| Objective To knock out integrin?v?3 gene in 4T1 cell by CRISPR/Cas9 system and to construct 4T1 cell line with knocking out integrin?v?3 gene stably;Futher more,to investigate the changes of PI3K,AKT,ERK,NF-?B p65 and I?Ba expression and its phosphorylation in the presence of BSP and integrin,And to explore the role of blocking integrin expression in inhibiting bone metastasis in breast cancer.Methods 1.Four sgRNAs targeting exons of integrinav or?3 gene were designed to construct lentiCRISPRv2-sgRNA recombinant plasmid and transformed into competent Stbl3.Then the recombinant plasmids were screened for sequencing and transfected into HEK293Ft cells to packaging into lentivirus.2.After infection of 4T1 cells with lentivirus,stable transfected cells were selected by puromycin,and monoclonal cells were isolated and cultured by limiting dilution method.3.The genomic DNA of monoclonal cells was extracted and sequenced.The expression of integrinavP3 mRNA was detected by qRT-PCR.The expression of integrinavP3 protein was detected by Western blot.4.The proliferation of mouse breast cancer 4T1 cells was detected by CCK-8 and the migration ability of mouse breast cancer 4T1 cells was detected by scratch and transwell.In addition,the adhesion ability of mouse breast cancer 4T1 cells was detected by Caclein-AM.5.After the cells were harvested with 200ng/ml bone sialoprotein,their total protein was collected at Oh,6h,12h and 48h.Then the level of intracellular protein and phosphorylation of PI3K,ATK,ERK,NF-?B p65 and I?Ba were detected by western blot.Results 1.The lentiCRISPRv2-sgRNA recombinant plasmid was successfully constructed,and a cell line of knocking out integrinav subunit with deletion 1bp and?3 subunit with deletion of 3bp were obtained.The expression level ofav or ?3 subunit mRNA was low,and there was almost no expression of integrinav or ?3 subunit protein in cell line.2.CCK-8 detection of cell proliferation activity results shows:knockout integrinav subunit or integrin?3 subunit inhibition of mouse breast cancer cell 4T1 proliferation,its inhibition than the corresponding single antibody blocking effect was more significant.In addition,in 3d and 5d the knockout integrinav subunit combined with integrin?3 antibody,knockout integrin?3 subunit combined with integrinav antibody further inhibited the proliferation of 4T1.3.Knockout of the integrinav subunit cell group(T5)and the knockout integrin?3 subunit cell group(T6)showed a decrease in cell migration in the scratch test compared to the normal 4T1 cell group.4.In transwell test,the number of migrating cells in the normal 4T1 cells and the empty vector cells was similar,and the number of migrating cells to the integrin subunit group was decreased.The number of cells migrated after 4T1 cells increased after treated by bone sialoprotein.5.In cell adhesion assay,normal 4T1 cells and cells containing empty carriers had a similarly adherence,and the cell adhesion was reduced after addition of the antibody.The adherence ability of knockout integrin subunit cells was significantly lower,and the adhesion ability of knockout integrinav subunit cells was significantly weaker than that of cells treated by integrinav antibody.6.After using with BSP,the expression of phosphorylated AKT and the expression of ERK1/2 increased,and the expression of total PI3K in the integrin av subunit cells increased,while the expression of I?Ba and NF-?B p65 and their's phosphorylation have on obvious change.Conclusions 1.Recombinanted plasmids targeting the integrinav or p3 subunit gene were obtained by CRISPR/Cas9 system,and the cell lines with stable knockout integrinav or ?3 subunit gene were screened out.2.After knockout of integrinav subunit or ?3 subunit,the cells' proliferation,the cells' migration ability and the adhesion ability to BSP were decreased.3.BSP can increase the migration and adhesion of mouse breast cancer 4T1 cells.4 Bone sialoprotein(BSP)can increase the phosphorylation of AKT and ERK1/2 in mouse breast cancer 4T1 cells. |
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