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

ABSTRACT: To determine the effects of RNAi-mediated inhibition of the growth hormone receptor (GHR) gene on tumors and colon cancer cells in vivo.Construction of a eukaryotic vector for human GHR expression, the pcDNA6.2-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 (G?), growth hormone (GH), 5-fluorouracil (FU), G?+FU or G?+FU+GH. Each nude mouse was subcutaneously inoculated with 1 × 10(7) 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.Compared to the control group, the weights of the inoculated nude mice on the 17(th) day after inoculation were: G?: 21.60 ± 0.71 g, GH: 21.64 ± 0.45 g, FU: 18.94 ± 0.47 g, FU+G?: 19.40 ± 0.60 g, G?+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: G?: 9.71 ± 3.82 mm(3), FU: 11.54 ± 2.42 mm(3), FU+G?: 11.42 ± 1.11 mm(3), G?+FU+GH: 10.47 ± 1.02 mm(3) vs NS: 116.81 ± 10.61 mm(3), P < 0.05. Compared to the GH group, the tumor volumes were significantly decreased in the experimental groups. The GHR protein expression (G?: 0.39 ± 0.02, FU: 0.40 ± 0.02, FU+G?: 0.38 ± 0.01, G?+FU+GH: 0.39 ± 0.01 vs NS: 0.94 ± 0.02, P < 0.05) and the GHR mRNA expression (G?: 14.12 ± 0.10, FU: 15.15 ± 0.44, FU+G?: 16.46 ± 0.27, G?+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.Inhibition of GHR in human colon cancer SW480 cells resulted in anti-tumor effects in nude mice.

SUBMITTER: Zhou D

PROVIDER: S-EPMC3848161 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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

Zhou Dong D Yang Jie J Huang Wei-Dong WD Wang Jun J Zhang Qiang Q

World journal of gastroenterology 20131101 44

<h4>Aim</h4>To determine the effects of RNAi-mediated inhibition of the growth hormone receptor (GHR) gene on tumors and colon cancer cells in vivo.<h4>Methods</h4>Construction of a eukaryotic vector for human GHR expression, the pcDNA6.2-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+F ...[more]

PMID: 24307807

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Project description:Coxsackievirus A9 (CV-A9) is a pathogenic enterovirus type within the family Picornaviridae. CV-A9 infects A549 human epithelial lung carcinoma cells by attaching to the ?V?6 integrin receptor through a highly conserved Arg-Gly-Asp (RGD) motif, which is located at the exposed carboxy-terminus of the capsid protein VP1 detected in all studied clinical isolates. However, genetically-modified CV-A9 that lacks the RGD motif (CV-A9-RGDdel) has been shown to be infectious in some cell lines but not in A549, suggesting that RGD-mediated integrin binding is not always essential for efficient entry of CV-A9.Two cell lines, A549 and SW480, were used in the study. SW480 was the study object for the integrin-independent entry and A549 was used as the control for integrin-dependent entry. Receptor levels were quantitated by cell sorting and quantitative PCR. Antibody blocking assay and siRNA silencing of receptor-encoding genes were used to block virus infection. Peptide phage display library was used to identify peptide binders to CV-A9. Immunofluorescence and confocal microscopy were used to visualize the virus infection in the cells.We investigated the receptor use and early stages of CV-A9 internalization to SW480 human epithelial colon adenocarcinoma cells. Contrary to A549 infection, we showed that both CV-A9 and CV-A9-RGDdel internalized into SW480 cells and that function-blocking anti-?V integrin antibodies had no effect on the binding and entry of CV-A9. Whereas siRNA silencing of ?6 integrin subunit had no influence on virus infection in SW480, silencing of ?2-microglobulin (?2M) inhibited the virus infection in both cell lines. By using a peptide phage display screening, the virus-binding peptide identical to the N-terminal sequence of HSPA5 protein was identified and shown to block the virus infection in both A549 and SW480 cell lines. HSPA5 was also found to co-localize with CV-A9 at the SW480 cell periphery during the early stages of infection by confocal microscopy.The data suggest that while ?V?6 integrin is essential for CV-A9 in A549 cell line, it is not required in SW480 cell line in which ?2M and HSPA5 alone are sufficient for CV-A9 infection. This suggests that the choice of CV-A9 receptor(s) is dependent on the tissue/cellular environment.

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

Publications

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

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