Experimental study on the contribution of costimulatory molecule B7-H3 in regulation of the biological function of human esophageal cancer cell line Eca-109

Abstract

Objective To investigate the role of B7-H3 in the regulation of biological function of human esophageal cancer cell by using RNA interference (RNAi) methods. Methods Obtaining LV-B7-H3-siRNA and negative control LV-NC-Eca-109 by lentivirus infecting and followed by fluorescence-activated cell sorter (FACS) Aria II cell sorting system. The expression level of B7-H3 in LV-B7-H3-siRNA-Eca-109 cells and LV-NC-Eca-109 was confirmed by using real-time quantitative polymerase chain reactionB (Real-time PCR), FACS and Western blotting methods. The cell proliferation was determined by cell counting kit-8 (CCK-8) assay, and the migration and invasion was determined by wound healing assay as well as transwell assay respectively. Results We constructed the recombinant lentivirus of siRNA targeting B7-H3. On day 2 (0.31±0.03 vs. 0.36±0.02), 4 (0.51±0.04 vs. 0.60±0.04), 5 (0.60±0.02 vs. 0.70±0.03) and day 6 (0.65±0.05 vs. 0.76±0.03), the proliferation rate of LV-B7-H3-siRNA group was significantly lower than that of LV-NC control group by using CCK-8 assay (P=0.011, 0.024), respectively. The wound healing assay on Eca-109 cells in LV-B7-H3-siRNA group as well as LV-NC group showed that, the cell-free area of the LV-B7-H3-siRNA group was significantly wider than that of LV-NC group at 12 h [(38.0±28.1) vs. (467.7±30.3) pix, P=0.042], 20 h [(509.7±6.2) vs. (403.3±16.7) pix, P=0.001] and 24 h [(454.7±19.9) vs. (355.0±32.1) pix, P=0.010] respectively after drawing the scratch line on the monolayer cells. The transwell invasion assay showed that at the time point of 24 h, the number of invaded cells in the LV-B7-H3-siRNA group (258.8±29.1) cells was significantly lower than that in the LV-NC group (474.0±85.3) cells (P=0.014). Conclusion We successfully constructed the recombinant lentivirus of siRNA targeting B7-H3, and the cellular studies showed that the down-regulation of B7-H3 could suppress the biological functions such as proliferation, migration and invasion of the Eca-109 cells. Thus the over-expression of B7-H3 may involve in the malignant transformation of human esophageal cancer. The B7-H3 molecule could be developed as the potential therapeutic targets for immunotherapy of this malignancy. Key words: Co-stimulatory molecule; B7-H3; Esophageal cancer; Invasion; Metastasis