Study On The Activity Of Bezafibrate Against Lung Adenocarcinoma Based On Gene Expression Profiling

Lung cancer is the leading cause of cancer death in the world.About one-third of lung cancer incidence is lung adenocarcinoma.Although the treatment of lung adenocarcinoma has made great progress,the prognosis of patients is poor.There are no obvious clinical symptoms in the early stage of lung adenocarcinoma.When it is discovered that metastasis has occurred or is in the advanced stage,only chemotherapy-based treatment can be adopted.However,traditional chemotherapeutic drugs usually have defects such as high toxicity and strong side effects.It is found that new high-efficiency and low-toxic therapeutic drugs have urgent clinical needs and extremely important social significance for prolonging the life of patients with lung adenocarcinoma and reducing mortality.The content of this study is mainly divided into two parts:Part Ⅰ: Analysis of key genes and pathways in lung adenocarcinoma1.Differentially expressed gene analysis: Three lung adenocarcinoma gene expression profile data sets,GSE27262,GSE75037 and GSE83213,were obtained from GEO database.The R language program and the limma installation package were used to perform differential gene expression analysis on the three data sets,and the RRA installation package was used to remove the sample differences between different platforms.Finally,172 common differentially expressed genes were obtained,of which 56 genes were up-regulated and the 116 genes was down-regulated(P<0.05,|log2Fold change|≥2).2.The acquisition of key genes in lung adenocarcinoma and its relationship with prognosis: The differentially expressed genes were constructed by Cytoscape to construct a network model of their interaction between proteins,and Cytohubba was used to select 10 representative models.Key genes TK1,EGFR,TPX2,RAF1,UBE2 C,AKT1,NUSAP1,TP53,TOP2 A and HIF1 A.The Oncomine and HPA databases verified that these 10 key genes were significantly different between lung adenocarcinoma and normal lung tissue at the transcriptome and protein levels(P<0.05),and 10 genes were associated with overall survival after calculated by SPSS(P<0.01).3.Acquisition of key signaling pathways in lung adenocarcinoma: functional annotation and pathway enrichment of differentially expressed genes using DAVID and TOPPGENE.After screening,some potential functions related to lung adenocarcinoma and related signaling pathways were obtained.The PPAR signaling pathway attracted our attention and the PPARα agonist bezafibrate was used as the research direction.Conclusion: The key genes of lung adenocarcinoma TK1,EGFR,TPX2,RAF1,UBE2 C,AKT1,NUSAP1,TP53,TOP2 A and HIF1 A were obtained.These genes are closely related to the development of lung adenocarcinoma.Both DAVID and TOPPGENE pathway analysis indicated that PPAR signaling pathway is closely related to lung adenocarcinoma,suggesting that PPAR signaling pathway agonists such as bezafibrate may be potential drugs for the treatment of lung adenocarcinoma.Part Ⅱ: Inhibition of proliferation of lung adenocarcinoma A549 cells by bezafibrate1.Inhibitory effect of bezafibrate on proliferation of lung adenocarcinoma A549 cells: The inhibitory effect of bezafibrate on proliferation of A549 was determined by CCK8 method.The experiment was divided into different concentration groups(25,50,100,200 and 400 μM),negative control group and blank control group.The drug treatment time was 24 h,48h and 72 h.The results showed that there were significant differences in the growth inhibition of A549 cells in different concentration groups at the same time point(P<0.05).The growth inhibition of A549 cells by bezafibrate showed a certain dose effect;Statistical difference(P<0.05),bezafibrate also showed a time-dependent inhibition of growth of A549 cells.2.Cell cycle arrest of bezafibrate on A549 cells: Cell cycle changes after 24 h of various concentrations of bezafibrate were analyzed by flow cytometry.The results showed that the ratio of G1/S phase cells in the 200 μM dose group was 88.46.% was statistically different from the control group(86.66%)(P=0.018);the ratio of 400 μM cells was 90.8%,which was statistically different from the control group(P=0.041),and there was also a significant difference between 200 μM and 400 μM.(P=0.023).3.Induction of apoptosis of A549 cells by bezafibrate: After treatment with bezafibrate for 24 h,the apoptosis of A549 cells was detected by Annexin-V and PI double staining.The results showed that bezafibrate could not induce apoptosis in A549,and its apoptosis-inducing effect was not related to the dose.This indicates that bezafibrate does not participate in the induction of apoptosis when it acts on lung adenocarcinoma cells and produces inhibition.4.The regulation of bezafibrate on risk genes: The regulatory effects of bezafibrate on EGFR,AKT1,TP53,RAF1 and HIF1 A after A549 cells were verified by real-time PCR.The results showed that bezafibrate significantly down-regulated EGFR,AKT1,TP53,RAF1 and HIF1 A,and there was a significant difference compared with the non-medicated group(P<0.05).Conclusion: Bezafibrate inhibits the proliferation of A549 cells,which leads to the arrest of A549 cells in G1/S phase,not involved in the apoptosis process,and bezafibrate can significantly down-regulate the representative key genes.