Fig 1: MiR‐552 targeted AJAP1 and inhibited AJAP1 expression. (A) The predicted binding sequences of miR‐552 and AJAP1 were obtained from TagetScanHuman 7.1. The sequences of wild‐type (WT) and mutated (MUT) AJAP1 are also shown. (B) A luciferase reporter gene assay was used based on the construction of WT AJAP1 3’‐UTR and MUT AJAP1 3′‐UTR. The relative luciferase activities in the Hep3B and HepG2 cell lines in the WT AJAP1 3′‐UTR group were found to be significantly lower than those of cells in the miR‐552 NC group, whereas the MUT AJAP1 3′‐UTR group differed insignificantly from its counterpart (the NC group) in both cell lines. *, P < 0.05 compared to the NC group. (C) RT‐qPCR results showed the expression levels of AJAP1 in Hep3B and HepG2 cells transfected with miR‐552 mimics and miR‐552 inhibitor. AJAP1 expression was found to be lower when miR‐552 was up‐regulated, yet higher expression was detected in the miR‐552 inhibitor group. *, P < 0.05 compared to the NC group. (D) Western blotting was used to measure the expression level of AJAP1 in Hep3B and HepG2 cells transfected with miR‐552 mimics and miR‐552 inhibitor. The miR‐552 mimics led to lower AJAP1 expression, while the miR‐552 inhibitor led to higher AJAP1 expression. (E) Among the 81 pairs of HCC and adjacent tissues, AJAP1 was shown to have lower expression in tumour tissues based on the RT‐qPCR experiment. ****, P < 0.0001 compared to the adjacent tissue group. (F) Relative expression of miR‐552 was negatively correlated with that of AJAP1 in HCC tissues, which was showed a very significant correlation (P < 0.0001)
Fig 2: Prognosis results of miR‐552 and AJAP1 of hepatocellular carcinoma (HCC) patients. (A) The survival rate of HCC patients after complete resection was higher in the high‐AJAP1‐level group. P < 0.05 compared to the low‐AJAP1‐level group. (B) The survival rate of HCC patients after complete resection was lower in the high‐miR‐552‐level group. P < 0.05 compared to the low‐miR‐552‐level group
Fig 3: MiR‐552 facilitated EMT and hepatocellular carcinoma (HCC) oncogenesis by downregulating AJAP1. (A, B) Using Western blot and RT‐qPCR experiments, we found the inhibitory effects of miR‐552 on the expression levels of E‐cadherin and ZO‐1 and promotive effects on the expression levels of N‐cadherin and Vimentin. AJAP1 upregulation resulted in higher E‐cadherin and ZO‐1 levels and lower N‐cadherin and Vimentin levels. Overexpression of miR‐552 and AJAP1 in combination did not influence these expression levels. *, P < 0.05 compared to the NC group. (C, D) A nude mice tumour transplantation assay showed that miR‐552 acted as a tumour promoter and that AJAP1 acted as an inhibitor in comparison with the NC group. The two in combination did not affect tumour size. *, P < 0.05 compared to the NC group
Fig 4: The mechanism of miR‐552 on altering HCC cell behaviours. Overexpression of miR‐522 binds to AJAP1 mRNA and inhibits its translation. As a result, the level of AJAP1 protein was attenuated, and the level of Src protein, which suppressed by AJAP1, was elevated. The downstream pathway was activated and further promoted EMT progression and cell proliferation. The highlighted elements of the model were verified in our study
Fig 5: MiR‐552 suppressed hepatocellular carcinoma (HCC) cell proliferation, migration and invasion by targeting AJAP1. (A) Hep3B and HepG2 cells transfected with empty vectors or AJAP1 plasmids were examined using RT‐qPCR. The relative mRNA levels of AJAP1 were significantly higher in the AJAP1 groups, indicating successful transfection. ***, P < 0.001 compared to the vector group. (B) The results from Western blot experiment showed higher relative expression levels in cells of the AJAP1 group than in cells of the vector group for the both Hep3B and HepG2 cell lines. This observation verified successful AJAP1 transfection. (C) CCK8 assay results showed that in both cell lines, AJAP1 overexpression suppressed HCC cell proliferation, whereas miR‐552 + AJAP1 cotransfection had no significant influence on cell proliferation. (D) In the Transwell assay to assess cell migration, fewer cells migrated when AJAP1 was overexpressed compared with the control group; however, the mimics+AJAP1 group had almost no difference compared to the control group. (E) In the Transwell assay to assess cell invasion, there was less invasion by AJAP1‐overexpressing cells compared to the control group; however, the mimics+AJAP1 group had almost no difference. *, P < 0.05 compared to the control group
Supplier Page from Abcam for Anti-AJAP1 antibody