The Establishment Of Lentiviral-vector With ShRNA Targeting Common Promoter Of HPV16E6and E7Oncogenes And Inhibition Of Cervical Cancer Cell Growth In Vitro And In Vivo

Cervical cancer (CC) is the second most common type of malignancy among women worldwide. Despite well organized screening, the occurrence of invasive cervical cancer is still high. Surgery and radiation therapy are the radical therapy to cervical cancer, but surgery only apply to the early cases. Radiation therapy is suitable for each period case, but could cause irreversible damage for ovary and vagina, and has serious impact on young patient's quality of life. Cervical cancer only has medium sensitive to chemotherapy, so chemotherapy is mainly used for the auxiliary treatment to locally adanced cases and the palliative treatment to late cases at present. So the prognosis of the invasive cervical cancer is not optimistic. Therefore, it is of important practical significance to seek new treatment besides of conventional surgery, radiotherapy and chemotherapy to improve the prognosis of cervical cancer.It is well known that the persistent infection of high-risk human papillomavirus (HR-HPV) is necessary cause of cervical carcinogenesis. Among all the HPV genotypes, HPV-16alone is responsible for development of above50%of cervical cancer. The continuous expression of both high-risk viral oncogenes, E6and E7, are essential for malignant transformation and the maintenance of the malignant phenotype of cervical cancer. Therefore, these viral oncogenes are considered to be the ideal targets for a novel block therapy for HR-HPV-positive cervical cancers.RNA interference (RNAi) has been proved to be a powerful tool to specifically silence gene expression and holds a great promise for the treatment of viral diseases, genetic disorders and cancer. Because of the exogenous gene of HPV in cervical cancer and has no homology to human genom, along with clear targets provide an ideal model for gene block therapy.RNAi can be divided into the transcriptional and post-translational level. Since the expression of HPV16E6and E7is controlled by a common promoter (P97), we offered a novel way of slencing gene, other than traditional method by degrading homologous mRNA at the post-transcriptional level. The mechanism of transcriptional gene silencing is that the siRNA which is homologous to the CpG island of promoter could via epignetic changes such as RNA-directed DNA methylation, histone modification and euchromatin formation, etc, to induce transcriptional repression of target genes. The advantage of this method is that with little variation of DNA sequence in epignetic changes, and hereditable to offspring.The right carrier is significant to conduct RNAi. As a gene delivery system, lentiviral vector has been explored for preclinical or clinical application and shown promising results. Compared to other virus carrier, LV is characterized by simple operation, high efficiency of infection, ability to infect multiple cell types, relatively large packaging capacity, stable transduction which is more suitable for application in cancer and good safety, etc, and become potent and versatile vector for gene therapy and transgenic animals.In current study, we first established lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes and E6transcript, then transduced the lentiviral construct into cervical HPV16positive cell lines Siha and Caski, selected and established stably transduced monoclonal cell lines, tested for E6and E7expression at mRNA and protein levels, along with E6-related proteins, MCM7and p53, as well as E7-ralated proteins, pRB and p16in the monoclonal cell lines. Then we observed the cell proliferation and invasiveness of the monoclonal cell lines compared to normal cell lines. Finaly, we subcutaneously injected into female BALB/c mice with Siha and Caski stably transduced monoclonal cell lines and related negative control, detail recorded tumorigenesis and tumor growth as well as body weight of each mouse. At the halfway point, euthanasia was used to part of BALB/c mice and excised xenografts were weighted, the remaining mice were continued to observe survival time. To prove the effectiveness of LV-shRNA induced TGS and explore the E6and E7role in malignant transformation and the maintenance of the malignant phenotype of cervical cancer, and provide more experimental evidence for the new strategy and targets in the treatment of cervical cancer. Part I The establishment of lentiviral-vector with shRNA targeting common promoter of HPV16E6and E7oncogenesObjective:To establishment of lentiviral-vector with designed shRNA which target to the commom promoter of human papillomavirus16E6and E7, and provide tools for proving the effectiveness of siRNA mediated transcriptional stable gene sliencing in subsequent study.Methods:shRNA specific target to the commom promoter of human papillomavirus16E6and E7was designed according to BLOCK-iTTM Lentiviral RNAi Expression System, and was synthesised by ShengGong, Shanghai, then took shRNA connected to pENTRRM/U6entry construct, mini extracted and sequenced plasmid, then took the right pENTRTM/U6entry construct connected to pLenti6/BLOCK-iTTM-DEST to generate the expression construct, Mid extracted plasmid, double enzyme cutted, identified by electrophoresis. Then produced a lentiviral stock by co-transfecting the ViraPowerTM packaging mix and the correct expression construct into the293FT producer cell line. Finally, titered our lentiviral stock with clasical HT1080cell line and concentrated the lentiviral stock.Results:1. The sequencing result of mini extracted plasmid revealed the insert sequence was right. 2. The result of expression construct plasmid by electrophoresis identification was correct.3. The titer of our lentiviral stock was2.2*106TU/ml.4. The minimum concentration of blasticidin required to kill untransducted Siha, Caski and HT1080were4ug/ml,6ug/ml and4ug/ml separately.Conclusions:1. Successfully establish a lentiviral-vector with shRNA targeting common promoter of HPV16E6and E7oncogenes.2. Successfully produce a lentiviral stock with relatively high titer. Part â…¡ Lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes inhibit cervical cancer cell growth in vitroObjective:To prove the effectiveness of LV-shRNA mediated TGS of E6and E7, and explore the E6and E7role in maintenance of the malignant phenotype of cervical cancer.Methods:We first transduced the LV into Siha, Caski cell lines, determined the expression of laminA/C with Western-blot, and got the suitable MOI for each cell line. Then the LV were transduced into Siha, Caski cell lines with the suitable MOI separately, stably transduced cells were selected with Blasticidin. The best stable transduced monoclonal cells were chosen by infinite dilution method, followed by examining the expression of E6/E7mRNA by Realtime-PCR. Finally, the expression of E6, MCM7, p53, E7, pRB, p16protein were determined by Western-blot, and cell growth and invation were separately tested by MTT and Transwell,Results:1. The suitable MOI for Siha and Caski was2and5separately.2. Successfully selected the best stably transduced Siha and Caski monoclonal cell lines.3. Lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes remarkably reduced the expression of E6/E7mRNA. 4. Lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes effectively reduced the expression of E6, MCM7, E7, p16proteins, meanwhile, significantly raised the expression of p53and pRB proteins.5. Lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes effectively inhibited cell proliferation and invation of the stably transduced monoclonal cell lines.Conclusions:1. Lentiviral vector is an ideal carrier to conduct RNAi.2. LV-shRNA can effectively transfer therapic gene into cervical cancer cell lines, and establish the stably transduced monoclonal cell lines.3. Lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes can specifically inhibit the expression of E6/E7mRNA and protein, and specifically restrain cell proliferation and invation of the stably transduced monoclonal cell lines. Part â…¢ Lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes inhibite cervical cancer transplant tumor growth in vivoObjective:To prove the lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes could effectively inhibit the growth of xenograft in BALB/c mice, and provide new treatment for cervical cancer.Methods:We subcutaneously injected into female BALB/c mice with the Siha and Caski LV-shRNA stably transduced monoclonal cell lines, observed the tumorigenesis in vivo, and detail recorded tumor growth, body weight of each mouse as well as surial data.Results:1. Successfully established a tumor-burdened model, and presented100%tumorigenesis.2. There were no obvious differences between experimental group and the control group in body weight changes.3. Tumor growth was significantly slower in experimental group compared to related control group, and the excised tumuor weights were obviously lighter than the corresponding control group.4. The survival time were significantly longer in experimental group compared to the corresponding control group. Conclusions:1. Lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes can effectively restrain the growth of xenograft in tumor-burdened BALB/c mice.2. Lentiviral-vector mediated shRNA targeting common promoter of HPV16E6and E7oncogenes can significantly prolong tumor-burdened survival time.