A Preliminary Study On The Effect Of TET1 On The Biological Behavior Of Cervical Cancer Cells And Its Mechanism

Objective: To use CRISPR/Cas9 in Si Ha and He La cells of cervical cancer to establish stable cell lines with knocked down TET1 gene,and to study the effect of TET1 gene on the biological behavior of cervical cancer cells and its related mechanism.Methods: Two pairs of sg RNA were designed for the TET1 gene using CRISPR/Cas9 technology: Oligo-TET1-F1/R1,Oligo-TET1-F2/R2,The plasmids of CRISPRv2 vector were digested by Bsm BI enzyme,then the two pairs of sg RNA were annealed and ligated to CRISPRv2 vector,then the linked products were transformed into the receptive cell DH5?,then the monoclonal colonies were selected for expansion c ?lture and sent to the detection sequence.According to the sequencing results,lenti CRISPRv2-sg RNA recombinant plasmids and CRISPRv2 plasmids were selected and transfected into cervical cancer cells by lentivirus packaging,and were used as TET1 knockdown experimental group and empty vector group,respecti vely.Cervical cancer cells without any treatment were used as the wild type group.The genomic DNA of cervical cancer cells in each group was extracted.The knock-down res?lts of TET1 were verified by T7E1 enzyme digestion,and the proteins of cervical cancer cells were further extracted,and the expression of TET1 was detected by Western blot.D uring the conventional c?lture period,observe the changes of cell morphology;detect the migration and invasion ability of cervical cancer cells in the wild type group,the vector group and the TET1 knockdown group by using wound healing experiment and tr answell invasion test;detect the proliferation ability of cervical cancer cell s in different groups by using real-time unlabeled dynamic cell analysis(RTCA)and MTT proliferation test;the colony formation assay was used to detect the colony formation ability of different groups of cervical cancer cells;the cell cycle of cervical cancer cells in different groups was detecte d by flow cytometry;the expression of E6 protein,p53 protein,Autophagy related molec ?lar indicators protein and molec ?lar markers associated with epithelial mesenchymal transformation protein was detected by Western blot in different groups of cervical cancer cells.Res?lts: 1)the res?lts of recombinant plasmid sequencing showed that lentiviral vectors expressing Cas9 and sg RNA were successf?lly constructed using the second pair of oligo-TET1-F2/R2 primers,and the res?lts of T7E1 enzyme digestion and Western blot indicated that TET1 was successf?lly knocked down.2)After the knocking down of TET1,the cell morphology of cervical cancer Si Ha and He La cells changed significantly,the cells became longer,and the cells showed more pseudopod changes.The corresponding experimental detection of TET1 knockdown cells showed that TET1 knockdown promoted the cell migration,invasion,proliferation and clonal formation,significantly reduced the G0/G1 phase cells,while the S phase and G2/M phase,namely the DNA synthesis phase and the DNA synthesis anaphase,significantly increased the cell cycle process and accelerated the cell growth.3)Western blot res?lts showed that knocking down TET1 in Si Ha cells co?ld promote the expression of E6 protein and inhibit the expression of p53 protein.4)Western blot results also showed that knocking out TET1 in Si Ha cells affected autophagy and promoted epithelial mesenchymal transformation(EMT)in cervical cancer cells.Conclusion: 1)Using CRISPR/cas9 technology,TET1 knockdown cell line were successf ?lly constructed;2)TET1 has the ability to reg ?late the biological behavior of cervical cancer cells;3)TET1 can affect the expression of E6 protein and p53 protein in cervical cancer cells;4)TET1 may have the ability to regulate epithelial mesenchymal transformation(EMT)and affect autophagy in cervical cancer cells.