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. 2009 Mar;19(1):81-8.
doi: 10.1089/oli.2008.0166.

Knockdown of caveolin-1 by siRNA inhibits the transformation of mouse hepatoma H22 cells in vitro and in vivo

Shujing Wang  1 , Li Jia, Huimin Zhou, Wei Shi, Jianing Zhang
Affiliations

Knockdown of caveolin-1 by siRNA inhibits the transformation of mouse hepatoma H22 cells in vitro and in vivo

Shujing Wang et al. Oligonucleotides. 2009 Mar.

Abstract

Caveolin-1 (Cav-1) is a main structural protein of caveolae and plays important roles in signal transduction and tumorigenesis. We previously showed that Cav-1 was highly expressed in mouse hepatoma cell lines and positively correlated with cell invasion capability. Thus, interfering with the expression and activity of Cav-1 might be a potential way to intervene with hepatoma progression. We used RNA interference to study the biological effects of silencing Cav-1 expression in hepatoma H22 cells, to validate its potential as a therapeutic target. Using small-interfering RNAs (siRNAs) targeting the mRNA region of Cav-1, we effectively suppressed Cav-1 mRNA and protein levels. This resulted in the decreased transformation ability of H22 cells in vitro and in vivo. In addition, downregulation of Cav-1 expression promoted the apoptosis of H22 cells in vitro and in vivo. These results suggest that the use of siRNA could be an efficient molecular therapeutic method for hepatoma with high expression of Cav-1.

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Figures

FIG. 1.
FIG. 1.
Downregulation of Cav-1 in H22 cells by small-interfering RNA (siRNA). H22 cells were transfected with Cav-1-specific siRNAs and control siRNAs, as described under “Materials and Methods”. (A) Efficiency of transfection of the oligonucleotides was evaluated by immunocytochemistry assay (×200). (B) Cav-1 mRNA content was analyzed by RT-PCR assay 72 hours after transfection. GAPDH was used as internal control. (C) Cell extracts were assessed by Western blot with anti-Cav-1 and anti-GAPDH antibodies. Each lane contains an equal amount of total protein. Relative signal intensities of Cav-1 mRNA and protein levels as compared with GAPDH were analyzed by LabWorks (TM ver4.6, UVP, BioImaging systems), respectively. (D) Cav-1 expression did not recover to original levels until 15 days after Cav-1-siRNA mix transfection.
FIG. 2.
FIG. 2.
Effect of Cav-1-siRNA on colony formation of H22 cells. Cells were seeded at 1000/well density in 6-cm plates. After 14 days, cell colonies were photographed. (A) Representative field photograph by colony-forming assay of H22, H22/controls, and H22/Cav-1-siRNA cells (×100). (B) Number of colonies were compared between the H22/Cav-1-siRNA and control cells. Each value is the mean ± standard deviation (SD) of 10 different fields (*p < 0.05). Data indicated that downregulation of Cav-1 in H22 cells suppressed the colony-formation capability in vitro.
FIG. 3.
FIG. 3.
Effect of Cav-1-siRNA on tumorigenesis of H22 cells in vivo. (A) The right flanks of 615 mice were subcutaneously injected with 2 × 107 cells in 0.1 mL of PBS of H22/Cav-1-siRNA and H22/control (n = 8). After 2 weeks, tumors were excised and weighed. Note that H22/Cav-1-siRNA cells formed smaller tumors than H22/control cells. (B) Tumors were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (×400). (C) Cav-1 protein expression was determined by immunohistochemistry staining in solid tumors derived from H22, H22/control, and H22/Cav-1-siRNA cells (×400). Differences in Cav-1 protein expression were shown (D, *p < 0.05). The data were obtained from three independent experiments. Results suggested that downregulation of Cav-1 in H22 cells attenuated tumorigenesis capability in vivo.
FIG. 4.
FIG. 4.
Silencing of Cav-1 increases the apoptosis level of H22 cells. (A) Representative images of TUNEL assays. H22/Cav-1-siRNA and control cells were assayed for their survival capability by TUNEL apoptosis detection kit. Cells were stained with DAPI for counting of total cell number. Apoptotic cells with DNA strand breaks were labeled with TUNEL. The results are representative of 30 different fields (×200). (B) Mean numbers of apoptosis cells per high-powered microscopic field (*p < 0.05). (C and D) Flow cytometry analysis with Annexin V–PI staining was performed to evaluate the percentage of Annexin V–PI positive cells. Apoptotic cells detected by Annexin V–PI staining assay were obviously increased after RNAi. Values were averaged from three independent experiments (*p < 0.05).
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