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RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells.

Strillacci A, Griffoni C, Spisni E, Manara MC, Tomasi V - Br. J. Cancer (2006)

Bottom Line: Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response.Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour.Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Biology, University of Bologna, and Center for Applied Biomedical Research (CRBA), St Orsola-Malpighi University Hospital, Bologna, Italy.

ABSTRACT
Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity.

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COX-2 specific knockdown by siRNAs in HUVE cells and evaluation of 6-keto-PGF1α production. HUVE cells were transiently transfected with siRNAs directed against COX-2 mRNA (sequences A–D; final concentration 200 pM): COX-2 levels (dark bars) and 6-keto-PGF1α production (bright bars) were analysed by Western blot and ELISA assay, respectively. All procedures are described under Materials and Methods. (A) Shows COX-2 and COX-1 expression after siRNAs treatment. After the evaluation of the bands intensity by Image Master VDS software, both COX-2 and COX-1 levels were normalised against β-actin expression. All samples (lanes 2–7) except control in lane 1 were treated with PMA 40 nM. Lanes 3–6: samples treated with siRNA A, B, C and D, respectively. Lane 7: HUVE cells transfected with siRNA-Scr (scrambled), representing a negative control. Data are expressed as % of PMA-stimulated control value (lane 2) and represent the mean±s.e.m. of three independent experiments. *(P<0.01); #(P<0.05).
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fig2: COX-2 specific knockdown by siRNAs in HUVE cells and evaluation of 6-keto-PGF1α production. HUVE cells were transiently transfected with siRNAs directed against COX-2 mRNA (sequences A–D; final concentration 200 pM): COX-2 levels (dark bars) and 6-keto-PGF1α production (bright bars) were analysed by Western blot and ELISA assay, respectively. All procedures are described under Materials and Methods. (A) Shows COX-2 and COX-1 expression after siRNAs treatment. After the evaluation of the bands intensity by Image Master VDS software, both COX-2 and COX-1 levels were normalised against β-actin expression. All samples (lanes 2–7) except control in lane 1 were treated with PMA 40 nM. Lanes 3–6: samples treated with siRNA A, B, C and D, respectively. Lane 7: HUVE cells transfected with siRNA-Scr (scrambled), representing a negative control. Data are expressed as % of PMA-stimulated control value (lane 2) and represent the mean±s.e.m. of three independent experiments. *(P<0.01); #(P<0.05).

Mentions: Considering the relevance of endothelial COX-2 in the angiogenic process, we used an in vitro angiogenesis experimental model, based on primary HUVEC, to detect whether siRNA molecules were capable of downregulating COX-2 expression and inhibiting COX-2-dependent angiogenesis. Four different siRNAs, directed against COX-2 mRNA, were transfected at 200 pM concentration, by using the Oligofectamine reagent, in HUVEC treated with PMA to enhance COX-2 expression. As shown in Figure 2, only two siRNAs (sequences B and C) were capable of reducing COX-2 protein levels by more than 50%, whereas a scrambled siRNA, used as a negative control, was found to be completely devoid of effects. Moreover, we demonstrated that the transient knockdown mediated by siRNAs in HUVEC was highly specific since COX-1 expression resulted unaffected (Figure 2A). In samples in which COX-2 was downregulated, also PGI2 production, evaluated by ELISA assay, significantly decreased up to more than 40% (Figure 2B). Thus, we chose siRNA sequence-B to perform an in vitro angiogenesis test (Figure 3). As reported in the literature (Ilan et al, 1998), HUVE cells were able to organise into capillary-like tubular structures when seeded on 3-D collagen gel and stimulated with PMA (compare PMA-stimulated cells in Figure 3B to control cells in A). We observed that transfection of siRNA-B in HUVEC strongly affected their ability to organise in tubular structures (Figure 3C), with a significant reduction of vessels number after PMA stimulation (as shown in Figure 3E). Cells transfected with scrambled siRNA (Figure 3D) were still able to differentiate in tubular structures with the same efficiency of PMA-stimulated control cells (as shown in Figure 3E), allowing to exclude toxicity and nonspecific effects of siRNA-B on angiogenesis. These results demonstrate that siRNAs are capable to affect the in vitro angiogenic process by downregulating COX-2 expression in a strong specific manner. We also evaluated whether the transfection of synthetic siRNAs in HUVE cells may activate the interferon-mediated Jak-STAT pathway, as previously reported for other siRNAs molecules (Sledz et al, 2003). Western blot analysis of phospho-STAT-1(Tyr701) (active form) levels, normalised against p85/p91 STAT-1 total protein levels, showed that only an high concentration (200 nM) of transfected siRNA-B is able to trigger the interferon system response, whereas a lower but effective dose of siRNA (200 pM) does not have any effect on STAT-1 phosphorylation (Figure 4A and B). Phorbol 12-myristate 13-acetate-treated samples were used as positive controls, as suggested by the literature (Cohen et al, 2005). Data from immunofluorescence analysis were in high agreement with these findings (Figure 4C). STAT-1 phosphorylation, followed by nuclear translocation, was strongly increased in samples transfected with siRNA 200 nM, while no relevant differences were detected between samples transfected with siRNA 200 pM and controls. In order to achieve a stable downregulation of COX-2 in cancer cells, we prepared a mammalian vector expressing an anti-COX-2 shRNA. We used the pSUPER.retro vector system, capable of integrating expression cassettes in human genome, to produce efficient and specific downregulation of COX-2 gene expression induced by a siRNA mechanism (Brummelkamp et al, 2002a, 2002b). We cloned the dsRNA sequence corresponding to siRNA-B into a pSUPER.retro vector containing also the expression cassette for the GFP. The recombinant vector was transfected into Phoenix packaging cells to produce retroviral ecotropic supernatant, used to infect HT29 cells. Infected cells were selected by using standard puromycin treatment (1 μg ml−1) for 48 h. Selected HT29 cells (HT29 pSUPER(+)) were analysed by Western blot for COX-2 expression. As shown in Figure 5(A and B), COX-2 levels were found to be significantly decreased (more than 70%) in HT29 pSUPER(+) when compared to control cells. The inhibition was still effective when the COX-2 gene expression was stimulated by PMA treatment. Cyclooxygenase-2 mRNA levels were analysed in HT29 pSUPER(+) by real-time PCR. Results were in strict accordance with data obtained by Western blot, confirming the specific COX-2 mRNA degradation by RNAi. In fact, we obtained an 80% reduction of COX-2 mRNA levels either in the absence or in the presence of PMA stimulation (Figure 5C). As a further demonstration of the efficiency of the COX-2 knockdown mediated by RNAi, we also found a significant decrease of PGE2 production in HT29 pSUPER(+) cells (Figure 5D). Since we found, as mentioned above, that the transfection of exogenous synthetic siRNAs is capable to activate the interferon system at high concentrations in HUVE cells, the following aim was to demonstrate whether an endogenous and constitutive production of shRNA in the HT29 pSUPER(+) model had a different effect. Surprisingly, we found that shRNAs, that strongly downregulate COX-2 expression in HT29 pSUPER(+) cells, did not trigger the interferon system response in the absence of PMA treatment, compared to the control. Both Western blot (Figure 6A and B) and immunofluorescence (Figure 6C) analysis of phospho-STAT-1(Tyr701) levels and localisation confirmed this evidence. Moreover, in order to obtain more data on the effects of the constitutive COX-2 downregulation in HT29 pSUPER(+) cells, we performed four different assays to evaluate the proliferation profile and the invasiveness of these clones, compared to two different controls: HT29 wild-type and HT29 pSUPER(−) cells. HT29 pSUPER(−) cells were selected with puromycin after infection with the retroviral vector, devoid of the anti-COX-2 shRNA expression cassette. Although the MTT proliferation assay (Figure 7A) and the cell cycle distribution analysis (Figure 7B) did not show significant differences between controls and HT29 pSUPER(+), data from migration assay performed with Boyden chambers (Figure 8A and B) and soft-agar colony formation assay (Figure 8C) suggest that the stable knockdown of COX-2 gene by RNAi promotes a significant reduction of the migratory ability as well as a strong inhibition of colony formation in soft agar in infected pSUPER(+) colon cancer cells. Interestingly, the loss of the malignant behaviour in vitro of pSUPER(+) HT29 cells did not seem to depend on an impairment of cell growth, since constitutive expression of anti-COX-2 shRNA in HT29 cells only slightly modified their proliferation profile and their cell cycle distribution, but it derived from a reduction of the ability to invade the extracellular matrix and to grow in anchorage-independent manner, which are indexes of an invasive and aggressive behaviour. In the light of future possible in vivo applications, we finally tested the efficiency of the pSUPER.retro infection system on HUVEC and different cancer cell lines. The infection efficiency on HUVE cells was very low (less than 5%), even if repeated attempts were performed. In contrast, HT29 and HCA7 colon cancer cell lines, compared to HeLa cells (used as positive control), were easily infected showing higher efficiency levels (Figure 9). The infection efficiency for both HT29 and HCA7 was around 45%, whereas it was around 35% for HeLa cells.


RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells.

Strillacci A, Griffoni C, Spisni E, Manara MC, Tomasi V - Br. J. Cancer (2006)

COX-2 specific knockdown by siRNAs in HUVE cells and evaluation of 6-keto-PGF1α production. HUVE cells were transiently transfected with siRNAs directed against COX-2 mRNA (sequences A–D; final concentration 200 pM): COX-2 levels (dark bars) and 6-keto-PGF1α production (bright bars) were analysed by Western blot and ELISA assay, respectively. All procedures are described under Materials and Methods. (A) Shows COX-2 and COX-1 expression after siRNAs treatment. After the evaluation of the bands intensity by Image Master VDS software, both COX-2 and COX-1 levels were normalised against β-actin expression. All samples (lanes 2–7) except control in lane 1 were treated with PMA 40 nM. Lanes 3–6: samples treated with siRNA A, B, C and D, respectively. Lane 7: HUVE cells transfected with siRNA-Scr (scrambled), representing a negative control. Data are expressed as % of PMA-stimulated control value (lane 2) and represent the mean±s.e.m. of three independent experiments. *(P<0.01); #(P<0.05).
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fig2: COX-2 specific knockdown by siRNAs in HUVE cells and evaluation of 6-keto-PGF1α production. HUVE cells were transiently transfected with siRNAs directed against COX-2 mRNA (sequences A–D; final concentration 200 pM): COX-2 levels (dark bars) and 6-keto-PGF1α production (bright bars) were analysed by Western blot and ELISA assay, respectively. All procedures are described under Materials and Methods. (A) Shows COX-2 and COX-1 expression after siRNAs treatment. After the evaluation of the bands intensity by Image Master VDS software, both COX-2 and COX-1 levels were normalised against β-actin expression. All samples (lanes 2–7) except control in lane 1 were treated with PMA 40 nM. Lanes 3–6: samples treated with siRNA A, B, C and D, respectively. Lane 7: HUVE cells transfected with siRNA-Scr (scrambled), representing a negative control. Data are expressed as % of PMA-stimulated control value (lane 2) and represent the mean±s.e.m. of three independent experiments. *(P<0.01); #(P<0.05).
Mentions: Considering the relevance of endothelial COX-2 in the angiogenic process, we used an in vitro angiogenesis experimental model, based on primary HUVEC, to detect whether siRNA molecules were capable of downregulating COX-2 expression and inhibiting COX-2-dependent angiogenesis. Four different siRNAs, directed against COX-2 mRNA, were transfected at 200 pM concentration, by using the Oligofectamine reagent, in HUVEC treated with PMA to enhance COX-2 expression. As shown in Figure 2, only two siRNAs (sequences B and C) were capable of reducing COX-2 protein levels by more than 50%, whereas a scrambled siRNA, used as a negative control, was found to be completely devoid of effects. Moreover, we demonstrated that the transient knockdown mediated by siRNAs in HUVEC was highly specific since COX-1 expression resulted unaffected (Figure 2A). In samples in which COX-2 was downregulated, also PGI2 production, evaluated by ELISA assay, significantly decreased up to more than 40% (Figure 2B). Thus, we chose siRNA sequence-B to perform an in vitro angiogenesis test (Figure 3). As reported in the literature (Ilan et al, 1998), HUVE cells were able to organise into capillary-like tubular structures when seeded on 3-D collagen gel and stimulated with PMA (compare PMA-stimulated cells in Figure 3B to control cells in A). We observed that transfection of siRNA-B in HUVEC strongly affected their ability to organise in tubular structures (Figure 3C), with a significant reduction of vessels number after PMA stimulation (as shown in Figure 3E). Cells transfected with scrambled siRNA (Figure 3D) were still able to differentiate in tubular structures with the same efficiency of PMA-stimulated control cells (as shown in Figure 3E), allowing to exclude toxicity and nonspecific effects of siRNA-B on angiogenesis. These results demonstrate that siRNAs are capable to affect the in vitro angiogenic process by downregulating COX-2 expression in a strong specific manner. We also evaluated whether the transfection of synthetic siRNAs in HUVE cells may activate the interferon-mediated Jak-STAT pathway, as previously reported for other siRNAs molecules (Sledz et al, 2003). Western blot analysis of phospho-STAT-1(Tyr701) (active form) levels, normalised against p85/p91 STAT-1 total protein levels, showed that only an high concentration (200 nM) of transfected siRNA-B is able to trigger the interferon system response, whereas a lower but effective dose of siRNA (200 pM) does not have any effect on STAT-1 phosphorylation (Figure 4A and B). Phorbol 12-myristate 13-acetate-treated samples were used as positive controls, as suggested by the literature (Cohen et al, 2005). Data from immunofluorescence analysis were in high agreement with these findings (Figure 4C). STAT-1 phosphorylation, followed by nuclear translocation, was strongly increased in samples transfected with siRNA 200 nM, while no relevant differences were detected between samples transfected with siRNA 200 pM and controls. In order to achieve a stable downregulation of COX-2 in cancer cells, we prepared a mammalian vector expressing an anti-COX-2 shRNA. We used the pSUPER.retro vector system, capable of integrating expression cassettes in human genome, to produce efficient and specific downregulation of COX-2 gene expression induced by a siRNA mechanism (Brummelkamp et al, 2002a, 2002b). We cloned the dsRNA sequence corresponding to siRNA-B into a pSUPER.retro vector containing also the expression cassette for the GFP. The recombinant vector was transfected into Phoenix packaging cells to produce retroviral ecotropic supernatant, used to infect HT29 cells. Infected cells were selected by using standard puromycin treatment (1 μg ml−1) for 48 h. Selected HT29 cells (HT29 pSUPER(+)) were analysed by Western blot for COX-2 expression. As shown in Figure 5(A and B), COX-2 levels were found to be significantly decreased (more than 70%) in HT29 pSUPER(+) when compared to control cells. The inhibition was still effective when the COX-2 gene expression was stimulated by PMA treatment. Cyclooxygenase-2 mRNA levels were analysed in HT29 pSUPER(+) by real-time PCR. Results were in strict accordance with data obtained by Western blot, confirming the specific COX-2 mRNA degradation by RNAi. In fact, we obtained an 80% reduction of COX-2 mRNA levels either in the absence or in the presence of PMA stimulation (Figure 5C). As a further demonstration of the efficiency of the COX-2 knockdown mediated by RNAi, we also found a significant decrease of PGE2 production in HT29 pSUPER(+) cells (Figure 5D). Since we found, as mentioned above, that the transfection of exogenous synthetic siRNAs is capable to activate the interferon system at high concentrations in HUVE cells, the following aim was to demonstrate whether an endogenous and constitutive production of shRNA in the HT29 pSUPER(+) model had a different effect. Surprisingly, we found that shRNAs, that strongly downregulate COX-2 expression in HT29 pSUPER(+) cells, did not trigger the interferon system response in the absence of PMA treatment, compared to the control. Both Western blot (Figure 6A and B) and immunofluorescence (Figure 6C) analysis of phospho-STAT-1(Tyr701) levels and localisation confirmed this evidence. Moreover, in order to obtain more data on the effects of the constitutive COX-2 downregulation in HT29 pSUPER(+) cells, we performed four different assays to evaluate the proliferation profile and the invasiveness of these clones, compared to two different controls: HT29 wild-type and HT29 pSUPER(−) cells. HT29 pSUPER(−) cells were selected with puromycin after infection with the retroviral vector, devoid of the anti-COX-2 shRNA expression cassette. Although the MTT proliferation assay (Figure 7A) and the cell cycle distribution analysis (Figure 7B) did not show significant differences between controls and HT29 pSUPER(+), data from migration assay performed with Boyden chambers (Figure 8A and B) and soft-agar colony formation assay (Figure 8C) suggest that the stable knockdown of COX-2 gene by RNAi promotes a significant reduction of the migratory ability as well as a strong inhibition of colony formation in soft agar in infected pSUPER(+) colon cancer cells. Interestingly, the loss of the malignant behaviour in vitro of pSUPER(+) HT29 cells did not seem to depend on an impairment of cell growth, since constitutive expression of anti-COX-2 shRNA in HT29 cells only slightly modified their proliferation profile and their cell cycle distribution, but it derived from a reduction of the ability to invade the extracellular matrix and to grow in anchorage-independent manner, which are indexes of an invasive and aggressive behaviour. In the light of future possible in vivo applications, we finally tested the efficiency of the pSUPER.retro infection system on HUVEC and different cancer cell lines. The infection efficiency on HUVE cells was very low (less than 5%), even if repeated attempts were performed. In contrast, HT29 and HCA7 colon cancer cell lines, compared to HeLa cells (used as positive control), were easily infected showing higher efficiency levels (Figure 9). The infection efficiency for both HT29 and HCA7 was around 45%, whereas it was around 35% for HeLa cells.

Bottom Line: Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response.Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour.Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Biology, University of Bologna, and Center for Applied Biomedical Research (CRBA), St Orsola-Malpighi University Hospital, Bologna, Italy.

ABSTRACT
Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity.

Show MeSH
Related in: MedlinePlus