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Lentiviral shRNA against KCa3.1 inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway.

Lin H, Zheng C, Li J, Yang C, Hu L - Sci Rep (2015)

Bottom Line: KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction.LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels.LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Otorhinolaryngology, Shanghai Jiao Tong University Affiliated Sixth People's hospital, Shanghai, China [2] Department of Otorhinolaryngology, Eye and ENT Hospital of Fudan University, Shanghai, China.

ABSTRACT
Calcium-activated potassium ion channel-3.1 (KCa3.1) plays a pivotal role in the potassium-calcium exchange involved in atopy. This study aimed to explore the impact of lentiviral-mediated shRNA silencing KCa3.1 on allergic response in a murine allergic rhinitis (AR) model. The BALB/c mice were divided into four groups: untreated AR group, negative control AR group, lentiviral KCa3.1-shRNA treated AR group and normal control group. Concentrations of ovalbumin (OVA)-specific IgE, histamine and leukotrienes C4 (LTC4) in serum, and IL-4, IL-9 and IL-17 in nasal lavage fluid (NLF) were analyzed. Goblet cells and mast cells were counted. KCa3.1 positive cells were counted after immunolabelling by immunofluorescence method. KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction. Furthermore, P815 cell line was used to explore the role and mechanism of lentiviral KCa3.1-shRNA on mast cells. The results showed that LV-KCa3.1-shRNA intervention effectively attenuated allergic responses in LV-KCa3.1-shRNA treated mice. LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels. LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

No MeSH data available.


Related in: MedlinePlus

Lentiviral vector system.(a–c) GV-118 vector encoding sustained RNA expression (a) pHelper 1.0 vector, encoding a viral structural protein (b) and pHelper 2.0 vector, encoding a viral capsid protein (c). Schematic diagram of a murine AR model and LV-KCa3.1-shRNA treatment (d).
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f6: Lentiviral vector system.(a–c) GV-118 vector encoding sustained RNA expression (a) pHelper 1.0 vector, encoding a viral structural protein (b) and pHelper 2.0 vector, encoding a viral capsid protein (c). Schematic diagram of a murine AR model and LV-KCa3.1-shRNA treatment (d).

Mentions: The shRNA targeting sequence for KCa3.1 (KCa3.1-shRNA) was 5′-TCATGATGGACATCCATTA-3′ and the scrambled RNA sequence (negative-shRNA) used as negative control was 5′-TTCTCCGAACGTGTCACGT-3′. The GV118 vector encoding KCa3.1-specific shRNA, pHelper 1.0 vector, and pHelper 2.0 vector (Fig. 6a–c, GeneChem, Shanghai, China) were co-transfected into the 293T cell line using lipofectamine 2000 (Invitrogen, Carlsbad, USA) to generate re-constructed lentiviruses according to the manufacturer’s instructions. After 48 hours, the supernatants were collected and re-constructed lentiviruses were harvested by centrifugation and then stored at −80 °C for further experiments.


Lentiviral shRNA against KCa3.1 inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway.

Lin H, Zheng C, Li J, Yang C, Hu L - Sci Rep (2015)

Lentiviral vector system.(a–c) GV-118 vector encoding sustained RNA expression (a) pHelper 1.0 vector, encoding a viral structural protein (b) and pHelper 2.0 vector, encoding a viral capsid protein (c). Schematic diagram of a murine AR model and LV-KCa3.1-shRNA treatment (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4536635&req=5

f6: Lentiviral vector system.(a–c) GV-118 vector encoding sustained RNA expression (a) pHelper 1.0 vector, encoding a viral structural protein (b) and pHelper 2.0 vector, encoding a viral capsid protein (c). Schematic diagram of a murine AR model and LV-KCa3.1-shRNA treatment (d).
Mentions: The shRNA targeting sequence for KCa3.1 (KCa3.1-shRNA) was 5′-TCATGATGGACATCCATTA-3′ and the scrambled RNA sequence (negative-shRNA) used as negative control was 5′-TTCTCCGAACGTGTCACGT-3′. The GV118 vector encoding KCa3.1-specific shRNA, pHelper 1.0 vector, and pHelper 2.0 vector (Fig. 6a–c, GeneChem, Shanghai, China) were co-transfected into the 293T cell line using lipofectamine 2000 (Invitrogen, Carlsbad, USA) to generate re-constructed lentiviruses according to the manufacturer’s instructions. After 48 hours, the supernatants were collected and re-constructed lentiviruses were harvested by centrifugation and then stored at −80 °C for further experiments.

Bottom Line: KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction.LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels.LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Otorhinolaryngology, Shanghai Jiao Tong University Affiliated Sixth People's hospital, Shanghai, China [2] Department of Otorhinolaryngology, Eye and ENT Hospital of Fudan University, Shanghai, China.

ABSTRACT
Calcium-activated potassium ion channel-3.1 (KCa3.1) plays a pivotal role in the potassium-calcium exchange involved in atopy. This study aimed to explore the impact of lentiviral-mediated shRNA silencing KCa3.1 on allergic response in a murine allergic rhinitis (AR) model. The BALB/c mice were divided into four groups: untreated AR group, negative control AR group, lentiviral KCa3.1-shRNA treated AR group and normal control group. Concentrations of ovalbumin (OVA)-specific IgE, histamine and leukotrienes C4 (LTC4) in serum, and IL-4, IL-9 and IL-17 in nasal lavage fluid (NLF) were analyzed. Goblet cells and mast cells were counted. KCa3.1 positive cells were counted after immunolabelling by immunofluorescence method. KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction. Furthermore, P815 cell line was used to explore the role and mechanism of lentiviral KCa3.1-shRNA on mast cells. The results showed that LV-KCa3.1-shRNA intervention effectively attenuated allergic responses in LV-KCa3.1-shRNA treated mice. LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels. LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

No MeSH data available.


Related in: MedlinePlus