The Mechanism Of METTL14^R298H Mutation On Cholangiocarcinoma Based On Whole Exome Sequencing Of Cholangiocarcinoma

Backgrounds:Cholangiocarcinoma(CCA)is a group of highly heterogenous malignant tumors originating from epithelial cells of the bile duct system.According to the anatomic location,CCA was classified into Intrahepatic Cholangiocarcinoma(ICCA),Perihilar Cholangiocarcinoma(PCCA)and Distal Cholangiocarcinoma(DCCA).Due to the occult clinical symptoms,most patients lose the chance of surgical resection at presentation and the 5-year survival outcome was poor because of the easy recurrence.specially in East Asian countries represented by China and Japan,the increasing incidence and mortality of CCA make it one of the most challenging problems in hepatobiliary surgery.At present,studies on the genomic characteristics of CCA,especially ICCA,have found some common mutations in CCA.However,regional and ethnic differences in the incidence of CCA subtypes suggest significant geographic and ethnic differences in their genomic characteristics,and studies on the genomic differences of CCA and its subtypes in Asian populations are lacking.Therefore,the integrated analysis of CCA genome data in Asian countries will help us to further identifed new CCA genome characteristics.At the same time,the analysis of heterogeneity between CCA subtypes will enable us to analyze the genomic differences between the iCCA and pCCA.Aims:In this study,we explored the genomic characteristics of CCA and its subtypes in Asian populations,especially the important genomic characteristics in the process of post-transcriptional modification,aiming to find new genomic characteristics of CCA and its subtypes,and provide new ideas for further research.Methods:We analyzed the whole-exon sequencing data of 348 CCA samples from three Asian centers,including 87 cases of pCCA(43 pairs of samples in our center)and 261 cases of iCCA(24 pairs of iCCA samples in our center).Results:We found that the non-synonymous mutation ratio and copy number alteration burden of iCCA were significantly higher than those of pCCA;CCA patients with high tumor mutation burden had poor prognosis,and copy number alteration burden had a good predictive effect on prognosis in pCCA patients.The enrichment results of COSMIC mutation signature showed that age-related mutation signature and mismatch repair defect-related signature accounted for a significant proportion in pCCA patients,and the overall proportion was higher than that in iCCA patients.Smoking-related mutation signature,aristolochic acid-related mutation signature,and liver tumor-related mutation signature accounted for a significant proportion in iCCA patients;The proportion of mutations associated with liver tumors in iCCA patients infected with hepatitis B was significantly increased.A total of 36 significant mutation genes were found in this study(12 of them were reported for the first time:MACF1,EPHA2,ATM,RBM10,PIK3R1,MLLT4,BRCA2,NACC1,SMARCA4,WHSC1,METTL14,AXIN1),among which the mutations in the key genes of posttranscriptional regulation RBM10 and METTL14 suggested that post-transcriptional regulation might play an important role in the occurrence and development of CCA.In 22 key regions identified for frequent copy number variations(6 copy number variation regions were first reported:amplification of 7q31.2 and 22q11.21,deletion of 1p36.13,2p24.1,7q35 and 12q24.33).After the pathway enrichment of the above CCA mutation characteristics,it was found that the mutations were mainly enriched in RTK-RAS,?catenin/Wnt,PI3K,cell cycle,TP53,TGF-beta,and HIPPO pathways.Conclusions:In this study,we integrated the whole-exon sequencing data of CCA from three Asian centers,and found new driving genes.Potential post-transcriptional modification affected the occurrence and development of CCA,as well as the difference in genomic characteristics between pCCA and iCCA.The above findings provide genomic characteristics for future CCA studies in Asian populations,and offer research basis and new ideas for further clinical transformation of CCA.Backgrounds:M6A methylation plays an important role in the development of tumors as a key pathway of post-transcriptional regulation.METTL14 is the key catalytic protein of the core m6A methyltransferase complex during RNA m6A methylation modification;Exploring the important role of METTL14and its mediated m6A methylation modification in the development of CCA and clarifying the function of METTLH14R298H in this process have a positive significance for the exploration of the pathogenesis of CCA.Aims:In this part,we focused on the important functional role of METTL14wt and its mediated m6A methylation modification in the development of CCA,and identified the impact of METTL14R298H mutation on the function of METTL14 and its mediated m6A methylation modification in the biological process of CCA.This study provided evidence for predicting the potential harmful variation of METTL14R298H mutation in CCA based on the above whole-exon sequencing.Methods:Sanger sequencing was used to confirm the presence of METTL14R298H in CCA tissues,and the genomic information of METTL14 in the CCA cell lines;Western blot and qRT-PCR were used to verify the expression of METTL14 in CCA tissues and stable transfected cell lines;Tissue microarray immunohistochemistry was used to explore the protein expression of METTL14,and to test the correlation between its expression level and the long-term prognosis of CCA;The m6A methylation level mediated by METTL14wt/METTL14R298H was determined by m6A colorimetric assay.CCK8 cell growth curve,EDU proliferation,and cloning growth were used to detect the proliferation ability of CCA cells;Transwell migration/invasion test and cell scratch test were used to determine the metastasis ability of CCA cells;The effects of METTL14wt/METTL14R298H on the proliferation and metastasis of CCA in vivo were determined by subcutaneous tumor formation in nude mice and lung metastasis model in nude mice.Results:Sanger sequencing confirmed the METTL14R298H mutation in CCA,and the mutation was also found in the external validation including extra CCA samples(1/42).METTL14wt and its mediated m6A methylation modification level was low expressed in CCA,and METTL14 low expression suggested poor 5-year survival outcome in patients.By comparing the m6A methylation level of METTL14 high expression and low expression groups,it was confirmed that METTL14 expression affected the m6A methylation level in CCA.Western blot and qRT-PCR results showed that the initial METTL14 expression levels of RBE and HCCC9810 CCA cell lines were low,and sanger sequencing results confirmed that there was no METTL14R298H mutation in these two cell lines;Lentivirus overexpression successfully established METTL 14wt and METTL 14R298H mutation stable cell lines;The results of m6A colorimetric assay suggested that METTL 14R298H could rEdUce the m6A modification ability of METTL 14wt.The results of CCK8 cell growth curve,EDU proliferation assay,and cloning growth assay suggested that METTL 14wt significantly inhibited CCA cell proliferation,while METTL14R298H mutation caused it to lose the obvious CCA proliferation inhibition.Transwell migration/invasion assay and cell scratch assay suggested that METTL 14R298H mutation significantly reversed the significant inhibitory effect of METTL 14wt on tumor metastasis in CCA cell lines.The results of nude mice subcutaneous tumor formation experiment and lung metastasis model showed that METTL 14R298H mutation significantly affected the CCA anti-tumor effect of METTL 14wt in tumor proliferation and metastasis.Conclusions:METTL14wt and its mediated m6A methylation play a significant tumor suppressor role in CCA;METTL14R298H mutation inhibits significant anticancer effect of METTL14WT in the development of CCA,which was identified as a loss-of-function mutation.Backgrounds:MACF1(Microtubule actin cross-linking factor 1)is a multidomain protein,which is closely related to the nuclear transport of ?-catenin dominated by transport complex in Wnt/b-catenin signaling pathway.Abnormal expression of MACF1 will affect the nuclear transport of ?-catenin.At present,there is no m6A methylation modification mediated by METTL14wt/METTL14R298H on MACF1/?catenin has not been reported.Aims:To explore the mechanism of m6A methylation modification mediated by METTL14wt/METTL14-298H on MACF1 in CCA,and further identify the effect of m6A methylation modification of MACF1 on the nuclear transport of ?-catenin.To further understand the specific mechanism of METTL14wt/METTL14R298H-mediated m6A methylation modification in the development of CCA.Methods:MeRIP(Methylated RNA immunoprecipitation)sequencing was used to explore the downstream target genes affected by METTL14wt/METTL14R298Hmediated m6A methylation modification;Based on the successful construction of MACF1 siRNA interfering CCA cell lines,the in vitro function experiments related to cell proliferation and metastasis verified the specific function of MACF1 in CCA.m6A-RIP assay verified the m6A methylation level of MACF1;RIP assay verified the difference in binding ability between METTL14wt/METTL14R298H and MACF1 mRNA;Western blot,qRT-PCR and immunofluorescence results suggested that MACF1 was differentially expressed in NC,METTL14wt and METTL14R298H;MACF1 actinomycin D mRNA lifetime assay verified the effect of m6 A methylation on the stability of MACF1 mRNA.Nucleoplasmic protein extraction assay was conducted to verify the effect of the difference in MACF1 expression mediated by METTL14wt/METTL14R298H on the nuclear transport of ?-catenin,and the expression changes of EMT pathway marker proteins(E-cadherin,N-cadherin and Vimentin)and value-added proteins(CyclinD1 and PCNA)were detected.Results:MeRIP sequencing results suggested that MACF1,a key gene in ?catenin/Wnt signaling pathway,was significantly regulated by METTL14wt/METTL14R298H-mediated m6A methylation modification;qRT-PCR result of CCA tissues and in vitro functional experiments showed that MACF1 played an oncogene role in the proliferation and metastasis of CCA.m6A-RIP assay suggested that METTL 14R298H had a significant methy lation modification for MACF1 compared with METTL 14wt;The results of RIP assay showed that the difference in methy lation modification of m6A might be related to the effect of METTL 14R298H mutation on the binding of METTL 14 to MACF1 mRNA.At the protein and mRNA levels,the expression of MACF1 in METTL 14wt cell line was significantly decreased,and the expression of MACF1 in METTL14R298H mutant cell line was higher than that in METTL14wt group.The lifetime assay of MACF1 mRNA and the knock-down of m6A reader YTHDF2 confirmed that the differential expression of MACF1 mRNA was mainly due to the difference in mRNA stability caused by the METTL14wt/METTL14R298H mediated m6A methy lation modification.The results of immunofluorescence and nucleoplasmic protein extraction assay showed that the expression of ?-catenin in the nucleus of METTL14R298H was higher than that of METTL14wt,which further affected the corresponding changes of downstream EMT pathway marker proteins(E-cadherin,N-cadherin,and Vimentin)and value-added proteins(CyclinD1 and PCNA).Conclusions:The above results showed that METTL 14R298H mutation affects the binding ability of METTL 14 to MACF1 mRNA,thereby causing the difference in m6A methylation of MACF1 mRNA,resulting in the change of MACF1 mRNA stability.Thus,by affecting the MACF1-mediated ?-catenin nuclear transport,it causes the change of CCA metastasis and proliferation-related proteins,and ultimately affects the occurrence and development of CCA.