Targeting Transforming Growth Factor-Beta1 Using RNA Interference Attenuates Melanoma Cell Growth in an ex-vivo Animal Model

Abstract

Objectives: The 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 using RNA interference on the growth of melanoma in C57BL/6 mice induced using the B16F0 cell line. 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 the 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 which served as the 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 cells and genetically modified murine melanoma cells were compared. 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 the 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 on the 14th day after the mice received B16F0 cells, B16F0/vector-control cells and B16F0/TGFβ1-RNAi cells, respectively. The pvalue was less than 0.05 using 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 was markedly reduced in the B16F0/TGFβ1-RNAi tumors. 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 using RNA interference technology inhibited the growth 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.