Lung cancer is the leading cause of cancer-related death worldwide, with approximately 80–85% of cases being non-small-cell lung cancer (NSCLC). The miRNAs are small non-coding RNAs that regulate gene expression at a post-transcriptional level by either degradation or inhibition of the translation of target genes. Evidence is mounting that miRNAs exert pivotal effects in the development and progression of human malignancies, including NSCLC. A better understanding of the role that miRNAs play in the disease will contribute to the development of new diagnostic biomarkers and individualized therapeutic tools. In the present review, we briefly describe the role of miRNAs in NSCLC as well as the possible future of these discoveries in clinical applications.
Lung cancer remains the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) accounts for approximately 80–85% of cases, with squamous cell carcinoma (SCC) and adenocarcinoma (AD) being the most common histological subtypes . Despite the advances in diagnostic and therapeutic techniques, the overall 5-year survival rate of lung cancer is <18% . The main reason for the poor prognosis is that most patients are diagnosed at an advanced stage due to the lack of specific biomarkers and tools for early detection. Moreover, optimization with better surgery, and chemo- and radio-therapies, has not altered the prognosis much, calling for novel biomarkers that can improve diagnosis and prognostic stratification, and serve further as individualized therapeutic tools.
Identified as new components of epigenetic gene-regulatory systems, miRNAs are evolutionarily conserved small non-coding RNAs that regulate gene expression post-transcriptionally mainly through 3′-untranslated region (3′-UTR) binding of target mRNAs, causing either degradation or inhibition of gene translation , so they are therefore involved in a wide range of fundamental cellular processes such as cell differentiation, proliferation, growth, mobility and apoptosis, as well as tumorigenesis . The identification of miRNA regulators of oncogenes and tumour-suppressor genes could have far-reaching implications for patients with NSCLC, including early detection and molecular classification, prognostic staging, prediction of treatment efficacy and the individualized therapies.
In the present review, we briefly describe the role of miRNAs in NSCLC as well as the possible future of these discoveries in diagnosis, prognosis and therapy of NSCLC. Both original and review articles published in English and fully available were searched for in PubMed (http://www.ncbi.nlm.nih.gov/pubmed/advanced) through use of the search term (miRNA OR microRNA OR miRNA-* OR microRNA-* OR miR-*) AND (non-small-cell lung cancer OR NSCLC), and over 230 articles were found dating from 2008 to 6 November 2014.
FEATURES OF miRNAs
Biogenesis and processing of miRNAs
The miRNAs are endogenous small (19–22 nucleotides) non-coding RNAs that have pivotal functions in various biological processes . The miRNA genes are transcribed by RNA polymerase II (pol II) into primary miRNA transcripts (pri-miRNAs), and then processed into shorter hairpin sequences of approximately 70 nucleotides (pre-miRNAs) by the nuclear microprocessor complex formed by the RNase III Drosha and DiGeorge syndrome critical region gene 8 (DGCR8). The pre-miRNAs are transported by the nuclear export protein Exportin-5 from the nucleus into the cytoplasm, where they undergo a processing step carried out by RNase III Dicer to form a dsRNA. In common cases, one of the strands is selectively bound by Argonaut proteins to enter the RNA-induced silencing complex (RISC) whereas the complementary strand is degraded. The miRNA in the RISC complex recognizes target genes based on sequence complementarity, primarily to the 3′-UTR of the target mRNAs. In brief, perfect or almost perfect base pairing leads to RISC-mediated cleavage and degradation of the target genes, whereas imperfect base pairing mainly induces translational repression and deadenylation of the target genes. In selected cases, miRNAs can bind to other regions of the target mRNAs, such as 5′-UTR , protein-coding sequences  or mRNA-binding protein , guiding translation activation, transcription regulation or decoy activity of the target mRNAs. As a result of the short sequence requirement for the imperfect base pairing in mammalian cells, a single miRNA could interplay with a multitude of target transcripts and alter gene expression across diverse biological processes, including cell differentiation, proliferation, growth, mobility and apoptosis. Accordingly, dysregulated miRNAs will contribute to a variety of pathological events, including cancer initiation and progression.
Mechanisms of miRNA dysregulation in cancer
With more than half the human miRNA genes located at chromosomal breakpoints, fragile sites, regions of loss of heterozygosity (LOH) or amplification, miRNAs are highly susceptible to genomic alterations . A fundamental example is that the frequent deletion of 13q14 from the chromosome in B-cell chronic lymphocytic leukaemias has been found to be the reason for the loss or down-regulation of miR-15 and miR-16, located in this region . Later studies proved that, on chromosome 19p13, a LOH region aligned with the hsa-miR-29a cluster was associated with lung, pancreatic and gynaecological cancers. Also, in lung cancer, a minimal LOH region of 17p13 was closely (1.9 Mb) aligned with the hsa-miR-22 cluster, and a region of homozygous deletion on chromosome 21q11 was close to (2.8 Mb) the hsa-let-7c cluster .
Epigenetic changes such as DNA methylation and histone modifications could lead to silencing of tumour-suppressor genes and contribute to cancer initiation and progression . Lujambio et al.  demonstrated that the DNA methylation of three miRNAs (miR-9, miR-34b/c and miR-148a) was associated with metastasis of human cancers including lung cancer. Other studies implied that miR-9/3, miR-193a and miR-34b/c were also targets of epigenetic silencing by DNA methylation in lung cancer [13,14]. As for histone modifications of miRNAs, Incoronato et al.  showed that miR-212 was silenced by histone H3 trimethylation at Lys27 (H3K27me3) and histone H3 dimethylation at Lys9 (H3K9me2) rather than DNA methylation in lung cancer. Our previous study showed that histone deacetylase (HDAC) 1/4 repression significantly increased miR-200b expression in chemoresistant lung AD cells by up-regulating the histone H3 acetylation level at the two promoters of miR-200b, partially via an Sp1-dependent pathway .
Polymorphisms of miRNAs
Dysregulation of miRNAs in cancer could occur through introduction of a single nucleotide polymorphism (SNP) into the miRNA sequence, affecting its transcription, processing and target recognition . As binding of an miRNA to its target gene is limited only to the ‘seed sequence’, even a change of one nucleotide would result in a different group of genes being regulated, e.g. functional SNPs of miR-499  and miR-196a2  have been found to be associated with cancer susceptibility, including lung cancer. Meanwhile, polymorphisms in the binding sites of miRNAs could also contribute to the susceptibility to lung cancer. A novel SNP (LCS6) in the 3′-UTR of the KRAS gene, which altered the binding affinity of let-7, was found to be associated with lung cancer risk in low-dose smokers by Chin et al. .
Mature miRNA processing starts with the pri-miRNA transcription, which can be regulated by transcription factors (TFs) or genes that are dysregulated in the cancer context, e.g. a series of miRNAs was found to be transcriptionally regulated by the fundamental tumour suppressor p53 and functioning as powerful effectors to control p53-mediated cell cycle arrest and apoptosis . Two other transcription factors, Myc and E2F transcription factor 1 (E2F1), were found to have an effect on the expression of the oncogenic miR-17-92 cluster .
On induction of the transforming growth factor β (TGF-β) pathway, the SMAD proteins can regulate transcription of miRNAs by binding to their promoters along with other proteins such as p68 , in a tissue-specific manner, e.g. TGF-β directly induced the expression of the miR-17-92 cluster in HEK-293 kidney, HepG2 liver and MCF7 breast cancer cell lines , and miR-21 in breast cancer cells , whereas it repressed the expression of miR-200a/b and miR-34 in gastric cancer cells and cervix epithelial AD HeLa cells, respectively [26,27].
Abnormal maturation pathways
The RNase III enzymes Drosha and Dicer could process immature pri-miRNAs into a pre-miRNA of approximately 70 nucleotides and an RNA duplex of approximately 22 nucleotides, respectively. The KH-type splicing regulatory protein (KSRP) was identified as enhancing both Drosha- and Dicer-mediated miRNA processing through interaction with specific sequences in the loop region of a group of pri-miRNAs. Knockdown of KSRP could repress the expression of specific mature miRNAs, such as let-7a and miR-206, and consequently affect cell proliferation and differentiation . A nucleocytoplasmic shuttling heterogeneous nuclear ribonucleoprotein, hnRNPA1, could facilitate the conversion of pri-miR-18a into pre-miR-18a and recognize the highly conserved loop region of miR-18a, resulting in a structural rearrangement of this hairpin to generate a more favourable cleavage site for Drosha [29,30]. Furthermore, additional frameshift mutations were discovered in Exportin-5, which was responsible for transporting pre-miRNAs to the cytoplasm for processing by Dicer .
Interaction with ceRNAs and proteins other than TFs
Competing endogenous RNAs (ceRNAs) are RNA sequences comprising multiple miRNA-binding sites, which act by competitively preventing the action of miRNAs on their mRNA targets . Identified as one of the most significantly up-regulated long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC) , the HULC (highly up-regulated in liver cancer) transcript contained miR-372-binding sites and could reduce miR-372 expression and activity in the HCC cell line Hep3B. Moreover, down-regulated miR-372 and up-regulated HULC expressions were also found in HCC tissue samples compared with the normal ones. In contrast to HULC over-expression in HCC, lncRNA PTCSC3 was found to be dramatically down-regulated in thyroid cancer cells, and PTCSC3 transfection led to a significant decrease in expression levels of the oncogenic miR-574/5p . Recently, it was proved that the forkhead box protein O1 (FOXO1) 3′-UTR could function as a ceRNA in competitive binding with miR-9 in breast cancer cells by producing a similar functional role to the miRNA inhibitor .
It is interesting that, in addition to binding to SMAD-binding elements (SBEs) in DNA, SMAD proteins can also bind to a so-called ‘RNA SBE (R-SBE)’ sequence within the stem region of pri-miRNA transcripts under the regulation of TGF-β signalling at the post-transcriptional level. Binding of these pri-miRNAs by SMADs was demonstrated to stabilize their interaction with Drosha in a p68-dependent manner, facilitating processing to the precursor forms . PUMILIO, an RNA-binding protein (RBP) not involved in miRNA biogenesis but rather in promoting miRNA activity, was reported to induce conformational change in the 3′-UTR of the cyclin-dependent kinase inhibitor p27Kip1 transcript and give miR-221/222 better access to the cognate target site within the p27Kip1 mRNA in MCF-7 breast cancer cell lines . The involvement of PUMILIO was also observed in bladder carcinoma cells, facilitating miR-502 and miR-125b to target E2F3 .
In brief, miRNA dysregulation in cancer can occur at genetic and epigenetic, transcriptional and post-transcriptional levels via miRNA genomic localization, epigenetic changes such as DNA methylation and histone modifications, introduction of miRNA SNP, defects in miRNA biogenesis, including transcriptional regulation, and abnormal maturation pathways, as well as interaction with ceRNAs and proteins other than TFs. The biogenesis pathways and possible mechanisms for dysregulation of miRNAs in cancer are summarized in Figure 1.
Biogenesis pathways of miRNAs and possible mechanisms for dysregulation
THE miRNAs AS ONCOGENES AND TUMOUR-SUPPRESSOR GENES IN NSCLC
Tumour-suppressor miRNAs in NSCLC
The first miRNA identified in humans was let-7 and it has been shown to inhibit the expression of oncogenes involved in cellular growth and proliferation, such as rat sarcoma (RAS), myelocytomatosis oncogene (Myc), the high mobility group AT-hook 2 (HMGA2), cyclin D2, cyclin-dependent kinase 6 (CDK6) and cell division cycle 25A (CDC25A) , resulting in suppression of cancer cell proliferation and cell cycle distribution. Intriguingly, let-7 can directly down-regulate Dicer expression , raising the possibility that it regulates the global production of miRNAs with profound effects for virtually every aspect of cell growth, proliferation and survival. In NSCLC, let-7 was proved to be a typical tumour inhibitor in both cell and animal studies [39,41]. Moreover, with its expression frequently down-regulated in tumour tissues compared with the corresponding normal tissues, let-7 showed a negative impact on the survival of patients with NSCLC who have undergone surgical treatment .
The transcription of the miR-34 family of genes, miR-34a and miR-34b/c, is directly induced by p53 in response to DNA damage, controlling cell cycle arrest and apoptosis in cancer cells . In fact, miR-34a was identified as one of the down-regulated miRNAs in NSCLC and that it worked as a tumour suppressor by inhibiting cancer cell proliferation in a p53-independent way . Recently, Wang et al.  proved that miR-34a could inhibit the formation of the NSCLC cell holoclone and clonogenic capacity, suggesting an anti-cancer stem cell (CSC) function of the miR-34 family. Expression of miR-34b/c was also significantly reduced in both NSCLC cell lines and tissue samples, and the restoration inhibited growth of NSCLC cells .
The miR-449 family (miR-449a, -449b and -449c) shares identical seed sequence and secondary structures with miR-34 and is thus assigned as part of the miR-34 family. In agreement with a putative tumour-suppressor role, miR-449a/b could repress E2F1 and promote p53 activity, strongly reducing proliferation and promoting apoptosis of cancer cells including lung cancer . A later study suggested that miR-449a/b may exert a tumour-suppressor function in lung cancer cells by directly targeting and down-regulating HDACs, which play a crucial roles in tumorigenesis .
Both miR-15a and miR-16/1 were found to be frequently down-regulated in NSCLC cells. Gene targets of this miRNA cluster included the cyclins D1, D2 and E1. Over-expression of the miR-15/16 cluster could induce G1 arrest in a retinoblastoma (RB) protein-dependent manner, and this induction was more profound when combined with miR-34a than the additive effect of treating cells with the two miRNAs separately [48,49], implying co-operation between the two tumour-suppressor miRNA clusters implicated in cell cycle control and the tumorigenesis of NSCLC.
The miR-200 family (miR-200a, miR-200b, miR-429, miR-200c and miR-141) together with miR-205 have been shown to be major determinants in epithelial–mesenchymal transition (EMT) by targeting zinc finger E–box-binding protein (ZEB) transcription factors, subsequently controlling the cluster of gene expression including E-cadherin and vimentin . In lung cancer cells, miR-200c over-expression led to reduced expression of ZEB1 and increased expression of E-cadherin . In vivo studies confirmed that ectopic expression of miR-200c could inhibit mesenchymal phenotypes of cancer cells and formation of metastases ; miR-200c has also been shown to repress Suz12 and Bmi1, two essential components of polycomb repressor complexes (PRCs) which are responsible for the maintenance of embryonic and adult stem cells . In addition, miR-200c has recently been proved to be a potent inhibitor of tumour progression and therapy resistance, by regulating a multitude of oncogenic signals including the RAS pathway . According to our miRNA microarray data, miR-200b was the most down-regulated miRNA in docetaxel-resistant human lung AD SPC-A1/DTX cells, compared with parental SPC-A1 cells . Ectopic miR-200b expression reversed docetaxel chemoresistance of lung AD cells through proliferative suppression, apoptosis enhancement and G2/M arrest . Further study suggested that HDAC1/4 negatively regulated the expression of miR-200b via an Sp1-dependent pathway, causing the up-regulation of E2F3, survivin and Aurora-A, and down-regulation of cleaved caspase-3 . Choi et al.  demonstrated that miR-200b could also negatively regulate vascular endothelial growth factor (VEGF) signalling by targeting VEGF and its receptors such as Fms-related tyrosine kinase 1 (Flt-1) and kinase insert domain-containing receptor (KDR).
Based on our previous study, miR-451 was also identified as a tumour suppressor in NSCLC. We analysed the miRNA expression profiles in NSCLC and the corresponding non-cancerous lung tissues and found that miR-451 was the most down-regulated miRNA in NSCLC. Its expression level correlated significantly with tumour differentiation, pathological stage and lymph-node metastasis. Over-expression of miR-451 in NSCLC cells led to decreased phosphorylation of Akt and increased Bax or Bad protein levels, which finally induced activation of caspase-3 and enhancement of apoptosis. Bioinformational and functional analysis indicated that RAS-related protein 14 (RAB14) was one of the target genes of miR-451. Down-regulation of RAB14 might be a mechanism by which miR-451 exerted its tumour-suppressor functions in NSCLC .
It has been found that members of the miR-29 family (miR-29a, miR-29b and miR-29c) are highly expressed in normal tissues and down-regulated in different types of cancers including lung cancer . These miRNAs could target DNA methyltransferases (DNMT3A and DNMT3B) and thereby restore patterns of DNA methylation and expression of silenced tumour-suppressor genes in lung cancer, such as the fragile histidine triad (FHIT) and the WW domain containing oxidoreductase (WWOX), leading to inhibited tumorigenicity both in vitro and in vivo . Other important targets that mediate miR-29 function as a tumour-suppressor include tristetraprolin (TTP)  and the Bcl-2 family member Mcl-1 .
Both miR-126 and its complementary species miR-126* were found to be down-regulated in NSCLC . By targeting VEGF-A, miR-126 decreased cancer cell growth, induced cell cycle arrest at the G1 phase, and impaired tumorigenicity both in vitro and in vivo . Of interest, miR-126 could also inhibit proliferation of NSCLC cells via negative regulation of its host gene epidermal growth factor-like domain 7 (EGFL7), suggesting a novel feedback minicircuitry comprising EGFL7 and miR-126 .
Other miRNAs that suppress NSCLC tumorigenesis and progression have been reported, e.g. miR-101 was found to be down-regulated in human lung cancers. Introduction of miR-101 into NSCLC cells led to reduced cell proliferation and invasion, and sensitized cells to paclitaxel-mediated apoptosis by inducing expression of the pro-apoptotic protein Bim  and inhibition of the anti-apoptotic protein Mcl-1 . Sun et al.  showed that ectopic expression of miR-182 significantly inhibited lung cancer cell proliferation via inhibition of the regulator of G-protein signalling-17 (RGS17), which was responsible for induced tumour cell growth through induction of phosphorylation of the cAMP-response-element-binding protein (CREB). Feng et al.  demonstrated that miR-192 was a tumour suppressor by targeting the RB1 gene, thereby inhibiting cell proliferation and inducing cell apoptosis in lung cancer cells. Incoronato et al.  identified miR-212 as a tumour suppressor by negatively regulating the protein expression of the anti-apoptotic phosphoprotein enriched in diabetes (PED) and increasing tumour necrosis factor (TNF)-related apoptosis in NSCLC cells. It was reported that miR-218 had a negative impact on NSCLC cell proliferation, invasion and colony formation by repressing paxillin (PXN), the over-expression of which may promote NSCLC progression and metastasis . By targeting CDK6, miR-129 repressed cell cycle progression and cell growth of NSCLC cells . A recent study revealed that miR-545, which was under-expressed in lung cancer, may function as a tumour suppressor by directly targeting cyclin D1 and CDK4 genes and suppressing cancer cell proliferation .
In summary, numerous tumour-suppressor miRNAs have been reported and validated in NSCLC in both in vitro and in vivo studies, functioning by either direct or indirect regulation of the key genes or proteins involved in cellular processes, such as cell proliferation, apoptosis, angiogenesis, EMT and CSC maintenance. Selected tumour-suppressor miRNAs and their targets in NSCLC are summarized in Figure 2.
Selected tumour-suppressor and oncogenic miRNAs and their targets in NSCLC
Oncogenic miRNAs in NSCLC
Apart from their roles as tumour suppressors, some miRNAs function as tumour promoters by targeting tumour-suppressor genes, e.g. the miR-17-92 cluster (miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a and miR-92a-1) has been proved to have important regulatory effects on cell proliferation in a variety of human cancers, including lung cancer . Members of this cluster could directly target the tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which participated in the cell-survival signalling pathway phosphoinositide 3-kinase (PI3K)/Akt and was usually eliminated or mutated in lung cancer . Hypoxia-inducible factor 1α (HIF-1α), which could transactivate genes involved in various biological processes including angiogenesis, apoptosis, pH regulation, glucose metabolism, extracellular matrix metabolism, cell proliferation, invasion and metastasis, was also reported as a direct target of miR-17-92 . Moreover, over-expression of miR-17-92 counterbalanced formation of γ-HAX2 foci and reactive oxygen species induced by knocking down the RB gene in RB wild-type lung cancer cells, suggesting a role for miR-17-92 in fine-tuning the DNA damage level in cancer cells to maintain genomic instability .
Both miR-221 and miR-222 share an identical seed sequence and have predicted overlapping targets. Garofalo et al.  showed that over-expression of miR-221/222, by targeting the PTEN and tissue inhibitor of metalloprotease-3 (TIMP-3) tumour suppressors, induced resistance of the TNF-α-related apoptosis-inducing ligand (TRAIL) and enhanced cellular migration via activation of the Akt pathway and metallopeptidases in aggressive NSCLC cells. They also demonstrated that miR-221/222, as well as miR-30b and miR-30c, had important roles in gefitinib-induced apoptosis and EMT of NSCLC cells both in vitro and in vivo by inhibiting the expression of the genes encoding Bim, apoptotic peptidase activating factor 1 (APAF-1), protein kinase C ε (PKC-ε) and the sarcoma viral oncogene homologue (SRC) . Zhang et al.  demonstrated that the p53-up-regulated modulator of apoptosis (PUMA) was another direct target of miR-221/222. Reduction of miR-221/222 inhibited cell proliferation and induced mitochondrially mediated apoptosis in both NSCLC and breast cancer cells.
Another well-studied oncogenic miRNA across human solid tumours, including lung cancers, is miR-21 . Its expression was proved to be correlated with phosphorylated epidermal growth factor receptor (pEGFR) and could be suppressed by EGFR tyrosine kinase inhibitors (EGFR-TKIs). Restrained miR-21 expression enhanced EGFR-TKI-induced apoptosis as well as growth inhibition of NSCLC cells . The tumour suppressor PTEN gene was identified as one of the direct targets of miR-21. In NSCLC, miR-21 post-transcriptionally down-regulated the expression of PTEN and stimulated cancer cell growth and invasion . In a mouse model-based study, Hatley et al.  showed that miR-21 drove NSCLC tumorigenesis through inhibition of negative regulators of the RAS/MEK [mitogen-activated protein kinase (MAPK)/ERK kinase]/ERK (ERK, extracellular-signal-regulated kinase) pathway, including sprouty 1 (SPRY1), sprouty 2 (SPRY2), B-cell translocation gene 2 (BTG2), and programmed cell death 4 (PDCD4), but also pro-apoptotic genes such as APAF-1, Fas ligand (FASLG) and ras homologue gene family member B (RHOB).
Studies on other oncogenic miRNAs have also extended the understanding of NSCLC tumorigenesis and progression, e.g. miR-31 was found to act as a tumour promoter in lung cancer by inhibiting the expression of the tumour-suppressor genes large tumour suppressor 2 (LATS2) and protein phosphatase 2A (PP2A) regulatory subunit-Ba isoform (PPP2R2A), in lung cancer cells, favouring cell growth and apoptosis inhibition . In human bronchial epithelial cells, miR-494 could function as an oncogene in carcinogenesis induced by the chemical carcinogen anti-benzo[α]pyrene-diol-epoxide via regulation of the post-transcriptional expression of PTEN . Another feedback circuit model was provided by Cao et al.  in which miR-301 could positively regulate expression of its host gene SKA2 through the ERK/CREB pathway in NSCLC cells, whereas inhibition of miR-301 or SKA2 facilitated cell proliferation and invasion.
Affecting multiple targets with a single hit is the most distinctive feature of miRNAs and, when the targets tend to be tumour-suppressor genes, miRNAs may also have an oncogenic role facilitating malignant behaviours of cancer cells via unlimited proliferation, blocked cell cycles, escape from apoptosis, accelerated invasion and resistance to growth inhibitory signals. Selected oncogenic miRNAs and their targets in NSCLC are summarized in Figure 2.
Oncogenic and tumour-suppressor functions of miRNAs in NSCLC
As in protein-coding genes, individual miRNAs may exhibit dual functions of an oncogene and tumour-suppressor gene in a context-dependent manner, e.g. miR-7 was proved to be a putative tumour suppressor in a large range of solid tumours, and was often down-regulated in NSCLC , suppressing tumorigenesis by targeting a number of important oncogenes such as EGFR, insulin receptor substrate 1 (IRS1), IRS2, v-raf-1 murine leukaemia viral oncogene homologue 1 (RAF1), p21/CDC42/RAC1-activated kinase 1 (PAK1) and BCL-2 [88–90]. However, Chou et al.  reported miR-7 as an important modulator of EGFR-mediated oncogenesis in lung cancers. EGFR activation, which occurs commonly in lung cancers with poor prognosis, could induce miR-7 expression through a RAS/ERK/Myc pathway, and consequently promoted proliferation and tumorigenicity of lung cancer cells by targeting the Ets2 transcriptional repressor (ERF).
The miR-183 family members (miR-183, miR-182 and miR-96) could repress zinc transporters that regulate zinc homoeostasis, and have been shown to have both oncogenic and tumour-suppressor functions in cancers . In lung cancers, miR-182 and miR-183 were demonstrated to inhibit lung tumour invasion and metastasis by targeting CTTN  and EZRIN , respectively. Moreover, miR-182 inhibited lung cancer cell proliferation and anchorage-independent growth by suppression of RGS17 . On the other hand, over-expression of the miR-183 family was reported as a risk factor in lung cancers by Zhu et al. , indicating an oncogenic role in tumorigenesis–probably in aspects other than invasion and metastasis, such as cell death control or angiogenesis.
Consideration of the time- and space-specific expression of miRNAs in the context of tumour heterogeneity indicates that it is not surprising that the expression profile and main function of miRNAs may differ profoundly across different histological subtypes of cancer or even different phases of the same subtype. In addition, the intertwined network of miRNAs, oncogenes and tumour-suppressor genes not only implicates ubiquitous involvement of miRNAs in almost all aspects of NSCLC pathogenesis, but also raises the possibility of developing novel methods for NSCLC interventions, including diagnosis, prognosis and individualized treatment using miRNAs.
CLINICAL APPLICATIONS OF miRNAs IN NSCLC
The miRNAs as diagnostic biomarkers of NSCLC
As mentioned above, miRNA expression is time- and space-specific, with the expression signature probably reflecting the developmental lineage and differentiation state of tumours and identifying poorly differentiated tumours successfully; this could suggest the potential value of miRNAs for early detection and screening of NSCLC , e.g. Yanaihara et al.  reported that 12 miRNAs (miR-17/3p, miR-21, miR-106a, miR-146, miR-155, miR-191, miR-192, miR-203, miR-205, miR-210, miR-212 and miR-214) could be used to discriminate NSCLC from non-cancerous lung tissues. Raponi et al.  showed that expression levels of 13 miRNAs (miR-210, miR-200c, miR-17/5p, miR-20a, miR-203, miR-200a, miR-106b, miR-93, miR-182, miR-83, miR-106a, miR-20b and miR-224) were higher, whereas expression levels of let-7e and miR-200a were lower in SCC compared with normal tissues.
Circulating miRNAs are frequently packaged in exosomes or microvesicles, or bound to specific proteins and resistant to RNase A digestion , and highly stable in body fluids such as plasma, serum, sputum, saliva, urine and milk [99–104]. The discovery of circulating miRNAs has opened a new avenue for NSCLC diagnosis because the tests are minimally invasive, have relatively low cost and can be repeated. Chen et al.  reported higher levels of miR-23 and miR-225 in the circulating exosomes of patients with NSCLC compared with healthy donors. Heegaard et al.  found that the expression of miR-146b, miR-221, let-7a, miR-155, miR-17/5p, miR-27a and miR-106a was significantly reduced, whereas that of miR-29c was increased in the serum of patients with NSCLC. Altered expression of the miRNAs in plasma, such as high expression of miR-29c, miR-155, miR-197, miR-182, miR-21 and miR-210 and low expression of miR-486/5p and miR-429, was detected in lung cancer patients compared with controls, providing potential blood-based biomarkers for NSCLC [99,107,108]. Sputum miRNAs can also serve as biomarkers for lung cancer detection. The combined over-expression of miR-21, miR-200b, miR-375 and miR-486 in both surgical tissues and sputum were reported as biomarkers in the prediction of lung AD . Further studies indicated that increased miR-21 expression produced 69.7% sensitivity and 100.0% specificity in the diagnosis of lung cancer by sputum cytology .
Certain miRNAs have been reported as helping in the molecular classification of NSCLC, e.g. six miRNAs (miR-205, miR-99b, miR-203, miR-202, miR-102 and pre-miR-204) were reported as exhibiting different expression profiles between lung AD and SCC . Xie et al.  found that over-expression of miR-21, miR-200b and miR-375, combined with a reduced level of miR-486, segregated patients with AD from those without, whereas down-regulation of miR-205, miR-210 and miR-708 could help to discriminate patients with SCC . Tan et al.  also reported a five-microRNA classifier (miR-210, miR-182, miR-486/5p, miR-30a and miR-140/3p) that could distinguish SCC from normal lung tissue. In a recent study, paired miRNA and mRNA profiling was performed in 44 NSCLC tissue samples. Of AD and SCC samples, six differentially expressed miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and eight differentially expressed target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated .
miRNAs can also function as biomarkers for histological differentiation of NSCLC from other lung tumour phenotypes. Du et al.  applied microarray technologies in a panel of six NSCLC and nine SCLC cell lines, and found that, among the overall 136 miRNAs, 19 were over-expressed in SCLC cell lines relative to NSCLCs whereas ten were under-expressed, suggesting the possibility of developing miRNA profiling as a diagnostic tool for differentiating NSCLCs from SCLCs. Gilad et al.  developed an assay that was effective in the histological typing of pathological and cytological lung cancer samples based on the expression of only a small set of miRNAs which differentiated the four main types of lung cancer: carcinoid, SCLC, SCC and AD. In brief, the miRNAs that were expressed at higher levels in NSCLC versus SCLC were miR-21 and miR-29b, and up-regulation of miR-375 and miR-205 could be used to differentiate SCC from AD.
Taken together, profiling studies of miRNA expression have led to improvement in the diagnostic accuracy of lung cancer screening and histological differentiation across subtypes in a non-invasive way. Nevertheless, the lack of reproducibility is quite common across existing studies due to methodological and quality control differences, as well as the limited number of miRNAs and samples studied, calling for better research strategies and more detailed work in the near future.
The miRNAs as prognostic biomarkers of NSCLC
A low level of let-7 expression was initially found to have significant association with an unfavourable prognosis in surgically resected NSCLC patients , suggesting a prognostic value for miRNAs in lung cancer. Soon after this, high expression of miR-155 and low expression of let-7a-2 were reported to correlate with poor survival in patients with lung AD . According to our studies, decreased miR-200b expression was detected in tumour tissues sampled from patients with lung AD who were treated with docetaxel-based chemotherapies; these proved to correlate with high expression of E2F3, decreased sensitivity to docetaxel and poor prognosis . Down-regulation of miR-451 in tumour tissues was also demonstrated to correlate with shorter overall survival of patients with NSCLC . Other miRNAs in which down-regulation demonstrates the predictive value for poor survival or high recurrence of NSCLC include miR-218 , miR-221 , miR-34a , miR-374a  and miR-181a . On the other hand, over-expression of miR-21 has been detected in a variety of human cancers, including NSCLC, and regarded as an independent negative prognostic factor for patient survival . A recent study suggested that patients with a high level of miR-19b and a low level of miR-146a in their serum achieved a lower overall response rate and shorter survival time . Our results showed that the expression level of miR-650 was significantly higher in lung AD than the corresponding non-tumour tissues and over-expression of miR-650 closely correlated with a high incidence of lymph-node metastasis, advanced clinical stage and poor prognosis of patients with lung AD . Other up-regulated miRNAs with a predictive value for poor survival of NSCLC include the miR-183 family (miR-182, miR-183 and miR-96) , miR-146b , miR-429 , miR-31 , miR-126 , miR-16  and miR-93 .
Tumour metastasis is one of the most important prognostic factors for advanced NSCLC, often resulting in treatment failure. Accumulating evidence suggests that miRNAs could serve as novel biomarkers for NSCLC invasion and metastasis, helping to distinguish high- and low-risk patients, and to select the most appropriate treatment strategies for them, e.g. Wang et al.  reported that miR-125a-5p expression negatively correlated with migration and invasion of lung cancer cells by regulating the expression of several downstream genes of EGFR signalling. Zheng et al.  found that the levels of miR-155 and miR-197 were higher in plasma from lung cancer patients with metastasis than from those without metastasis, and were significantly decreased in responsive patients during chemotherapy. By using blood samples collected from the different stages of NSCLC in patients, Lin et al.  demonstrated a statistical difference for miR-126 and miR-183 expression between stage I/II and stage IV patients, indicating that the serum levels of the two miRNAs could be identified as an indicator for metastatic NSCLC.
The studies described above clearly suggest that miRNA-based techniques are being actively explored as prognostic tools in NSCLC. However, it is still very necessary to carry out additional validation in large patient populations to avoid potential biases and limitations.
The miRNAs as new approaches and targets for NSCLC therapy
The therapeutic application of miRNAs involves mainly two strategies: one is to reintroduce a tumour-suppressor miRNA to restore the loss of function; the second is to restrain oncogenic miRNAs directly or indirectly. In actual practice, strategies include, but are not limited to, lipid-formulated mimics, DNA constructs coding for specific miRNAs, miRNAs packaged in viral carriers, targeted delivery by nanoparticles, antagomirs, locked nucleic acids (LNAs), miRNA sponges and small molecules to reduce miRNA expression .
Synthetic miRNA mimics and plasmids expressing miRNAs, modelled after endogenous tumour-suppressor miRNAs, could reactivate cellular pathways which drive a therapeutic response and provide a new opportunity to treat NSCLC as monotherapies. Wiggins et al.  reported that lipid-based delivery of a chemically synthesized miR-34a blocked NSCLC growth and down-regulated the anti-apoptosis protein survivin expression, without induction of immune responses in mouse models. Reintroduction of let-7 by adenovirus vectors significantly reduced NSCLC growth both in vitro and in vivo . In our previous study, mice xenografts derived from miR-200b-transfected docetaxel-resistant lung AD SPC-A1 (SPC-A1/DTX) cells grew substantially more slowly compared with the control group under the pressure of docetaxel, indicating that ectopic miR-200b expression in SPC-A1/DTX cells could significantly enhance the in vivo response to docetaxel . Immunostaining analysis showed that the positive rate of proliferating cell nuclear antigen (PCNA) of tumours from miR-200b-transfected SPC-A1/DTX cells was decreased compared with the control, indicating a down-regulation of the in vivo cell-proliferating ability attributed to miR-200b .
To achieve therapeutic effect by targeting oncogenic miRNAs, miRNA inhibitors such as antagomirs, LNAs and miRNA sponges have been developed, e.g. in miR-21 inhibitor-transfected NSCLC cells, the level of tumour suppressor PTEN protein was increased and tumour cell growth and invasive characteristics were largely reduced . An anti-miR-150 expression vector was reported to inhibit NSCLC cell proliferation both in vitro and in nude mice xenografts . Obad et al.  developed a method that enabled antagonism of miRNA function using seed-targeting octameric LNA oligonucleotides. Transfection of these ‘tiny LNAs’ into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed, with concomitant up-regulation of direct targets.
The miRNAs for overcoming treatment resistance of NSCLC
Except for the direct anti-cancer effects, another important clinical application for miRNAs is to break the resistance mechanisms by negative regulation of the target genes involved in drug transport, drug metabolism and repair of DNA damage, and alterations of apoptosis and cell cycle progress, making current treatment regimens more effective.
Cisplatin is one of the classic chemotherapy agents used in NSCLC. Wang et al.  reported that up-regulation of miR-138 gave rise to increased cell apoptosis and chemosensitivity of cisplatin-resistant A549 cells through down-regulation of the excision repair cross-complementation group 1 (ERCC1). Zhang et al.  found that transfection of cisplatin-resistant A549 cells with miR-513a-3p resulted in resensitization to cisplatin by targeting the GST P1 (GSTP1), which promoted cisplatin resistance. In a recent study, Yin et al.  demonstrated that over-expression of miR-101 sensitized A549 cells to cisplatin via the activation of caspase-3-dependent apoptosis. Galluzzi et al.  demonstrated that miR-181a enhanced cisplatin-triggered cell death by stimulating Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3, whereas miR-630 diminished the sensitivity of A549 cells to cisplatin by inducing cell arrest in the G0/G1 phase and increasing the cell cycle inhibitor p27(Kip1). Another study  indicated that miR-192 conferred resistance to cisplatin and inhibited apoptosis of A549 cells via negative targeting of the pro-apoptotic protein Bim. Wei et al.  found that plasma miR-21 levels in platinum-responding patients were lower than in patients with stable or progressive disease. In accordance with this result, Gao et al.  reported that miR-21 expression significantly increased the resistance of A549 cells to platinum, whereas reduced miR-21 decreased the resistance of A549 cells to cisplatin. In validation, the tissue samples of patients with NSCLC showed further that increased miR-21 expression was associated with a shorter disease-free survival. A couple of years ago, a prospective study revealed a two-miRNA signature (miR-149 and miR-375) that could be helpful in predicting the response to cisplatin and vinorelbine, as well as a prognostic four-miRNA signature (miR-200c, miR-424, miR-29c and miR-124) in patients with advanced NSCLC .
Paclitaxel and its semi-synthetic analogue, docetaxel, are first-line chemotherapy regimens for aggressive NSCLC, with genotoxic effects attributed to microtubule-stabilizing apoptotic induction via microtubule bundling and Bcl-2 blocking [139,140]. A subset of miRNAs has been shown to be differentially expressed in docetaxel-resistant NSCLC, showing up-regulation of miR-98, miR-192 and miR-424, and down-regulation of miR-194, miR-200b and miR-212 . Xie et al.  compared the miRNA expression levels between benign and malignant effusion samples from patients with lung cancer and found that cells isolated from effusions rich in cell-free miR-152 were more sensitive to docetaxel. Chatterjee et al.  found that over-expression of miR-17/5p sensitized paclitaxel-resistant lung cancer cells to paclitaxel-induced apoptotic cell death by directly targeting the beclin 1 (BECN1) gene, one of the most important autophagy modulators. Our previous work demonstrated that down-regulation of miR-200b and miR-100 resulted in E2F3 and Plk-1 over-expression, respectively, and in turn contributed to the chemoresistance of lung AD cells to docetaxel. Restoration of miR-200b and miR-100 led to resensitization of NSCLC cells to docetaxel both in vitro and in vivo, along with suppression of their cell-proliferating ability, up-regulation of the apoptosis rate, as well as an increased percentage of cells in the G2/M phase and a decreased population in the S phase [56,144]. Our experimental data also indicated that decreased let-7c was critical for chemo- and radio-resistance as well as the chemotherapy-induced EMT phenotype of lung AD cells. Restoration of let-7c reversed chemo- and radio-resistance as well as the mesenchymal features of docetaxel-resistant lung AD cells both in vitro and in vivo via direct Bcl-xL targeting and inactivation of Akt phosphorylation . A recent study demonstrated that up-regulation of miR-7 enhanced the paclitaxel sensitivity of NSCLC cells by suppressing cell proliferation and promoting cell apoptosis, whereas the inhibition of miR-7 abrogated the anti-proliferative pro-apoptotic effects of paclitaxel, and re-treatment of miR-7 mimics enhanced the paclitaxel-mediated down-regulation of EGFR in NSCLC cells, providing evidence of the potential utility of miR-7 as a sensitizer in paclitaxel therapy for NSCLC .
The EGFR-TKIs erlotinib and gefitinib are standard treatments for NSCLC and have striking effects in patients harbouring activating EGFR mutations. However, the secondary mutations of EGFR and Met amplification are the two most important factors underlying EGFR-TKI resistance in NSCLC [147,148]. Weiss et al.  reported that miR-128b could directly regulate EGFR. The LOH of miR-128b occurred frequently in NSCLC and correlated significantly with clinical response and survival after gefitinib. Webster et al.  found that miR-7 could not only down-regulate EGFR expression and induce cell cycle arrest and cell death of NSCLC cells, but also attenuate activation of protein kinase B (Akt) and ERK1/2, two critical effectors of EGFR signalling. Dacic et al.  investigated the miRNA expression in lung ADs with different oncogenic mutations and demonstrated that miR-155, miR-25 and miR-495 were up-regulated only in the EGFR/KRAS-negative, EGFR-positive and KRAS-positive lung ADs, respectively.
PTEN loss was proved to facilitate EGFR-TKI resistance by partially uncoupling mutant EGFR from downstream signalling and activating EGFR . Both miRNA-21 and miR-221/222 participated in the gefitinib resistance of NSCLC cells via direct PTEN regulation, suggesting the therapeutic value of these miRNAs as EGFR-TKI sensitizers [77,78]. Met protein expression and phosphorylation were also closely associated with both primary and acquired resistance to EGFR-TKI therapy in NSCLC [148,152]. Through use of the resistant clones of the gefitinib-hypersensitive, EGFR exon 19, mutant NSCLC cell lines, Garofalo et al.  demonstrated that this resistance could be overcome by Met inhibitors or anti-miR-221/222 and anti-miR-30c, which recovered the expression of the pro-apoptotic protein Bim and increased the gefitinib sensitivity of NSCLC both in vitro and in vivo. Zhou et al.  showed that miR-130a was down-regulated in gefitinib-resistant NSCLC cells. Ectopic expression of miR-130a contributed to gefitinib resensitization by targeting the Met 3′-UTR and down-regulating Met protein levels.
EMT has been shown to have a role in acquired resistance to both gefitinib and erlotinib in NSCLC cells, indicating that a mesenchymal phenotype is associated with the ‘inherent resistance’ to EGFR-TKI in NSCLC [154,155]. Bryant et al.  reported that expression of 13 miRNA genes could predict response to EGFR inhibition in NSCLC cell lines and tissues. In particular, ectopic expression of miR-200c down-regulated expression of EMT proteins and deceased resistance to erlotinib as well as the metastatic behaviour of lung cancer cells. It has recently been found that miR-147 could induce NSCLC cells to undergo mesenchymal–epithelial transition and induce cell cycle arrest. In addition miR-147 strikingly recovered sensitivity to gefitinib in NSCLC cells by inhibition of Akt and EMP-1 .
Notably, in a recent study, Zhao et al.  used a panel of NSCLC cell lines with primary or acquired erlotinib resistance and found that miR-34a and erlotinib co-operated synergistically at dose levels that induce maximal cancer cell inhibition, suggesting that most cases of NSCLC previously not suited to erlotinib may be sensitized to the drug when used in combination with miR-34a-based therapy.
Advances in research have brought recognition that miRNAs also have an important role in cancer therapy by modulating the response to radiotherapy. The mechanism of miR-34 up-regulation was first shown to be due to altered transcription from a well-known radiation response gene, p53 . Restoration of miR-34a expression enhanced radiation-induced apoptosis partly by suppressing the LyGDI signalling pathway, suggesting miR-34a as a radio-sensitizer for NSCLC therapy . In addition, EGFR can also function in radiation resistance in tumour cells with multiple downstream pathways, such as RAS/RAF/MEK/MAPK, PI3K/Akt and signal transducer and activator of transcription (STAT), activated [91,160], e.g. Lee et al.  demonstrated that miR-7 may be a useful therapeutic target for overcoming the radio-resistance of human cancers including NSCLC by directly suppressing the activation of EGFR/PI3K/Akt. Weidhaas et al.  found that over-expression of let-7a and let-7b conferred radio-sensitization whereas over-expression of let-7g caused radio-protection in A549 cells, attributed to the alteration in target genes such as components of the DNA damage-response pathway and RAS. It is interesting that, in A594 cells harbouring activated KRAS signalling, either ectopic over-expression of let-7a or inhibition of Lin28, a post-transcriptional repressor of let-7, could decrease expression of KRAS and radio-sensitize A549 cells . Salim et al.  demonstrated that miR-214 was up-regulated in radio-resistant NSCLC cells. Knockdown of miR-214 radio-sensitized the cells by stimulation of senescence, whereas its over-expression protected radio-sensitive NSCLCs against radiotherapy-induced apoptosis, at least partially mediated by p38 MAPK.
Altogether, these studies suggest that knowledge of specific miRNA signatures has the potential to provide information about resistance to chemo-, radio- and targeted therapies, paving the way for individualized clinical treatment. Although promising, the study of the association between miRNAs and treatment resistance is still in its cradle. Larger sample size and validated methodologies are indispensable before translating these fundamental research advances into medical practice.
CONCLUSIONS AND FUTURE DIRECTIONS
Over the past decade, emerging evidence has suggested that miRNAs could act as potent oncogenes and tumour suppressors by modulating the expression of key regulators of cellular proliferation, growth, survival and signalling in a wide variety of cancers including NSCLC. A more detailed regulatory role of miRNAs in tumorigenesis and progression may allow accurate definition of the clinical application of specific miRNAs in diagnosis, prognosis and treatment of NSCLC.
Currently, the main challenges to the use of miRNAs as biomarkers are technical, including the limited number of samples compared with the number of analyses, variations in control usage and normalization, and lack of consistency between laborato-ries and across platforms, e.g. the association of miRNAs with cancer chemosensitivity was first raised by Meng et al.  in 2006, suggesting over-expressed miR-200b as a spoiler in gemcitabine-induced apoptosis of human malignant cholangiocytes. However, according to our experimental results and those of others, miR-200b seems to be a tumour suppressor and chemosensitivity restorer in cancers including NSCLC, mainly via signalling pathways of EMT, CSC maintenance, angiogenesis, apoptosis and cell cycle distribution [56,57,166,167]. Another interesting research field is the use of miRNAs as therapeutic agents. In spite of the unique advantages of miRNAs, such as a mild and multi-targeted regulation of genes in key signalling pathways, a stable, specific and non-toxic delivery system capable of transporting these agents selectively to the tumour site remains the major hurdle in the translation of miRNA-based gene therapy to the clinic.
To accelerate the process of moving miRNA research from the bench to the bedside, strict quality control, better reagents, and standard guidelines for sample preparation and data analysis are required. Additional validation in large patient populations is still necessary. For the future, miRNAs from body fluids, especially blood plasma, might be the centre of attention. As discussed above, miRNA expression levels in plasma may be used as a non-invasive confirmatory screening test, complementary to low-dose helical computed tomography (CT). Moreover, particular attention should be paid to the design of prospective trials in large well-defined patient groups. To date, a number of clinical trials using miRNA profiling for patient prognosis and clinical response are under way (http://clinicaltrials.gov). The first miRNA-based drug against hepatitis C virus infection–LNA-anti-miR-122 (Miravisen, Santaris Pharma)–is successfully undergoing phase II trials (http://www.santaris.com) [168,169]. The first miRNA-based therapy specifically for cancer–MRX34–is now in a phase I trial for primary liver cancer and liver metastases .
In conclusion, the in-depth exploration of miRNAs in NSCLC has extended our understanding of carcinogenesis and provided potential healthcare professionals with promising cancer biomarkers as well as target agents, bearing the hope of developing individualized medicine and improving prognosis in the battle against NSCLC.
apoptotic peptidase-activating factor 1
cell division cycle
competing endogenous RNA
cancer stem cell
epidermal growth factor-like
epidermal growth factor receptor
heterogeneous nuclear ribonucleoprotein
highly up-regulated in liver cancer
KH-type splicing regulatory protein
locked nucleic acid
long non-coding RNA
loss of heterozygosity
mitogen-activated protein kinase
non-small-cell lung cancer
phosphatase and tensin homologue deleted on chromosome 10
regulator of G-protein signalling
RNA-induced silencing complex
squamous cell carcinoma
single nucleotide polymorphism
transforming growth factor β
tyrosine kinase inhibitor
tumour necrosis factor
vascular endothelial growth factor
zinc finger E–box-binding protein
This work was funded by National Natural Science Foundation of China (81301913) and Natural Science Foundation of Jiangsu Province of China (BK20130698).