FTO Promotes Proliferation, Migration And Invasion Of Lung Adenocarcinoma Cells By MRNA Demethylation

Lung cancer is the leading cause of cancer incidence and mortality.In2018,2.1 million lung cancer cases are newly diagnosed and 1.8 million deaths are predicted.Accounting for nearly 1 in 5(18.4%)cancer deaths.80% of lung cancer is non-small cell lung cancer(NSCLC),which covers lung adenocarcinoma(LUAD,50-60%)and lung squamous cell carcinoma(LUSC,30%).Although target and anti-PD1 therapy have shown promise in the treatment of lung cancer,5-year survival rate is still very low.It is an urgent need to evaluation of the biological mechanism for the regulation of lung cancer.FTO is the first identified through genome wide association studies(GWAS)to be associated with an increased risk of obesity.Recently,FTO was found acting as m6 A demethylases.Several reports have indicated a potential association between aberrant m6 A modification and lung cancer development.It has been proved that FTO overexpressed in many cancers including NSCLC,gastric cancer,cervical carcinoma,pancreatic cancer and acute myeloid leukemia.Given the strong association of FTO and various cancers,however little is known about the role of FTO in LUAD.In the present study,we sought toevaluate the prognostic role of FTO and LUAD.By data mining TCGA database,we first determined the relationship between FTO expression and survival rate in LUAD.Then we performed a loss-and gain-of-function study in vitro to explore the function of FTO in LUAC cells,A549 and H1299.Last,the underlying molecular mechanism through identification of critical mRNA targets of FTO was investigated by mRNA sequence.Part I FTO expression level in LUAC Objective:To analyze the expression level of FTO in LUAC,further to examine the roles of FTO in LUAC.Method:FTO mRNA and protein expression levels in different human tissues were analyzed on the Human Protein Atlas portal(http://www.proteinatlas.org/).The relationship between FTO expression levels and the survival proportion in LUSC and LUAC were analyzed in TCGA cohort by UALCAN(http://ualcan.path.uab.edu/index.html).Result:1.Data from The Human Protein Atlas portal showed that,the expression of FTO mRNA level was higher in lung,even high than that in adipose tissue.2.Data from UALCAN showed that,compared with the adjacent normal tissues,the expression of FTO was lower in LUSC and LUAC.3.Furthermore,the down regulation of FTO was significantly correlated with higher survival rate in LUAC.Conclusion:The expression of FTO was lower in LUAC.Furthermore,the down regulation of FTO was significantly correlated with higher survival rate in LUAC.Part II Construction and identification of LUAC cells with different FTO expression levels Objective:To establish FTO overexpression,R96 Q overexpression and FTO down-expression model in A549 and H1299.Method:The lentiviral vectors with different FTO expression levels and R96 Q overexpression were constructed.After lentiviral vector transfection,qRT-PCR and Western blot were used to determine the infection effect of FTO.Result:1.In A549 and H1299 cells,the expression of FTO mRNA in FTO overexpression and R96 Q overexpression group was significantly higher than that in the control group,while the FTO mRNA in the FTO down-regulated group was significantly decreased(P<0.05).2.In A549 and H1299 cells,FTO overexpression and F96 protein expression in R96 Q overexpression group were significantly higher than those in the control group,while FTO protein in FTO down-regulated group was significantly decreased(P<0.05).Conclusion:FTO overexpression,R96 Q overexpression and FTO down-expression model were successfully established in A549 and H1299.Part III effects of FTO demethylation on the proliferation,migration,invasion and apoptosis of LUAC Objective:To analyze the effects of FTO demethylation on the proliferation,migration,invasion and apoptosis of LUAC.Method:CCK8 method,scratch test and transwell chamber method were used to detect the changes of proliferation,migration and invasion.Flow cytometry was performed to analyze the changes of cell apoptosis.The m6 A semi-quantitative kit was used to detect the m6 A of cells.Result:FTO overexpression promoted cells proliferation,migration,invasion and apoptosis.As expected,FTO knockdown suppressed cells proliferation,migration,invasion and apoptosis.Compared with control cells,FTO overexpress resulted in the mRNA m6 A expression increased,while FTO knockdown decreased the m6 A expression levels.The demethylase activity of FTO was required for LUAD cells growth,survival,and invasion.For further verifying demethylase activity of FTO in LUAD,R96 Q mutation A549 or H1299 cells were generated,R96 Q missense induced FTO lack of demethylase activity.Interesting,overexpress R96 Q blunted the FTO effect on promoted cells proliferation,migration,invasion and apoptosis,meanwhile R96 Q overexpress had no effected on decrease cellular m6 A level.Conclusion:The demethylase activity of FTO played a key role in LUAC cellsproliferation,migration and invasion.Part IV RNA-seq analysis Objective:To explore the mechanism of FTO demethylation on proliferation,migration and invasion of LUAC.Method:First,RNA-seq analysis was performed on overexpression of FTO and overexpression of R96 Q cells.Next,Gene ontology(GO)enrichment analysis and Kyoto Gene and Genomic Encyclopedia(KEGG)analysis were performed.Finally,the mRNA level of SPARC was detected on A549 cells.Result:1.Sequencing results showed that 500 genes were significantly expressed,of which 244 genes were up-regulated and 256 down-regulated.2.GO enrichment analysis identified categories 11,15 and 13 in the cellular component(CC),molecular function(MF)and biological process(BP)groups,respectively.3.In the KEGG analysis,a total of 204 differentially expressed pathways were identified between the FTO overexpressing group and the R96 Q overexpressing group,of which 20 pathways showed significant differences(P<0.05).These pathways were primarily involved in the metastasis and invasion of tumor cells.4.We found that IL1 B,FN1,PTGS2,MMP2,SPARC,TLR4,ITGA2,APOE,BMP4 and STAT1 genes may be key genes in LUAC,and SPARC wasselected as our target gene by screening.5.SPARC mRNA level analysis,overexpression of FTO increased the expression of SPARC on A549,and overexpression of R96 Q reduced the expression of SPARC on A549.Conclusion:SPARC may be one of the major regulators on proliferation,migration and invasion of LUAC.