Construction of a lentiviral vector carrying the casitas B-lineage lymphoma (Cbl) -b short hairpin RNA (shRNA) and its effect on the biological behavior of A375 melanoma cells in vitro

Abstract

Objective To construct a recombinant lentiviral vector carrying the casitas B-lineage lymphoma (Cbl) -b short hairpin RNA (shRNA) , and to evaluate its effect on the biological behavior of A375 melanoma cells in vitro. Methods Three specific shRNAs targeting Cbl-b gene and a negative control shRNA were designed and synthesized, and recombinant lentiviral vectors were constructed. A375 cells were divided into 5 groups to be transfected with 3 kinds of lentiviral vector expressing Cbl-b gene-specific shRNAs (CBLB-shRNA-1 group, CBLB-shRNA-2 group and CBLB-shRNA-3 group) , a lentiviral vector containing negative control shRNA (negative control group) , and an empty vector (blank control group) . Real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine the silencing efficiency at 72 hours after transfection. Cell counting kit-8 (CCK-8) assay was conducted to evaluate cellular proliferative activity at 24, 48, 72 and 96 hours after transfection, flow cytometry to detect cell apoptosis and cell cycle at 72 hours after transfection, and Transwell invasion assay to assess cellular invasive activity at 72 hours after transfection. Results Three recombinant lentiviral vectors containing Cbl-b shRNA were constructed successfully. As Western blot analysis revealed, the CBLB-shRNA-3 showed the highest silencing efficiency. CCK-8 assay indicated that the proliferative activity of A375 cells was significantly lower in the CBLB-shRNA-3 group than in the negative control group and blank control group at 72 and 96 hours after transfection (all P < 0.01) . Flow cytometry showed that the apoptosis rate of A375 cells was significantly higher in the CBLB-shRNA-3 group (22.73% ± 6.58%) than in the negative control group (6.08% ± 1.35%, P < 0.01) and blank control group (6.34% ± 1.07%, P < 0.01) . The CBLB-shRNA-3 group showed a significantly higher proportion of A375 cells at G1 phase, but a significantly lower proportion of A375 cells at S phase compared with the negative control group and blank control group (all P < 0.01) . Transwell assay showed that there were significant differences in the number of A375 cells crossing the artificial basement membrane (matrigel) at 72 hours after transfection among the negative control group, blank control group and CBLB-shRNA-3 group (76.60 ± 1.82, 73.20 ± 3.83, 19.60 ± 1.14, respectively; F = 794.50, P < 0.01) . Conclusions A recombinant CBLB-shRNA-3-expressing lentiviral vector which can efficiently silence Cbl-b gene has been successfully constructed. It can inhibit the proliferation, cell cycle progression and invasive activity of A375 cells, but promote the apoptosis of A375 cells. Key words: Melanoma; Proto-oncogene proteins c-cbl; RNA, small interfering; Cell proliferation; Apoptosis; Cell cycle; Gene, Cbl-b; A375 cells; Short hairpin RNA