| In recent years,RNA epigenetic transcription modification has become one of the hot research fields.N6-methyladenine(m6A)is the most abundant posttranscriptional modification on mammalian mRNAs,which is jointly regulated by methyltransferases,demethylases,and their binding proteins.It participates in various physiological and pathological regulation by influencing mRNA metabolism.This dynamic and reversible regulation is associated with the occurrence of various diseases,and abnormal m6A modification may lead to the occurrence of tumors and neurodegenerative diseases.ALKBH5 is one of the major RNA m6A demethylases and plays multiple biological functions in diseases.As reported,ALKBH5 is involved in the progression of glioblastoma,pancreatic cancer,colon cancer and other tumors.In addition,ALKBH5 also plays important functions in the nervous system,reproductive system,etc.Our research group focuses on investigation of RNA m6A methylation in brain tumors and neuronal diseases.Here,we mainly focus on ALKBH5 to study its associated mechanism of tumorigenesis of glioblastoma and neurodegeneration of the mouse cerebellum.Glioblastoma multiform(GBM)is one of the most malignant tumors in the central nervous system.In recent years,various efforts have been paid to improve its therapeutic efficiency and clinical outcomes of the patients,which requires a comprehensive understanding of its mechanism.It has been reported that when iron metabolism is disrupted,oxidative stress reactions will occur and accelerate the progression of glioblastoma and reduce the survival rate of patients.So far,detailed molecular mechanism is unclear yet and await further investigation.Previously,ALKBH5 is found to play a role in maintaining tumorigenicity of glioblastoma in m6A-dependent manner.On the other hand,we previously found that ALKBH5 regulates iron metabolism in pancreatic ductal adenocarcinoma.Therefore,we speculate that if overexpression of ALKBH5 in the glioblastoma may also lead to the disorder of iron metabolism,thus triggering tumor development of glioblastoma.To verify this hypothesis,we first performed m6A sequencing and bioinformatics analysis on clinical patient samples of multiple glioblastomas,and found significant changes in the methylation levels of iron metabolism related genes in the tumor samples.Meanwhile,ALKBH5 knockdown caused significant decrease of the content of iron ions in glioblastoma cells.We further compared the transcriptome-wide m6A changes in glioblastoma cell lines before and after knockdown ALKBH5.We found that the methylation levels of various iron metabolism-related genes changed,with the most significant increase observed in ferritin-encoding FTH1 RNA methylation levels.Therefore,we further investigated the regulatory effect of ALKBH5 on iron metabolism through FTH1 RNA demethylation in glioblastoma.We investigated the effects of m6A on FTH1 target genes from the perspectives of RNA metabolism,including RNA splicing,RNA stability,and translation.We found that the demethylation effect of ALKBH5 on FTH1 did not affect its RNA splicing or stability,but mainly participated in translation regulation.In addition,we found that m6A methylation of FTH1 can enhance the binding of IRP protein to FTH1.Those results suggest that overexpression of m6A demethylase ALKBH5 and the resulting abnormal m6A methylation can affect the occurrence and development of GBM through the FTHI iron metabolism pathway,providing new ideas and theoretical basis for exploring the novel therapeutic targets of glioblastoma.In the second part,we investigated the effects of RNA m6A demethylase Alkbh5 gene knockout on cerebellar morphology and function in elderly mice.Our group previously found that under hypobaric hypoxia stimulation,the loss of Alkbh5 affects the balance of RNA m6A methylation,leading to impaired postnatal cerebellar development.We further investigated here whether ALKBH5 affects cerebellar neuropathy in the elderly mice.We found that with the aging of mice,the expression of ALKBH5 protein in cerebellum gradually increased,and the expression in Purkinje cell was higher than that in other types of neural cells.At the same time,the body weight and whole brain weight of elderly Alkbh5-/-mice(KO)decreased compared to age-matched wild-type mice(WT),and the cerebellar weight of elderly KO mice was significantly reduced.We also detected the expression of NeuN,Calbindin D28K,MAP2,GFAP and other proteins in the middleaged and elderly WT and KO mice through immunohistochemistry experiments.The number of Purkinje cell and neuron dendrites in aged KO mice decreased significantly.The time for the elderly KO mice to pass through the balance beam was significantly longer than that of the wild type mice of the same age,and their gait was unstable.However,we failed to observe similar phenotypes in the middle-aged mice.In summary,the deficiency of demethylase ALKBH5 caused cerebellar atrophy,loss and damage of Purkinje neurons in the elderly mice,thereby impairing their motor coordination and balance abilities.The above results suggest that imbalance in RNA m6A methylation can lead to neurodegenerative lesions in the mouse cerebellum.In conclusion,we explored the function and mechanism of RNA m6A demethylase ALKBH5 in glioblastoma and cerebellar neurodegeneration from two different viewpoints,thus providing new idea for the treatment of nervous system disease. |
