Effects Of DEC1 Gene ShRNA Interference On Biological Behavior Of Glioma Cells And Its Chemotherapy Sensitivity To Temozolomide

DEC1(differentiated embryo-chondrocyte expressed gene 1)by Shen in 1997 for the first time isolated from human embryonic cartilage tissue,is a bHLH(basic helix-loophelix)transcription factor family members.DEC1 is involved in many important physiological processes,such as cartilage formation,immune response,biological rhythm,fat formation and so on.The results showed that DEC1 was highly expressed in colorectal cancer,lung cancer,renal cell carcinoma,breast cancer and other tumors,and was positively correlated with the malignant grade of some tumors.The expression of DEC1 in tissue specific tumor can be induced by a variety of harmful stimuli,is closely associated with tumor hypoxia,these findings suggest that DEC1 is closely related to the malignant phenotype of tumor,and plays an important role in the development of tumor.However,the role of DEC1 in glioma has not been reported.In this study,we successfully constructed the lentiviral interference plasmid vector of DEC1 gene,psi-LVRU6MP-DEC1-shRNA,and co transfected 293 T cells with the auxiliary plasmid to obtain the virus solution.The T98 G and U87 glioma cells were infected by virus solution.By puromycin screening,and by the Western blot test,stably transfected glioma cell lines of their gene DEC1 shRNA interference and control of the DEC1-shRNA-1 group and DEC1-shRNA-2 group and Negative-shRNA ofT98 G and U87 was successfully constructed.On the basis of this,the MTT assay,plate clone formation experiment,Transwell chamber experiment,scratch test,nude mouse tumorigenesis test and CCK-8 cytotoxicity test were performed to detect the changes of biological behavior of glioma cells in vivo and in vitro after DEC1 gene knockdown and the effect on chemotherapy reactivity.The results showed that DEC1 gene inhibited the proliferation of glioma cells in vivo and in vitro after shRNA interference.In vitro,the ability of colony formation and invasion and migration were also decreased.CCK-8 cytotoxicity test showed that the sensitivity of glioma cells to temozolomide increased after DEC1 gene knockout.GBM(glioblastoma multiforme)is the most malignant central nervous system tumor(WHO IV),although after surgical resection,radiotherapy and chemotherapy,the median survival is only 12-15 months.TMZ(Temozolomide)is a new generation of alkylating agent,is currently the most effective treatment of GBM chemotherapy,mainly to multiple DNA sites methylation to exert cytotoxic effects.Although TMZ adjuvant therapy can significantly prolong the survival time of patients with GBM,but the degree is limited,only about 3 months.The reason for this limited treatment is that GBM cells have some resistance to alkylating agents.One of the important mechanisms is the inherent resistance of MGMT(O6-Methylguanine-DNA methyltransferase,).Therefore,to find a key factor that can affect the drug resistance and to clarify its specific role in the mechanism,to solve the GBM resistance,improve the treatment of TMZ effect,to extend the survival of GBM patients has a very important value.BER(base excision repair)is also one of the causes of chemotherapy resistance to temozolomide.The DNA double strand break(DSB)in the BER repair system is preferentially repaired by the homologous recombination(HR)system.HR system-deficient cells are highly sensitive to TMZ-induced damage.RAD51 is a key component of the HR pathway and is highly consistent with the integrity of HR.RAD51 knockdown is associated with enhanced TMZ chemotherapy.In this study,80 cases of primary pleomorphic GBM patients underwent primary surgical resection of paraffin histopathological sections of DEC1 and MGMT in glioma tissue by immunohistochemistry(IHC)in the expression of relevance.There was a positive correlation between the expression of glioblastoma and glioblastoma.The expression of MGMT and RAD51 protein in glioma cells after DEC1 knockdown was also detected by Western blot.This suggests that the increased sensitivity of glioma cells to temozolomide in DEC1 gene shRNA interference may be the result of the effect of DEC1 and MGMT or RAD51.In conclusion,DEC1 may be a potential target for gene therapy for glioma patients,especially for patients with glioma resistant to temozolomide chemotherapy.