MicroRNA in Lung Cancer Metastasis

Tumor metastasis is a hallmark of cancer, with distant metastasis frequently developing in lung cancer, even at initial diagnosis, resulting in poor prognosis and high mortality. However, available biomarkers cannot reliably predict cancer spreading sites. The metastatic cascade involves highly complicated processes including invasion, migration, angiogenesis, and epithelial-to-mesenchymal transition that are tightly controlled by various genetic expression modalities along with interaction between cancer cells and the extracellular matrix. In particular, microRNAs (miRNAs), a group of small non-coding RNAs, can influence the transcriptional and post-transcriptional processes, with dysregulation of miRNA expression contributing to the regulation of cancer metastasis. Nevertheless, although miRNA-targeted therapy is widely studied in vitro and in vivo, this strategy currently affords limited feasibility and a few miRNA-targeted therapies for lung cancer have entered into clinical trials to date. Advances in understanding the molecular mechanism of metastasis will thus provide additional potential targets for lung cancer treatment. This review discusses the current research related to the role of miRNAs in lung cancer invasion and metastasis, with a particular focus on the different metastatic lesions and potential miRNA-targeted treatments for lung cancer with the expectation that further exploration of miRNA-targeted therapy may establish a new spectrum of lung cancer treatments.


Introduction
Lung cancer constitutes the leading cause of cancer death worldwide [1], with most patients presenting advanced disease stage at initial diagnosis. Although early screening by computed tomography (CT) reduces the associated mortality [2], tumor invasion and migration-mediated disease progression represents the leading cause of cancer-related death despite standard treatment.
Numerous studies regarding tumor invasion and migration depict the interaction between tumor cells and adjacent tissues or microenvironments, reporting different mechanisms and various signal pathways related to tumor spreading. Specifically, the critical role of the epithelial-to-mesenchymal transition (EMT) in cancer invasion, migration, and metastasis provides a clue to prevent cancer spread and identify possible therapeutic targets [3].
MicroRNAs (miRNAs), a group of small non-protein-coding RNAs (20-25 nucleotides), suppress gene expression primarily through direct interaction with the 3 -untranslated region (3 UTR) of corresponding target messenger RNAs (mRNAs) [4]. Target mRNA fate depends on the seed match architecture between the miRNA binding and mRNA seeding sequences. mRNA degradation is induced upon perfect miRNA complementary with the seeding sequence, whereas imperfect or partial In epithelial-to-mesenchymal transition (EMT), uncontrolled epithelial cells first reduce dependence on their normal tissue microenvironment and proliferate [18]. Polarized epithelial cells lose epithelial cell junctional proteins, such as E-cadherin, claudins, and zona-occludens 1 (ZO-1), gaining mesenchymal markers including N-cadherin, vimentin, and fibronectin, with cytoskeletal reorganization. EMT, which engages different molecular process, transcription factor (TF) activation, and alternative miRNA expression [16], occurs in various physiological or pathological processes and is categorized according to biological and functional consequences: fertilized oocyte implantation and embryonic gastrulation (Type 1); inflammation and fibrosis (Type 2); and cancer cell invasion In epithelial-to-mesenchymal transition (EMT), uncontrolled epithelial cells first reduce dependence on their normal tissue microenvironment and proliferate [18]. Polarized epithelial cells lose epithelial cell junctional proteins, such as E-cadherin, claudins, and zona-occludens 1 (ZO-1), gaining mesenchymal markers including N-cadherin, vimentin, and fibronectin, with cytoskeletal reorganization. EMT, which engages different molecular process, transcription factor (TF) activation, and alternative miRNA expression [16], occurs in various physiological or pathological processes and is categorized according to biological and functional consequences: fertilized oocyte implantation and embryonic gastrulation (Type 1); inflammation and fibrosis (Type 2); and cancer cell invasion and metastasis (Type 3) [19]. In cancers, epithelial malignant cells acquire mesenchymal characteristics to Table 1. Epithelial-to-mesenchymal transition-related transcription factors and associated microRNAs in Non-Small Cell Lung Cancer (NSCLC).

Twist TF-Related miRNAs
Twist, an EMT TF, is a target of miR-98, as their expression levels inversely correlates in clinical NSCLC tissue specimens. MiR-98 up-regulation suppresses cell invasion and migration by impeding Twist-induced EMT [56]. Bioinformatics analysis and luciferase-reporter assay revealed that miR-92b suppresses Twist to reduce NSCLC metastasis [50]. MiR-33a targets Twist family BHLH transcription factor 1 (Twist 1) to inhibit NSCLC invasion and metastasis in vitro and in vivo [43].

MiRNAs Modulate Other EMT-Associated Signaling Genes and Related Downstream Proteins
Numerous EMT-associated signaling modulators are also regulated by miRNAs [123]. TGF-β, a well-known EMT activator, mediates cell proliferation, apoptosis, inflammation, tissue repair, and carcinogenesis [124]. Upon receptor binding, TGF-β induces receptor complex formation and activates downstream signals. TGF-β signaling includes Smad-dependent and Smad-independent pathways. In the former, Smad2/3/4 protein complex formation inhibits E-cadherin expression and induces fibronectin and matrix metalloprotease (MMP) expression, which regulates EMT processes. The latter involves RAS/RAF/ERK and PI3K/AKT signaling pathways, which are crucial for cell proliferation. [125,126]. Up-regulated TGF-β1 promotes lung adenocarcinoma invasion and metastasis via an EMT-associated mechanism [127]. Aberrant TGF-β up-regulation is critical to the development of targeted therapy resistance and disease progression in NSCLC [128][129][130][131]. MiRNAs target TGF-β pathway downstream factors to modulate EMT: miR-155 targets RhoA or Smad2/3, and miR-148a targets Rho-associated protein kinase I (ROCK1) [67,89,132]. MMPs digest most protein components in the ECM and participate in cell migration and invasion in physiological and pathological conditions, such as tissue remodeling and cancer cell progression [133].
MiR-136 (miR-136-5p) inhibits lung cancer cell metastasis and EMT by directly targeting Smad2 and Smad3 [79]. Among a cohort of 1242 samples from the Gene Expression Omnibus and The Cancer Genome Atlas (TCGA) datasets, miR-136-5p was up-regulated in lung adenocarcinoma versus normal tissues. Bioinformatics analysis using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Protein Analysis Through Evolutionary Relationships (PANTHER) pathways demonstrated that claudin-18, sialophorin, and syndecan 2, which function in cell adhesion and focal adhesion, likely comprise miR-136-5p target genes [135]. Because of the intrinsic complexity and sophistication of tumor initiation and progression, miR-136-5p might exhibit disparate dysregulation and functions in various cancers [135].
In NSCLC and hepatocellular carcinoma, MET oncogene activated miR-221 and miR-222 by activating the c-JUN TF. These miRNAs suppress phosphatase and tensin homolog (PTEN) and tissue inhibitor of metalloproteinases 3 (TIMP3), and promote cellular invasion and migration by activating the Akt (Protein kinase B, PKB) murine thymoma viral oncogene homolog (AKT) pathway and metallopeptidase [69]. Somatic PTEN mutation occurs in 4-8% of NSCLC [136,137], whereas PTEN overexpression inhibits lung cancer cell invasion and metastasis by inhibiting integrin αVβ6 signaling [138]. In patients with NSCLC, decreased PTEN expression constitutes a poor prognosis factor [139]. PTEN is also a target of miR-19 and miR-26a to regulate EMT in NSCLC [44,57]. MiR-664 regulates tumorigenesis and malignant progression in lung cancer cell lines, with up-regulated miR-664 promoting cell invasion and migration by targeting PTEN [102].
Transmembrane serine protease 4, a membrane-anchored protease, mediates cell invasion and migration in a variety of cancers including lung cancer. This protein suppresses miR-205 (miR-205-5p) expression to promote EMT. In vivo, miR-205-5p expression inhibits cell growth, migration, and metastasis formation. MiR-205-5p directly targets integrin α5, a pro-invasive protein in NSCLC. Down-regulated integrin α5 expression in lung cancer cells completely abrogates cell migration, decreases the fibronectin adherence capacity, and reduces tumor growth in vivo [52].
In summary, the EMT plays a crucial role in tumor invasion and metastasis, and it is also complex, multifunctional, and tightly regulated developmental program. Accumulating evidence suggests that microRNAs tightly regulate EMT in lung cancer cells. MicroRNAs act as pro-or anti-EMT through different targets and signal pathways, which regulates lung cancer invasion and metastasis.

Role of miRNAs in Different Metastasis Sites (Bone, Brain and Lymph Nodes) in Lung Cancer
In addition to predicting patient survival and tumor relapse, patients with NSCLC with and without metastasis exhibit different miRNA profiles [143,144]. Numerous studies have investigated the association between miRNA expression profile and lung cancer metastatic sites [145] (Figure 2).

Role of miRNAs in Lung Cancer Bone Metastasis
Bone metastasis occurs in approximately 15 to 30 percent of patients with lung cancer [146], representing one of the most deleterious clinical consequences [147]. However, the exact mechanism of bone metastasis remains unknown. The miRNAs associated with lung cancer bone metastasis are listed in Table 2.
A high-throughput sequencing study to explore the candidate bone metastasis-related miRNAs in lung adenocarcinoma generated two small RNA (corresponding to 18-30 nucleotides) libraries from the blood of patients with lung adenocarcinoma with and without bone metastasis. Expression profiling revealed 7 down-regulated and 21 up-regulated miRNAs in lung adenocarcinoma with bone metastasis. Bioinformatics analysis identified putative associated signaling pathways including MAPK, Wnt, and nuclear factor kappa light chain enhancer of activated B cells (NF-κB), along with pathways involving cytoskeletal proteins, angiogenesis factors, and MMP [148].
Moreover, 18 patients with NSCLC and vertebral column metastasis exhibited higher miR-21 expression levels than that in 20 patients with bone tuberculosis [149]. MiR-21 promotes cell proliferation and inhibits apoptosis in H2170 NSCLC cells through overexpression of cytochrome C oxidase assembly homolog 19 (COX19) [149], which affects COX subunit assembly by increasing COX activity. Reducing COX activity increases cytochrome C content, activating cell apoptosis signaling pathways and finally leading to apoptosis [150,151]. MiR-21 also mediates tumorigenesis and osteoclastogenesis by targeting PDCD4, which regulates osteoclastogenesis [152].
Some viruses regulate their own and/or host gene expression via aberrant miRNA expression [153,154]. Microarray analysis to compare miRNA expression in bone metastasis (n = 10) from lung cancer with that of primary lung cancers (n = 24) identified and validated a candidate viral miRNA, Hsv2-miR-H9-5p, encoded by herpes simplex virus type 2 latency-associated transcript [155]. Hsv2-miR-H9-5p expression is significantly higher in bone metastasis lesions than primary lung cancers. Hsv2-miR-H9-5p increases lung cancer cell migration and invasion in vitro by directly targeting suppressor of cytokine signaling 2 (SOCS2), inhibiting Jak2 kinase activity and Jak2-signal transducer and activator of transcription 3 (STAT3) binding [156]. SOCS2 expression is down-regulated in lung cancer [157].

Role of miRNAs in Lung Cancer Bone Metastasis
Bone metastasis occurs in approximately 15 to 30 percent of patients with lung cancer [146], representing one of the most deleterious clinical consequences [147]. However, the exact mechanism of bone metastasis remains unknown. The miRNAs associated with lung cancer bone metastasis are listed in Table 2.
A high-throughput sequencing study to explore the candidate bone metastasis-related miRNAs in lung adenocarcinoma generated two small RNA (corresponding to 18-30 nucleotides) libraries from the blood of patients with lung adenocarcinoma with and without bone metastasis. Expression profiling revealed 7 down-regulated and 21 up-regulated miRNAs in lung adenocarcinoma with bone metastasis. Bioinformatics analysis identified putative associated signaling pathways including MAPK, Wnt, and nuclear factor kappa light chain enhancer of activated B cells (NF-κB), along with pathways involving cytoskeletal proteins, angiogenesis factors, and MMP [148].
Moreover, 18 patients with NSCLC and vertebral column metastasis exhibited higher miR-21 expression levels than that in 20 patients with bone tuberculosis [149]. MiR-21 promotes cell proliferation and inhibits apoptosis in H2170 NSCLC cells through overexpression of cytochrome C oxidase assembly homolog 19 (COX19) [149], which affects COX subunit assembly by increasing COX activity. Reducing COX activity increases cytochrome C content, activating cell apoptosis signaling pathways and finally leading to apoptosis [150,151]. MiR-21 also mediates tumorigenesis and osteoclastogenesis by targeting PDCD4, which regulates osteoclastogenesis [152].
MiR-139-5p serum levels from patients with lung adenocarcinoma and osteolytic bone metastasis are lower than those in patients with other organ metastasis. MiR-139-5p expression in mesenchymal stem cells (MSCs) significantly increases during osteogenic differentiation. Notch homolog 1, translocation-associated (Drosophila) (Notch1), a direct miR-139-5p target, exhibits significant down-regulation during MSC osteogenesis [159]. Tumor transfer of miR-192-enriched exosome-like vesicles to the endothelial compartment of the osseous milieu in vivo reduced bone metastases burden. MiR-192 overexpression confers anti-osseous metastatic activity in vivo and limits tumor-induced angiogenesis [160]. MiR-203/TGF-β/Smad2 expression represents an important tumor suppressor signaling pathway for bone metastasis in NSCLC, as patients with bone metastasis exhibited lower tumor tissue miR-203 expression than those without bone metastasis [161].

Role of miRNAs in Lung Cancer Brain Metastasis
Brain metastasis affects approximately 25% of patients with NSCLC during their lifetime [162]. However, no molecular biomarkers or effective indices are available to reduce brain metastasis risk. The mechanism of brain metastasis is also not completely clear owing to the limited available tissue specimens. Table 3 lists lung cancer brain metastasis-related miRNAs.
MiRNA microarray-based comparison of expression profiles in five primary lung adenocarcinoma tumors versus three brain metastatic lung adenocarcinoma samples reveals obvious miR-145 down-regulation in brain metastatic samples, albeit no relationship between miR-145 and lymph node metastasis [163]. Among miRNAs from 527 patients with stage I NSCLC, miRNA microarray analysis identified 10 miRNAs associated with brain metastasis including miR-145 [164]. Promoter methylation-mediated miR-145-5p down-regulation promotes lung adenocarcinoma cell brain metastasis, whereas miR-145-5p expression reduces cancer cell migration [165]. MiR-21 is a target of STAT3 [179,180]. In patient-derived stem cell lines from lung-to-brain metastasis, miR-21 down-regulation attenuates brain metastasis-initiating cell self-renewal and migration comparably to STAT3 knockdown [166]. Compared to parental cells, miR-95-3p is down-regulated in brain metastasis cells generated through injection of lung adenocarcinoma cells into a left ventricle of nude mice. MiR-95-3p overexpression suppresses cell invasion, proliferation, and colony formation. Cyclin D1 was identified as a direct miR-95-3p target [167].
MiR-4317 is significantly down-regulated in tumor tissues compared with that in paired normal tissues, whereas patients with early stages and non-lymph node metastasis exhibit higher miR-4317 levels. MiR-4317 up-regulation significantly suppresses cell proliferation, colony formation, invasion, and migration. It also hampers NSCLC cell cycling by directly targeting fibroblast growth factor 9 (FGF9) and cyclin D2 (CCND2). In mouse xenograft model, miR-4317 suppresses tumor growth and brain and lung metastasis [178]. MiRNA microarray analysis identified miR-328 as related to brain metastasis by comparing samples from patients with (n = 7) and without (n = 8) brain metastasis. MiR-328 overexpression in A549 cells significantly promotes cell migration concomitant with protein kinase C alpha (PRKCA) up-regulation [171].
Overexpression of mir-423-5p, selected using microarray analysis of brain metastasis-related miRNAs and validated by quantitative PCR, promotes NSCLC cell colony formation, cell motility, migration, and invasion by direct targeting metastasis suppressor 1 (MTSS1). In clinical samples, lung adenocarcinoma tissues without brain metastasis exhibit positive staining for MTSS1 expression [176]. Microarray analysis between patients with and without brain metastasis revealed that a three-miRNA (including miR-210, miR-214, and miR-15a) signature predicts the brain metastasis of patients with lung adenocarcinoma with high sensitivity and specificity [170].
Recently, increasing evidence revealed that exosomes play important roles in the tumor microenvironment and the mechanism of malignant tumor metastasis. Exosomes, consist of a phospholipid bilayer, which is composed mainly of proteins, lipids, carbohydrates, and nucleic acids [181,182]. Exosome carries miRNAs, termed "exomiRs", to acceptor cells to promote non-adjacent intercellular communication, which involves in cell differentiation, immune response, antigen presentation, and cell invasion/migration [183][184][185]. The transfer of exosomal miRNA can modulate gene expression in acceptor cancer cells to facilitate metastasizing cancer cell settlement in pre-metastatic organs, suggesting these exosomal miRNAs prepare the pre-metastatic niche [186].
Astrocytes oppose brain metastasis via exosome-delivered miR-142-3p, which directly binds to the suppressing transient receptor potential ankyrin-1 (TRPA1) 3 UTR. TRPA1 also directly targets the FGF receptor 2 C-terminal proline-rich motif, thereby constitutively activating the receptor and increasing lung adenocarcinoma progression and metastasis [168]. Transferring miR-142-3p from astrocytes to lung cancer cells suppresses TRPA1 in the latter, promoting brain metastasis. MiR-184 and miR-197 are also overexpressed in patients carrying EGFR mutation with brain metastasis; their expression level may serve to stratify the brain metastasis risk in this subpopulation [169].

Role of miRNAs in Lung Cancer Lymph Node Metastasis
Lymphatic metastasis comprises an important mechanism in tumor spreading in addition to metastasis via blood vessels. The primary epithelial cancer cells enter into the lymphatic drainage system and spread to local or distal lymph nodes after penetrating the basement membrane [187]. For patients with early stage lung cancer, lymphatic invasion or lymph node involvement represents a key prognostic factor. Regional lymph node status is important for lung cancer staging and treatment planning [188]. However, traditional image examination (chest CT) sensitivity is poor. Micro-metastasis or occult lymph node metastasis is still found in approximately 20% of early stage (T1/T2) lung cancer tumors [189,190]. Positron emission tomography (PET) scanning and endobronchial ultrasound-guided transbronchial needle aspiration can decrease the high false-negative rate and provide greater sensitivity and specificity for mediastinal lymph node assessment [191][192][193].
The identification of molecular biomarkers expressed in tumor tissue or patient serum is helpful to predict lymph node metastasis. Table 4 lists the different miRNAs related to lymph node metastasis. The combination of Let-7g and miR-21 profiling and KRAS mutational status may be considered a useful biomarker for clinical management of NSCLC patients.
Capodanno et al. [194] miR-1 PIK3CA Suppressor Lung tumors Low expression of miR-1 and overexpression of PIK3CA in NSCLC tissues may be useful predictors of lymph node metastasis and postoperative recurrence in patients with NSCLC.
Zhao et al. [195] miR-7 Bcl-2 Suppressor Lung tumor Overexpressed CDR1as in NSCLC functioned to promote tumor progression via miR-7 signals. Up-regulated miR-7 increased the sensitivity of lung adenocarcinoma cells to CDDP by inducing apoptosis.
Zhang et al. [196], Cheng et al. [197] miR-9s Oncogene Lung tumors Involved in NSCLC progression and could serve as a promising biomarker.
Muraoka et al. [198], Xu et al. [199]       Li et al. [337] * There were 24 putative target genes for Let-7g after analysis by miRanda, TargetScan, Pictar and miRDB prediction algorithms. # TargetScan software were applied for in silico prediction of miR-31 targets. ¶ There were 44 co-regulated target genes of both miRNA-126-3p and miRNA-126-5p by using twelve target gene prediction software programs (TargetScan, miRWalk, Microt4, miRDB, miRanda, miRBridge, miRMap, miRNAMap, PITA, PicTar2, RNA22 and RNAhybrid). § Identified by predicting by online database, miRecords and mining of the data from Gene Expression Omnibus (GEO) and TCGA. @ Total of the fourteen prediction programs were used for screened the putative target genes. STRING database was used for the selection of hub genes which were probably involved in the strategic pathway related to lung adenocarcinoma. Bcl The role of miR-200c in lung cancers is controversial. MiR-200c inhibits NSCLC cells invasion and migration, and expression of the miR-200c targets USP25 in NSCLC correlates with clinical stage and lymphatic node metastasis [115]. Lower miR-200c expression also significantly correlates with poor differentiation grade, lymph node metastasis, and lower E-cadherin expression [115,275]. However, higher tumor miR-200c expression was reportedly associated with poor survival in patients with NSCLC [209,276].
MiR-130 also plays a controversial role in NSCLC. MiR-130 is significantly down-regulated in NSCLC tumor tissues and cell lines. High miR-130 expression inversely correlates with lymph node metastasis and late stages. MiR-130 up-regulation significantly suppresses NSCLC cell growth and enhances cell apoptosis by directly targeting PTEN [243]. MiR-130 family consists of miR-130a and miR-130b, and they have nearly identical sequences, although miR-130a and miR-130b come from chromosome 11 and chromosome 22, respectively. MiR-130a functions as a proangiogenic miRNA and antagonizes the inhibitory effect of growth arrest homeobox transcription factor and homeobox A5 (HoxA5) on endothelial cell proliferation, migration, and tube formation [338]. MiR-130a is also overexpressed in NSCLC tissues, with higher expression being strongly associated with lymph node metastasis and poor prognosis [244]. Further studies are thus needed to clarify the role of miR-130 in NSCLC.
Serum miRNA levels also serve as biomarkers of NSCLC metastasis or prognosis [209,213]. High serum miR-21 correlates with advanced stages and lymph node metastasis. MiR-21 promotes cell proliferation, metastasis, and chemo-radio-resistance in NSCLC cells by targeting PTEN [211]. High serum miR-19a and miR-19b also significantly correlate with tumor-node-metastasis (TNM) stage and lymph node metastasis [207,208]. Patients with NSCLC exhibit significantly increased serum miR-494 levels compared with those in healthy controls, with the levels markedly decreasing when patients receive effective therapy. MiR-494 up-regulation in serum or tumor tissues significantly associates with higher incidence of lymph node metastasis, advanced clinical stage, and higher histological grade [317,318]. MiR-210, miR-421, and miR-411 levels in tumor tissues or serum of patients with lung cancer significantly positively correlate with lymph node metastasis and poor prognosis [279,280,300,301]. Patients with NSCLC exhibit lower serum miR-138 than that of healthy controls. Low miR-138 expression correlates with positive lymph node metastasis and poor prognosis [248]. MiR-138 suppresses NSCLC proliferation, metastasis, and autophagy by targeting sirtuin 1 (Sirt1) [249]. MiR-138 also targets Yes-associated protein 1 (YAP1) [250].
MiRNAs are detected in sputum and plasma [313]. Between lung cancer tissues with adjacent non-cancerous specimens, the former show lower miR-486-5p expression, with the reduced expression being associated with advanced clinical stage and lymph node metastasis of NSCLC [312][313][314]. In vitro, miR-486-5p down-regulation promotes tumor progression and metastasis by targeting Rho GTPase-activating protein 5 (ARHGAP5). MiR-486-5p expression in sputum and plasma specimens could provide a diagnostic approach for early lung cancer detection [314]. Moreover, miR-486-5p up-regulation in cancer cells reduces expression of Pim-1, a direct target. Pim-1 kinase, a proto-oncogene, is overexpressed in 66.2% of lung tumor tissues by immunohistochemical staining. Pim-1 expression is significantly higher in NSCLC tissues than in adjacent normal tissues [315].
Meta-analysis from the TCGA database demonstrated that lower miR-133a-3p correlates with negative lymph node metastasis and might act as a tumor suppressor [245]. In lung adenocarcinoma, miR-452-5p expression is obviously lower than that in adjacent normal tissues, and negatively correlates with lymph node metastasis and TNM stage [310]. MiR-145-5p shows similar findings among 125 paired NSCLC tissues and the TCGA database, indicating that both miRNAs function as tumor suppressors [255]. Among 372 NSCLC and 42 adjacent normal lung tissues from the Gene Expression Omnibus dataset, miR-101-3p showed higher expression in normal than NSCLC tumor tissues. Low miR-101-3p expression significantly correlated with lymph node metastasis and shorter OS [228].
Gene promoter methylation generally results in down-regulation of gene expression. Aberrant miR-200c promoter methylation obviously negatively correlates with miR-200c expression and is associated with lymph node metastasis and poor clinical outcome [275]. Histone methylation-mediated (H3K27me3) miR-139 silencing enhances NSCLC invasive and metastatic phenotype, with down-regulated miR-139 expression being significantly associated with lymph node metastasis and tumor invasiveness [251].

Potential of miRNA as a Therapeutic Target and Tool in Patients with Lung Cancer
Increasing evidence demonstrates that miRNAs play pivotal roles in lung cancer invasion and metastasis. Studies using miRNA profiling to predict prognosis and clinical treatment response indicate that miRNA expression profiles can predict patient cancer relapse and clinical outcome in NSCLC [143]. The European Lung Cancer Working Party (ELCWP) prospective study, initiated to identify a miRNA-based signature for treatment response and survival for NSCLC treated with cisplatin and vinorelbine, revealed that a four-miRNA signature (miR-200c, miR-424, miR-29c, and miR-124) could predict treatment response of first-line cisplatin and vinorelbine and act as a prognostic factor in patients with NSCLC [341]. The combination of a plasma immune-related microRNA-signature classifier and immunohistochemical stain of programmed death-ligand 1 in tumor specimens could predict poor treatment response and OS in patients with NSCLC treated with immune-checkpoint inhibitors [342].
For predicting disease prognosis, gain-and loss-of-function studies of miRNAs have provided a rationale and innovative insight toward precision medicine by targeting miRNA to prevent tumor progression or spreading of cancer cells, because miRNAs can stably modulate gene networks [343]. Possible approaches include: (i) miRNA-based treatment (Direct strategy). Introduction of synthetic miRNA analogs (miR mimics) to mimic tumor suppressor miRNAs that are down-regulated in cancer cells, or antisense oligonucleotides (known as anti-miRs or antagomiR) to silence oncogenic/metastasis-promoting miRNAs [344,345]. Although ectopic expression of synthetic miRNAs mimics was accomplished in vitro, there was little in vivo data using miRNA mimics delivered by intravenous injection. The expression of miRNAs is also restored by inserting genes coding for miRNAs into viral constructs, such as the adenovirus-associated vectors (AAV) [346][347][348]. These vectors do not integrate into the genome and have high efficiency of transduction. Kota and colleagues cloned miR-26a into an AAV vector and viral particles were tested in a mouse model of liver cancer. Systemic administration of miR-26a results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity [348]. Besides, miRNA-based treatment involves various strategies, including miR-mask and miRNA sponges which interrupt the interaction between target and miRNAs [346]. (ii) Induction of miRNA expression (Indirect strategy). Some drugs were developed to modulate the expression of miRNAs by regulating activation or repression of upstream transcription factors. By screening for more than 1000 small molecular compounds, diazobenzene 1 promoted transcription of miR-21 and produced a 250% increase of miR-21 relative to the untreated cells [349].
A critical challenge of targeted miRNA therapy is how to introduce the synthetic oligonucleotide or miRNA mimic into the cancer cells. Viral and non-viral vectors comprise commonly used vectors for miRNA delivery [350]. However, viral vector introduction into the host system can trigger an immune response [351]. Systemic treatment with miR-10b antagomir, a 2'-O-methyl-group (OMe)-modified, cholesterol-conjugated antisense miR, and miR-34a mixed with atelocollagen could suppress breast and colon cancer metastasis, respectively, in animal studies [352,353]. Systemic delivery of miR-34a into experimental lung metastasis of murine B16F10 melanoma using a liposome-polycation-hyaluronic acid nanoparticle formulation modified with tumor-targeting single chain antibody fragment (scFv) reduces tumor load in the lung [354].
Several pre-clinical and clinical trials of miRNA as targeting therapy for lung cancer are ongoing ( Table 5). Let-7 suppresses lung tumor via KRAS in vivo, and exogenous lentivirus-mediated let-7 delivery significantly reduces the tumor burden in mouse models of NSCLC [355]. Systemic let-7 or miR-34a delivery by injection of neutral lipid emulsion also significantly attenuates tumor burden in the KRAS autochthonous NSCLC mouse model [272,356]. For EGFR mutant NSCLC, combinatorial treatment with let-7b and miR-34a provides synergistic treatment effect with erlotinib, an EGFR tyrosine kinase inhibitor, to suppress NSCLC proliferation [357]. Plasmid-mediated miR-126 plasmid inhibits A549 cell proliferation in vitro and inhibits tumor growth in vivo by increasing expression of EFG-like domain 7 [358]. MiR-145 inhibits NSCLC proliferation by directly targeting c-Myc in vitro [359]. Cationic polyurethane-short branch polyethylenimine (PEI) -mediated delivery of miR-145 inhibits xenograft tumor growth, EMT, and metastasis, and prolongs the survival times of a lung adenocarcinoma mouse model [360]. Efficient systemic delivery of miR-133-b and miR-29 by cationic lipoplexes inhibits tumor growth in vitro and in vivo [361,362]. The first miRNA mimic-based therapy, MRX34 (Mirna Therapeutics Inc., Austin, TX, USA.), a liposomal miR-34 mimic, entered phase I clinical trial of liver cancer therapy in 2013 [363], demonstrating acceptable safety and antitumor activity of MRX34 in a subset of patients with refractory advanced solid tumors [364], along with positive results of lung cancer in vitro and in animal studies [356,365]. However, the further phase I/II clinical trials (ClinicalTrials.gov identifiers: NCT01829971, NCT02862145) were terminated or withdrawn because the suitability of associated serious immune-related adverse events for clinical application was questioned. MesomiR 1 (NCT02369198), a first-in-man, phase I clinical trial, enrolled patients with NSCLC and malignant pleural mesothelioma to assess the safety and activity of TargomiRs as the second and third line of treatment [367]. TargomiRs (TargomiRs; EnGeneIC Ltd., Sydney, Australia) comprise minicells loaded with a miR-16-based mimic, which acts as an anti-EGFR specific antibody. The MesomiR 1 study intended to specifically deliver miR-16 to suppress tumor development, as the family of this miRNA is associated with tumor suppression in several cancers. The clinical trial demonstrated the acceptable safety profile in patients with malignant pleural mesothelioma [366]. Future research is necessary to address clinical treatment efficacy.
Argonaute-2 (AGO2) mediates post-transcriptional gene silencing, as an essential component of the RNA-induced silencing complex (RISC). After miRNA assembles into RISC, the activation complex silences and degrades the target mRNA transcripts [368]. However, when a double-strand RNA loads into AGO2, the AGO2-bound RNAs can activate transcription in the nucleus, paradoxically increasing mRNA expression [343,369]. According to this mechanism, a phase 1 trial (NCT02716012) of hepatocellular carcinoma was launched for a small activating RNA (saRNA) drug to increase CCAAT/enhancer-binding protein α (C/EBPα) expression. The dsp21 (saRNA) is designed to activate p21WAF1/CIP1 gene expression, and it inhibits cell proliferation, and induces apoptosis in lung cancer H441 and A549 cells [370,371]. More importantly, dsp21 increased the chemo-sensitivity to cisplatin of lung cancer cells in vitro and in vivo [371]. This synergistic effect of saRNA and chemotherapy may provide a reasonable concept for developing a treatment strategy in lung cancers. In addition to chemotherapy, aberrant activity of oncogenic pathways are the characteristics of carcinogenesis, it is worth noting that the combined treatment strategy of miRNA and saRNA to concurrently silence and activate opposing pathways of cancer. The combination strategies may develop more potent precision therapies of cancers [372].
There are several advantages of miRNA-based therapeutic in lung cancer over other treatment strategies, such as targeting growth factor receptors or enzymatic proteins. MiRNA-based therapies have emerged as promising therapeutic tools for cancer management due to highly specific in tissues and tumors. In addition, the advantage of using miRNA approaches is based on the ability to concurrently target multiple effectors of pathways involved in cell differentiation, proliferation, and survival. Therefore, miRNAs therapies have extremely efficiencies in regulating distinct biological cell processes relevant to malignant cell homeostasis [346,373]. The ability of miRNAs to regulate multiple genes in a molecular pathway makes them excellent candidates for novel molecular-targeting treatments.
However, there are still some problems with miRNA therapies. Therapeutic miRNAs is difficult to cross through cell membranes resulting in poor cellular uptake of oligonucleotides because of the size and negative charge of miRNAs. In addition, delivering a therapeutic miRNA to the associated target tissues also challenges. MiRNAs are relatively unstable and reduce their half-life substantially in the blood circulation due to subject to rapid degradation by RNases [272]. It is an obstacle to achieve a sufficient amount of synthetic oligonucleotides to sustain the inhibitory effect [346,374,375]. Finally, the potential off-target effects of miRNA therapeutics are major concerns because of concurrent regulation of many genes. In addition, recent reports revealed the toxicity related to miRNA therapies [376]. These pharmacokinetic and pharmacological drawbacks of RNA-based therapeutics, such as off-targeting, low serum stability, and innate immune responses, require more research.
Although miRNA replacement therapy remains challenging with numerous problems needing to be resolved, several clinical trials with miRNA mimics have already been initiated. By developing more specific carriers and expression models, regulation of miRNA function will likely become more specific and effective for cancers. Cancer therapy through miRNA regulation may thus engender a new era for cancer patients in the near future.

Conclusions
Lung cancer remains a major cause of cancer-associated deaths globally. The aggressive behavior of lung cancer involved in invasion and migration caused disease rapid progression despite standard treatment. MiRNAs regulate multiple genes and different signal pathways. Increasing studies suggested that miRNAs reveal discrete expression patterns in lung cancers. Dysregulation of miRNA expression regulates EMT and cancer metastasis by targeting various genes. Different miRNA expression in tumor tissues or sera is associated with different metastatic sites. These miRNA profiles also correlate with prognosis and clinical treatment response in lung cancers and could be potential targets of lung cancer treatment. More research on miRNA targeted therapies is necessary to increase the target specificity and potency and decrease the off-target effects and toxicity. Exploring miRNA-targeted therapy may establish a new spectrum of lung cancer treatments.