| Pancreatic cancer is the deadliest malignant tumor among digestive tract tumors,and its mortality rate is very close to the prevalence rate.The morbidity and mortality of pancreatic cancer have increased slightly in recent years.Pancreatic cancer is characterized by low initial symptoms and rapid progression,and most patients are diagnosed with advanced disease.Due to strong invasiveness and metastases,as well as resistance to multiple chemotherapeutic drugs,the 5-year survival rate of pancreatic cancer patients who undergo surgical resection is less than 10%.Malignant metastasis of cancer cells occurs in the early stages of pancreatic cancer development,which is the main reason for the difficulties in the diagnosis and treatment of pancreatic cancer.Hypoxia is an important feature of most solid tumors,which activates signaling pathways in cancer cells or various cells in the tumor microenvironment in a cell-autonomous and non-cell-autonomous manner to promote tumor progression.Enhanced glycolysis is the most prominent feature of the metabolic response of cancer cells to hypoxia.This hypoxia-induced metabolic switch shunts glucose metabolites from mitochondria to glycolysis,maintains ATP production and prevents the generation of toxic ROS.This process not only provides energy source for hypoxic cancer cells,but also protects cancer cells from hypoxic-induced apoptosis,thus promoting cancer cell survival and metastasis.Therefore,an in-depth study of the molecular mechanisms involved in regulating the hypoxic response in pancreatic cancer will provide new powerful targets for future treatments of pancreatic cancer.Epitranscriptomics refers to the chemical modifications that occur on coding and non-coding RNAs.Currently,research into the function of these modifications is emerging and has been shown to be involved in the regulation of self-renewal and survival after DNA damage in embryonic stem cells and cancer cells.N6-methyladenosine(m6A)is the most abundant internal modification in mRNA,which is dynamically regulated by methyltransferases and demethylases,and is recognized and bound by specific RNA-binding proteins for its function.m6A modification affects the decay,splicing,nuclear localization and translation of mRNA,and participates in many biological processes.More and more evidence showed that m6A methylation modification is closely related to malignant proliferation,metastasis and chemotherapy resistance of tumors.HDAC4(histone deacetylase 4)belongs to class Ⅱa histone deacetylase,which is involved in the regulation of epigenetic remodeling of chromatin.The HDAC family is responsible for the deacetylation of histone lysine residues,and is involved in the regulation of a variety of biological processes and the occurrence and development of many diseases by causing tight binding of DNA and histone octamer and inhibiting gene transcription.In addition,HDAC4 can also mediate the post-translational modification of non-histone proteins,regulating their expression and function.Studies have shown that the abnormal high expression of HDAC4 is related to the occurrence and development of gastric cancer,lung cancer,ovarian cancer and other tumors,but the biological significance of HDAC4 in pancreatic cancer has not been systematically reported.This topic mainly studies the changes and biological significance of hypoxia-mediated mRNA m6A modification in pancreatic cancer cells,and explores the molecular mechanism of m6A modification regulating the biological functions of hypoxic pancreatic cancer cells.The research content can be divided into the following three parts:(1)Biological significance of m6A methylation modification in hypoxic pancreatic cancer cells:Through Dot blot,m6A quantification,qRT-PCR and Western blot,it was preliminarily clarified that hypoxia is mainly through ALKBH5 down-regulates the m6A modification of pancreatic cancer cell mRNA;MeRIP-seq combined with RNA-seq data analysis,extracellular acidification rate(ECAR)detection and Transwell methods were used to clarify that ALKBH5-mediated down-regulation of m6A modification is related to hypoxia-induced glycolysis and migration of pancreatic cancer cells.(2)The molecular mechanism of ALKBH5-mediated m6A modification regulating the biological function of hypoxic pancreatic cancer cells:The combined analysis of MeRIP-seq of hypoxic pancreatic cancer cells and RNA-seq analysis of ALKBH5 knockdown pancreatic cancer cells was used to screen out the candidate genes with significant changes,and qRT-PCR and Western blot were used to preliminally clarify the regulatory effect of ALKBH5 on HDAC4 expression;MeRIP-qPCR,dual luciferase reporter experiments and actinomycin D experiments were used to confirm that ALKBH5 promoted the stability and regulated the expression of HDAC4 mRNA by down-regulating the m6A methylation modification of HDAC4 mRNA under hypoxia conditions;qRT-PCR,ECAR detection and Transwell experiments were used to confirm that HDAC4 promoted glycolysis and migration of pancreatic cancer cells induced by hypoxia.(3)ALKBH5 promotes hypoxic-induced glycolysis and migration of pancreatic cancer cells by regulating HDAC4:Pancreatic cancer cell with overexpression of ALKBH5 were constructed and the expression or activity of HDAC4 was inhibited.The role of ALKBH5 in promoting hypoxia-induced glycolysis and migration of pancreatic cancer cells by regulating HDAC4 was clarified by detecting the expression of glycolysis-related enzyme genes,glucose and lactate levels,ECAR and Transwell experiments under hypoxic conditions.In conclusion,this study found that m6A demethylation enzyme ALKBH5 significantly overexpressed in hypoxic pancreatic cancer cells,and ALKBH5 promoted the expression of HDAC4 mRNA by down-regulating m6A methylation modification,thus promoting glycolysis and migration of pancreatic cancer cells.Our findings help to elucidate novel molecular mechanisms by which hypoxia mediates pancreatic cancer progression and may provide new powerful targets for future treatments of pancreatic cancer. |
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