Knockout Of MAGE-C2 Gene Using Cas9 RNP Method And Its Influences On Proliferation And Apoptosis Of Lymphoma Cells

Objective: The MAGE-C2 gene is a proto-oncogene and belongs to the cancer-testis antigen(CTA)MAGE family.MAGE-C2 is not expressed in human normal tissues except testicular tissues,but is expressed in a variety of malignant tumor tissues including lung cancer tissues,and its expression is closely related to the occurrence and development of tumors.In order to study the role of MAGE-C2 as a target protein in the treatment of lymphoma,and as the initial part of the establishment of a CAR-T cell research project targeting the MAGE-C2 antigen,this work first focused on the human T cell lymphoma cell line Jurkart.The MAGE-C2 gene was knocked out using CRISPR / CAS9 RNP gene editing technology,and the effects of MAGE-C2 gene knockout on inducing apoptosis,inhibiting proliferation,and enhancing the effects of the chemotherapeutic drug Methotrexate(MTX)were investigated.Method: 1.The PMJ915-CAS9(69090)plasmid expressing the CAS9 gene was constructed,amplified and expressed in E.coli BL21(DE3),and the purified CAS9 protein was harvested.2.Design and synthesize adapted g RNAs in the CRISPR system that knocks out the MAGE-C2 gene.3.Transfer the CAS9 protein / MAGE-C2 specific g RNA complex into Jurkart cells by electroporation,and knock out the MAGE-C2 gene.4.To observe the spontaneous apoptosis of Jurkart cells after MAGE-C2 gene knockout,the change of the appreciation rate and the effect on the effect of chemotherapy drugs.Results: The CAS9-expressing vector was successfully constructed and the expressed CAS9 protein was purified.After being transfected with MAGE-C2 specific g RNA for 48 hours,quantitative RT-PCR and Westorn blot detection were performed,and the MAGE-C2 gene was successfully knocked out.Cells developed spontaneous apoptosis,a significantly reduced proliferation rate(P<0.05).Methotrexate(MTX)treatment of cells knocked out of MAGE-C2 gene 48 hours after treatment,compared with non-knockout cells,it has a significantly enhanced killing effect(P<0.05).Conclusion: MAGE-C2 gene plays an important role in maintainingthe stable proliferation of lymphoma cells.CRISPR / CAS9 gene editing technology can successfully knock out its expression,and induce apoptosis of lymphoma cells and enhance the killing effect of chemotherapeutics.This research lays the foundation for innovations in therapies targeting the MAGE-C2 gene,and opens up a preliminary technical approach for establishing CAR-T that recognizes MAGE-C2 using CRISPR /CAS9 gene editing technology.