| Objective: Pancreatic cancer is the deadliest malignancy of the digestive system.The occurrence of pancreatic cancer is a gradual process with multiple factors and multiple steps,which involves genetic alterations and disorders of epigenetics.As a vital component of epigenetics,N6-methyladenine(m6A)modification affects the regulation of almost every step of the RNA life cycle,including mRNA transcription,splicing,nuclear export,localization,translation,stability,and degradation.m6 A modification is a methylation modification catalyzed by methyltransferase on the 6th N atom of RNA adenine.Evidences have revealed that the expression of m6 A modification and its regulators is often disordered in different cancers,and is crucial to cancer formation,proliferation,invasion and metastasis,recurrence,and resistance to chemotherapy and radiotherapy.In pancreatic cancer,it has been reported that m6 A RNA modification promote the maturation of oncogenic micro RNA-25,thereby inhibiting the PH domain of leucine-rich repeat protein phosphatase 2,activating the AKT-p70S6 K signaling pathway and triggering the malignancy of pancreatic cancer cells.Alk B homologue 5(ALKBH5)was proved to inhibite pancreatic cancer cell motility by demethylating m6 A modification of the long noncoding RNA KCNK15-AS1.Moreover,ALKBH5 demethylates the m6 A modification of period circadian regulator 1 and inhibits its mRNA degradation,leading to the activation of the ATM-CHK2-P53/CDC25 C signaling pathway,thereby inhibiting the growth of pancreatic cancer cells.In addition,ALKBH5 reduces the m6 A level of Wnt inhibitory factor 1(WIF1)and inhibit the activation of Wnt signaling pathway,thus inhibiting the proliferation,migration and invasion of pancreatic cancer cells and improving its sensitivity to chemotherapy.m6 A recognition protein YTH domain family protein(YTHDF)2 was proved to promote pancreatic cancer cell proliferation through the AKT/GSK3?/Cyclin D1 pathway.However,knockout of YTHDF2 induces epithelial-mesenchymal transition(EMT)by upregulating the expression of YAP,thus promoting the migration and invasion of pancreatic cancer cells.However,the role and regulatory mechanism of m6 A RNA modification in pancreatic cancer are still unknown.Therefore,we designed and carried out this study to investigate the expression and specific regulatory mechanism of m6 A RNA modification in pancreatic cancer,and to explore the role of this regulatory network in the malignant biological behavior of pancreatic cancer,providing a new potential biological target for the diagnosis and treatment of pancreatic cancer.Materials and methods: 1.This protocol was approved by the Ethics Committee of Shengjing Hospital of China Medical University,and written informed consent was obtained from all patients.All the pancreatic cancer tissues and their comparing adjacent normal tissues were obtained from patients who underwent resection at Shengjing Hospital of China Medical University.None of these patients received any preoperative chemotherapy or radiotherapy.The fresh frozen samples were obtained within 10 min after tumor excision and immediately stored at-80°C until use in experiments.All samples(16 pairs fresh frozen samples and 50 pairs archival formalin-fixed paraffin-embedded samples)were histologically diagnosed with pancreatic adenocarcinoma(PAAD)by pathologic examination.The collection of follow-up data was carried out completely,and overall survival was defined as the time interval from the date of surgery to the date of death or the end-point of follow-up(December 30,2020).Human pancreatic cancer cell lines and human immortalized pancreatic duct epithelial cell line HPDE6-C7 were cultured for this study.Epi Quik m6 A RNA Methylation Quantification Kit was used to detect the m6 A content in the total RNA of PAAD tissues,corresponding para-tumor tissues,pancreatic cancer cell lines and HPDE6-C7 cell.2.Bioinformatics investigation and real-time quantitative polymerase chain response(RT-qPCR)were performed to detect the expression of 10 major m6 A regulators in PAAD tissues,comparing para-tumor tissues,five pancreatic cancer cell lines and HPDE6-C7 cell,and identified that the key m6 A regulator causing m6 A modification clutter in pancreatic cancer was m6 A demethylase fat mass and obesity-associated protein(FTO).Next,immunohistochemistry examination of paraffin specimens of 50 pairs of PAAD tissues and corresponding para-tumor tissues was evaluated to explore the clinicopathological role of FTO in pancreatic cancer.Furtherly,the Incucyte Zoom Live-Cell Imaging System analysis,Cell Counting Kit-8(CCK8)assays,wound healing migration assays,transwell cell invasion assays were used to detect the effect of FTO on the malignant biological behavior of pancreatic cancer cells.3.Bioinformatics analysis,construction and transfection of small interfering RNA(si RNA)knocking down FTO,construction and transfection of wild type and demethylase inactivated mutant plasmids up-regulating FTO,RT-qPCR,Western blotting(WB),and RNA stability assays were performed to explore the downstream target genes of FTO in pancreatic cancer.Furtherly,we investigated the effect of the target gene PJA2 on the malignant biological behavior of pancreatic cancer cells,and conducted rescue experiments.4.The downstream related pathways of FTO in pancreatic cancer were detected by ransfection of wild type plasmids in pancreatic cancer cells to up-regulate FTO and screening stable cell lines,transfecting si RNA to down-regulate PJA2,RT-qPCR and WB,so as to explore the function and regulatory mechanism of m6 A RNA modification in the malignant biological behavior of pancreatic cancer.5.Statistical analyses were performed utilizing the statistical software in Graph Pad Prism version 8.0.Two-tailed Student's t-tests were used for continuous variables.For statistical correlation,Pearson's and Spearman's correlation coefficient was used according to requirements.Kaplan-Meier analysis and log-rank test were used to evaluate the differences in patient's survival.Results were presented as means±standard error of the mean or standard deviation for at least three independent biological replicates.Data were considered statistically significant as follows: * means P < 0.05;** means P < 0.01;and *** means P < 0.001.ns represents no statistical significance.Results: 1.m6 A RNA modification is upregulated in pancreatic cancer.We found that the m6 A levels were essentially upregulated in PAAD tissues compared with their corresponding para-tumor tissues.Besides,m6 A levels of five human pancreatic cancer cell lines were found to be significantly higher than those of HPDE6-C7 cells.2.Reduced m6 A demethylase FTO expression is responsible for the high level of m6 A RNA modification in pancreatic cancer.2.1 In order to find the key regulator for m6 A modification disorders in pancreatic cancer,we systematically analyzed the changes of 20 major m6 A regulators in pancreatic cancer from The Cancer Genome Atlas(TCGA)and other open databases utilizing an assortment of biological information analysis methods.We found that m6 A regulators had broad genetic alterations in pancreatic cancer,and compared with corresponding para-tumor tissues,the mRNA expression of nearly all m6 A regulators in PAAD tissues have changed.Furtherly,Kaplan-Meier curve and log-rank test analyses uncovered that genetic alterations in m6 A regulators were related with poorer disease/progression-free survival(DFS/PFS)and overall survival(OS)in pancreatic cancer patients(log-rank test,P = 0.035 and 0.007,respectively).2.2 Next,we detected the expression of 10 major m6 A regulators in PAAD tissues,corresponding para-tumor tissues,pancreatic cancer cell lines and HPDE6-C7 cells.The results showed that m6 A demethylase FTO was essentially down-regulated in pancreatic cancer.Knockdown of FTO in pancreatic cancer cells extraordinarily increased the m6 A levels,and overexpression of wild type FTO significantly decreased the m6 A levels.Therefore,FTO was determined to be the key regulator resulting in m6 A modification clutter in pancreatic cancer.Besides,we found that the expression of FTO was not only closely connected with individual cancer stages and nodal metastasis status,but also significantly affected the prognosis of PAAD patients.PAAD patients with reduced FTO expression had poor survival outcome.Furtherly,we found that knockdown of FTO altogether advanced the proliferation,invasion and metastasis capabilities of pancreatic cancer cells;overexpression of wild type FTO essentially restrained the proliferation,metastasis and invasion capabilities of pancreatic cancer cells.3.FTO enhances praja ring finger ubiquitin ligase 2(PJA2)mRNA stability in an m6A-YTHDF2-dependent manner.3.1 Linked Omics,UALCAN,and c Bio Portal databases were utilized to investigate FTO co-expressing genes in PAAD tissues.In each database,the top 20 genes significantly positively or negatively related to FTO in PAAD tissues were chosen,and their intersections were taken separately.Overall,five(AKT3,PJA2,PLEKHM3,ZYG11 B,and WDR47)and three(NUDT8,YDJC,and RASSF7)genes were found to be positively and negatively related to FTO,respectively.mRNA expression analysis of these eight candidate genes in pancreatic cells lacking or overexpressing FTO was then performed,and the results showed only the expression of PJA2 followed the same trend as FTO.Therefore,PJA2 was determined to be a downstream target of FTO in pancreatic cancer.3.2 Moreover,knockdown of YTHDF2 reduced the decay rate of PJA2 and increased the mRNA and protein expression of PJA2,whereas YTHDF1 knockdown had no significant effect.Taken together,reduced FTO expression upregulated the relative PJA2 m6 A levels,which in turn induced the degradation of PJA2 mediated by YTHDF2,thereby stifling PJA2 mRNA and protein expression in pancreatic cancer cells.4.FTO-dependent demethylation of m6 A modification in pancreatic cancer regulates Wnt signaling via PJA2.FTO and PJA2 significantly decreased the expression of Wnt family member 5a(Wnt5a),lymphoid enhancing factor 1(LEF1),and ?-catenin,whereas increased AXIN1 and WIF1,and phosphorylated glycogen synthase kinase-3?(GSK-3?),thereby inhibiting the Wnt signaling pathway in pancreatic cancer cells.Conclusion: 1.m6 A RNA modification is upregulated in pancreatic cancer tissues and cell lines due to the low expression of m6 A demethylase FTO.2.Reduced FTO expression is closely related to poor prognosis of PAAD patients.Knockdown of FTO significantly promotes the proliferation,migration and invasion of pancreatic cancer cells;while overexpression of wild-type FTO plasmids significantly inhibites the proliferation,migration and invasion of pancreatic cancer cells.3.FTO enhances PJA2 stability in an m6A-YTHDF2-dependent manner.In summary,low FTO expression triggeres the upregulation of m6 A levels in PJA2,promoting its degradation via YTHDF2,which ultimately leads to low PJA2 expression,consequent activation of the Wnt signaling pathway,and promoting the proliferation,migration and invasion of pancreatic cancer cells.The described FTO-m6A-YTHDF2-PJA2-Wnt signaling axis plays an important role in the regulation of malignant biological behavior of pancreatic cancer. |
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