Abstract: Objective To explore the potential mechanism of HBV HBx protein on liver cancer cell proliferation regulation. Methods After HBx overexpression, the proliferation of HEPG2 liver cancer cells was detected. HBx was immunomotized co-precipitated in the Flag-HBx overexpressed HEPG2 cells, and then protein mass spectrometry was performed, the SCORE threshold was set to be greater than 50, and the Coverage threshold was set to be greater than 10. In HBx overexpressed HEPG2 cells, siRNAs were used to knock down the corresponding genes identified by the mass spectrometry, and the proliferation of cells was detected. Results The proliferation level of HEPG2 cells was increased after the expression of Flag-HBX, ( P < 0. 05). Compared with the control group, the proliferation of HEPG2 cells was decreased after the knock-down of Mcur1 (P< 0. 05). Compared with the control group, the overexpression of Mcur1 and HBx at the same time can promote the proliferation of HEPG2 (P< 0. 05), and the overexpression of MCUR1 only has no effect on the proliferation of HEPG2. Compared with the HBx group, the proliferation of HEPG2 cells with overexpressed HBx and lowly expressed MCUR1 was decreases (P< 0. 05). Compared with the control group, the ROS level of HEPG2 after HBx overexpression was increased (P < 0. 05), and it had no significant change in HEPG2 with overexpression of HBx and knock-down of MCUR1 at the same time. Compared with the control group and the HBx group, the overexpression of MCUR1 and HBx at the same time can increase the ROS level of HEPG2 (P < 0. 05). Compared with the HBx group, the proliferation of HEPG2 was decresed when HBx was overexpressed and the MCUR1 was knock down (P< 0. 05), however, this decreasing could be restored by H2O2 treatment. Compared with the control group, the positioning of nuclear transcription factor Nrf2 was increases in the HBx overexpressed group (P< 0. 05), while it had no significant change in cells with HBx overexpression and MCUR1 knock-down at the same time. Compared with the control group, the activated form of NOCD1 in the nucleus was increased, and it had no significant change after using ML385 in HBx overexpressed cells. Compared with the HBx group, the proliferation level of HEPG2, HEPG2 after ML385 expressed HBX at the same time as HBX (P<0. 05). Compared with the HBX group, the proliferation of HEPG2 was decreased after treatment of NOTCH inhibitor IMR-1 or ML385. Conclusion HBx promotes HepG2 cell proliferation through the MCUR1/ ROS/ Nrf2/ Notch axis

Key words: HBX, liver cancer, proliferation, ROS, MCUR1