Fig 2: DTL is involved in the OPN-induced invasiveness of liver cancer cells. Cells were transfected with DTL siRNA or OPN expressing vector or treated with rhOPN for 24 h and then the cells were assessed using the Transwell assay. (A) DTL knockdown slightly inhibited invasion by both Huh7 cells and HepG2 cells, but markedly decreased the rhOPN-induced invasiveness of both Huh7 cells and HepG2 cells. These results indicated that DTL is also involved in the rhOPN-induced promotion of the invasiveness of both Huh7 cells and HepG2 cells. The data are presented as the mean ± SD of three independent experiments. (B) Similar effects were also demonstrated in the groups transfected with the vector expressing OPN. The results further indicated DTL is involved in the invasiveness of both Huh7 cells and HepG2 cells, serving a vital role in OPN-induced invasiveness of both Huh7 cells and HepG2 cells. *P<0.05. OPN, osteopontin; DTL, denticleless E3 ubiquitin protein ligase homolog; NC, negative control; rh, recombinant human.

Fig 3: OPN mediates the protein expression level of DTL in liver cancer cells. (A) Treatment of Huh7 cells with rhOPN induced the expression of DTL in a dose-dependent manner. After 6 h of treatment with 5 and 20 nM rhOPN, DTL expression was significantly upregulated in Huh7 cells. (B) Similar effects were demonstrated in HepG2 cells; rhOPN also significantly induced the expression of DTL in the HepG2 cells. (C) An OPN-overexpression vector and OPN siRNAs were used to regulate the OPN level in Huh7 cells. The DTL level was then detected using western blotting at 48 h after transfection of the cells, which demonstrated that OPN knockdown by OPN siRNAs led to significant downregulation of the expression of DTL, whereas OPN overexpression led to a significant increase in the DTL protein expression level. Western blotting was also performed to assess the effects of knockdown of DTL on OPN-induced elevation of DTL level and the effect of knockdown of DTL on OPN expression. (D) Similar effects were observed in HepG2 cells. Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs. control. OPN, osteopontin; DTL, denticleless E3 ubiquitin protein ligase homolog; OE, overexpression; NC, negative control; Ctrl, control; rh, recombinant human.

Fig 4: PI3K/AKT signaling is involved in the regulation of DTL by OPN in liver cancer cells. (A) Western blotting indicated that treatment with the PI3K/AKT inhibitors LY294002 and wortmannin led to a significant decrease in the expression of DTL induced by rhOPN (5 nM) in Huh7 cells. Western blotting was also performed to assess the effects of LY294002 and wortmannin on the AKT pathway in Huh7 cells. The relative protein expression level has been semi-quantified for each group. (B) Similar effects were observed in HepG2 cells, and the rhOPN-induced enhancements of DTL expression were inhibited by treatments with LY294002 (5 µM) and wortmannin (5 µM). *P<0.05 vs. Ctrl or as indicated. OPN, osteopontin; DTL, denticleless E3 ubiquitin protein ligase homolog; OE, overexpression; NC, negative control; Ctrl, control; rh, recombinant human.

Fig 5: DTL mediates the effects of OPN on the proliferation of liver cancer cells. (A and B) The levels of OPN and DTL were significantly decreased following transfection with the corresponding siRNAs, whereas the OPN level was significantly increased after transfection with the vector overexpressing OPN in liver cancer cells. The relative protein expression level was semi-quantified for each group. *P<0.05 and **P<0.01 vs. NC. (C and D) DTL knockdown attenuated the effects of OPN-induced proliferation in liver cancer cells. Data are presented as the mean ± SD of 3 independent experiments performed in quadruplicate. P<0.05 for the OPN OE vs. OPN OE + DTL siRNA for the 48 and 72 h time points. OPN, osteopontin; DTL, denticleless E3 ubiquitin protein ligase homolog; OE, overexpression; NC, negative control; Ctrl, control.