Construction of shRNA-Bmi-1 plasmid and its effect on proliferation of ECA109 cells

Objective: To explore the role of Bmi-1 gene in the proliferation of squamous carcinoma cells and whether the silencing Bmi-1 can inhibit the growth of squamous cell carcinomas cells. Methods: The expressions of Bmi-1 in primary cultured Fibroblasts, karatinocyte cell line Hacat, squamous carcinoma cell line A431, and ECA109 were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Recombinant plasmid inserted with Bmi-1 gene short hairpin RNA (shRNA) expression vector PGPU6/GFP/Neo-shBmi-1 was constructed and transfected into ECA109 cells with control set. After transfection for 48 and 72 hours, the mRNA and protein levels of Bmi-1 were examined with RT-PCR and Western blot analysis, respectively. The proliferation of the ECA109 cells was evaluated by MTT method and flow cytometry. Results: Bmi-1 was highly expressed in A431 and ECA109 cells than in Fibroblast cells and Hacat cells. The mRNA and protein expressions of Bmi-1 were significantly silenced in ECA109 cells after recombinant expression vector PGPU6/GFP/Neo-shBmi-1 transfection (P < 0.05). Compared with the control groups, the proliferation of ECA109 transfected with PGPU6/GFP/Neo-shBmi-1 was significantly inhibited (P < 0.05), and cells in G1 phase increased while in S phase decreased (P < 0.05). Conclusions: Bmi-1 is involved in the proliferation of squamous carcinoma cells. After the silencing of Bmi-1 expression, the proliferation ECA109 cells is suppressed due to the altered cell cycle.