviral infection and screening results show that recombinant HPV-16E6-E7lentiviral liquid and empty vector control lentivirus infection after48hours with a fluorescent microscope view of the investigation, bright green fluorescent protein seen in cells infected with the virus. It showed that stably transfected with the recombinant HPV-16E6-E7Lentiviral FaDu cells stably transfected FaDu cells transfected with empty vector control lentivirus.4. FaDu cells were collected after infection of recombinant lentivirus. PCR was used to identify the E6and E7genes.1.5%agarose gel electrophoresis showed a bright strip near at500bp and474bp consistent with the theoretical value of the HPV-16E6gene sequence; also have a bright strip near the250bp consistent with the theoretical value of297bp of the sequence of HPV-16E7.5. RT-PCR confirmed that the lentiviral stability infected FaDu cell E6, E7mRNA were expressed at high levels. The experimental group E6, E7mRNA relative expression level (2.6±0.22and1.8±0.12) was significantly higher than the vector control group (0.003±0.0001and0.003±0.0002) and untransfected group (0.002±0.0002and0.005±0.0001). This lentiviral stable infection FaDu cell E6, E7showed a high level of expression (P<0.05).6. Western blot analysis showed significantly E6, E7protein blot bands of the lentiviral stability infected FaDu cells. Consistent with the results of the RT-PCR. Preparation of recombinant HPV-16E6-E7infection of human pharyngeal squamous cell carcinoma cells FaDu cells capable of efficient express E6, E7protein.7. CCK-8cell proliferation assay kit determined the absorbance value of the cells in each group hole (A). Experimental group in2d,3d,4d,5d absorbance values were0.6418±0.0391,1.0808±0.0878,1.3598±0.0464,1.5936±0.1107. Comparing with vector control group (0.5442±0.01490,0.8952±0.0358,1.2002±0.0549,1.3848±0.0388) and blank control group (0.5268±0.0320,0.888±0.03302,1.2162±0.0320,1.4228±0.0444), there is a significant difference (p <0.05).8. Cell apoptosis was detected by flow cytometry (FCM). It showed that the experimental group (7.246±0.815) comparing with the vector control group (13.464±0.609) and blank control group (13.298±1.324) the apoptosis rate the were significantly reduced, which have a significant difference (p<0.01). Comparing with the blank control group and vector control group, the average rate of apoptosis was no significant difference (p>0.05). Pharyngeal squamous cell carcinoma FaDu cell genomic integrated with HPV E6-E7can inhibit pharyngeal squamous cell carcinoma FaDu cell apoptosis.9. ELISA was used to detect the Caspase-3, Caspase-9activity:Caspase-3relative activity of the experimental group, vector control group and blank control group were1.398±0.106,2.538±0.24and2.587±0.19. Caspase-9relative activity were1.268±0.083,1.992±0.13and1.984±0.11. Compared with the experimental group and the control group (blank control group and vector control group), the Caspase-3, caspase-9relative activity was significantly reduced, there were significant differences (p<0.01).10. Flow cytometric analysis of cell cycle:percentage of experimental group cell cycle:G0～G1phase, S phase and G2～M phase were53.816±1.665,28.836±1.013and17.348±0.878. Vector control group were62.284±1.609,22.292±0.970and15.424±1.236; blank control group were62.262±2.139,22.664±1.551and15.074±1.451. Blank control group and vector control group in S phase and G2/M phase and G0/G1, the difference was not significant (p>0.05). The experimental group compared with the control group (blank control group and vector control group), was increasing in S phase and G2/M phase, reducing the proportion of G0/G1phase, there was a significant difference (p<0.05). It suggested that Pharyngeal squamous cell carcinoma FaDu cells genomic integration of HPV E6-E7can increase the proportion of S phase and G2/M phase cells, reduce the proportion of cells in G0/G1phase, promote cell division and proliferation.11. The Transwell cell experimental results showed:the average number of cells in vitro invasion assay within the field were:the experimental group was117.4± 14.8, and the vector control group and blank control group were68.4±8.2and72±8.1. Comparing with the experimental group and two control groups (vector control groupblank control group), the mean cell number of penetrate the Matrigel was increased significantly, and there were significant differences (p <0.01). It suggested that Pharyngeal squamous cell carcinoma FaDu cell genomic integration of HPV-16E6-E7can significantly increase pharyngeal squamous cell carcinoma FaDu cell.Part three Regulation of HPV16infection squamous cell carcinoma of the hypopharynx and FaDu cell miRNA expressionMethods1. Stable cultured the integration of HPV-16E6-E7FaDu cells and uninfected lentiviral FaDu cells. To confirm three only HPV-16infection and twenty-one HPV-negative fresh frozen hypopharynx squamous cell carcinoma specimen.2. Extracted human hypopharyngeal squamous cell carcinoma tissue and cellular total RNA.3. qRT-PCR was used to detect the total cellular RNA of miR-363, miR-15a and miR-155expression levels in each group of specimens and cell.Results1. Hypopharyngeal squamous cell carcinoma tissue Only infected with HPV-16express miR-363, miR-15a and miR-155. The expression levels were5.62±3.48,1.97±0.48and0.34±0.08. Hypopharyngeal squamous cell carcinoma of HPV-negative specimensin miRNA expression levels were0.90±1.53,1.09±1.31and1.92±1.47. Each miRNA expression levels in the experimental group were6.75±0.17,4.15±0.13and0.16±0.01. miRNA expression levels in the vector control group cells were0.09±0.01,0.20±0.01and7.01±1.04. And the blank control group cells miRNA expression levels were0.09±0.003,0.21±0.003and4.14±0.02.2. In HPV-16positive human hypopharyngeal squamous cell carcinoma, the miR-363, miR-15a expression were significantly raised, while miR-155 expression was no significant change. Hypopharyngeal squamous cell carcinoma FaDu cells transfected with the HPV-16E6-E7genes, the miR-363, miR-15a expression were significantly raised. While the miR-155expression was no significant change. There was no significant difference between HPV positive and HPV-16E6-E7infected human hypopharyngeal squamous cell carcinoma FaDu cells in miR-363and miR-15a and miR-155expression levels. And slo, There was no significant difference between HPV negative human hypopharyngeal squamous cell carcinoma and FaDu cells in miR-363and miR-15a and miR-155expression levels.Conclusions1. Hypopharyngeal squamous cell carcinoma in the presence of HPV infection, HPV-positive specimens is25.0%(7/28), HPV-16is the most common infection type.2. The subject includ28cases of hypopharyngeal squamous cell carcinoma patients. Compare with HPV-negative patients, less HPV-positive patients have heavy consumption of alcohol and/or tobacco use history. There are significant differences.3. Human hypopharyngeal squamous cell carcinoma FaDu cell genome integrated with HPV-16E6-E7, the cell proliferation and invasiveness are enhanced, apoptosis is suppressed, the proportion of S phase and G2/M phase is increased, and G0/G1phase is reducded.4. Comparing with HPV-16positive hypopharyngeal squamous cell carcinoma and FaDu cells transfected with the HPV-16E6-E7genes, there have significantly raised in miR-363, miR-15a expression, while there has no significant change in miR-155. |
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