The Role And Mechanism Of YTHDF3 In β-catenin Mutated Hepatocellular Carcinoma

Background and Aims:Hepatocellular Carcinoma（HCC）is a malignant tumor with the eighth largest number of new cases and the second largest number of new deaths in the world,which seriously threatens people’s health.Since most of the risk factors for HCC will lead to chronic hepatitis and cirrhosis in the first place,most patients with HCC are usually diagnosed with the relatively advanced stage of liver cancer,leading to the high recurrence rate and high mortality rate of HCC.The occurrence and development of HCC are often accompanied by gene mutations,among whichβ-catenin is one of the most common types with an incidence of up to 30%.YTH N⁶-methyladenosine RNA binding protein 3（YTHDF3）is one of the readers for RNA N⁶-methyladenosine（m⁶A）modification,which is involved in many important physiological and pathological processes.Therefore,the present research aims to explore the role of YTHDF3 inβ-catenin mutant HCC and elucidate its specific mechanism.Methods:In this study,the Ythdf3 knockout mice(Ythdf3^-/-)was constructed at first and the transposon plasmid expressingβ-catenin mutated protein was optimized.By the means of fluid dynamic injection through mice tail vein,transposase plasmid expressing Sleeping Beauty transposase as well as transposon plasmids expressingβ-catenin mutant and c-Met were injected into wild-type（WT）and Ythdf3^-/-mice,and the mouseβ-catenin mutated HCC model was successfully constructed.The difference of tumors from the two groups in this model was statistically compared,and the tumor tissues were stained with Ki67 and TUNEL.Next,the YTHDF3knockout cell line was constructed by CRISPR/Cas9 system in Hep G2 cell line,which has been provedβ-catenin mutated HCC cell line.Meanwhile,the YTHDF3overexpressed Hep G2 cell line was successfully constructed by lentivirus infection.The effects of YTHDF3 knockout or overexpression on the proliferation of Hep G2cell line were analyzed by cell function assays,including colony formation assay,CCK-8 assay and Ed U assay.By taking advantage of database analysis and m⁶A-seq analysis,we found zinc finger E-box binding homeobox 2（ZEB2）as the target of YTHDF3 inβ-catenin mutated HCC,which was confirmed by RNA immunoprecipitation（RIP）-q PCR.Then,ZEB2 expression was knocked down by sh RNA in YTHDF3 overexpressed Hep G2 cell line,and the proliferation of Hep G2cell line was assessed by functional experiments.Additionally,the Hep G2 cell line expressing YTHDF3 mutant protein was constructed,which was m⁶A-binding defective,and the binding of YTHDF3 protein and ZEB2 m RNA was evaluated by RIP-q PCR.The proliferation ability of mutant YTHDF3 expressing Hep G2 cell line was also compared with that of normal YTHDF3 overexpressing Hep G2 cell line.METTL3 was also knocked down in normal and YTHDF3 overexpressing Hep G2cell lines by sh RNA,and the expression of ZEB2 was detected.Besides,cicloheximide（CHX）and MG-132 inhibition assays were performed to verify how YTHDF3 regulated ZEB2 expression.At last,the correlation between YTHDF3 andβ-catenin mutation in human HCC was confirmed by analyzing the tissue samples from HCC patients by immunohistochemistry,western blotting,quantitative fluorescence PCR（q PCR）and immunofluorescence.Results:In this research,we constructed the mouseβ-catenin mutated HCC model in WT and Ythdf3^-/-mice by mouse tail vein fluid dynamic injection,and found that Ythdf3knockout significantly inhibited the growth ofβ-catenin mutated HCC.Moreover,the results of Ki67 and TUNEL staining indicated that YTHDF3 affected the tumor growth by regulating the proliferation ability ofβ-catenin mutated HCC cells rather than apoptosis.The results of functional assays performed in YTHDF3 knockout or overexpressing Hep G2 cell lines further demonstrated that YTHDF3 could regulate the proliferation ofβ-catenin mutated HCC cell lines.In addition,by analyzing the m⁶A-seq results of YTHDF3 knockout Hep G2 cell line as well as the published sequencing data,it was suggested that YTHDF3 played a regulatory role inβ-catenin mutant HCC by targeting ZEB2.The results of RIP-q PCR proved the direct binding of YTHDF3 protein and ZEB2 m RNA,and knockdown of ZEB2 in YTHDF3overexpressing Hep G2 cell line significantly impaired the enhancement of proliferation ability of Hep G2 cells by YTHDF3 overexpression.Besides,by analyzing the difference between normal and mutant YTHDF3 overexpressing Hep G2cell lines,we demonstrated that YTHDF3 targeted ZEB2 in an m⁶A-dependent manner and knocking down of METTL3 strongly impaired the binding of YTHDF3protein and ZEB2 m RNA.CHX and MG-132 inhibition assays suggested that YTHDF3 regulated ZEB2 expression by facilitating translation efficiency.Finally,a significant correlation between YTHDF3 andβ-catenin mutation in HCC was identified by analyzing the tissue samples from HCC patients,which was consistent with the conclusion above.Conclusion:In summary,this research found that YTHDF3 was significantly correlated withβ-catenin mutated HCC,and YTHDF3 deficiency inhibited tumor growth in the mouseβ-catenin mutated HCC model.Besides,it was demonstrated that YTHDF3targeted ZEB2 m RNA in an m⁶A-dependent manner and regulated the proliferation ofβ-catenin mutated HCC tumor cells by facilitating the translation efficiency of ZEB2.By exploring the role and mechanism of YTHDF3 inβ-catenin mutated HCC,our research proposed a new idea for understanding the mechanism of HCC and provided a novel potential target for the clinical treatment of HCC.