RESULTS: We found that the
mRNA and
protein levels of PA2G4 were higher in HCC samples than in 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. Moreover, the expression of PA2G4 was elevated in HCC patients with
metastases than those
metastasis-free. Cell migration, invasion,
phalloidin staining and western blot analyses demonstrated that PA2G4 promoted 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
mRNA half-time indicated that PA2G4 increased FYN expression by stabilizing its
mRNA transcript. Recovering the impaired FYN level induced by PA2G4 knockdown rescued the impeded cell mobilities. Furthermore, 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. In addition,
RNA binding protein immunoprecipitation (RIP) and anti-
N6-methyladenosine immunoprecipitation (MeRIP) assays demonstrated that FYN
mRNA was
N6-methyladenosine (m6A) modified and bound with PA2G4, as well as YTHDF2. Moreover, 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: These results indicate that PA2G4 plays a pro-metastatic role by increasing FYN expression through binding with YTHDF2 in HCC. PA2G4 may become a reliable prognostic marker or therapeutic target for HCC patients.