The Effects Of TET1 On The Biological Behavior Of Cervical Cancer Hela Cells

Objective: Through the CRISPR/Cas9(clustered regularly interspaced short palindromic repeat)technology to build TET1 knockout Hela cells line,to explore the effects of TET1 on Hela cells proliferation,migration,invasion and tumorigenicity,for clinical targeted therapy of cervical cancer.Methods:The TET1knockout-Hela cell lines were constructed by CRISPR/Cas9 technology(clustered regularly interspaced short palindromic repeat),which were defined as the experimental group.First,two single chain oligo DNA were designed,and then they were synthetized by dsDNA to connect with pGK1.2 plasmid.The plasmid was transfected into Hela cells by liposome chemical transfection method,and then Hela-cell DNA was extracted to detect the efficiency of TET1 knockout.Finally,they were sent to do DNA sequencing by Shenggong Company,and then Hela-cell protein was extracted to detect the expression of TET1 protein by Western blot method.The wild Hela cells were defined as control group.The proliferation ability of the experimental and control group Hela cells were explored by the real-time dynamic label free cell analysis technology(RTCA,Real Time Cellular Analysis).In the xCELLigence RTCA S16 system,each group cells were cultured for 90 H(n=8)in the same cell number,and then the each group CI values(Cell Index,CI)were analyzed.The migration ability of the experimental and control group Hela cells were studied by scratch assay.In the 6-well plates,each group cells were cultured for 24h(n=3),and then we make scratches in each well.Cell scratch healing rates were calculated and recorded.The migration and invasion ability of the experimental and control group Hela cells were studied by Transwell(Transwell chamber,Transwell insert)assay.In the Transwell chamber,each group cells were cultured for 24 h or 48h(n=3)in the same cell number,and then the number of each group which passed through the Transwell chamber were calculated and recorded.The invasion ability of theexperimental and control group Hela cells were studied by tumor formation in nude mice.The cells of logarithmic growth phase were inoculated into nude mice subcutaneous inguinal(n=3).After we continuous observed 6 weeks,the tumor in nude mice was removed for measuring,and photographing and analyzing the cells ability of tumor formation.Results: After the synthesis of Case9 vector,it was found that only TET1(cas9)-2F/2R had the target band at 100 bp,some of the bacteria containing the target band were sent to measure the DNA sequencing.After transfection,Hela cells transfected with blank plasmid was not fluorescent reaction,but the cells transfected with TET1-gRNA plasmid Hela cells emitted more and more green fluorescence reaction as the time.It showed that the TET1-gRNA plasmid was transfected into Hela cells successfully.Hela cells transfected with TET1-gRNA plasmid(No.1 and No.2)and wild-Hela cells which were selected as the control group.Genomic DNA was extracted and verified by T7E1 method.The knockout cells cutting efficiency was 54% and 30%,respectively.The results of Western blot in each group showed that TET1 protein was expressed normally in wild group Hela cells,but the expression level of TET1 protein in the knockout group was significantly decreased or absent.Gene sequencing results also demonstrated that we successfully obtained the TET1 knockout monoclonal Hela cells line.The 90 h CI value of the TET1 knockout cells and the wild-Hela cells were 1.04±0.86 and1.47±1.01 respectively Results showed that the CI value of TET1 knockout-Hela cells were greater than that of wild-Hela cells group(P<0.05).The migration rate of TET1knockout-HeLa cells at 24 h and 48 h were 29 ± 4% and 51 ± 7%,while the wild type group migration rate of HeLa cells were 19 ± 4% and 31 ± 4%.Results showed that the migration rate of TET1 knockout-Hela cells were greater than that of wild-Hela cells group(P<0.05).The invasion number of TET1 knockout-HeLa cells through Transwell chamber at 24 h and 48 h were 158 ± 6 and 206 ± 12,while the wild type HeLa cells were 109 ± 4 and 142 ± 7.Results showed that the number of TET1knockout-Hela cells passed through Transwell chamber were greater than that of wild-Hela cells group(P<0.05).The formation-tumor size of TET1 knockout-HeLa cells was 362 ± 41 mm3 in mice,while wild-HeLa cells were 215 ± 23 mm3.Resultsshowed that the volume of tumor formed from TET1 knockout-Hela cells were bigger than that of wild-Hela cells group(P<0.05).Conclusion: The TET1 knockout cervical cancer Hela cells lines were successfully constructed by CRISPR/Cas9 technology.The proliferation,migration,invasion,and invasion ability of TET1 knockout group was higher than that of wild Hela cells.TET1 gene knockout may promot the proliferation,migration,invasion,and invasion of Hela cells.