The Roles of MicroRNAs and Protein Components of the MicroRNA Pathway in Lung Development and Diseases

Dicer1 in Mouse Lung Development

An important role of the miRNA pathway in lung development was first shown by the conditional deletion of Dicer1 in the endoderm of primary lung bud (64). At Embryonic Day 15.5 (15.5 d post coitum), the mutant lungs showed the formation of a large epithelial sac within each lobe instead of tree-like airway structures, likely due to significant reduction of branching activities in early stages of development (64). Epithelial cells in these mutant lungs continued to grow; however, aberrant cell death was observed in the epithelium, where Dicer1 was inactivated (64). Although Dicer1 was deleted in the epithelium, expression of FGF10 in the mesenchyme was up-regulated. This likely leads to changes in the expression of other genes, such as BMP4 and Sprouty. These observations suggest that genes downstream of the miRNA pathway in lung epithelium play critical roles in epithelial–mesenchymal interactions essential for proper lung morphogenesis (64).

DICER1 in Lung Cancers

Significantly, germline mutation of DICER1 is associated with human pleuropulmonary blastoma (PPB), a rare, inherited pediatric lung tumor (65). Recently, DICER1 mutations have been associated with cystic nephroma, medulloepithelioma, and Sertoli-Leydig cell tumors (65–67). The early stages of PPB feature cystic expansion of airways lined by benign-appearing epithelium. This is followed by the malignant transformation of mesenchymal cells adjacent to abnormal epithelium, and the formation of sarcoma (65). These phenotypes resemble the epithelial sac and mesenchymal expansion observed in Dicer1 conditional mutant mice (64). Although most patients with PPB harbor a heterozygous germline mutation of DICER1, expression of DICER1 protein was undetectable in segments of epithelium of PPB tumors, likely due to additional somatic mutation. However, heterozygous DICER1 expression was retained in the malignant mesenchyme. This combination of heterozygous germline mutation together with segmental loss of DICER1 in epithelium is different as compared with endoderm deletion of Dicer1 in mutant mice. This likely explains why PPB lungs are functional at birth, as compared with mouse lungs of Dicer1 mutant. Together, these results suggest that loss of DICER1 in the epithelium of human developing lungs alters the expression of diffusible factors that are important for epithelial–mesenchymal interactions, which leads to abnormal growth of mesenchymal cells in PPB. Although findings from mouse genetic studies shed light on the pathogenesis of PPB, more sophisticated genetic manipulation is required to potentially generate an animal model for PPB.

Heterozygous deletion of Dicer1 in airway epithelium enhances tumor development in a mouse model of K-Ras–induced lung cancer (68), strongly suggesting that Dicer1 functions as a haploinsufficient tumor suppressor (69). This is further supported by observations of reduced expression of DICER1 and other miRNA pathway components, and a global down-regulation of miRNAs in human tumor tissues, including those of lung cancers (70–72).

miR-17-92 Clusters in the Proliferation of Epithelial Progenitors

miRNAs from the miR-17-92 cluster and its two homologous clusters (miR106a-363 and miR-106b-25) are highly expressed during early stages of lung development, and decrease in late stages or adult lungs (73). Expression of miR-17-92 can be detected in both mesenchyme and epithelium. Overexpression of miR-17-92 in distal lung endoderm under the control of the mouse surfactant protein C (Sftpc) promoter resulted in increased proliferation and expansion of the distal progenitor population. However, the number of club and ciliated cells was reduced, together with an abnormality in the formation of terminal sacs in Embryonic Day–18.5 lungs (73). One of the direct targets of miR-17-92 in distal lung epithelial cells is Retinoblastoma-like 2 (Rbl2) (73), and down-regulation of Rbl2 in transgenic mice at least partially contributes to the increased epithelial proliferation (Figure 2). In another report, miR-17-20–106a was shown to regulate epithelial branching by suppressing signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase 14 (MAPK14), the downstream components of FGF10-FGFR2 signaling. Inhibition of miR-17 paralogs leads to the upregulation of Stat3 and MAPK14 in embryonic lung epithelium cultured with FGF10 (74). Furthermore, miR-17-92 has been shown to target CCAAT/enhancer binding protein and GATA binding protein 6 in a newly isolated human lung progenitor population (E-cadherin/leucine-rich repeat containing G protein-coupled receptor 6 positive) to maintain the balance between differentiation and self-renewal (75).

Mice deficient for miR-17-92 are smaller and die shortly after birth. The most significant phenotype was severe hypoplastic lungs, as compared with other organs, such as heart. However, no specific branching or other developmental abnormality was found in these lungs (76). Moreover, results from mice with double or triple deletion of miR-17-92, miR-106b-25, and miR-106a-363 clusters showed that they cooperate functionally in regulating embryonic growth and apoptosis (76). This proves a role for miR-17-92 and its paralog clusters in promoting proliferation and growth of embryonic lungs as well as other organs in vivo. Due to its ubiquitous expression in both developing epithelium and mesenchyme, conditional knockout mice are required to dissect their activities in different cell types of embryonic lungs.

miR-17-92 Clusters in Lung Cancer

Dysregulation of developmental programs is associated with tumorigenesis. Extensive studies have shown an oncogenic role for miR-17-92 in the pathogenesis of different human cancers, including lung cancer (77, 78). Amplification and overexpression of miR-17-92 cluster is observed in lung cancer cells (77, 79, 80). In tumors, expression of miR-17-92 cluster is induced by oncogenes, such as v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (NYMC) and E2F family members (81). It promotes cell growth by targeting genes critical for growth arrest, such as cyclin-dependent kinase inhibitor 1A and retinoblastoma 1-related genes (82). Furthermore, studies have shown that inhibition of miR-17-5p and miR-20a with antisense oligonucleotides induce apoptosis selectively in lung cancer cells overexpressing miR-17-92 (83). Although the proliferation activities of miR-17-92 clusters in development are consistent with their oncogenic roles in tumors, the prevalence of miR-17-92 cluster amplification/up-regulation in lung cancers is still unclear. Because miR-106a-363 and miR-106b-25 knockout animals are viable and grow to adulthood without obvious phenotype, they are useful to test whether deletion of miR-106a-363 and miR-106b-25 in vivo will slow the initiation or progression of K-Ras–induced lung cancer.

miR-302/367 Cluster in the Proliferation and Apical–Basal Polarity of Lung Endoderm

miR-302/367 cluster is identified as a direct target of Gata6 in mouse lung endoderm (84). Similar to the miR-17-92 cluster, levels of miR-302/367 are high at early stages of lung development, but decline rapidly as development proceeds (84). Overexpression of miR-302/367 driven by a human SFTPC promoter leads to a thickening of distal epithelium and reduction in sacculation (84). In the airways of these transgenic mice, numbers of both Sox2-positive and Sox9-positive progenitor cells are significantly increased, whereas markers for both distal and proximal airways, including CC10 and Sftpc, are dramatically reduced (84). This was further supported by the reduced proliferation, together with increased differentiation, in transgenic mice, in which miR-302/367 activities were reduced by the expression of a miR-302/367 sponge, which consisted of multiple miR-302/367 binding sites (84). In the lung endoderm, miR-302/367 promotes the proliferation of undifferentiated progenitor cells, partially by targeting Rbl2 and cyclin-dependent kinase inhibitor 1A (Figure 2). miR-302/367 also regulates apical–basal polarity of lung epithelium by suppressing T-cell lymphoma invasion and metastasis 1 and platelet-activating factor acetylhydrolase, isoform 1b, subunit 1 (Lis1 or Pafah1b1) (Figure 2) (84). Little is known about the miR-302/367 cluster in human lung diseases. A recent report showed that high miR-367 is associated with unfavorable prognosis in resected non–small cell lung cancer (85).

miR-34/449 in Ciliogenesis of Airway Epithelium

miR-449a/b/c (referred to as miR-449) is transcribed from a polycistronic cluster. miR-449 is highly expressed in tissues with multiciliated cells, such as lungs (86–88). Its expression is significantly induced in the process of differentiation of both human airway mucociliary epithelial cells (HAECs) grown on an air–liquid interface and mucociliary epidermis of Xenopus embryos (88, 89). In both systems, expression of miR-449 is selectively enriched in ciliated cells. Loss or knockdown of miR-449 expression in both HAECs and Xenopus embryonic epidermis leads to significant reduction of ciliated cells. miR-449 promotes ciliogenesis by directly targeting Notch1 and δ-like 1 (Figure 2) (88). Conversely, miR-449 null mice developed normally, without gross defects. One of the explanations for the lack of phenotype is the compensatory role of miR-34 family members that share identical seed sequence and similar expression pattern with miR-449 (90, 91). miR-34 family members are highly expressed in lungs and also increased in the process of ciliogenesis of HAECs (88, 91). In miR-449 null testis, levels of miR-34 are up-regulated, suggesting a compensatory expression and function (90). Most recently, their redundant and significant roles in ciliogenesis were reported in mice deficient for all miR-34/449. Deletion of all three loci of miR-34/449 in mice resulted in a significant decrease in cilia length and cilia number, due to defective basal body maturation and apical docking. Derepression of Cp110, a centriolar protein that negatively regulates primary cilia assembly, partially contribute to the abnormal ciliogenesis in miR-34/449 null mice (Figure 2) (92).

Interestingly, levels of all miR-34/449 family members are significantly reduced in HAECs of patients with asthma and after IL13 stimulation (93). It is known that high Notch activity in airway epithelium is associated with increased numbers of mucous cells together with reduced numbers of ciliated cells (94). Furthermore, treatment of adult HAECs with Notch antagonists resulted in increased ciliated cells and decreased numbers of mucous cells (94). In addition, Notch antagonists blocked IL13-induced mucous metaplasia. Together, this suggests that reduced miR-34/449 in asthmatic airway epithelium might lead to increased Notch activity, which might contribute to the increased number of mucous cells (93).

miR-34/449 Function as a Tumor Suppressor

miR-34 is known to be a direct target of p53 in blocking cell proliferation and inducing apoptosis (40). Lost or reduced expression of miR-34 or hypermethylation of its promoter have been observed in multiple cancers, including both small cell and non–small cell lung cancers (95–97). Overexpression of miR-34 in lung cancer cells or animal models leads to inhibition of the initiation or progression of cancer or sensitization to radiotherapy (98, 99). Interestingly, the p53 pathway remains intact in miR-34 null mice. This is likely due to the redundancy of miR-449 or other p53 downstream effectors, such as Cdkn1a and BCL2-associated X protein that are working in parallel with miR-34 (91). Indeed, miR-34a deficiency strongly promotes tumorigenesis in a K-Ras–induced lung cancer when p53 is haploinsufficient, strongly suggesting a tumor suppressor activity for miR-34 (100). Unlike miR-34, expression of miR-449 is induced by the cell cycle regulatory transcription factor, E2F1, a positive regulator for cell proliferation (101). It has been suggested that miR-449 functions as a negative feedback regulator of E2F1 to avoid excessive E2F1-induced proliferation (101). Expression of miR-449 is down-regulated in non–small cell lung cancers (102). Similar to miR-34, overexpression of miR-449 leads to growth arrest of lung cancer cells by targeting myelocytomatosis oncogene and met proto-oncogene (102, 103). Although the activity of miR-449 in ciliogenesis is mainly assigned to the suppression of Notch or centriolar protein, Cp110 (Figure 2) (88), miR-449/34 might play an important role in promoting cell cycle exit, which is essential to initiate the differentiation of airway epithelial cells. It is unknown whether miR-449 deficiency alone or in combination with miR-34 deletion will promote lung tumorigenesis, similar to what has been observed in miR-34 null mice.

miR-4423 in Ciliogenesis of Human Airway Epithelium and Lung Cancer

Recently, a primate-specific miRNA, miR-4423, was found to be selectively expressed in human mucociliary epithelium. Similar to miR-449, overexpression of miR-4423 promotes ciliogenesis. Intriguingly, the seed sequence and predicted mRNA targets of miR-4423 overlap with those of miR-449/miR-34. However, whether miR-449/34 and miR-4423 synergistically regulate ciliogenesis of HAECs remains unknown. The expression of miR-4423 is also significantly reduced in a large fraction of lung cancers, including both squamous carcinomas and adenocarcinomas, relative to matched adjacent normal tissues. In addition, overexpression of miR-4423 in several lung cancer cell lines reduces their anchorage-independent growth and the size of tumors formed in xenograft model (104). The finding of primate-specific miRNAs, or large-animal–specific response or function of miR-124 (105), presents a need to develop additional model systems other than murine models.

miR-326 as Part of the Shh Signaling Pathway in Developing Lung Mesenchyme

In a genome-wide profiling to identify miRNAs associated with the Shh pathway in early developing lungs, expression of both miR-326 and its host gene, Arrestin β1, are found to be selectively enriched in embryonic mesenchymal cells, and are specifically regulated by Shh activity (106). Furthermore, functional analysis showed that miR-326 acts as a negative modulator for Shh signaling by directly targeting smoothened and GLI-Kruppel family member 2. This suggests a miR-326–mediated negative feedback loop in modulating Shh pathway during lung development (Figure 2) (106). In brain tumor progenitor cells, miR-326 was also shown to modulate Shh signaling by targeting smoothened (107, 108).

Recent studies showed a reactivation of the Shh pathway in bleomycin-induced fibrotic lungs and in human IPF lungs (109–111). This might lead to aberrant epithelial–fibroblast interaction and induction of fibroblast cell proliferation. In a separate report, miR-326 was shown to regulate TGFb1, E26 avian leukemia oncogene 1, 5′ domain, SMAD family member 3, and matrix metallopeptidase 9 in fibroblast cells and in a bleomycin-induced pulmonary fibrosis (112). In addition, levels of miR-326 were found to be significantly lower in IPF lung tissues as compared with control (112). This contradicts findings in early developing lung mesenchyme in which high Shh activity led to up-regulation of miR-326; however, it will be advantageous to investigate how miR-326 is down-regulated, and whether this relates to reactivation of Shh in human IPF lungs.

miR-142-3p Modulates Wnt Signaling in Embryonic Lung Mesenchyme

In another array profiling of miRNAs selectively enriched in epithelium or mesenchyme of early developing lungs, miR-142-3p was found to be selectively expressed in mesenchyme (113). Disruption miR-142-3p expression in cultured embryonic lungs using vivo-morpholino, an octa-guanidinium dendrimer–conjugated morpholino, resulted in reduced branching and ectopic expression of α-smooth muscle actin in distal lung mesenchyme. miR-142-3p influences smooth muscle cell (SMC) progenitor proliferation and differentiation by directly regulating adenomatous polyposis coli, a negative regulator of Wnt signaling (Figure 2) (113).

Dicer1, Drosha, and DGCR8 in Proliferation and Differentiation of Vascular SMCs

Although the role of the miRNA pathway in the development of vascular SMCs (vSMCs) has been extensively examined in systemic arteries, such as aortas, little is known regarding its activity in airway SMCs and pulmonary vSMCs, two components of lung mesenchyme important for development and diseases. SMCs from different organs or structures share core regulatory networks; however, they are regulated by distinct environmental signals during development, and they differ in their expression of ion channels, hormone receptors, and cell-signaling pathways. Here, we review recent findings on miRNAs in vSMCs, and discuss the need to study whether airway SMCs and pulmonary vSMCs are regulated by the same or distinct mechanisms.

The important role of the miRNA pathway in vSMCs was demonstrated by the deletion of Dicer1 specifically in vSMCs. Deletion of Dicer1 by transgelin (SM22a)-Cre resulted in reduced proliferation and disrupted differentiation of vSMCs, leading to abnormal vessel wall formation and extensive hemorrhage (114, 115). SM22α-Cre is mainly expressed in vSMCs, with little or no expression in visceral SMCs. This prevents the analysis of the role of Dicer in SMCs of other organs, such as smooth muscle of lungs. Recently, Dicer1 was deleted by an inducible myosin, heavy polypeptide 11–CreERT2 in 3- to 4-week-old mice that resulted in significant lower blood pressure due to reduced expression of contractile proteins and loss of vascular contraction function (116). Myosin, heavy polypeptide 11–CreERT2 is expressed in visceral SMCs, and deletion of Dicer1 in intestinal SMCs leads to a relaxed state of gut smooth muscle (116). These observations were supported by similar defects in vSMC development in animals in which Drosha or DGCR8, two other key components in pri-miRNA processing, are specifically deleted in SMCs (117, 118). However, how deletion of Dicer1, Drosha, or DGCR8 affects airway SMCs or pulmonary vSMCs is unknown.

The defects of protein components of the miRNA pathway in SMCs of pulmonary diseases, such as asthma and pulmonary hypertension, are still poorly understood. However, there is evidence that hypoxia, which contributes to the pathogenesis of various human lung diseases, including pulmonary artery hypertension, can enhance the overall activity of the miRNA pathway in pulmonary arterial SMCs (PASMCs). Hypoxia induces the prolyl-hydroxylation and accumulation of Ago2, a key component of miRISC (119). In addition, TGF/BMP signaling regulates the expression of miR-21 and a subset of miRNAs post-transcriptionally by modulating the activity of pri-miRNA processing machinery in SMCs (41). Together, these results strongly suggest that the expression or function of protein components of the miRNA pathway can be modulated in association with developmental or disease conditions.

miR-145/143 in Regulating SMC Differentiation

Several individual miRNAs are shown to play pivotal roles in regulating vSMC differentiation. miR-145 and miR-143 are evolutionary conserved and transcribed from a polycistronic transcription unit without homology to each other (120). In adult mice, expression of the miR-145/143 cluster is regulated by serum response factor (Srf) and myocardin, and selectively enriched in SMCs, including bronchial SMCs and pulmonary vSMCs (120). The smooth muscle layers of arteries were noticeably thinner in miR-145 single-knockout or miR-145/143 double-knockout mice, likely due to a reduction in cell size (120). Results from several mouse genetic or disease models showed that loss of miR-143/miR-145 leads to disarray of actin stress fibers, diminished migratory activity of SMCs, and significantly compromised vSMC contraction (42, 120–122). Results from cell culture and tissues of knockout mice showed that genes involved in cytoskeleton organization (Ssh2, Add3, and Srgap1/2), podosome formation and migration (PDGF-R, Pkc and Fascin), and SMC proliferation and differentiation (Klf4/5, Elk1, Ace1, Tpm-4 and CamkII-δ) are under the control of miR-145 and miR-143 (42, 120–122), thus suggesting a synergetic role of these two miRNAs in regulating proper SMC phenotype (Figure 2). These defects in SMC differentiation and proliferation within vessel walls led to the development of hypotension in mutant mice. In cultured vSMCs, expression of miR-145/143 is also transcriptionally activated by TGF-β and BMP4 that promotes a contractile phenotype by targeting Klf4 (123). Interestingly, the phenotype of miR-143/miR-145 null mice is much milder as compared with that of SMC-specific deletion of Dicer1, suggesting that additional miRNA must be required for proper differentiation of vSMCs in vivo (114, 116).

miR-145/143 in Pulmonary Hypertension and Fibrosis

A significant up-regulation of miR-145/143 in response to hypoxia was observed both in the lung and right ventricle, but not in other organs (121). In response to chronic hypoxia, miR-145 knockout animals displayed no increase in systolic right ventricular pressure and right ventricular hypertrophy, unlike wild-type mice that showed significant increase of right ventricular pressure and right ventricular hypertrophy as expected (121). In addition, knockdown of miR-145, but not miR-143, leads to a milder response to chronic hypoxia, suggesting a specific role of miR-145 in this process (121). In human tissue, miR-145 was significantly elevated in both idiopathic or heritable pulmonary arterial hypertension (iPAH or hPAH) conditions as compared with controls. Levels of miR-145 in PASMCs isolated from patients with PAH with bone morphogenetic protein receptor, type II mutation is much higher compared with control PAMSCs from normal subjects. In addition, knockdown of bone morphogenetic protein receptor, type II in normal PAMSCs resulted in significant up-regulation of both miR-145 and miR-143. Together, these data suggest that miR-145 is downstream of BMP signaling in PAMSCs, and may represent a novel target for PAH treatment (121).

Although miR-145/143 expression is enriched in SMCs, miR-145 is shown to promote myofibroblast differentiation. In contrast, miR-145 deficiency diminished TGF-β1–induced α-smooth muscle actin expression. miR-145 expression is also increased in lungs of patients with IPF as compared with healthy control lungs. miR-145 deficiency is protective against bleomycin-induced pulmonary fibrosis (124). Overexpression of miR-145/143 also can inhibit the growth of lung adenocarcinoma cells, likely due to the misexpression of an miRNA that normally does not present in epithelial-derived cells (125–127). miR-145/143 is also abundantly expressed in airway SMCs. However, it is unknown whether airway SMCs are properly differentiated in miR-145/143 null mice or whether miR-145/143 null mice are protected from the development of asthma in response to ovalbumin challenge.

miR-133 in SMC Differentiation

miR-1 and miR-133 play crucial roles in the differentiation and function of skeletal and cardiac muscle. In vivo, miR-1 and miR-133 synergistically specify cardiomyocytes by suppressing myocardin during the development of heart (128–130). In cultured SMCs, miR-1 also suppressed SMC differentiation by targeting myocardin; however, the level of miR-1 in vSMCs in vivo is negligible (131, 132). In contrast, miR-133 is expressed in vSMCs in vivo and is regulated by platelet-derived growth factor–induced activation of MAPK/extracellular signal–regulated kinase 1/2. Overexpression of miR-133 by adenovirus significantly reduced vSMC proliferation, whereas knockdown of miR-133 by anti–miR-133 increased growth (132). miR-133 influences the proliferation and migration of vSMCs by targeting transacting transcription factor-1 and moesin. Furthermore, miR-133 plays a significant role in vSMC proliferation and phenotype switch after balloon injury of carotid arteries in vivo. These results suggest that miR-133 is a key regulator of vSMC phenotype (132). The role of miR-133 in airway SMCs and pulmonary vSMCs remains unknown.

miR-206 in the Innervation of Airway SMCs

miR-206 is important for regeneration of neuromuscular synapses after acute nerve injury of skeletal muscle (133). In mouse developing lungs, brain-derived neurotrophic factor (Bdnf) is required for airway smooth muscle innervation. Interestingly, Bdnf is directly targeted by miR-206, the expression of which is significantly suppressed by Shh activation (Figure 2). This suggested that the Shh pathway increases Bdnf expression post-transcriptionally by suppressing miR-206 (127). This is further supported by increased expression of Bdnf and innervation in embryonic lungs of miR-206 null mice (134). Little is known about the expression and function of miR-206 in human or animal asthmatic airway SMCs. It is also unknown whether miR-206 null mice will respond to ovalbumin challenge differently. These are important and interesting questions for future investigation.

miR-206 in the Proliferation and Differentiation of vSMCs

miR-206 directly targets Notch3, which plays a critical role in the development of the pulmonary vessel walls (Figure 2). Levels of miR-206 are significantly reduced in PASMCs isolated from hypoxia-induced PAH mice, which negatively correlates with increased levels of Notch3. Reduction of miR-206 in human PASMCs causes increased proliferation and reduced apoptosis, which are key histopathological features of PAH. These results suggest that miR-206 is a potential regulator of proliferation, apoptosis, and differentiation of PASMCs (135). Although the role of miR-206 in SMC proliferation has not been examined in early developing lungs, reduced expression of miR-206 by Shh might not only be responsible for the increased innervation of SMCs, but also for the promotion of SMC proliferation. Despite this progress, it is unknown whether miR-206 null mice will develop PAH with or without challenge, such as hypoxia.