The Mechanism Of Knockouting Cxcr4 Gene To Inhibit The Invasion Of 4T1 Cell By CRISPR/Cas9 System

Background and ObjectiveEvery year,approximately 500 thousand women die from breast cancer,which is a malignant tumor often occurs on women.The obstacle to cure the disease is the metastasis of cancer cells.Many studies have shown that tumor cells will spread to other tissues through the lymph nodes or vascular after cancer cells proliferate at the origin.Furthermore,The tumor cells themselves can secrete vascular endothelial growth factor,that facilitates the vascularization,accelerating its migration.bone is the main position in breast cancer metastasis.Recent years,it has been found that CXCR4 is highly expressed in breast cancer cells,and the expression rate of CXCR4 in high metastatic breast cancer cell lines is higher than that in low metastatic breast cancer cell lines.It has been confirmed that CXCR4 can promote cell migration of hemopoietic stem cell,as the specific ligand for SDF-1.There are numerous studies showing that the SDF-1/CXCR4 axis can activate intracellular signaling pathways and its downstream effector can regulate cell proliferation,chemotaxis and metastasis.regulate cell proliferation,chemotaxis,metastasis and other physiological functions.Therefore,the CXCR4 knocking out of 4T1 cell lines is established by CRISPR/Cas9 system,trying to find out the roles of SDF-1/CXCR4 signal axis in the proliferation and migration of breast cancer cells.At the same time,it's being explored the interaction between SDF-1/CXCR4 signal axis and the integrin,?v?3.Methodsa.Two sgRNA were designed,and LentiCRISPRv2 was used as the carrier to construct the LentiCRISPRv2-sgRNA recombinant plasmid and transformed into the competent cells Stb13.b.The recombinant plasmids were transfected into 293T cells and packaged into lentivirus.The suspend containing lentiviruses was collected and infected the 4T1 cells.The monoclonal cells were isolated and purified by puromycin.The expression of CXCR4 was detected by RT-qPCR,western blotting.c.The proliferation activity of 4T1 cells after knockout was detected by CCK-8,and the migration ability of the cells was detected by scratch test and trans well assay.In addition,the chemotactic effect of SDF-1 on cell migration was detected.d.After being treated with SDF-1 at 100ng/ml for 12 hours,the expression level of intracellular protein and phosphorylation of Akt,Erk and NF-?B were detected by western blot.e.After 4T1 cells with different concentrations of SDF-1 treated 4h,8h,12h,24h;and after MAD-MB-231 cell with different concentrations of SDF-1 treated 24h,the expression of integrin alpha v subunit and beta 3 subunit protein were detected by WB.Resultsa.LentiCRISPRv2-sgRNA recombinant plasmid was successfully constructed.b.The lentivirus was successfully packaged,and the monoclonal cells were successfully screened by puromycin after the virus infected 4T1 cells.The 4T1-C cell line,which had 27 bases missing from the CXCR4 gene was successfully obtained.c.The results of cell proliferation by CCK-8 showed that the cell proliferation rate of the cells(4T1-C)that knocked out the CXCR4 gene was significantly inhibited after the growth of 24h(P<0.05),and the inhibitory effect was similar to that of the cells treated with AMD3100.d.In the scratch test,the cell migration rate was significantly reduced compared with the normal group cells(4T1-C)except the CXCR4 gene.The Transwell assay showed that SDF-1 had obvious chemotaxis(P<0.05)on cell migration,but the chemotaxis effect of SDF-1 on the cell was not statistically significant when the cells were knocked out of the CXCR4 gene.After analyzing of all the final experimental results,it showed that different concentrations of SDF-1 had significant effect on chemotactic migration of 4T1-F and 4T1-L,but not the 4T1-C,and there is significant difference on the number of cell migration among 4T1-F,4T1-C and 4T1-L.Difference;in the concentration range of 200ng/ml,the higher the concentration of SDF-1 is,the more obvious the chemotaxis effect on 4T1-F and 4T1-L cell migration.e.WB results showed that there was no significant changes in the expression of integrin avP3 in 4T1-F cells and MAD-MB-231 cells treated at different concentrations of SDF-1.After 4T1-F and 4T1-C were treated with SDF-1,there was no significant changes of the total Akt protein expression in the cells(P>0.05),the expression of Erk and NF-?B total protein in 4T1-F cells increased(P<0.05),and the expression of Erk total protein in 4T1-C cells was unchanged,and the expression of total protein was decreased.The expression of p-Akt,p-Erk and p-NF-?B in 4T1-F cells increased(P<0.05).The expression of p-Akt,p-Erk and p-NF-?B in 4T1-C cells unchanged.Conclusionsa.Recombinant plasmids targeting the CXCR4 gene were obtained by CRISPR/Cas9 gene editting system,and the cell strain with stablely knockout CXCR4 gene was successfully obtained.b.The cell proliferation and migration ability of knockout CXCR4 gene was significantly decreased.c.The combination of SDF-1 and CXCR4 can activate of the downstream PI3K-AKT,MAPKs/ERK,and NF-?B signaling pathway,while the cells that knock out the CXCR4 gene can not effectively activate the downstream PI3K-AKT,MAPKs/ERK,NF-?B signaling pathway.d.In breast cancer bone metastasis,there is no significant correlation between SDF-1/CXCR4 pathway and integrin alpha v beta 3 pathway.