Effects Of Knocking Out HPV16E6/E7by TALEN On The Malignant Properties Of Cervical Cancer Caski Cells

Background It has been proved that the high risk human papillomavirus (HR-HPVs), especially HPV16, have a close etiology relationship with the development and progression of cervical cancer by more researches. Consistent expressions of E6and E7viral oncoproteins are the key to forming and maintaining tumors malignancy phenotype. In addition, E6and E7viral genome derived from HPVs which exist only in infected cells, are ideal targets for gene therapy. Transcription activator-like effectors nucleases (TALEN) is a new genetic engineering technology. It can avoid the defects of incomplete, poor stability interference of RNA interference (RNAi). In this study, it was intended to knock out HPV16E6/E7by TALEN recombined plasmid and observe the changes of various biological behaviors in CaSki cells.Objectives Studying the effects of knocking out HPV16E6/E7by TALEN on the malignant properties of cervical cancer CaSki cells that attempt to clarify the treatment feasibility for cervical carcinoma and provide new clues and theoretical evidence for the treatment of HPV16-related cancer after knocking out HPV16E6/E7by TALEN technology.Methods1. Taken HPV16-positive human cervical cancer CaSki cells as targets, TALEN left arm and right arm targeting HPV16E6and E7were designed based on principles of TALEN plasmid designing and synthesized following the kit of TALEN plasmid. They were named as HPV16E6-L, HPV16E6-R and HPV16E7-L, HPV16E7-R, respectively.2. TALEN empty vectors were transfected into HPV16-positive human cervical cancer CaSki cells and recombinant plasmids HPV16E6-L, HPV16E6-R were transfected into other CaSki cells, which were screened by puromycin and five monoclonies were picked and amplified. Cell clones knocked out HPV16E6were screened by reverse transcription-polymerase chain reaction (RT-PCR). Then HPV16E7-L, HPV16E7-R plasmids were co-transfected to the selected cell clone. Cell clone HPV16E6and E7were knocked out was screened through dilution method and RT-RCR. And E6/E7protein expressions were detected by Western blotting in the above cells.3. The flat cloning experiments, flow cytometry, Annexin V-FITC/PI combined with flow cytometry experiments, Transwell and Caspase-3activity assay experiments were used to investigate the proliferation, apoptosis, migration and invasion of CaSki cervical cancer cell after knocking out HPV16E6/E7.Results1. CaSki cells transfected with HPV16E6-L and HPV16E6-R and empty TALEN plasmid were named:CaSki-TALEN-vect cells and CaSki-TALEN-E6cells. RT-PCR showed that clone2and clone5did not express E6mRNA in five CaSki-TALEN-E6cell clones. The HPV16E7-L and HPV16E7-R were co-transfected into CaSki-TALEN-E6cell clone5and five monoclonies were selected by dilution method. RT-PCR results showed that clone1did not express E7mRNA in the above five cell clones and was named CaSki-TALEN-E6/E7. Western blotting was used to detect E6and E7expression in CaSki cells, CaSki-TALEN-vect and CaSki-TALEN-E6/E7cells at the protein level, which results showed E6and E7proteins were expressed in CaSki cells and CaSki-TALEN-vect cells, the difference was not significant (P>0.05), while there was no expression of E6and E7in CaSki-TALEN-E6/E7cells.In view of the above findings, CaSki-TALEN-E6/E7cells were taken as the experimental group, CaSki-TALEN-vect cells as the control group in the following experiments.2. Effects of TALEN Knocking out HPV16E6/E7on the Malignant Properties of CaSki cells (1) The impact on proliferation of cervical cancer CaSki cell:â‘ the results of cell growth curve and the doubling time suggested that compared with CaSki-TALEN-vect cells, the growth rate of CaSki-TALEN-E6/E7cells were significantly slowed down(P <0.01), doubling time were significantly longer(P<0.01).â‘¡Colony formation assay results showed cell colony formation of CaSki-TALEN-E6/E7cells were lower than CaSki-TALEN-vect cells (P<0.01), and the clone volume were small.â‘¢Flow cytometric analysis of cell cycle distribution showed compared with control groups, CaSki-TALEN-E6/E7cells in Gl phase increased (P<0.01), but significantly decreased S phase cells (P<0.01), appeared G2/M phase arrest (P<0.01).(2) Impact on cervical cancer CaSki cell apoptosis:â‘ Annexin V-FITC/PI results revealed apoptosis was higher in CaSki-TALEN-E6/E7cells compared with CaSki-vect cells (P<0.01).â‘¡Caspase-3activity assay showed that compared with control cells, the activity of caspase-3in CaSki-TALEN-E6/E7cells were increased (P<0.01).(3) Impact on cervical cancer CaSki cell migration and invasion:Transwell migration and invasion assay results displayed that compared with CaSki-vect cells, CaSki-TALEN-E6cells and CaSki-TALEN-E7cells reduce the number of cells in a small room at the end membranes significantly (P<0.01).Conclusions1. TALEN eukaryotic expression recombinant plasmids of targeting HPV16E6and E7were constructed successfully and cell clones knocking out HPV16E6and E7were screened.2. The effects on the malignant behavior of CaSki cells after TALEN knocking out HPV16E6/E7include:â‘ the proliferation in cervical cancer CaSki cell was inhibited,â‘¡the apoptosis of cervical cancer CaSki cell was promoted,â‘¢the ability of migration and invasion in cervical cancer CaSki cell was decreased.