Objective:To screen the interaction proteins of WW domain containing protein 1 (WWP1), and explore the effects of WWP1 and etoposide induced 24 (EI24) on cell proliferation in hepatocellular carcinoma (HCC).Methods:Yeast two-hybrid screening system was used to identify the interaction proteins of WWP1. The interaction was further validated by co-immunoprecipitation. WWP1 and EI24 stably over-expressing or deleted HepG2 cells were established by using the lentivirus transduction method. Colony forming assay and cell counting kit-8 (CCK8) assay were performed to identify the effects of WWP1 and EI24 on cell proliferation. In addition, the role of WWP1 in the tumorigenicity of liver cancer in vivo was examined by subcutaneous injection of different level of WWP1 expressed HepG2 into nude mice. Results:WWP1 can interact with EI24 and ubiquitin-degrade EI24 protein. The WWP1 and EI24 over-expressing or deleted HepG2 cell lines were successfully generated. Overexpression of WWP1 decreased while knockdown of WWP1 increased the protein level of EI24. The results of CCK-8 assay showed that the relative proliferation activities of WWP1 overexpressed (WWP1-OE) group and WWP1 knockdown (shWWP1) group on 36 hours were (347.00±8.15)% and (187.08±4.86)%, respectively, significantly different from (270.33±15.01)% of control group (both P<0.05). The relative proliferation activities of EI24 overexpressed (EI24-OE) group and EI24 knockdown (shEI24) group on 36 hours were (183.75±8.11)% and (317.33±9.60)%, respectively, significantly different from (270.33±15.01) % of control group (both P<0.05). The results of colony formation assay showed that the colony numbers of control group, WWP1-OE group and shWWP1 group were (52±7)/visual field (VF), (76±4)/VF, (19±3)/VF, respectively. Overexpression of WWP1 significantly increased while knockdown of WWP1 significantly decreased the colon formation ability of HepG2 cells (both P<0.05). The colon number of control group, EI24-OE group and shEI24 group were (38±4)/VF, (10±3)/VF, (69±7)/VF, respectively. Overexpression of EI24 significantly decreased while knockdown of EI24 significantly increased the colony formation ability of HepG2 cells (both P<0.05). The results of xenograft mice model showed that the tumor volumes of control, WWP1-OE, and shWWP1 group were (1 400.00±43.71)mm 3, (2 636.67±290.45) mm 3 and (642.17±36.00)mm 3, respectively, with significant differences ( P<0.05). The tumor weight for these three groups were (1.23±0.08)g, (2.05±0.17)g, and (0.88±0.09)g, respectively, with significant differences ( P<0.05). The tumor volumes of control, EI24-OE, and shEI24 group were (1 245.17±93.10)mm 3, (662.17±60.88)mm 3 and (1 986.67±226.75)mm 3 respectively, with significant differences ( P<0.05). The tumor weight for these three groups were (1.15±0.04)g, (0.85±0.02)g and (1.73±0.05)g respectively, with significant difference ( P<0.05). Conclusion:WWP1 promote the cell proliferation of liver cancer through ubiquitin-degradation of EI24.

Objective:To screen the interaction proteins of WW domain containing protein 1 (WWP1), and explore the effects of WWP1 and etoposide induced 24 (EI24) on cell proliferation in hepatocellular carcinoma (HCC).Methods:Yeast two-hybrid screening system was used to identify the interaction proteins of WWP1. The interaction was further validated by co-immunoprecipitation. WWP1 and EI24 stably over-expressing or deleted HepG2 cells were established by using the lentivirus transduction method. Colony forming assay and cell counting kit-8 (CCK8) assay were performed to identify the effects of WWP1 and EI24 on cell proliferation. In addition, the role of WWP1 in the tumorigenicity of liver cancer in vivo was examined by subcutaneous injection of different level of WWP1 expressed HepG2 into nude mice. Results:WWP1 can interact with EI24 and ubiquitin-degrade EI24 protein. The WWP1 and EI24 over-expressing or deleted HepG2 cell lines were successfully generated. Overexpression of WWP1 decreased while knockdown of WWP1 increased the protein level of EI24. The results of CCK-8 assay showed that the relative proliferation activities of WWP1 overexpressed (WWP1-OE) group and WWP1 knockdown (shWWP1) group on 36 hours were (347.00±8.15)% and (187.08±4.86)%, respectively, significantly different from (270.33±15.01)% of control group (both P<0.05). The relative proliferation activities of EI24 overexpressed (EI24-OE) group and EI24 knockdown (shEI24) group on 36 hours were (183.75±8.11)% and (317.33±9.60)%, respectively, significantly different from (270.33±15.01) % of control group (both P<0.05). The results of colony formation assay showed that the colony numbers of control group, WWP1-OE group and shWWP1 group were (52±7)/visual field (VF), (76±4)/VF, (19±3)/VF, respectively. Overexpression of WWP1 significantly increased while knockdown of WWP1 significantly decreased the colon formation ability of HepG2 cells (both P<0.05). The colon number of control group, EI24-OE group and shEI24 group were (38±4)/VF, (10±3)/VF, (69±7)/VF, respectively. Overexpression of EI24 significantly decreased while knockdown of EI24 significantly increased the colony formation ability of HepG2 cells (both P<0.05). The results of xenograft mice model showed that the tumor volumes of control, WWP1-OE, and shWWP1 group were (1 400.00±43.71)mm 3, (2 636.67±290.45) mm 3 and (642.17±36.00)mm 3, respectively, with significant differences ( P<0.05). The tumor weight for these three groups were (1.23±0.08)g, (2.05±0.17)g, and (0.88±0.09)g, respectively, with significant differences ( P<0.05). The tumor volumes of control, EI24-OE, and shEI24 group were (1 245.17±93.10)mm 3, (662.17±60.88)mm 3 and (1 986.67±226.75)mm 3 respectively, with significant differences ( P<0.05). The tumor weight for these three groups were (1.15±0.04)g, (0.85±0.02)g and (1.73±0.05)g respectively, with significant difference ( P<0.05). Conclusion:WWP1 promote the cell proliferation of liver cancer through ubiquitin-degradation of EI24.