The Effect Evaluation And Mechanism Study Of Trametinib In Treating Glioma And Improving Its Drug Sensitivity In Combination With JQ1 In Vitro.

Research background and objective:Glioma is the most common primary and invasive brain tumor of the central nervous system,with high morbidity and disability and poor prognosis.Temozolomide(TMZ),as the first-line chemotherapeutic drug for its treatment,is very prone to the generation of inherent or acquired drug resistance of the tumor,which significantly reduces its response rate,so it is urgent to find new effective methods for the treatment of glioma.Previous studies have found that Trametinib(TRA)can improve the prognosis of patients with melanoma brain metastases,and can inhibit the proliferation,migration and invasion of glioma cells.However,how to enhance the anti-tumor effect of trametinib and replace temozolomide as an effective inhibitor for the treatment of glioma remains to be further studied.Therefore,the purpose of this study is to clarify the therapeutic effect of trametinib on glioma and the potential mechanism of enhancing its drug sensitivity.On this basis,the synergistic effect of combined bromodomain and extraterminal domain(BET)inhibitor JQ1 on the treatment of glioma was preliminarily explored.Research methods:1.To clarify the effect of trametinib on cell proliferation,migration and invasion phenotype: U251 and U87 cells were used as the research objects,temozolomide singledrug treatment was used as the reference,and the cell proliferation activity was detected by the CCK-8;Edu Staining and colony formation assay were used to detect the proliferation activity of the cells.Grouping was set according to the concentration gradient,and the effects of drugs on cell migration and invasion were clarified by scratch wound assay and Transwell invasion test.2.Explore the key molecules that affect the sensitivity of trametinib: U251 cells treated with dimethyl sulfoxide(DMSO)were used as controls,and after 48 h of TRA single-drug treatment,RNA sequencing(RNA-seq)was used to screen the differentially expressed gene DDR1 that may affect the sensitivity of trametinib treatment was identified;Real-time q PCR and Western Blot were used to verify the expression of this target.Using bioinformatics analysis(TIMER 2.0 database analysis)to explore the correlation between DDR1 and various tumors,especially the occurrence and development of glioma.3.To explore the effect and mechanism of DDR1 on the sensitivity of trametinib in the treatment of glioma: Knock down the expression of DDR1 in U251 cells by transient transfection of si RNA,and use the NC group as the control,the changes of cell proliferation phenotype were detected by CCK-8,Edu staining;Real-time q PCR and Western Blot were used to verify the expression changes of autophagy pathwayrelated markers such as AKT-m TOR,LC3,and P62,and senescence-related markers P16,P21,and P53.4.To explore the regulation of JQ1 on DDR1 and the effect of combining trametinib on cell proliferation and invasion phenotype: The inhibitor JQ1 with synergistic effect with TRA was obtained by screening different therapeutic combinations in our small molecule compound library.U251 cells treated with DMSO were used as the control group,and after JQ1 treatment for 48 h,the differentially expressed gene discoidin domain receptor 1(DDR1)was screened by RNA-seq,and verified by Real-time q PCR and Western Blot.The DMSO treatment group,trametinib monotherapy group(TRA)and JQ1 monotherapy group were used as controls,and the combined drug group(TRA+ JQ1)produce synergistic inhibitory effect on the proliferation and invasive phenotypes of glioma cells.5.Statistical method: The data comparison among groups was analyzed by t test,and the statistical software Graphpad prism 9 was used for statistical analysis.Research results:1.For both U251 and U87 cells,the IC50 of trametinib was significantly lower than that of temozolomide(U251: 0.3568 μM VS.22.24 μM;U87: 4.544 μM VS.104.3μM).At the same concentration,the ability of TRA to inhibit the proliferation of U251 and U87 was more obvious than temozolomide.Trametinib can effectively reduce the proliferation,migration and invasion of glioma cells in a dose-dependent manner.2.RNA-seq analysis and screening showed that the differentially expressed gene DDR1 was up-regulated in TRA-treated cells;RT-PCR and Western Blot verified the above results and found that DDR1 was also up-regulated in trametinib-resistant cells.Bioinformatics analysis showed that the expression of DDR1 was increased in tumor tissues such as glioma,head and neck cancer,gastric cancer,breast cancer and liver cancer,especially in glioma.3.U251 cells after DDR1 knockdown were more sensitive to Trametinib(IC50 in NC group: 3.118 μM,IC50 in si-DDR1 group: 1.516 μM),and their proliferation ability was significantly decreased.The expression of autophagy-related protein LC3Ⅱ in siDDR1-U251 cells increased,while P62 decreased,and AKT inhibited the activation of m TOR to promote autophagy.The expression levels of senescence-related markers P16,P21 and P53 in si-DDR1-U251 cells were increased,and the number of senescent cells was less than that in the NC group.4.For U251 cells,JQ1 IC50 is 0.2547 μM,and the CCK-8 proliferation curve suggests that 100 n M JQ1 can inhibit the proliferation of U251 cells.The results of RNA-seq indicated that the expression of DDR1 decreased after JQ1 treatment,which was consistent with the confirmed results at the protein and gene levels,and it was doseand time-dependent.The combined effect index of trametinib combined with JQ1 was less than 0.9,the expression level of DDR1 decreased in the combination group,and the proliferation and invasion abilities of U251 cells further decreased.Research conclusions:1.The sensitivity of glioma cells to trametinib was significantly higher than that of temozolomide.Trametinib significantly inhibited the proliferation,migration and invasion of glioma cells,showing a good tumor inhibitory effect.2.The up-regulation of DDR1 can reduce the inhibitory effect of trametinib on glioma cells.The possible mechanism of action is to promote the proliferation of glioma cells,induce cell senescence and inhibit the occurrence of autophagy to reduce the sensitivity to trametinib.3.JQ1 can inhibit the growth of glioma cells and reduce the expression of DDR1.JQ1 combined with trametinib can increase the sensitivity of glioma to the latter and play a synergistic anti-tumor effect.