Mechanical Stretch Up-regulates MicroRNA-26a and Induces Human Airway Smooth Muscle Hypertrophy by Suppressing Glycogen Synthase Kinase-3β

Abstract

Airway smooth muscle hypertrophy is one of the hallmarks of airway remodeling in severe asthma. Several human diseases have been now associated with dysregulated microRNA (miRNA) expression. miRNAs are a class of small non-coding RNAs, which negatively regulate gene expression at the post-transcriptional level. Here, we identify miR-26a as a hypertrophic miRNA of human airway smooth muscle cells (HASMCs). We show that stretch selectively induces the transcription of miR-26a located in the locus 3p21.3 of human chromosome 3. The transcription factor CCAAT enhancer-binding protein ␣ (C/EBP␣) directly activates miR-26a expression through the transcriptional machinery upon stretch. Furthermore, stretch or enforced expression of miR-26a induces HASMC hypertrophy, and miR-26 knockdown reverses this effect, suggesting that miR-26a is a hypertrophic gene. We identify glycogen synthase kinase-3␤ (GSK-3␤), an anti-hypertrophic protein, as a target gene of miR-26a. Luciferase reporter assays demonstrate that miR-26a directly interact with the 3-untranslated repeat of the GSK-3␤ mRNA. Stretch or enforced expression of miR-26a attenuates the endogenous GSK-3␤ protein levels followed by the induction of HASMC hypertrophy. miR-26 knockdown reverses this effect, suggesting that miR-26a-induced hypertrophy occurs via its target gene GSK-3␤. Overall, as a first time, our study unveils that miR-26a is a mechanosensitive gene, and it plays an important role in the regulation of HASMC hypertrophy. MicroRNAs (miRNAs)2 are an evolutionarily conserved novel class of small non-coding RNAs that have achieved status as potent regulators of gene expression. According to their genomic location relative to protein-coding gene locus, miRNAs can be divided into intergenic miRNAs and intragenic miRNAs. The former have independent transcriptional units, including promoter, transcript sequence, and terminator; therefore, they do not overlap with other genes (1, 2). The later are found in the introns of protein-coding host genes, and they generally share the same regulatory motifs with their host genes (3-5). Most of the miRNAs are transcribed by RNA polymerase II as primary miRNAs (1, 2) and processed by the RNase III enzymes Drosha and Dicer to produce 21-to 23-nucleotide double-stranded RNA duplexes (6, 7). These smaller RNAs are then exported to the cytoplasm by Exportin 5 (8, 9), where they are subsequently processed into mature miRNAs by Dicer. The mature miRNAs are loaded into the miRNA-induced silencing complex (7), where they recognize their target protein-coding mRNAs to inhibit mostly mRNA translation or degradation (10) by base pairing to complementary sequences within the 3Ј-untranslated region (3Ј UTR). With respect to miRNAs functions, they play pivotal roles in the pathophysiological processes such as apoptosis, cell differentiation, cell proliferation, and organ development (4, 11). Functionally speaking, several human diseases have now been associated with dysregulated miRNAs expression.Airway remodeling is a characteristic featu...

Changes in microRNA expression in rat lungs caused by sevoflurane anesthesia: a TaqMan^|^reg; low-density array study

“…Some of the differentially expressed 28 miRNAs are known to be involved in inflammatory lung diseases and PAH. For example, miR-126, -21, and -26a are important in asthma (17,18,21); let7d, miR-21, -155, and -29 are important in IPF (26); and miR-146a, -155, -21, -25, and -26a are important in PAH (14). Here, we found that expression of each of these miRNA was significantly changed by sevoflurane anesthesia; therefore, sevoflurane anesthesia may affect lung diseases such as asthma, IPF, and PAH.…”

Desmin Regulates Airway Smooth Muscle Hypertrophy through Early Growth-responsive Protein-1 and MicroRNA-26a

“…The pSilencer 4.1-CMV miR-26a expression construct was prepared as described previously (14). Mouse GSK-3␤ cDNA 3Ј-UTR was synthesized and cloned into pcDNA 3.1D/V5-His-TOPO vector (Invitrogen) according to the manufacturer's instructions.…”

“…Real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Expression Analysis-Real-time RT-PCR assays were performed as described earlier (14). The amount of amplified transcripts (2 Ϫ ⌬⌬CT ) was estimated by the comparative CT (⌬ CT ) method and normalized to an endogenous reference (GAPDH) relative to a calibrator.…”

“…Solution Hybridization Detection Analysis-The expression levels of mature miRNAs were measured by solution hybridization detection method as described earlier (14).…”

“…Recently, we have shown the role of miR-26a in the induction of hypertrophy in human ASMC (14). However, whether desmin can regulate the ASMC phenotype through a miRNA-regulated pathway is unknown.…”