PART ?:Study On The Function And Molecular Mechanism Of METTL7A And METTL7B In Colorectal Cancer PART ?:YAP Mediates Protease-activated Receptor 2 Signaling To Enhance The Anti-apoptotic Ability Of Colon Tumor Stem Cells

PART ?:Study on the function and molecular mechanism of METTL7A and METTL7B in colorectal cancerColorectal cancer(CRC)is the third-most common type of cancer and with a high mortality rate in China.Hence,to resherch the mechanism of colorectal cancer may help to find new targets or biomarkers for cancer therapy.Proteins are subjected to different post-translational modifications(PTMs)in vivo,and abnormal PTMs promote tumorigenesis.Protein methyltransferases catalyze the methylation of proteins involved in histone epigenetics or non-histone activity.METTL7A and METTL7B are members of the methyltransferase-like family,and their protein structures have conserved methyltransferase domains,but their function and mechanism in colorectal cancer has not been reported.Here,we analyzed animal models of colon cancer and various public databases of colorectal cancer(GEO and TCGA data),and found that METTL7A and METTL7B were reduced in tumor tissues.In addition,the low expression of METTL7B in tumor tissues was verified by IHC.To further confirm the role of METTL7A and METTL7B in colorectal cancer,we established HT-29,SW480 and DLD1 overexpression and knockdown cell lines.After the overexpression of METTL7A and METTL7B,the colony formation of different cell lines,migration ability and the growth of xenograft tumor in vivo were significantly inhibited.Conversely,knocking down METTL7A and METTL7B promoted cell migration and colony formation.In order to explore the tumor inhibition mechanism of METTL7A and METTL7B,we obtained the interacting proteins with METTL7A and METTL7B by mass spectrometry,and predicted the biological functions by GO function enrichment.The results showed that the METTL7B interacting proteins were mainly involved in protein synthesis and mitochondrial function.Puromycin labeling assay show overexpression of METTL7B inhibited protein synthesis.Finally,it was confirmed that METTL7B inhibits the activation of mTORC1 signaling pathway and the phosphorylation of downstream translation-related proteins S6K1 and 4EBP1,thus inhibiting mRNA translation and reducing protein synthesis.I-TASSER online analysis showed that METTL7B contained conserved methyltransferase domain.The function of METTL7B inhibited mTOR downstream kinases p-S6K1 and p-4EBP1 was abolished after mutating the predicted methylase active site.These results prove that METTL7B may regulate mTOR signaling pathway through methyltransferase activity,inhibit protein synthesis,and ultimately inhibit tumor formation.At the same time,predicted data shows METTL7A may involve in peptide chain elongation,but need further confirmation.We used AOM-DSS to induce colon cancer in Mettl7a1 or Mettl7b knockout mice.The results of animal models showed that deletion of Mettl7al or Mettl7b gene significantly promoted tumor formation,including increased tumor number and tumor volume.In conclusion,METTL7A and METTL7B inhibit the migration and colony formation of tumor cells;METTL7B regulates protein synthesis in vivo by inhibiting mTOR signaling pathway;the tumor suppressive function of METTL7B may depend on the activity of methyltransferase.Our reshearchs of METTL7A and METTL7B may provide a new theoretical basis for the diagnosis and treatment of colorectal cancer.PART ?:YAP mediates protease-activated receptor 2 signaling to enhance the anti-apoptotic ability of colon tumor stem cellsTumor stem cells(CSCs)have the ability to initiate tumor growth and play critical roles in the formation,recurrence and metastasis of cancer.High concentration of reactive oxygen species(ROS)in the inflammatory microenvironment can induce stem cell apoptosis,but improving the anti-apoptotic ability of colon CSCs can induce tumor formation.In addition,the inflammatory microenvironment is enriched with a large number of proteases,some of which can selectively activate protease-activated receptor 2(PAR2)to promote tumor development.We confirmed the high expression of PAR2 in colorectal cancer tissues by IHC,and the survival rate of patients with high expression of PAR2 was significantly reduced.Blocking PAR2 signal by shRNA or inhibitors significantly inhibited colon CSC sphere formation,flow cytometry showed that knockdown PAR2 reduced the anti-apoptotic ability of tumor stem cells.Our previous study found that PAR2 regulates the stability and transcriptional activity of YAP protein,and YAP plays important role in tumor stem cells.We overexpressed different mutant forms of YAP in PAR2 knockdown cells,and functional experiments showed that rescue YAP did not affect stem cell renewal ability,but significantly enhanced the anti-apoptotic ability.On the other hand,PAR2-YAP signaling not only inhibits intracellular ROS production,but also enhances the resistance of stem cells to NO-induced apoptotic.Finally,mRNA microarray analysis showed that PAR2-YAP signal regulated antioxidant gene CAV1 expression,and which positively correlated with anti-apoptotic gene MCL1 and BCL2.In conclusion,we demonstrate that PAR2 activation promotes colon tumor stem cell properties and PAR2-YAP signaling enhances the resistance of colorectal CSC to nitric oxide-induced apoptotic;elucidating a new mechanism for tumor stem cell survival in the inflammatory microenvironment.