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Silencing of CD47 and SIRP α by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells

View Article: PubMed Central - PubMed

ABSTRACT

Background: In the context of tumor immunology, tumor cells have been shown to overexpress CD47, an anti-phagocytic signal directed to macrophages to escape from phagocytosis by interacting with Signal Regulatory Protein α SIRPα.

Background: In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRPα in macrophages with the aim to eliminate tumor cells by macrophages in co-culture experiments.

Methods: THP-1 cells were differentiated to macrophages with PMA. The mRNA levels of differentiation markers CD14 and Mcl-1 mRNA and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRPα was evaluated at the mRNA level by qRT-PCR and at the protein level by Western Blot. Macrophages were co-cultured with tumor cells in the presence of PPRHs to silence CD47 and/or SIRPα. Cell viability was assessed by MTT assays.

Results: THP-1 cells differentiated to macrophages with PMA showed an increase in macrophage surface markers (CD14, Mcl-1) and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRPα expression in macrophages at the mRNA and protein levels. In the presence of PPRHs, MCF-7 cells were eliminated by macrophages in co-culture experiments, whereas they survived in the absence of PPRHs.

Conclusions: Our data support the usage of PPRHs to diminish CD47/SIRPα interaction by decreasing the expression of both molecules thus resulting in an enhanced killing of MCF-7 cells by macrophages, which might translate into beneficial effects in cancer therapy. These results indicate that PPRHs could represent a new approach with immunotherapeutic applications.

No MeSH data available.


Related in: MedlinePlus

CD47 and SIRPa levels upon PPRHs transfection. a MCF-7 cells (60,000) were plated one day before transfection with PPRHs against CD47. RNA was extracted 24 h after transfection. mRNA levels were determined by qRT-PCR using untransfected cells as control. b THP-1 cells (15,000) were plated and transfected with PPRHs against SIRPα. Other conditions were as in A). Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005). c MCF-7 cells (60,000) were plated one day prior to transfection of PPRHs against CD47 and total protein was extracted 3 h after transfection. A representative Western blot image of CD47 protein levels upon PPRHs transfection is shown. d THP-1 cells (15,000) were plated and transfected with PPRHs against SIRPα and total protein was extracted 24 h after transfection. Control corresponded to untransfected cells. A representative Western blot image of SIRPα protein levels upon PPRHs transfection is shown
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Fig4: CD47 and SIRPa levels upon PPRHs transfection. a MCF-7 cells (60,000) were plated one day before transfection with PPRHs against CD47. RNA was extracted 24 h after transfection. mRNA levels were determined by qRT-PCR using untransfected cells as control. b THP-1 cells (15,000) were plated and transfected with PPRHs against SIRPα. Other conditions were as in A). Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005). c MCF-7 cells (60,000) were plated one day prior to transfection of PPRHs against CD47 and total protein was extracted 3 h after transfection. A representative Western blot image of CD47 protein levels upon PPRHs transfection is shown. d THP-1 cells (15,000) were plated and transfected with PPRHs against SIRPα and total protein was extracted 24 h after transfection. Control corresponded to untransfected cells. A representative Western blot image of SIRPα protein levels upon PPRHs transfection is shown

Mentions: Two types of PPRHs against CD47 were used in the experiments, targeting either the promoter (HpCD47Pr-T) or intron 1 (HpCD47I3-T) in the template strand of the DNA (Table 1). To explore the ability of PPRHs to silence CD47, CD47 mRNA levels were analyzed in MCF-7 cells either in the absence or in the presence of PPRHs. Both HpCD47I3-T and HpCD47Pr-T were able to decrease CD47 mRNA levels down to 2-fold in MCF-7 cells compared to the control (Fig. 4a). To target SIRPα, HpSIRPαPr-C, designed against the promoter sequence and HpSIRPαI7-T, designed against the intron 7 sequence (Table 1) were transfected in THP-1 cells. A decrease in SIRPα expression was achieved with both PPRHs (Fig. 4b). The effects of PPRHs on CD47 and SIRPα were also determined at the protein level in MCF-7 and THP-1 cells, respectively (Fig. 4c,d). Both PPRHs targeting CD47 decreased the protein level by 2.5 fold (Fig. 4c). Likewise, SIRPα protein levels were reduced by both HpSIRPαPr-C and HpSIRPαI7-T (Fig. 4d).Fig. 4


Silencing of CD47 and SIRP α by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells
CD47 and SIRPa levels upon PPRHs transfection. a MCF-7 cells (60,000) were plated one day before transfection with PPRHs against CD47. RNA was extracted 24 h after transfection. mRNA levels were determined by qRT-PCR using untransfected cells as control. b THP-1 cells (15,000) were plated and transfected with PPRHs against SIRPα. Other conditions were as in A). Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005). c MCF-7 cells (60,000) were plated one day prior to transfection of PPRHs against CD47 and total protein was extracted 3 h after transfection. A representative Western blot image of CD47 protein levels upon PPRHs transfection is shown. d THP-1 cells (15,000) were plated and transfected with PPRHs against SIRPα and total protein was extracted 24 h after transfection. Control corresponded to untransfected cells. A representative Western blot image of SIRPα protein levels upon PPRHs transfection is shown
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Fig4: CD47 and SIRPa levels upon PPRHs transfection. a MCF-7 cells (60,000) were plated one day before transfection with PPRHs against CD47. RNA was extracted 24 h after transfection. mRNA levels were determined by qRT-PCR using untransfected cells as control. b THP-1 cells (15,000) were plated and transfected with PPRHs against SIRPα. Other conditions were as in A). Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005). c MCF-7 cells (60,000) were plated one day prior to transfection of PPRHs against CD47 and total protein was extracted 3 h after transfection. A representative Western blot image of CD47 protein levels upon PPRHs transfection is shown. d THP-1 cells (15,000) were plated and transfected with PPRHs against SIRPα and total protein was extracted 24 h after transfection. Control corresponded to untransfected cells. A representative Western blot image of SIRPα protein levels upon PPRHs transfection is shown
Mentions: Two types of PPRHs against CD47 were used in the experiments, targeting either the promoter (HpCD47Pr-T) or intron 1 (HpCD47I3-T) in the template strand of the DNA (Table 1). To explore the ability of PPRHs to silence CD47, CD47 mRNA levels were analyzed in MCF-7 cells either in the absence or in the presence of PPRHs. Both HpCD47I3-T and HpCD47Pr-T were able to decrease CD47 mRNA levels down to 2-fold in MCF-7 cells compared to the control (Fig. 4a). To target SIRPα, HpSIRPαPr-C, designed against the promoter sequence and HpSIRPαI7-T, designed against the intron 7 sequence (Table 1) were transfected in THP-1 cells. A decrease in SIRPα expression was achieved with both PPRHs (Fig. 4b). The effects of PPRHs on CD47 and SIRPα were also determined at the protein level in MCF-7 and THP-1 cells, respectively (Fig. 4c,d). Both PPRHs targeting CD47 decreased the protein level by 2.5 fold (Fig. 4c). Likewise, SIRPα protein levels were reduced by both HpSIRPαPr-C and HpSIRPαI7-T (Fig. 4d).Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: In the context of tumor immunology, tumor cells have been shown to overexpress CD47, an anti-phagocytic signal directed to macrophages to escape from phagocytosis by interacting with Signal Regulatory Protein &alpha; SIRP&alpha;.

Background: In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRP&alpha; in macrophages with the aim to eliminate tumor cells by macrophages in co-culture experiments.

Methods: THP-1 cells were differentiated to macrophages with PMA. The mRNA levels of differentiation markers CD14 and Mcl-1 mRNA and pro-inflammatory cytokines (IL-1&beta;, IL-18, IL-6, IL-8 and TNF-&alpha;) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRP&alpha; was evaluated at the mRNA level by qRT-PCR and at the protein level by Western Blot. Macrophages were co-cultured with tumor cells in the presence of PPRHs to silence CD47 and/or SIRP&alpha;. Cell viability was assessed by MTT assays.

Results: THP-1 cells differentiated to macrophages with PMA showed an increase in macrophage surface markers (CD14, Mcl-1) and pro-inflammatory cytokines (IL-1&beta;, IL-18, IL-6, IL-8 and TNF-&alpha;). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRP&alpha; expression in macrophages at the mRNA and protein levels. In the presence of PPRHs, MCF-7 cells were eliminated by macrophages in co-culture experiments, whereas they survived in the absence of PPRHs.

Conclusions: Our data support the usage of PPRHs to diminish CD47/SIRP&alpha; interaction by decreasing the expression of both molecules thus resulting in an enhanced killing of MCF-7 cells by macrophages, which might translate into beneficial effects in cancer therapy. These results indicate that PPRHs could represent a new approach with immunotherapeutic applications.

No MeSH data available.


Related in: MedlinePlus