Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Sep 9;29(1):123.
doi: 10.1186/1756-9966-29-123.

Silencing of RhoA and RhoC expression by RNA interference suppresses human colorectal carcinoma growth in vivo

Haibo Wang  1 , Gang Zhao, Xiangping Liu, Aihua Sui, Kun Yang, Ruyong Yao, Zongbao Wang, Qiang Shi
Affiliations

Silencing of RhoA and RhoC expression by RNA interference suppresses human colorectal carcinoma growth in vivo

Haibo Wang et al. J Exp Clin Cancer Res. .

Abstract

Background: RhoA and RhoC have been proved to be over-expressed in many solid cancers, including colorectal cancer. The reduction of RhoA and RhoC expression by RNA interference (RNAi) resulted growth inhibition of cancer cells. The present study was to evaluate the effect of silencing of RhoA and RhoC expression by RNAi on growth of human colorectal carcinoma (CRC) in tumor-bearing nude mice in vivo.

Methods: To establish HCT116 cell transplantable model, the nude mice were subcutaneously inoculated with 1.0 × 10(7) HCT116 cells and kept growing till the tumor xenografts reached 5-7 mm in diameter. Then the mice were randomly assigned to three groups(seven mice in each group): (1) normal saline(NS) group, (2)replication-defective recombinant adenovirus carrying the negative control shRNA (Ad-HK) group and (3)replication-defective recombinant adenovirus carrying the 4-tandem linked RhoA and RhoC shRNAs (Ad-RhoA-RhoC) group. Ad-HK (4 × 10(8) pfu, 30 ul/mouse), Ad-RhoA-RhoC (4 × 10(8) pfu, 30 ul/mouse) or PBS (30 ul/mouse) was injected intratumorally four times once every other day. The weight and volumes of tumor xenografts were recorded. The levels of RhoA and RhoC mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR) and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the death of cells.

Results: The xenografts in mice could be seen at 5th day from the implantation of HCT116 cells and all had reached 5-7 mm in size at 9th day. After injection intratumorally, the growth speed of tumor xenografts in Ad-RhoA-RhoC group was significantly delayed compared with those in NS and Ad-HK group(P < 0.05). The results of QRT-PCR showed that mRNA levels of RhoA and RhoC reduced more in Ad-RhoA-RhoC group than those in NS and Ad-HK group. The relative RhoA and RhoC mRNA transcripts were decreased to 48% and 43% respectively (P < 0.05). Immunohistochemical analyses of tumor xenograft sections also revealed the decreased RhoA and RhoC expression in Ad-RhoA-RhoC group. TUNEL assay also showed higher death of tumor xenograft tissue cells in Ad-RhoA-RhoC group.

Conclusion: Recombinant adenovirus mediated RhoA and RhoC shRNA in tandem linked expression may inhibit the growth of human colorectal tumor xenografts in vivo. These results indicate that RhoA and RhoC might be potential targets for gene therapy in colorectal cancer.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Tumor-bearing nude mice with 100% of tumor implantation rate.
Figure 2
Figure 2
Growth curve of subcutaneous implanted tumors in nude mice treated with NS, Ad-HK, or Ad-RhoA-RhoC. Tumor volume is plotted against time elapsed. A significant delay in tumor growth is seen in the group treated with Ad-RhoA-RhoC. Data are presented as means±SD (n = 7). * P < .05, from this point onwards.
Figure 3
Figure 3
Comparison of the size of harvested implanted tumors in nude mice treated with NS, Ad-HK, or Ad-RhoA-RhoC. A: fresh anatomized B: formalin-fixed.
Figure 4
Figure 4
Tumor tissues in nude mice in different treated groups (HE, ×200) A: NS group; B: Ad-HK group; C: Ad-RhoA-RhoC group. Tumor cells were intensely stained with hematoxylin and showed smaller sizes. Necrotic regions were mainly eosin stained.
Figure 5
Figure 5
Immunohistochemistry reaction for RhoA and RhoC protein in implanted tumor tissues of nude mice in different treated groups (RhoA, ×400, RhoC, ×200). Fig 5 also showed the integrated optical density (IOD) values of the implanted tumor tissues. A: NS group; B: Ad-HK group; C: Ad-RhoA-RhoC group. The positive cells were stained brown, using antibodies to RhoA or RhoC.
Figure 6
Figure 6
Cell death in implanted tumor tissues. Cell death was detected by TUNEL assay in implanted tumors treated with NS(A), Ad-HK(B), or Ad-RhoA-RhoC(C and D). Original magnification, ×200. The nuclei of positive cell were stained brown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin. 2007;57:43–66. doi: 10.3322/canjclin.57.1.43. - DOI - PubMed
    1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ. Cancer statistics, 2008. CA Cancer J Clin. 2007;58:71–96. doi: 10.3322/CA.2007.0010. - DOI - PubMed
    1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74–108. doi: 10.3322/canjclin.55.2.74. - DOI - PubMed
    1. Wilke HJ, Van Cutsem E. Current treatments and future perspectives in colorectal and gastric cancer. Ann Oncol. 2003;14:ii49–55. doi: 10.1093/annonc/mdg730. - DOI - PubMed
    1. Fritz G, Just I, Kaina B. Rho GTPases are over-expressed in human tumors. Int J Cancer. 1999;81:682–787. doi: 10.1002/(SICI)1097-0215(19990531)81:5<682::AID-IJC2>3.0.CO;2-B. - DOI - PubMed

Publication types

MeSH terms