N6-Methyladenosine Promotes The Glycolysis Of Cisplatin-resistant A549 Lung Cancer Cells Via Facilitating Translation Of PDK4

Objective: Cisplatin resistance is a major obstacle in the treatment of lung cancer.One of the basic characters in drug resistant cancer is the abnormal metabolism,especially for the unusual glycolysis.However,its mechanism has not been fully expounded.N6-Methyladenosine(m6A)mRNA modification plays a vital role in various biological functions and in progression of cancer cells.But its functions,particularly for their roles in metabolic reprogramming of cancer,remain largely unknown.m6 A modification of mRNAs was found increased in the cisplatin-resistant process of lung cancer.Deletion of Methyltransferase like 3(Mettl3)led to the down-regulation of m6 A level and inhibited the metabolic capacity of cisplatin-resistant in lung cancer,especially the glycolysis capacity.Pyruvate Dehydrogenase Kinase 4(PDK4)is an important upstream regulator of aerobic glycolysis in cancer cells,which is considered to be a key drug resistance gene.To explore the mechanism of m6 A regulating the glycolysis process and PDK4 translation of cisplatin resistant in A549 cells,we provide a new idea and direction for effectively overcoming chemotherapy resistance.Methods: 1.A549 cells and cisplatin-resistant in lung cance cells(A549/CDDP)were routinely cultured in vitro.By detecting the glucose consumption,lactate production rate,ATP levels of the cells to compare the metabolic reprogramming of both cells,respectively.The extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)were analyzed by using the Seahorse Extracellular Flux(XFe96)analyzer to further determine the main production of A549/CDDP cells.2.By using LC-MS/MS,the m6 A levels of mRNAs isolated were identified from A549 and A549/CDDP cells.Western blot was used to detect the expression of Mettl3 and ALKBH5 in A549 and A549/CDDP cells,which further proved the increased m6 A modification in the progress of cell resistance.3.To prove that m6 A affected the metabolic program of A549/CDDP cells and regulated its glycolysis ability,the expression of Mettl3 was interfered or ALKBH5 overexpressed in A549/CDDP cells to detect the glucose consumption,lactate production rate and ATP levels.4.The western blot was used to detect the expression of PDK4 in A549 and A549/CDDP cells,with Mettl3 overexpressed or silenced,the glucose consumption,lactate production rate and ATP levels,cell growth rate of the corresponding cells were detected.5.The effects of m6 A on the transcription,translation,intracellular distribution and degradation of PDK4 were investigated by m6A-RT-PCR,nuclear and cytoplasmic separation and Polysome profiling.6.Luciferase reporter assay and immunoprecipitation were used to investigate the potential mechanisms involved in m6 A regulated expression of PDK4.7.The expression of Mettl3,YTHDF1 and PDK4 mRNA in lung cancer were analyzed from public datasets of lung cancer database gene expression profiles.Results: 1.Compared with A549 cells,A549/CDDP cells had significantly higher glucose consumption,lactate production rate and ATP levels.Meanwhile,A549/CDDP production mainly depended on glycolysis.2.Compared with A549 cells,the mRNA m6 A level of A549/CDDP cells was significantly increased.Western blot showed that the protein expression of Mettl3 was increased in A549/CDDP,while the methylated transferase ALKBH5 had no significant difference.3.After knockdown the Mettl3 or overexpressed the ALKBH5 in A549/CDDP cells,the metabolic capacity was reduced.At the same time,the glycolysis capacity of A549/CDDP was reduced after knockdown Mettl3,while the resistance to cisplatin was reversed.4.PDK4 mediated Mettl3 regulating glycolysis in A549 lung cancer cells.Western blot results showed that PDK4 expression was up-regulated in A549/CDDP shM3 cells compared to the control.Western blot results showed that the X expression of PDK4 was increased in A549/CDDP,while the expression of PDK4 was significantly decreased or increased after the cells were knocked out or overexpressed Mettl3.Interfered with the expression of PDK4 could suppress the metabolism and proliferation of A549/CDDP.Meanwhile,overexpressed the PDK4 could reverse the lower metabolism and proliferation of A549/CDDP cells after decreased Mettl3.5.m6 A could up-regulate stability and translation of PDK4 in A549/CDDP lung cancer cell.The expression of both precursor(pre-)and mature(mat-)mRNA of PDK4 had no significantly difference in A549/CDDP shNC and A549/CDDP shM3 cells.There was no difference on subcellular localization of PDK4 on promoter activity or by separating nuclear and cytoplasmic RNA and protein in A549/CDDP shNC and shM3 cells.We treated control and A549/CDDP shM3 cells with Act-D to block transcription.Results showed that half-life of both precursor and mature mRNA in wild type cells were had no significantly difference than in A549/CDDP shM3 cells.It indicated that m6 A modification may had no effect on the splicing of precursor mRNA and the degradation of mature mRNA of PDK4 in cancer cells.And then,both A549/CDDP shNC and A549/CDDP shM3 cells were treated with protein translation inhibitor cycloheximide(CHX).Results from western blot analysis showed that half-lives of PDK4 protein in A549/CDDP shM3 cells were lower than A549/CDDP shNC cells,suggested that m6A-induced PDK4 expression was related to protein stability.Further,we pretreated with MG-132 to inhibit proteasome activity of A549/CDDP shNC and A549/CDDP shM3 cells,the data showed that had no significantly difference between them.Polysome profiling supported the decreases of polysomes(>80S)in A549/CDDP shM3 cells.RT-PCR showed that PDK4 mRNA in translation active polysomes(>80S)of A549/CDDP shM3 cells were significantly lower than in A549/CDDP shNC cells,suggested that m6A-induced PDK4 expression was related to protein translation.6.m6 A methylated 3'UTR regulated translation of PDK4.From the prediction website(http://www.cuilab.cn/sramp),we obtained the peaks of m6 A modified PDK4: one m6 A peaks in 5'UTR regions and three peaks in 3'UTR regions.PGL3-PDK4-5'UTR was constructed,and no change was found in the translation efficiency.We generated luciferase reporters containing a firefly luciferase,followed by the wild type PDK4 3'UTR,or mutant 1/2/3 3'UTR(“GGAC” to “GGCC”).The result showed that PDK4 WT-3'UTR had significantly less luciferase in A549/CDDP shM3 than that in A549/CDDP shNC cells,interestingly,the mut-1 could partially reverse this down-regulation of translation efficiency.It indicated that m6 A in mut-1-3'UTR of PDK4 positively regulated its mRNA translation.The result of RIP-PCR showed that YTHDF1 was enriched in PDK4 mRNA remarkably,while this relative enrichment was significantly reduced in A549/CDDP shM3 cells.After transfecting the XII si-YTHDF1 in A549/CDDP,the result showed that si-YTHDF1 attenuated PDK4 expression in A549 CDDP cells.we measured the variation of eEF-1 and eEF-2 binding PDK4 mRNA in A549/CDDP shM3 cells.Our data exposed that the binding between PDK4 mRNA and eEF-1,not eEF-2,in A549/CDDP shM3 cells was significantly less than that in A549/CDDP shNC cells.Further,Co-IP analysis indicated that the binding between YTHDF1 and eEF-1,not eEF-2,was significantly decreased in A549/CDDP shM3 cells compared with that in A549/CDDP shNC cells.This data suggested that YTHDF1 and eEF-1 were likely responsible for m6 A induced translation elongation of PDK4 mRNA in A549/CDDP.7.The m6A/PDK4 axis in lung cancer tissues.In lung microarray datasets from the GEO database,a microarray dataset(GSE40791)was identified in 194 cases.Increased expression of Mettl3 and YTHDF1 were observed in lung cancer tissues as compared to matched adjacent normal tissues.Significant rising expression levels of PDK4 from T1 to T4 stage of lung cancer tissues were observed.implying an increasing tendency of PDK4 expression during malignant transformation.Conclusions: m6 A level was significantly increased during drug resistance of lung cancer A549 cells,after the expression of Mettl3 was knocked out,the glycolysis ability of cisplatin-resistant in lung cancer A549/CDDP cells was significantly reduced by inhibiting the expression of PDK4.In addition,our results further confirm that m6 A promotes PDK4 mRNA translation by modifying PDK4 3'UTR,and that YTHDF1 and eEF-1 mediate the process of m6 A promoting PDK4 translation.