Abstract: Objective The present paper aimed to inhibit the expression of tumor suppressor p53-binding protein 2 (TP53BP2) in liver cancer by short hairpin RNAs(shRNAs) with lentiviral vector. Methods Two pairs of RNA interference sequences targeting to TP53BP52 gene were designed, and their corresponding shRNA sequences were synthesized. After annealing of shRNA to form double-stranded oligo sequences, the recombinant plasmid was constructed by gene recombination technique. The correct recombinant plasmid was used after PCR and sequencing identification of the colony for lentivirus packaging and titer determination. The interference effect of lentivirus lenti-shTP53BP2 on TP53BP2 gene in HepG2 cells was observed by Western Blot, qRT-PCR and laser confocal technique. Results The sequencing alignment results showed that each recombinant lentiviral vector was consistent with the designed reference sequence, indicating that each recombinant lentiviral vector was successfully constructed; the titers of pHS-ASR-LW429, pHS-ASR-LW512 and pHS-ASR-LW513 were 9.7×10⁸ TU/mL, 6.1×10⁸ TU/mL and 6.4×10⁸ TU/mL, respectively; the HepG2 cells were infected with lentivirus lenti-shTP53BP2 (pHS-ASR-LW512 and pHS-ASR-LW513), and the results of Western blot, qRT-PCR and laser confocal technique showed that the two lenti-shTP53BP2 significantly down-regulated the TP53BP2 RNA level and its protein expression in HepG2 cells as compared with the control lentivirus (PHS-ASR-LW429). Conclusion In this study, we successfully construct the shRNA lentiviral vector targeting to TP53BP2 gene with the capacity of effectively down-regulation of TP53BP2 expression in HepG2 cells, which might lay a foundation for further research on the mechanism of TP53BP2 in the hepatocarcinogenesis.

Key words: HepG2 cells, Tumor suppressor p53-binding protein 2, Short hairpin RNA, Lentivirus, In vitro