MiR-23b controls TGF-β1 induced airway smooth muscle cell proliferation via direct targeting of Smad3.

MicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation. Here we report that miR-23b inhibited airway smooth muscle cells (ASMCs) proliferation through directly targeting of Smad3. We obtained ASMCs by laser capture microdissection of normal and asthmatic mice lung tissues. Mice ASMCs were cultured and induced by TGF-β1. The implication between TGF-β1 and miR-23b in ASMCs were detected by RT-PCR. The effects of miR-23b on ASMCs proliferation and apoptosis were assessed by transient transfection of miR-23b mimics and inhibitor. The expression of Smad3 in ASMCs were detected by RT-PCR and Western blotting analysis. Dual-Luciferase Reporter Assay System will be applied to identify whether Smad3 is a target gene of miR-23b. TGF-β1 and miR-23b mRNA expression of in-situ bronchial ASMCs collected by laser capture microdissection were increased in asthmatic mice compared to non-asthma controls. This is accompanied by an increase in miR-23b mRNA expression in TGF-β1 induced ASMCs. miR-23b up-regulation significantly inhibited TGF-β1-induced ASMCs proliferation and promoted apoptosis. MiR-23b negatively regulates the expression of Smad3 in ASMCs. Dual-Luciferase Reporter Assay System demonstrated that Smad3 was a direct target of miR-23b. MiR-23b may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via direct targeting of Smad3.

Chen M ，Shi J ，Zhang W ，Huang L ，Lin X ，Lv Z ，Zhang W ，Liang R ，Jiang S ... -  《-》

MiR-23b controls TGF-β1 induced airway smooth muscle cell proliferation via TGFβR2/p-Smad3 signals.

Abnormal proliferation of ASM (airway smooth muscle) directly contributes to the airway remodeling during development of lung diseases such as asthma. Here we report that a specific microRNA (miR-23b) controls ASMCs proliferation through directly inhibiting TGFβR2/p-Smad3 pathway. The expression of miR-23b in ASMCs was detected by quantitative real-time polymerase chain reaction (RT-PCR). The effects of miR-23b on cell proliferation and apoptosis of ASMCs were assessed by transient transfection of miR-23b mimics and inhibitor. The target gene of miR-23b and the downstream pathway were further investigated. Overexpression of miR-23b significantly inhibited TGF-β1-induced ASMCs proliferation and promoted apoptosis. RT-PCR and Western blotting analysis showed miR-23b negatively regulates the expression of TGFβR2 and p-Smad3 in ASMCs. Subsequent analyses demonstrated that TGFβR2 was a direct and functional target of miR-23b, which was validated by the dual luciferase reporter assay. MiR-23b may function as an inhibitor of airway smooth muscle cells proliferation through inactivation of TGFβR2/p-Smad3 pathway.

Chen M ，Huang L ，Zhang W ，Shi J ，Lin X ，Lv Z ，Zhang W ，Liang R ，Jiang S ... -  《-》

MiR-143-3p controls TGF-β1-induced cell proliferation and extracellular matrix production in airway smooth muscle via negative regulation of the nuclear factor of activated T cells 1.

MicroRNAs (miRNAs) are small noncoding RNAs that function in diverse biological processes. However, little is known about the precise role of microRNAs in the functioning of airway smooth muscle cells (ASMCs). Here, we investigated the potential role and mechanisms of the miR-143 -3p on proliferation and the extracellular matrix (ECM) protein production of ASMCs. We demonstrated that miR-143-3p was aberrantly lower in ASMCs isolated from individuals with asthma than in individuals without asthma. Meanwhile, TGF-β1 caused a marked decrease in a time-dependent manner in miR-143-3p expression in ASMCs from asthmatics. Additionally, the overexpression of miR- 143-3p robustly reduced TGF-β1-induced ASMCs proliferation and downregulated CDK and cyclin expression, whereas the inhibition of miR-143-3p significantly enhanced ASMCs proliferation and upregulated the level of CDKs and cyclins. Re-expression of miR-143-3p attenuated ECM protein deposition reflected as a marked decrease in the expression of type I collagen and fibronectin, whereas miR-143-3p downregulation caused an opposite effect on the expression of type I collagen and fibronectin. Moreover, qRT-PCR and western blot analysis indicated that miR-143-3p negatively regulated the expression of nuclear factor of activated T cells 1 (NFATc1). Subsequent analyses demonstrated that NFATc1 was a direct and functional target of miR-143-3p, which was validated by the dual luciferase reporter assay. Most importantly, the overexpression of NFATc1 effectively reversed the inhibition of miR-143-3p on TGF-β1-induced proliferation, and strikingly abrogated the effect of miR-143-3p on the expression of CDK4 and Cyclin D1. Together, miR-143-3p may function as an inhibitor of asthma airway remodeling by suppressing proliferation and ECM protein deposition in TGF-β1-mediated ASMCs via the negative regulation of NFATc1 signaling, suggesting miR-143-3p as a potential therapeutic target for asthma.

Cheng W ，Yan K ，Xie LY ，Chen F ，Yu HC ，Huang YX ，Dang CX ... -  《-》

Airway smooth muscle cells from ovalbumin-sensitized mice show increased proliferative response to TGFβ1 due to upregulation of Smad3 and TGFβRII.

Shi J ，Chen M ，Ouyang L ，Huang L ，Lin X ，Zhang W ，Liang R ，Lv Z ，Liu S ，Jiang S ... -  《-》

TGF-β1-Induced Airway Smooth Muscle Cell Proliferation Involves TRPM7-Dependent Calcium Influx via TGFβR/SMAD3.

TRPM7 in mast cells plays a key role in asthma. However, the effect of TRPM7 on TGF-β1-induced airway smooth muscle cell (ASMC) proliferation remains unclear. Therefore, we designed this study to explore whether TRPM7 is involved in TGF-β1-induced ASMC proliferation. An asthmatic mouse model was established, and the expressions of TGF-β1 and TRPM7 in mice lungs were detected using enzyme-linked immunosorbent assay (ELISA) and western blotting. In addition, murine ASMCs were cultured and stimulated with TGF-β1. Possible TGF-β1 and TRPM7 interactions were examined using RT-PCR and western blotting analyses with ASMCs. The effect of TRPM7 knockdown on ASMC calcium influx was assessed by the fluorescent indicator indo-1. MTT and flow cytometry were applied to evaluate the effects of TRPM7 knockdown on ASMC proliferation and apoptosis. TGF-β1 elevated the expression of TRPM7 via TGFβR/SMAD3. Knocking down TRPM7 decreased the intracellular Ca2+ concentration and cellular proliferation of ASMCs, although apoptosis remained unaffected. Our initial findings suggest that TRPM7 inhibition may prevent asthma-induced airway remodeling.

Chen M ，Zhang W ，Shi J ，Jiang S ... -  《-》