The Role And Mechanism Of RNA N6-methyladenosine Modification On The Biological Behaviour Of Papillary Thyroid Cancer

Background:Thyroid cancer is the most common malignancy of endocrine system.In recent years,the incidence of thyroid cancer has been increasing year by year,and it ranks the 7^thamong malignant cancers in China.More than 95%of the pathological types of thyroid cancers are differentiated thyroid cancers(DTC),and papillary thyroid cancer(PTC)is the most common pathological subtype in DTC.Most PTCs have a good prognosis after standardized treatment,but about 15%of patients still arise recurrence,metastasis and iodine resistance after operation,which significantly reduces the survival rate.Therefore,it is urgent to further clarify the molecular mechanism of PTC.RNA methylation modification is the most common chemical modification of RNA in higher eukaryotes,including m6A,m5C,m1A,etc.N6-methylpurine(m6A)is the most common of RNA methylation modification,it regulates all stages of RNA life cycle dynamically and reversibly through theRNA methyltransferases(Writer),theRNA demethylases(Eraser)and m6A-binding proteins(Reader).More and more evidence supports that m6A is closely related to the occurrence and development of many tumors,such as breast cancer,intestinal cancer,leukemia,etc.However,whether the m6A modification could play a biological role in PTC is still unclear.Our study aims to explore the role of RNA m6A modification in PTC,find the m6A modifying enzyme that plays a key role,and clarify the effect of this key enzyme on the biological behavior of PTC and its specific regulatory mechanism.Methods:First,we detected the overall level of m6A in 5 pairs of PTC tissues and paired adjacent tissues by m6A level quantitation kit.Then,the expression of m6A modified enzymes in 30 PTC tissues and paired adjacent tissues were detected by q RT-PCR,and screened the key enzyme of m6A modification in PTC.After confirming that the demethylase FTO is the key enzyme of m6A modification in PTC,we expanded the tissue sample size to 100 pairs and detected the mRNA expression of FTO in PTC tissues and paired adjacent tissues by q RT-PCR,then analyzed the relationship with clinicopathological features.The protein expression of FTO was detected by western blot and immunohistochemistry in 20 pairs of PTC tissues and paired adjacent tissues.Secondly,we detected the protein expression of FTO in the normal thyroid cell line Nthy-ori3-1 and four PTC cell lines by western blot,and selected the PTC cell lines whose FTO protein expression was significantly lower than that of Nthy-ori3-1 cell line for experiment.Then,we used si-RNA to down-regulate the expression of FTO and lentivirus to over-express FTO,and constructed over-expressed FTO-mut group cell lines with mutated m6A domain.The transfection and overexpression efficiency was determined by q RT-PCR and western blot.The effect of FTO on PTC cell proliferation was detected by CCK8 assay and plate cloning experiments,and its effect on the growth of PTC xenograft tumor was detected by PTC nude mice xenograft.The effect of FTO on PTC cellular glycolysis metabolism was detected by Seahorse XF Glycolysis Test,Glucose Uptake and L-Lactate Colorimetric assay.Glycolytic metabolism-related proteins were detected by western blot.¹⁸F-FDG micro PET imaging was used to detect the effect of FTO on glucose uptake in PTC xenograft tumors.Finally,the target gene with m6A modification downstream of FTO was screened by MeRIP-seq and mRNA-seq.Detect the effects of FTO on the mRNA and protein expression of target gene APOE by q RT-PCR and western blot.Subsequently,DAA inhibitor,MeRIP-qPCR and dual luciferase reporter gene were used to verify the m6A modification effect of FTO on APOE.QRT-PCR was used to detect the effect of down-regulation of IGF2BP family on APOE expression,and RNA stability assay was used to detect the influence of FTO and IGF2BP2 on the mRNA stability of APOE.Then,we detected the expression of APOE in PTC tissues and paired adjacent tissues by q RT-PCR and immunohistochemistry,and its relationship with clinicopathological features and the correlation with FTO expression in PTC were also analyzed.The effect of APOE on PTC proliferation and glycolytic metabolism were tested in vitro and vivo experiments.In addition,we used GSEA to analyze the common downstream signal pathways of FTO and APOE,and detect the expression of signaling pathway-related proteins by western blot.Results:We found that the overall level of RNA m6A modification in PTC is up-regulated,and the expression of demethylase FTO is significantly down-regulated in PTC tissues,which is negatively related with extrathyroidal extension and lymph node metastasis.The protein expression of FTO in PTC is also down-regulated.The expression of FTO in TPC1 and K1 cell lines was significantly lower than that of normal thyroid cell line Nthy-ori3-1.Therefore,we selected these two cell lines for experiments.Si-FTO could significantly down-regulate the expression of FTO and increase the overall level of m6A in PTC cells.Down-regulating FTO could promote the proliferation of K1and TPC1 cells,and it also could promote the tumor growth of PTC in vivo.Overexpression of FTO could reduce the overall level of m6A in PTC cells and inhibit the proliferation of PTC in vitro and vivo,while the overexpression of FTO-mut group had no effect on the overall level of m6A and proliferation of PTC.Meanwhile,down-regulating FTO could promote the glycolytic metabolism of PTC cells,and inhibit its mitochondrial oxidative phosphorylation.Glucose consumption and lactate production of PTC cells were increased,and the expression of glycolysis-related proteins GLUT1,HK2 and LDHA were also up-regulated.The overexpression of FTO could inhibit the glycolytic metabolism of PTC cells,but the overexpression of FTO-mut had no effect on their glycolytic metabolism.Moreover,the glycolysis inhibitor 2-DG could inhibit cell proliferation caused by down-regulation of FTO,indicating that FTO affects cell proliferation through glycolytic metabolism.¹⁸F-FDG micro PET imaging revealed that FTO could inhibit the glucose uptake of PTC xenograft tumors in nude mice.MeRIP-seq and RNA-seq data were combined to identify the target gene APOE of m6A modification downstream of FTO.Down-regulation of FTO will promote the expression of APOE,and over-expression of FTO will inhibit the expression of APOE.Methylation inhibitor DAA could inhibit the up-regulation expression of APOE caused by down-regulation of FTO.MeRIP-qPCR found that m6A antibodies could enrich m6A-modified mRNA of APOE,and down-regulation of FTO could significantly increase the degree of enrichment.The dual luciferase reporter gene assay found that down-regulation of FTO could increase the luciferase activity of wild-type APOE,while it has no effect on the luciferase activity of mutant-type APOE.Subsequently,we found that down-regulating FTO could increase the stability of APOE mRNA and promote its expression,while down-regulating IGF2BP2 will down-regulate the expression of APOE by reducing its mRNA stability.At the same time,down-regulation of FTO will increase the binding of IGF2BP2 to APOE mRNA.We found that the expression of APOE was significantly up-regulated in PTC tissues,and was positively related to the diameter?2cm and extrathyroidal extension.Moreover,the expression of APOE and FTO in PTC were significantly negatively correlated.Down-regulation of APOE could inhibit the proliferation,glycolytic metabolism and the expression of GLUT1 protein.2-DG could inhibit cell proliferation caused by overexpression of APOE,indicating that APOE also affects cell proliferation through glycolytic metabolism.Co-transfection of si-FTO and si-APOE found that down-regulation of APOE could significantly reduce the proliferation,glycolytic metabolism,glucose consumption and lactate production caused by si-FTO.In vivo,we also found that down-regulation of APOE could significantly reduce sh-FTO-induced proliferation and glucose uptake.Finally,GSEA analysis found that both FTO and APOE are highly enriched with downstream IL-6/JAK/STAT3signaling pathways.Down-regulating FTO will promote the expression of p JAK2 and p STAT3 proteins,and down-regulating APOE could inhibit p JAK2 and p STAT3proteins expression.Conclusion:The overall level of RNA m6A is up-regulated in PTC,and the expression of demethylase FTO is significantly down-regulated.FTO inhibits the expression of APOE through m6A modification mediated by IGF2BP2 and may inhibit the glycolytic metabolism of PTC through the IL-6/JAK2/STAT3 signaling pathway,thereby affecting its proliferation.