Long non‐coding RNAs: How to regulate the metastasis of non–small‐cell lung cancer

Abstract Non–small‐cell lung cancer (NSCLC) has become the most lethal human cancer because of the high rate of metastasis. Hence, clarifying the molecular mechanism underlying NSCLC metastasis is very important to improve the prognosis of patients with NSCLC. Long non‐coding RNAs (LncRNAs) are a class of RNA molecules longer than 200 nucleotides, which can participate in diverse biological processes. About 18% of human LncRNAs were recently found to be associated with tumours. Many studies indicated that aberrant expression of LncRNAs played key roles in the progression and metastasis of NSCLC. According to the function in tumours, LncRNAs can be divided into two classes: oncogenic LncRNAs and tumour‐suppressor LncRNAs. In this review, we summarized the main molecular mechanism of LncRNAs regulating NSCLC metastasis, including three aspects: (a) LncRNAs interact with miRNAs as ceRNAs; (b) LncRNAs bind with target proteins; and (c) LncRNAs participate in the transduction of different signal pathways. Then, LncRNAs can exert their function to regulate the metastasis of NSCLC through influencing the progression of epithelial‐mesenchymal transition (EMT) and the properties of cancer stem cell (CSC). But, it is necessary to do some further research to demonstrate the LncRNAs particular regulatory mechanism of inhibiting the metastasis of NSCLC and explore new drugs targeting LncRNAs.


| INTRODUC TI ON
The lung cancer is the leading cause of cancer-related death in China and is responsible for more than 1 million deaths around the world annually. 1,2 Despite there are many methods to treat the lung cancer cases, the overall survival is still poor because patients were at advanced stages when diagnosed. 3 Non-small-cell lung cancer (NSCLC), including adenocarcinoma, squamous cell carcinoma and large cell carcinoma, composes about 85% of lung cancers, and more than half of patients with newly diagnosed NSCLC have metastatic disease. 4 Because of the high rate of metastasis, NSCLC has become the most lethal human cancer. 5 Thus, understanding the molecular basis underlying NSCLC progression and metastasis is important to improve the treatment and prognosis of patients with NSCLC.
Long non-coding RNAs (LncRNAs) are a class of RNA molecules longer than 200 nucleotides, which can participate in diverse biological processes, including cell differentiation, modulation of apoptosis and invasion, reprogramming stem cell pluripotency and parental imprinting. 6 The main characteristics of LncRNAs are that these RNAs are able to be transcripted, but cannot be translated into proteins, so they exert their respective biological functions at RNA level. 7 Meanwhile, their abnormal expression is closely related to a variety of diseases, especially tumours. 8 About 18% of human LncRNAs were recently found to be associated with tumours. 9 Many studies demonstrated aberrant expression of LncRNAs played key roles in the progression and metastasis of NSCLC. [10][11][12] According to the function in tumours, LncRNAs can be divided into two classes: oncogenic LncRNAs and tumour-suppressor LncRNAs. Nowadays, LncRNAs have become a new therapeutic target for treating NSCLC metastasis.
More and more evidence indicated that LncRNAs were involved in tumour invasion and metastasis. Hence, our main purpose is to review recent literature on the relationship between LncRNAs and the metastasis of NSCLC.

| LncRNAs interact with miRNAs as ceRNAs
Many researchers have proved that LncRNAs could regulate the level of miRNAs, which in turn regulate the expressions of miRNA's target genes. LncRNAs might compete with miRNAs as miRNA sponges in tumour progression. Competing endogenous RNA (ceRNA) theory indicated that RNA transcriptions included LncRNAs communication through a new manner mediated by microRNA response elements (MREs). 13 Here, we listed some LncRNAs functioned as ceRNAs, including oncogenic LncRNAs and tumour-suppressor LncRNAs.

| Oncogenic LncRNAs
LncRNA histocompatibility leucocyte antigen complex P5 (HCP5), which is transcriptional regulated by SMAD3 in NSCLC cells, contains miR-203 response elements. MiR-203 expression was down-regulated in NSCLC and negatively correlated with clinical tumour-node-metastasis (TNM) stages. 14 19 Tian et al discovered that ZFAS1 was up-regulated in NSCLC tissues and higher expression in more advanced tumour tissues. 20 It was demonstrated that ZFAS1 exerted as ceRNA to enhance the expression of proliferation, invasion and metastasis-related genes, such as ZEB1, MMP-14, MMP16, BMI1, Sp1 and ZEB2 by competitively sponging miR-150, miR-200b or miR-200c. 21,22 HMMR-AS1, as a carcinogen, is involved in advanced TNM staging, greater tumour volume and positive lymph node metastasis because of its high expression. Cai et al found HMMR-AS1 functioned as a ceRNA of miR-138, and the high expression of miR-138 caused the repression of its endogenous target SIRT6. 23 SIRT6 is a direct target of miR-138, and knockdown of SIRT6 in NSCLC cells could increase the paclitaxel sensitivity. 24 HMMR-AS1 could be considered as a potential target for the diagnosis and treatment of NSCLC.
SUMO1P3, located at human chromosome 1q23.2, is firstly identified for its high expression in gastric cancer tissues. 25 Zhang et al confirmed SUMO1P3 expression was significantly increased in NSCLC cancer tissues and cell lines. Meanwhile, the expression level of SUMO1P3 in metastatic lymph node specimens was up-regulated in comparison with primary NSCLC tissue specimens. 26 They also found SUMO1P3 promoted NSCLC cell migration and invasion by repressing miR-136, 26 which directly targeted Smad2 and Smad3 to inhibit epithelial-mesenchymal transition (EMT) process in lung cancer cells. 27 LncRNA H19 was the first identified LncRNA in 1990. 28 Although it was reported that H19 was frequently overexpressed in lung cancer and related to cell proliferation, 29  High NNT-AS1 expression was associated with advanced tumour stage and lymph node metastasis of patients with NSCLC. 13 They also identified NNT-AS1 could function as a ceRNA by sponging miR-129-5p in lung cancer. Other research discovered that miR-NA-129-5p suppressed lung cancer cell proliferation and invasion through targeting microspherule protein 1, E-cadherin and vimentin. 34 In addition to the above LncRNAs, many studies revealed that other oncogenic LncRNAs, which could play their roles as ceRNAs, were associated with the NSCLC TNM stage and had higher expression in NSCLC than normal tissues. In Table 1, we listed some other oncogenic LncRNAs competed with miRNAs as miRNA sponges, thereby regulating the downstream targets to promote NSCLC metastasis.

| Tumour-suppressor LncRNAs
LncNONHSAT081507.1 (LINC81507) was first identified by Peng et al using Agilent Human LncRNA Array. 44 Recently, Peng et al found reduced expression of LINC81507 resulted in cell growth, proliferation, migration and EMT in NSCLC cells, whereas ectopic overexpression of LINC81507 resulted in the opposite effects both in vitro and in vivo. 45 LINC81507 acted as a ceRNA for miR-199b-5p through directly binding and interfering miR-199b-5p-mediated regulation of CAV1 to reduce migration and invasion. In conclusion, LINC81507 served as a tumour suppressor gene in NSCLC.
The LncRNA growth arrest-specific transcript 5 (GAS5), a tumour suppressor gene, was significantly down-regulated in NSCLC tissues and cell lines, and elevated expression of GAS5 inhibited cell proliferation and induced apoptosis in NSCLC cells. 46

| LncRNAs bind with target proteins
LncRNAs can promote or inhibit the metastasis of NSCLC by directly binding with the target proteins.

| Oncogenic LncRNAs
BCYRN1 (brain cytoplasmic RNA 1, also known as BC200), a 200-nucleotide LncRNA, was found highly expressed in some carcinomas of the breast, cervix, oesophagus, lung, ovary, etc, but normally not detectable in the corresponding normal tissues. 48 Hu et al found BCYRN1 was the target gene of c-MYC and could mediate cell migration and invasion in NSCLC via influencing the expressions of MMP9 and MMP13. 49 In conclusion, BCYRN1 LINC00511 was a newly identified LncRNA, which was up-regulated in human breast cancer as an oncogene. 55 Sun et al demonstrated that LINC00511 was highly up-regulated in both NSCLC tissues and cell lines. Furthermore, LINC00511-mediated oncogenic effects were partially through its epigenetically silencing of the p57 expression via directly binding with enhancer of zeste homolog 2 (EZH2). 56 In addition to these above LncRNAs, there were many other oncogenic LncRNAs, which could play their inducing NSCLC metastasis by indirectly influencing signal pathways transduction via binding with proteins. In Table 2, we listed some other oncogenic LncRNAs and their target proteins. LncRNA NORAD was discovered to exploit a novel mechanism for regulating protein function. 65, 66 Tan et al indicated that LncRNA NORAD was down-regulated in lung cancers and that NORAD low expression was associated with lymph node metastasis and poor prognosis. 67 Mechanistically, NORAD exploited its multiple repeated sequences to function as a multivalent platform for binding and sequestering S100P, thereby suppressing the associated pro-metastatic signalling network of S100P.

| Tumour-suppressor LncRNAs
LncRNA AK126698, a tumour suppressor in NSCLC progression, was found that its expression was lower in cisplatin-resistant A549/DDP cells compared with parental A549 cells. 68

| LncRNAs participate in the transduction of different signal pathways
Signal pathways play very important role in regulating progression and metastasis of tumour, such as Wnt/β-catenin,

| Wnt/β-catenin signal pathway
Clinically, Wnt/β-catenin pathway activation predicts increased risk of tumour recurrence in patients with NSCLC. 75 MIR31HG was a LncRNA, identified as >2166 nucleotides in length. 76 Recent studies showed that increased MIR31HG expression increased gefitinib resistance in NSCLC lines through the EGFR/PI3K/AKT signalling pathway. 77

| PTEN/AKT signal pathway
PTEN/AKT signal pathway is a kind of classic intracellular transduction pathway, and the abnormal activation of this pathway is also related with the development of diseases, such as tumours, autoimmune diseases and diabetes mellitus. 83 LncRNA ASAP-IT1, located in chromosome 8q24.21 in whole length of 1179bp, was initially found in ovarian cancer for its abnormal expression. 84  FER1L4, a novel LncRNA, was first identified that its down-regulated expression in human gastric cancer. 99 Gao et al also found that FER1L4 was down-regulated in NSCLC in vivo and in vitro, and overexpression of FER1L4 could inhibit cell metastasis through regulating the PI3K/Akt signal pathway. 100 Many studies indicated that the PI3K/Akt signalling was aberrantly activated in human malignancies and was associated with tumour metastasis and drug resistance. 101

| Regulate the progression of epithelialmesenchymal transition (EMT)
The EMT is closely related to the high invasiveness and metastasis of cancer cells, including NSCLC. 102 More and more evidence indicates that LncRNAs are involved in tumour invasion and metastasis by regulating EMT.
LncRNA HOX transcript antisense RNA (HOTAIR) exhibited significantly higher expression in the tumour tissues than the adjacent non-tumour tissues in patients with NSCLC. 103 In lung cancer cells, HOTAIR was required for the expression of matrix metalloproteinases that break down the extracellular matrix to pave the path. 104,105 Taken together, HOTAIR was induced by EMT stimuli, and such an induction in turn promoted the gene expression programme that resulted in EMT. 106 HOTAIR also could potentially regulate lung cancer metastasis through physical interactions with E3 ubiquitin ligases and their corresponding substrates. 106 LncRNA activated by TGF-β (ATB) was first identified in liver cancer. 107 Many studies showed that ATB promoted malignancy in many kinds of cancers, including breast cancer, glioma and colon cancer. 108 Linc00662, a newly discovered LncRNA, had a strong correlation with lower overall survival rate and higher lymph node metastasis rate of patients with lung cancer. Lin28, as an RNA-binding protein and a reprogramming factor, can promote the tumourigenesis and progression in many human cancers. 118 Evidence indicated that Lin28 also could participate in CSC regulation. 119 Recently, Gong et al demonstrated that Linc00662 not only induced cell migration and invasive ability but also elevated the CSC percentage in NSCLC cells by the interaction with its downstream factor Lin28. 120 According to our review, the molecular mechanism of LncRNAs regulating NSCLC metastasis mainly includes three aspects

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict interests.