| Objective:Initial preliminary studies found that HBx can activate Notch1 signaling pathwag of L02 cells and participate in the malignant transformation of liver cells.This study to construct recombinant plasmids containing short hairpin RNA (shRNA) targeting the Notch1 gene and investigate the effects on the growth of HBx-expressing L02 cell.To study the molecular mechanism of Notch1 signaling in this process.Methods:Three shRNAs were designed according to the coding sequence of the Notch1 gene and used to construct recombinant plasmids. The recombinant plasmids were transfected into HBx-expressing L02 cell using Lipofectamine 2000. The Notch1 mRNA and protein expression after transict thransfection were detected by qRT-PCR and Western blotting analysis;and samely the Notch1 signaling components,P16 and Bcl-2 after stable transfection were detected.Then,CCK-8 assay and flow cytometry were adopted to measure the cell proliferation, cycle and apotosis.Finally,the mRNA binding ration of the NF-κB was detected by EMSA.Results:Three recombinant plasmids were successfully constructed. The expression of Notch1 mRNA and protein was obviously downregulated in HBx-expressing L02 cell transfected with the recombinant plasmids. The best silencing effect was achieved in cells transient transfected with the pshRNA1 plasmid,the mRNA inhibition ration is 78.83%(p<0.05),and by stable transfected with pshRNA1 plasmid the mRNA inhibition ration is 91.68%(p<0.01). The RNAi with Notch1 signaling attenuated cell proliferation(P<0.05), increased the G0/G1 phase of cell cycle(P<0.01),reduced the S phase of cell cycle(P<0.05)and promoted apoptosis of HBx-expressing L02 cell(P<0.05), in addition, upregulated P16(p<0.05) , downregulated BCl-2(p<0.001) and reduced the mRNA binding ration of the NF-κB.Conclusions:The recombinant plasmids containing shRNA targeting the Notch1 gene can specifically block Notch1 signaling,and HBx could promote the growth of human non-tumor hepatic cell line L02 cells via activation of Notch1 signaling and crosstalk with the NF-κB. RNAi with Notch1 signaling could inhibit proliferation,prevent cell cycle progression and promote apoptosis of the L02/HBx cells. It make clear that HBx could promote the malignant transformation of L02 cells via activation of Notch1 signaling. it perhaps take effect on this process via up-regulation with P16 and down-regulation with Bcl-2. Notch1signaling maybe crostalking with NF-κB signaling participated in HBx inducing malignant transfomation of L02 cells and the mechanism of carcinogenesis. |
