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Inhibitory effects of adenovirus mediated tandem expression of RhoA and RhoC shRNAs in HCT116 cells.

ABSTRACT: BACKGROUND: RhoA and RhoC are deregulated by over expression in many human tumors, including colorectal cancer. Some reports show that they play a pivotal role in the carcinogenesis, tumor development and infiltration metastasis. In this study, for the first time we constructed recombinant adenovirus to investigate the inhibitory effects of RhoA and RhoC shRNAs in tandem expression on the cell proliferation and invasion of colorectal cancer HCT116 cells. METHODS: The recombinant adenovirus carrying RhoA and RhoC shRNAs in tandem expression was transfected into HCT116. The mRNA transcription and protein expressions of RhoA and RhoC were examined by RT-FQPCR and Western blot respectively. Cellular proliferation inhibitory activity was determined by methyl thiazolyl tetrazolium (MTT) assay and invasive and migrating potential was detected through in vitro Matrigel coated invasion and migration assay. RESULTS: Both mRNA and proteins Levels of RhoA and RhoC were significantly reduced in HCT116 cells transfected with Ad-A1+A2+C1+C2 than those in Ad-HK group and control one. The relative RhoA and RhoC mRNA transcriptions were decreased to 40% and 36% (P < 0.05), while proteins expression reducing 42% and 35%, respectively (P < 0.05). Growth curves analysis showed that alive cell number in the Ad-A1+A2+C1+C2 group was lower than others in the third to sixth day and transwell chamber analysis showed that migration/invasion activity was significantly suppressed in Ad-A1+A2+C1+C2 group. CONCLUSION: Our results indicate recombinant adenovirus carrying RhoA and RhoC shRNAs in tandem expression may inhibit the growth and invasion of HCT116 cells. Application of such vector to inhibit one or more genes may be a new method to cancer therapy.

SUBMITTER: Liu XP

PROVIDER: S-EPMC2672922 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Inhibitory effects of adenovirus mediated tandem expression of RhoA and RhoC shRNAs in HCT116 cells.

Liu Xiang-Ping XP Wang Hai-Bo HB Yang Kun K Sui Ai-Hua AH Shi Qiang Q Qu Shen S

Journal of experimental & clinical cancer research : CR 20090418

<h4>Background</h4>RhoA and RhoC are deregulated by over expression in many human tumors, including colorectal cancer. Some reports show that they play a pivotal role in the carcinogenesis, tumor development and infiltration metastasis. In this study, for the first time we constructed recombinant adenovirus to investigate the inhibitory effects of RhoA and RhoC shRNAs in tandem expression on the cell proliferation and invasion of colorectal cancer HCT116 cells.<h4>Methods</h4>The recombinant ade ...[more]

PMID: 19374769

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Project description:Dysregulation of microRNAs is observed in many cancers, including breast cancer. In particular, miR-10b appears to play an important role in tumor cell invasion and breast cancer progression. In this study, we investigated hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with c-Src kinase and the transcriptional factor, Twist, in breast tumor cells (MDA-MB-231 cells). Our results indicate that HA binding to CD44 promotes c-Src kinase activation, which, in turn, increases Twist phosphorylation, leading to the nuclear translocation of Twist and transcriptional activation. Further analyses reveal that miR-10b is controlled by an upstream promoter containing the Twist binding site(s), whereas ChIP assays demonstrate that stimulation of miR-10b expression by HA/CD44-activated c-Src is Twist-dependent in breast tumor cells. This process results in the reduction of a tumor suppressor protein (HOXD10), RhoA/RhoC up-regulation, Rho-kinase (ROK) activation, and breast tumor cell invasion. Treatment of MDA-MB-231 cells with PP2 (a c-Src inhibitor) or Twist-specific siRNAs effectively blocks HA-mediated Twist signaling events, abrogates miR-10b production, and increases HOXD10 expression. Subsequently, this c-Src/Twist signaling inhibition causes down-regulation of RhoA/RhoC expression and impairment of ROK-regulated cytoskeleton function (e.g. tumor cell invasion). To further evaluate the role of miR-10b in RhoGTPase signaling, MDA-MB-231 cells were also transfected with a specific anti-miR-10b inhibitor in order to silence miR-10b expression and block its target functions. Our results demonstrate that anti-miR-10b inhibitor not only enhances HOXD10 expression but also abrogates HA/CD44-mediated tumor cell behaviors in breast tumor cells. Taken together, these findings indicate that the HA-induced CD44 interaction with c-Src-activated Twist plays a pivotal role in miR-10b production, leading to the down-regulation of tumor suppressor protein (HOXD10), RhoGTPase-ROK activation, and tumor cell invasion. All of these events are critical prerequisite steps for the acquisition of metastatic properties by human breast cancer cells.

Dataset Information

Inhibitory effects of adenovirus mediated tandem expression of RhoA and RhoC shRNAs in HCT116 cells.

Publications

Inhibitory effects of adenovirus mediated tandem expression of RhoA and RhoC shRNAs in HCT116 cells.

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