siRNA-targeted inhibition of growth hormone receptor in human colon cancer SW480 cells

Abstract

AIM: To determine the effects of RNAi-mediated inhibition of the growth hormone receptor (GHR) gene on tumors and colon cancer cells in vivo.

METHODS: Construction of a eukaryotic vector for human GHR expression, the pcDNA™6.2-GW/EmGFP-small interfering RNAs (siRNAs)-GHR plasmid, was used to inhibit GHR expression. Thirty-six BALB/c nude mice were randomly divided into groups and treated with normal saline (NS), recombinant plasmid (G₂), growth hormone (GH), 5-fluorouracil (FU), G₂+FU or G₂+FU+GH. Each nude mouse was subcutaneously inoculated with 1×10⁷ human colon cancer SW480 cells; the nude mice were weighed before inoculation and on the 2^nd, 5^th, 8^th, 11^th, 14^th and 17^th day after inoculation. All nude mice were sacrificed after 17 d. Each subcutaneous tumor was removed and studied. Tumor volume was measured on the 5^th, 8^th, 11^th, 14^th and 17^th day after inoculation. The expression of GHR protein in the tumor tissue was detected by Western blotting analysis, and the differences in GHR mRNA expression in the tumor tissue were detected by real-time quantitative reverse transcription-polymerase chain reaction.

RESULTS: Compared to the control group, the weights of the inoculated nude mice on the 17^th day after inoculation were: G₂: 21.60 ± 0.71 g, GH: 21.64 ± 0.45 g, FU: 18.94 ± 0.47 g, FU+G₂: 19.40 ± 0.60 g, G₂+FU+GH: 21.04 ± 0.78 g vs NS: 20.68 ± 0.66 g, P < 0.05; the tumor volumes after the subcutaneous inoculation were: G₂: 9.71 ± 3.82 mm³, FU: 11.54 ± 2.42 mm³, FU+G₂: 11.42 ± 1.11 mm³, G₂+FU+GH: 10.47 ± 1.02 mm³vs NS: 116.81 ± 10.61 mm³, P < 0.05. Compared to the GH group, the tumor volumes were significantly decreased in the experimental groups. The GHR protein expression (G₂: 0.39 ± 0.02, FU: 0.40 ± 0.02, FU+G₂: 0.38 ± 0.01, G₂+FU+GH: 0.39 ± 0.01 vs NS: 0.94 ± 0.02, P < 0.05) and the GHR mRNA expression (G₂: 14.12 ± 0.10, FU: 15.15 ± 0.44, FU+G₂: 16.46 ± 0.27, G₂+FU+GH: 15.37 ± 0.57 vs NS: 12.63 ± 0.14, P < 0.05) were significantly decreased and increased, respectively, in the experimental groups.

CONCLUSION: Inhibition of GHR in human colon cancer SW480 cells resulted in anti-tumor effects in nude mice.

Keywords: Growth hormone receptor, Small interfering RNAs, Colon cancer, Gene therapy, Signaling pathway

Core tip: Human growth hormone receptor (GHR) is highly expressed in colon cancer tissues. GH/GHR plays an important role in colon cancer emergence and development. After specific binding of GH to GHR in tumor tissues, the JAK-STAT signaling pathway is activated, resulting in improved cell growth and proliferation. small interfering RNAs (siRNAs)-targeted inhibition of the human GHR gene was used to investigate its impact on the emergence and development of colon cancer and to determine how human colon cancer cells respond to GHR suppression. The siRNA-containing plasmid could suppress GHR expression in colon cancer cells and exhibited anti-tumor effects in nude mice.