CRISPR/Cas9 Mediated ?Np63? Knock In Can Promote The Mouse Pancreatic Cancer Cells' Stemness And Increase Their Sensitivity To Gemcitabine

Background and ObjectivePancreatic cancer is one of the most lethal malignancies and has been the fourth leading cause of cancer death worldwide.It has no effective means to diagnosis in the early stage and the median survival has only 6 to 8 months with a five-year survival <5%.Recent studies have found multiple genetic abnormalities which have been implicated in the complex process of pancreatic cancer and constructed the KC(LSL-KrasG12D/+,Pdx-1-Cre)and KPC(LSL-KrasG12D/+,Trp53R172H/+,Pdx-1-Cre)genetically engineered mice to mimic the pancreatic cancer.P63 is a member of the p53 transcription factors family which has been considered as a tumor suppressor gene in many cancers.Due to the alter promoter,p63 can be divided into TAp63 and?Np63 two subtypes.?Np63? is the primary subtype of p63 which plays a key role in cancer.?Np63? competes with p53 in the way of dominant negatively and inhibits the expression of p53 downstream gene and considered to be oncogene,but some studies demonstrate that it can combine the mutants of p53 and play a role of cancer suppression gene.In gastric cancer,?Np63? inhibits tumor cell proliferation wheras it can promote tumor cell proliferation in breast cancer.In lung cancer,?Np63? inhibits cell migrations,however,it can promote cell migration in breast cancer.In patients with breast cancer,head and neck squamous cell carcinoma and lung cancer,tumor with highly expressed ? Np63? exhibition a sensitive to chemotherapeutic dr?gs;whereas in bladder cancer,ovarian cancer and prostate cancer,highly expressed ?Np63? will reduce the sensitivity of the chemotherapeutic agent.In order to solvethese above problems,this study use the CRISPR / Cas9 gene editing technique to target the ?Np63? gene,knocking into mouse pancreatic cancer cell line DT6606.To study its role in tumor cell proliferation,migration,stemness and chemotherapeutic drug sensitivity.Methods1.The construction of px330-Rosa26-sgRNA cleavage vector and homologous recombination targeting vector.The sgRNA sequence targeting on mouse gene Rosa26 is designed by the ZIFIT website and ligated into the px330 plasmid to construct the px330-Rosa26-sgRNA vector.The target gene fragment is got by PCR.After the CMV promoter and zeocin antibiotic marker obtained on the pcDNA3.1 plasmid,the fragment is ligated into the pRosa26-1 homologous arm vector to construct the homologous recombination targeting vector pRosa26-? Np63?.2.Cell transfection and positive cells screening.The mouse pancreatic cancer cell line DT6606 is co-transfected with pRosa26-?Np63? and sgRNA vector by Lip2000 liposome transfection reagent.The transfected cells are obtained by antibiotic marker Zeocin,PCR is used to identify whether ?Np63? has been knocked in.The qPCR is used to detecte the fragment of Rosa26 gene to test wheather?Np63? has been knocked in prospective position.3.The expression of ?Np63? and its effects on the proliferation,migration,stemness and chemosensitivity of pancreatic cancer cells.The expression of ?Np63? is detected in RNA and protein levels by qPCR and Immunocytochemistry.The proliferation of cell pop?lation is observed by long-term living cell imaging system.The proliferation ability of single cell is detected by cloning formation experiment.Cell migration ability is measured by stem cell spheroid formation experiments.MTS is used to detect the sensitivity of cells to chemotherapeutic dr?gs.Res?lts1.The CRISPR/Cas9 system px330-Rosa26-sgRNA cleavage vector and homologous recombination targeting vector pRosa26-?Np63? have been constructed successfully.2.Screening of monoclonal cells by limiting dilution,we have successfully construct mouse pancreatic cancer cell sublines 1B9 and 2D5,which are knocked in the ?Np63? gene at the Rosa26 site,while 1B9 has a higher expression of ?Np63?than 2D5.3.Compared to DT6606 cells,the morphology of 1B9 cells are changed,the cellscould be oval and grow into groups.The boundaries between cells are unclear but there are no significant differences in 2D5 cells.4.Compared to DT6606 cell,2D5 cell has a stronger proliferative and migration ability and the proliferation ability of 1B9 cells has a significant decrease and the migration ability is not changed.In tumor stem cells sphere-formation assay,1B9 and2D5 have increased spheres,but 1B9 cells have more spheres(P<0.001).5.Compared to DT6606 cells,1B9 and 2D5 have increased sensitivity to gemcitabine which is pancreatic cancer chemotherapeutic drugs;1B9 is less sensitive than 2D5(P<0.001).Conclusions1.The cell sublines 1B9 and 2D5 of mouse pancreatic cancer cell line DT6606 which are inserted at the Rosa26 site and highly expressed ?Np63? have been successfully constructed.2.The mouse pancreatic cancer cells exist heterogeneity in the cell subline formation,proliferation,migration,cell stemness and chemotherapeutic drug sensitivity.3.The stemness of mouse pancreatic cancer cells and sensitivity to chemotherapeutic drugs gemcitabine can be promoted by knocking ?Np63? in prospective site of Rosa26 gene.