DEC1’s Function,mechanism And Clinical Significance In Temozolomide Resistant Glioblastoma

Glioblastoma multiforme(GBM)is one of the most common and malignant tumors of the central nervous system.GBM has strong proliferative ability and is easy to invade and recur.It belongs to WHO pathological grade IV.The prognosis of the patients is poor.The average survival period is only about 12-15 months.The growth mode of GBM is mainly infiltrative growth.It is difficult to remove GBM completely by operation alone,and it often needs to be combined with radiotherapy and chemotherapy.However,chemotherapy or radiotherapy resistance often exists in the treatment of GBM.Even after standardized comprehensive treatment,the clinical prognosis of GBM patients is still poor.The diagnosis and treatment of GBM has become a worldwide problem in the field of neurosurgery.Temozolomide(TMZ)It is a new generation of alkylating agent anticancer drug.It can induce DNA methylation of cancer cells to cause cell damage and then kill tumors.At the same time,because of its liposoluble property,TMZ can work through the blood-brain barrier.Therefore,TMZ is one of the most widely used chemotherapy drugs in the clinical treatment of GBM,but the increasingly serious production of TMZ chemotherapy resistance greatly reduces its clinical efficacy,and A large number of clinical studies have shown that TMZ standard treatment can only effectively prolong the survival of GBM patients for 3 months,so it is necessary to study the TMZ resistance and its mechanism of GBM patients.Differentiated embryochondrite expressed gene 1,DEC1 is one of the members of b HLH superfamily.It can participate in circadian rhythm formation,energy metabolism,immune stress and other physiological processes.At the same time,the activity of DEC1is closely related to the degree of tissue hypoxia and the ability of vascular regeneration.The expression level of DEC1 can be used as an important index to evaluate the prognosis of tumor,but the role and mechanism of DEC1 in TMZ resistance have not been clearly reported.Objective:To study the role of DEC1 in TMZ resistance in GBM,further improve the molecular mechanism of TMZ resistance,and lay a theoretical foundation for exploring new therapeutic targets of TMZ resistant GBM patients.Method:1.The potential TMZ resistance related genes were screened by GEO database and literature review.The primary TMZ sensitive cell line GBM normal and TMZ resistant cell line GBM13 were constructed.RT-PCR was used to detect the difference of Mrna expression level and GEPIA,CGGA database was used to verify the relationship between them and the total survival time of patients.2.To construct stable cell lines of T98G,LN18 and U251 with DEC1 overexpression and knockdown.After TMZ treatment,蛋白质免疫印迹实验,flow cytometry and CCK8 test were used to detect the role of DEC1 in TMZ resistance.3.Immunohistochemistry was used to compare the expression of DEC1 and O6-methylguanine-DNA methyltransferase(MGMT)in normal brain tissue and GBM tissue,and the expression relationship between MGMT and DEC1 was determined.4.Construct T98G and LN18 cell lines with DEC1 overexpression MGMT knockdown and DEC1 knockdown MGMT overexpression.After TMZ treatment,Western blot,flow cytometry and CCK8 were used to detect cell viability and apoptosis related indexes,and clarify the relationship between the resistance of TMZ mediated by DEC1 and MGMT.5.Western blot and RT-PCR were used to detect the regulation of DEC1 on MGMT and its upstream molecule SP1 expression in DEC1 overexpression/knockdown cell line,and promoter reporter gene was used to explore the molecular mechanism.6.To construct MGMT overexpression T98G and LN18 stable cell lines,using small interfering RNA to reduce SP1 expression.After TMZ treatment,Western blot,flow cytometry and CCK8 were used to detect apoptosis related indicators,and to study the role and mechanism of Sp1 in TMZ resistance.Results:1.MAPK10,CTSS and DEC1 were highly expressed in GBM13.The expression level of CTSS and DEC1 was negatively correlated with the clinical prognosis.2.DEC1 can significantly increase the resistance to TMZ and inhibit the apoptosis induced by TMZ in cell lines(T98G and LN18)with positive expression of MGMT,while in U251 cell line with negative expression of MGMT,DEC1 has no effect on the apoptosis induced by TMZ.3.MGMT overexpression can restore the TMZ sensitivity induced by DEC1 knockdown,but after MGMT expression is reduced,the enhancement of DEC1 resistance to TMZ is cancelled.4.Overexpression of DEC1 was up-regulated,while knockdown of DEC1 was down regulated.DEC1 and MGMT were highly expressed in GBM,and the positive correlation was confirmed by spearsman linear regression.5.DEC1 can promote the expression of MGMT and SP1,while SP1 is positively regulating MGMT and the regulation of DEC1 on MGMT expression is offset with the knock down of SP1.6.With the decrease of SP1 expression,the sensitivity of tumor cells to TMZ increased,and MGMT overexpression could reverse the sensitivity of glioma cells to TMZ after SP1knockdown Conclusion:1.MAPK10,CTSS and DEC1 may be potential TMZ resistance genes.2.The high expression of CTSS and DEC1 was negatively correlated with the clinical prognosis.3.DEC1 relies on sp1-mgmt to regulate the sensitivity of cells to TMZ.4.DEC1 regulates the transcription and expression of MGMT through SP1.5.SP1 relies on MGMT to regulate cell sensitivity to TMZ.