PA2G4 Promotes The Metastasis Of Hepatocellular Carcinoma By Stabilizing FYN MRNA In A YTHDF2-Dependent Manner

Objective: Hepatocellular carcinoma(HCC)is one of the most common cancers worldwide with high mortality.Advanced stage upon diagnosis and cancer metastasis are the main reasons for the dismal prognosis of HCC in large part.The proliferation-associated protein2G4(PA2G4),alternatively named Erb B3-binding protein 1(EBP1),exerts versatile functions as a general signaling molecule in cell physiological processes,involving in regulating cell proliferation,differentiation,immune response and apoptosis.The role of PA2G4 in tumorigenesis and cancer progression has been widely investigated in multiple cancer types.Despite that,the role and mechanism of PA2G4 in the metastasis and progression of hepatocellular carcinoma is still unclear.Methods and Results: To explore the clinical relevance of PA2G4 for HCC progression,TCGA database was used to evaluate its m RNA expression pattern.The m RNA levels of PA2G4 were significantly higher in HCC specimens than in normal liver tissues.Moreover,the m RNA and protein levels of PA2G4 were also upregulated in HCC samples compared with normal liver tissues,and high expression of PA2G4 in HCC was correlated with a poor prognosis,by an integrative analysis of immunohistochemistry(IHC),western blot and bioinformatic approach.Additionally,the expression of PA2G4 was elevated in HCC patients with metastases than those metastasis-free.Multivariate Cox regression analysis also demonstrated that PA2G4 was an independent risk factor for recurrence.Cell migration,invasion,phalloidin staining and western blot analyses demonstrated that PA2G4 promoted migration,invasion and epithelial to mesenchymal transition(EMT)of HCC cells in vitro.And a lung metastasis animal model exhibited that PA2G4 enhanced metastatic ability of HCC cells in vivo.RNA-sequencing combined with dual luciferase reporter assay and evaluation of m RNA half-time indicated that PA2G4 increased FYN expression by stabilizing its m RNA transcript.FYN is a non-receptor tyrosine kinases(RTK)member of the SRC family kinase(SFK).In cancers,it was reported to play a pro-tumorigenic and prometastatic role by promoting proliferation.Recovering the impaired FYN level induced by PA2G4 knockdown rescued the impeded cell mobilities.Endogenous immunoprecipitation(IP)and in-situ immunofluorescence(IF)showed that YTH N6-methyladenosine RNA binding protein 2(YTHDF2)was the endogenous binding patterner of PA2G4.RNA binding protein immunoprecipitation(RIP)and anti-N6-methyladenosine immunoprecipitation(Me RIP)assays demonstrated that FYN m RNA was N6-methyladenosine(m6A)modified and bound with PA2G4,as well as YTHDF2.Furthermore,the m6A catalytic ability of YTHDF2 was found indispensable for the regulation of FYN by PA2G4.At last,the correlation of expression levels between PA2G4 and FYN in HCC tissues was verified by IHC and western blot analysis.Conclusions:PA2G4 plays a pro-metastatic role by increasing FYN expression through binding with YTHDF2 in HCC.At the same time,PA2G4 is highly expressed in HCC and elevated expression of PA2G4 in HCC predicted poor prognosis indicate that PA2G4 may become a reliable prognostic marker or therapeutic target for HCC patients.