Targeting Transforming Growth Factor-Beta1 by RNA Interference Attenuates Melanoma Cell Growth and Inhibits Pulmonary Metastasis in an ex-vivo Animal Model

Abstract

Background: Transforming growth factor-beta (TGF-β) is the key molecule implicated in impaired immune function in human patients with malignant melanoma. We examined the effects of TGF-β1 protein knockdown by RNA interference on the growth and metastasis of melanoma in C57BL/6 mice induced by the B16F0 cell line. Aim and Objectives: The purpose of this work is to provide preclinical assessment of the therapeutic potential of TGF-β1 protein knockdown by RNA interference in melanoma. Materials and Methods: The TGF-β1 hairpin oligonucleotide was cloned into retroviral vector pSM2. The resulting plasmid (TGF-β1 -RNAi/pSM2) was stably introduced into murine melanoma cell line, B16F0, and designated as B16F0/TGF-β1-RNAi cells. The vector plasmid was transfected into B16F0 cells and designed as B16F0/vector-control cells served as a control. Five million B16F0 cells, B16F0/vector-control cells and B16F0/TGF-β1-RNAi cells were injected subcutaneously into the right flanks of adult female syngeneic mice C57BL/6 respectively. The growth rate of the parental cell and genetically modified murine melanoma cells were compared. C57BL/6 mice were also evaluated for pulmonary metastasis following tail vein injection of two million B16F0 cells, B16F0/vector-control cells and B16F0/TGF-β1-RNAi cells. Results: TGF-β1 expression was reduced in B16F0/TGF-β1-RNAi cells compared with B16F0 cells and B16F0/vector-control cells. The proliferation rate of B16F0/TGF-β1-RNAi cells was similar to that of the B16F0 cells and B16F0/vector-control cells in vitro. The tumor sizes were 495.32±77.25 mm^3, 516.65±73.71 mm^3 and 326.72±97.34 mm^3 at the fourteenth day in the mice receiving B16F0 cells, B16F0/vector-control cells and B16F0/TGF β1-RNAi cells respectively. The p value was less than 0.05 by one-way ANOVA. TGF-β1 knockdown in B16F0 cells enhanced the infiltration of CD4(superscript +) and CD8(superscript +) T cells in the tumor regions. The blood vessel density of the tumors markedly reduced in B16F0/TGF β1-RNAi tumors. The pulmonary metastasis also reduced significantly on 21day in mice injected with B16F0/TGF β1-RNAi tumors compared with mice injected with B16F0 cells and B16F0/vector-control cells. Conclusions: We found that TGF-β1 protein expression was significantly reduced from B16F0 cells after TGF-β1 hairpin oligonucleotide transduction. Silencing of TGF-β1 expression in B16F0 cells by RNA interference technology can inhibit the growth and metastasis of this tumor after being transplanted to C57BL/6 mice. Overall, our results have important implications for the clinical use of RNA interference targeting TGF-β1 as cancer gene therapy.