Effect of semaphorin4D gene silencing on malignant behavior of human ovarian cancer SKOV3 cells

Abstract

To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude mice. Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blot were used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SKOV3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0.401 ± 0.051, 0.120 ± 0.035 and 0.014 ± 0.015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ± 0.019, 0.536 ± 0.040,respectively, P < 0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0.196 ± 0.023, 0.074 ± 0.015 and 0.040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.275 ± 0.009, 0.282 ± 0.015, respectively, P < 0.05). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR (inhibitory rate) of pshRNASema 4D-B group was (61.0 ± 3.3)% (P < 0.05). Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.