Role of Non-Coding RNAs in Lung Cancer

Abstract

Lung cancer is the most common cancer worldwide, and the leading cancer killer in both men and women. Globally, it accounts for 11.6% of all cancer cases and is responsible for 18.4% of cancer-related deaths. The mechanisms underlying lung cancer development and progression have been widely studied, and roles for non-coding RNAs (ncRNAs) have been identified. Non-coding RNAs are a type of RNA molecules that are not translated into proteins. The main types of ncRNAs include transfer RNAs (tRNAs), microRNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), small nucleolar/nuclear RNAs (snoRNAs, snRNAs), extracellular RNAs (exRNAs), tRNA fragments, and long non-coding RNAs (lncRNAs). In the past few years, there has been an increased interest in the role of ncRNAs in oncology, and lung cancer tumorigenesis specifically. Multiple ncRNAs were identified as tumor suppressors: tRNA fragments, snoRNAs, and piRNAs while others were reported to have tumor-promoting functions: circular RNAs (circRNAs), snoRNAs, piRNAs, YRNAs, natural antisense transcripts (NATs) and pseudogene transcripts. In this chapter, we discuss the latest body of knowledge regarding the role of ncRNAs in lung cancer pathogenesis as well as their potential use as biomarkers or therapies against lung cancer.

1. Introduction

Lung cancer is responsible for 19% of cancer-related deaths, making it the number one cause of cancer-related mortality worldwide. A recent global cancer statistics report has estimated that approximately 2.2 million new cases and 1.8 million deaths were reported in 2020 [1, 2]. Similarly, in the United States, 230,000 new cases of lung cancer and 132,000 deaths were reported in the same year [1].

While smoking is a well-investigated major risk factor, responsible for approximately 80% of total lung cancer fatalities, not all lung cancers are associated with smoking, particularly in women [3]. Other environmental and occupational factors have been linked to lung cancer development, including second hand smoke exposure, ionizing radiation, arsenic in drinking water, and radon, silica, asbestos, heavy metals, and air pollution exposures. Moreover, genetic factors and other comorbidities such as chronic obstructive pulmonary disease (COPD) have been identified as risk factors for lung cancer [4].

Lung cancer is defined as a cancer that starts in lung tissue. The condition is divided into two main types, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The NSCLC type represents around 85% of all lung cancer cases, whereas the SCLC represents the remaining 15% [5, 6]. The NSCLC is further divided into several subtypes which include lung adenocarcinoma (LAC), squamous cell carcinoma (SCC), and large cell carcinoma (LCC). The World Health Organization (WHO) has classified lung tumors into multiple subtypes. This classification relies on the use of immunohistochemical characterization and microscopy and provides standardized criteria and terminology for diagnosis on small biopsies and cytology [7]. It also provides guidance for molecular testing, recognizing the therapeutic importance of targetable genetic alterations.

The molecular basis of lung cancer development is complex and involves a combination of genetic and epigenetic alterations that regulate tumor development. Multiple pro-oncogenic and tumor suppressor genes, as well as growth factors, transcription factors and other regulatory molecules are involved. Recent studies describing the pathways involved in lung cancer tumorigenesis have identified non-coding RNAs (ncRNAs) as important players in its pathogenesis regulation. Non-coding RNAs are a subtype of RNA molecules that regulate gene expression at the transcriptional and post-transcriptional level but are not translated into proteins. These ncRNAs are divided into two categories, short non-coding RNAs (less than 200 bp) an long non-coding RNAs (lncRNAs, more than 200 bp) [8]. The category of short ncRNAs consists of microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), tRNA-derived stress-induced RNA (tiRNA), YRNA and transfer RNA-derived RNA fragment (tRF). Further, the lncRNA makes up a heterogeneous category, which includes long intergenic non-coding RNAs (lincRNAs), bidirectional lncRNAs, intronic lncRNAs, overlapping sense lncRNAs, circular intronic RNAs (ciRNAs), T-UCR lncRNAs (transcribed from ultra-conserved regions), antisense transcripts, and enhancer RNAs [9, 10]. The lncRNAs display additional subtypes, and can also act as precursors to smaller ncRNAs such as snoRNAs, miRNAs, piRNAs, or siRNAs [10]. A summary of known non-coding RNA subtypes is provided in Table 1.

ncRNA	Full name
miRNA	microRNA
piRNA	PIWI interacting RNA
snoRNA	small nucleolar RNA
snRNA	small nuclear RNA
lncNRA	long non-coding RNA
circRNA	circular RNA
siRNA	small interfering RNA
tRNA	transfer RNA
tRF	tRNA fragments
tiRNA	tRNA-derived stress-induced RNA
TERC	Telomerase RNA Component
NAT	Natural Antisense Transcript
T-UCR	Transcribed ultra-conserved noncoding RNA

Table 1.

Common ncRNAs and standard nomenclature.

2. Role of ncRNAs in non-small cell lung cancer

2.1 Role of ncRNAs in NSCLC development

Lung adenocarcinoma is the most common lung cancer subtype, representing 40% of all lung NSCLC cases [11]. It is also the most common subtype diagnosed in never smokers. This subtype of lung cancer usually occurs in the lung periphery, evolving from mucosal glands, but can also be found in scars or areas of chronic inflammation [12].

In early 2000, Hanahan and Weinberg proposed a set of molecular and biochemical principles, that the most of human cancers share based on the knowledge accumulated at the time. The initial list of six traits included apoptosis evasion, self-sufficiency in growth signaling, insensitivity to anti-growth signals, sustained angiogenesis, limitless replicative potential with tissue invasion and metastasis [13]. Two decades later, new traits and principles were introduced in the list of hallmarks with the latest set containing emerging hallmarks and cancer enabling characteristics (Figure 1). The enabling characteristics involve unlocking phenotypic plasticity, non-mutational epigenetic reprogramming, senescent cells, and polymorphic microbiomes.

Figure 1.

The schematic representation of hallmarks of cancer (from Hanahan and Weinberg) [14].

The last few decades have seen a steep increase in interest and consequent number of studies related to ncRNAs role in lung cancer development. Significant attention is given to main ncRNA groups such as miRNAs, lncRNAs, snoRNAs, piRNAs and as well as tRNAs with its derivatives. In the next few sections, we will be discussing the roles that some groups within ncRNAs families play in lung cancer tumorigenesis, diagnosis, prognosis, and therapy.

2.1.1 MicroRNAs

MiRNAs are the most studied group of ncRNAs involved in lung cancer. MiRNAs make up a family of small noncoding RNAs with length of 21–25 nucleotides that can promote mRNA degradation by inhibiting their translation [15].

Several miRNAs can act on the same target mRNA, but a single miRNA can also regulate several different target mRNAs. Studies estimate that 30% of genes are regulated by miRNAs, and they participate various processes, such as gene regulation, apoptosis, cell differentiation and hematopoietic development [15, 16]. One of the hallmarks of cancer is sustained cell proliferation and unsuppressed cell growth [14]. Proteins such as kinases and kinase receptors play an important role in cell proliferation processes. For example, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) can attach to EGF receptor (EGFR), activating it, and causing further signaling pathway activation downstream. This leads to activation of the Ras/Raf/MEK/ERK and PI3K/Akt/mTOR pathways, two major signaling pathways, that play role in cell cycle progression and proliferation [17]. The EGFR is a direct target of numerous miRNAs, thus they play an important role in cell proliferation processes. These include, miR-146, miR-149, miR-7, miR-133, miR-27a-3p, miR-30, miR-34, miR-134, miR-145, miR-218, and miR-542-5p [15, 18, 19, 20, 21].

The fusion proteins EML4 and ALK are also able to induce PI3K/Akt/mTOR and Ras/Raf/MEK/ERK pathways. The EML4 and ALK getting more interest as potential targets in NSCLC therapy, and miR-96 was shown to suppress ALK post-transcriptionally in a cell model [22]. The miR-760 had negative impact on cell proliferation in NSCLC cell lines by downregulating ROS1 expression, another potential therapeutic target. Same as previous kinase proteins, ROS1 promotes cell proliferation and survival via SHP-1/SHP-2, JAK/STAT, PI3K/Akt/mTOR, and MEK/ERK pathways [23, 24].

The Kirsten rat sarcoma 2 virus (KRAS) gene, encodes for K-Ras, which also acts through EGFR, ALK and ROS to activate Ras/Raf/MEK/ERK pathway [25, 26]. In a recent study, miR-193a-3p was reported to inhibit KRAS-mutated lung tumor growth by targeting KRAS directly [27]. Similarly, miR-181a-5p was reported to suppress A549 lung epithelial cells proliferation and migration by downregulating KRAS [28]. In addition, miR-148a-3p was found to suppress cell proliferation in NSCLC by downregulating SOS1, a protein involved in the Ras signaling pathway [29]. The miR-1258 was reported to suppress tumor progression by targeting GRB2/Ras/Erk pathway, while miR-520-3p was found to be involved in PI3K/Akt/mTOR pathway in vitro [30, 31].

The highly expressed miR-15a and miR-16 suppress cyclin D1, an important regulator of cell cycle progression. Cyclin D1 acts upstream of retinoblastoma (RB) pathway, and its suppression leads to RB upregulation and cell cycle arrest, which is a necessary step leading to cell senescence or apoptosis [32]. The E2F3 gene, which encodes for transcription factor E2F3, is a target of miR-449a and has low expression levels in tumor tissues. The miR-449a upregulation suppresses E2F3 with consequent cell cycle blockage and cell senescence [33]. Finally, miR-641 and miR-660 were reported to promote lung cancer cells apoptosis through suppressing the p53 pathway by targeting MDM2 [34, 35]. Figure 2 summarizes the list of miRNAs involved in pathways associated with cancer hallmarks.

Figure 2.

A schematic diagram of miRNAs and their involvement in cancer hallmarks pathways (from Wu [15]).

In addition to regulating genes involved in cancer-related pathways, miRNAs are also involved in enabling cancer cells with unlimited replicative immortality. The miRNAs from the miR-29 family demonstrated tumor-suppressing effect in lung cancer cells in vitro by targeting DNMT3A and DNMT3B [36]. These DNA methyltransferases (DNMT) play a role in controlling telomere length similar to telomerase, being involved in cell replicative potential.

Another critical cancer feature that involves miRNAs is metastasis. An important factor in cancer metastasis is epithelial-to-mesenchymal transition (EMT), that normally takes place in the embryonic development stage. The EMT involves the loss of E-cadherin-mediated cell adhesion and increased cell motility, which in turn promotes tumor invasion and metastasis. The miRNA 15a was reported to inhibit metastasis in NSCLC by targeting BCL2L2, and its overexpression significantly inhibited cell viability, invasion and migration [37]. There are several main transcription factors (TFs) related to epithelial-to-mesenchymal (EMT) transition that are directly targeted by miRNAs. These TFs are Snail, Slug, ZEB1, ZEB2 and Twist [38]. For example, miR-126 was reported to regulate NSCLC cell invasion and migration suppressing EMT by directly targeting PI3K/AKT/Snail pathway [39]. MiR-346 is upregulated in NSCLC and it promotes cell proliferation, metastasis and hinders apoptosis by targeting XPC/ERK/SNAIL/E-cadherin pathway [40]. Interestingly, miR-22 and miR-30a are downregulated in NSCLC, and they regulate EMT by directly targeting Snail [41, 42, 43].

In order to grow and spread, tumor cells require nutrients and oxygen, and that requires angiogenesis. Vascular endothelial growth factors (VEGFs) are the most studied group involved in angiogenesis process. MiRNAs from the miR-200 family were reported to suppress neovascularization in lung cancer cell lines by targeting the VEGF [44, 45]. Additionally, miR-126 and miR-128 were observed to target VEGF-A and VEGF-C, respectively, and inhibit angiogenesis in vitro when upregulated [46, 47]. When overexpressed, miR-497 was found to suppress angiogenesis in lung cancer by targeting an HDGF, and inducing a independent pathway [48]. However, there are miRNAs that promote angiogenesis as well. The miR-494 enhances angiogenesis by targeting a VEGF suppressor PTEN [49, 50]. Besides that, miR-23a activates VEGF pathway by targeting PHD1 and PHD2, inducing HIF-1α, and consequently promoting neovascularization [50].

Apoptosis, a programmed cell death process, is another cancer hallmark that miRNAs are involved in. The dysregulation of miRNAs can lead to downregulation of genes involved in apoptosis, as well as other tumor suppressor genes. Apoptosis can take place via two major pathways, extrinsic and intrinsic; the intrinsic pathway is triggered by genotoxic agents, metabolic aberrations, and transcriptional signals, while the extrinsic pathway is triggered by extracellular apoptotic signals [15]. These mentioned cues are picked up by BH3-only proteins, which results in deactivation of BCL-2 regulator proteins, with consequent activation of caspases, and eventual cell death [51]. In this context, there are miRNAs that can have antiapoptotic or proapoptotic effects. As central regulators of apoptosis, members of the BCL-2 family can be targets of several miRNAs in lung cancer cells. For example, BCL-2 is a direct target of miR-7 in NSCLC cells [52]. Another member of BCL-2 family, BCL-W was identified as a direct target of miR-335 in A549 and NCI-H1299 cells [53]. The miRNA-15a, that was mentioned earlier, besides inhibiting metastasis, also induces cell apoptosis by targeting BC2L2 [37]. In addition to BCL-2 related miRNAs, miR-608 has been identified as BCL-XL-induced miRNA and was found to be involved in regulating apoptosis in A549 and SK-LU-1 cells [54]. Another miRNA that induces apoptosis in lung cancer cells (A549, H460, and 95D cell lines) is miR-192. This miRNA acts by targeting the retinoblastoma 1 (RB1) gene [55].

When it comes to miRNAs with antiapoptotic effect, the activation of PPAR-γ/VEGF pathway by miR-130b leads to indirect Bcl-2 targeting and consequent suppression of NSCLC cell apoptosis [56]. A study showed that miR-214 modulates NSCLC cells radiotherapy response through regulation of p38 MAPK, apoptosis, and senescence [57]. Similarly, miR-197 is upregulated in cancer tissue, and miR-197 knockdown in NIH-H460 and A549 cells promotes apoptosis induction. It was also shown that miR-197 can act on the p53 pathway on various levels to hinder apoptosis, and to promote cell proliferation [58]. Other miRNAs known to have antiapoptotic properties in lung cancer are miR-21, miR-212, miR-17-5p and miR-20a. The miR-21 targets negative regulators of the RAS pathway and targets proapoptotic genes in NSCLC [59].

MiRNAs also participate in cellular energetics deregulation processes in cancer cells. For example, a decreased expression of miR-144 in vitro can increase glucose uptake by upregulating the glucose transporter (GLUT1) expression [60]. Besides, miR-33b was found to suppress cell growth in NSCLC by downregulating an enzyme participating in glucose metabolism lactate dehydrogenase A (LDHA) [61]. MiR-199a impacts glycolytic pathways by suppressing HIF-1α and consequently inhibits cancer growth [62]. MiR-31-5p also supports glycolysis through downregulation of the inhibitor of HIF-1α (FIH) gene, thus promoting cancer development [63].

2.1.2 Long noncoding RNAs

Long noncoding RNAs include transcripts with sizes that exceed 200 nt and they make up the majority of ncRNAs [64]. There are several subgroups of lncRNAs which are involved in lung cancer. Among the lncRNAs that have gained more attention among investigators, natural antisense transcripts (NATs), transcribed ultra-conserved region RNAs (T-UCR), telomerase RNA components (TERC), circular RNAs (circRNAs), and pseudogenes transcripts are the ones that stand out.

LncRNAs cover a wide range of functions such as cell and tissue differentiation, gene imprinting, interaction with transcription mechanisms, regulation of mRNA splicing and degradation, protein translation modulation and sponging of miRNAs to name a few [65]. Given a wide range of biological functions, lncRNAs are also involved in lung cancer mechanisms as well, with members having tumorigenic or tumor suppressor properties.

One of the most studied lncRNAs in lung cancer is MALAT1 and it is known to have carcinogenic properties through modulation of miR-124/STAT3. It is reported to be overexpressed in lung cancer and to promote carcinogenesis via miR-124 sponging which is a STAT3 suppressor [66]. The MALAT1 was also reported to promote epithelial-mesenchymal transition and metastasis through miR-204/SLUG axis [67]. Another closely studied lncRNA is XIST, which was reported to inhibit NSCLC development when downregulated by activating miR-335/SOD2/ROS signal pathway [68]. The XIST was also reported to control NSCLC proliferation and invasion by modulating miR-186-5p [69]. Its silencing suppressed cell proliferation, migration, and invasion, and promoted apoptosis through regulation of miR449a and Bcl-2 [70]. Another reported lncRNA with pro-tumor effect is HOTAIR, which is overexpressed in a various cancer types, including NSCLC [71].

Natural antisense transcripts (NATs), are a group of lncRNAs that have transcript complementarity with other RNA transcripts [72]. The NATs NKX2–1-AS1, WRAP53, FAM83A and AFAP1-AS1 were found to be upregulated in lung cancer, with first two increasing lung malignant cell proliferation rates [73, 74], FAM83A promoting lung cancer cell progression [75], and AFAP1-AS1 promoting cell migration in NSCLC [76]. On top of that, pseudogene transcripts such as DUXAP8 and DUXAP10 were reported to be upregulated while SFTA1P was found to be downregulated in NSCLC [77, 78, 79]. Other lncRNAs that have been reported as involved in lung cancer are T-UCRs. For example, Uc.338 and Uc.339 increase malignant cell cycle progression and migration in cells, and they are upregulated in NSCLC, while Uc.454, having the same biological role is downregulated [80, 81, 82].

CircRNAs make up a large group of ncRNAs and are produced through an unconventional splicing event called back-splicing. CircRNAs are part of the lncRNAs with coding potential, they possess important biological functions such as acting as miRNA and protein inhibitors and regulating protein function [83]. Circular RNAs can have several action mechanisms: interact with chromatin histones, act as miRNA sponges, entrap transcription factors to prevent gene transcription, attach themselves to RNA polymerase, and encircle protein-coding exons [84].

The role of the interaction between circRNA, miRNA and mRNA in lung cancer development is gaining more attention. A study reported that circRAD23B is overexpressed in lung tumors compared to healthy adjacent tissue. In these tumors, the circRAD23B sponged miR-593-3p and miR-653-5p, and normalized the expression levels mRNAs CCND2 and TIAM1, respectively [85]. Similarly, the circRNA100146 sponged miR-361-3p and miR-615-5p as a result of its overexpression. The sponging of miRNAs leads to upregulation of their respective targets, miR-361-3p targeting COL1A1, NFAT5, and TRAF3, and miR-615-5p targeting MEF2C in H460 NSCLC cells [86]. Another finding shows how an apoptotic resistance takes place in NSCLC due to sponging of miR-497, a BCL-2 (anti-apoptotic gene) suppressor by circPVT1 [87]. On the other hand, some circular RNAs such as circHIPK3 can have tumor-promoting effect by binding miR-193, miR-124, miR-654, and miR-379. The non-coding RNA hsa_circ_0007385 behaves as a competing endogenous RNA to miR-181 and is an oncogenic circRNA. One of the contributors to the cancer proliferation and improved invasion capacity in lung adenocarcinoma is the upregulation of hsa_circ_001358 [88]. Another circRNA that acts as an oncogene is circ_0000735. It is upregulated in lung cancer tissue compared to normal tissue, and it sponges tumor suppressor miRNAs miR-11,179 and miR-1182. The association of circ_0000735 with the lung cancer cells capacity to self-renew was also reported [89].

Table 2 provides a list of studies that investigated the role of circRNA-miRNA-mRNA axis in the context of lung cancer. There are circular RNAs that can convey tumor-suppressing capabilities utilizing their ability to sponge pro-carcinogenic microRNAs. One example is circ-ITCH, a circRNA with tumor suppressing ability that stems from its interaction with miR-7 and miR-214 [97].

circRNA	Targeted miRNA(s)	Indirect target(s)	Biological effect(s)	Reference
circ-RAD23B	miR-593-3p	CCND2	↑ Cell invasion	[85]
	miR-653-5p	TIAM1
circRNA 100,146	miR-361-3p	NFAT5, COL1A1, TRAF3	↑ Cell invasion; ↑ proliferation	[86]
	miR-615-5p	MEF2C
circPVT1	miR-497	BCL-2	↑ apoptosis; ↓ cell proliferation	[87]
	miR-125b	E2F1	↑ tumorigenesis	[90]
circFGFR3	miR-22-3p	Gal-1, p-AKT, and p-ERK1/2	↑ cell invasion	[91]
circ_0004015	miR-1183	PDPK1	↓ survival; ↑ cell viability; ↑ proliferation; ↑ cell invasion; ↓ drug (gefitinib) resistance	[92]
circPUM1	miR-326	CCND1 and BCL-2	↑ cell proliferation; ↑ cell invasion; ↑ cell migration	[93]
circFLI1	miR-584-3p	ROCK1	↑ Metastasis	[94]
circABCB10	miR-1252	FOXR2	↑ cell proliferation; ↑ cell migration	[95]
circHIPK3	miR-124	SphK1, STAT3 and CDK4	↑ Cell proliferation; ↓ apoptosis	[96]

Table 2.

Oncogenic circRNAs acting as miRNA sponges in NSCLC, and their biological effects (adapted from Braicu et al. [84]).

2.1.3 Transfer RNAs

Transfer RNAs are a type of small ncRNA that plays a major role in protein synthesis by serving as a link between mRNA and the growing chain of amino acids [98]. Besides its known conventional function, tRNA dysregulated expression has been found in some malignancies. Transfer RNAs can be cleaved by ANG (angiogenin) and form tRNA-derived stress-induced RNAs (tiRNAs), which in turn can be further changed into tRNA-derived fragments (tRFs) [99]. As an example of tRNA dysregulation, the tiRNAs ts-3676 and ts-4521 were found to be downregulated in a lung tumor tissue when compared with healthy tissue [100]. Also, ts-46 and ts-47, tiRNAs with tumor suppressing properties were found to be downregulated in lung cancer. The introduction of mentioned tiRNAs in two lung cancer cell lines demonstrated their ability to negatively impact cell proliferation rate and self-renewal capacity in tumor cells.

An association between the tiRNAs ts-101 and ts-53 and PiwiL2, a protein that plays an important role in silencing transposons, was also found in lung cancer [101]. The tRNAs-Leu and tRNAs-Val were found to be overexpressed in lung tumor tissue, in 37% and 26% of samples, respectively. On the other hand, tRF-Leu-CAG was found to be overexpressed in not only lung tumor tissue, but also in serum and cell lines. The involvement of tRF-Leu-CAG in cell cycle progression and cell proliferation was reported, and its overexpression was shown to be associated with NSCLC progression. This tRF seems to interact with protein AURKA and its role in lung cancer needs further investigation [102].

2.1.4 Small nucleolar RNAs

Small nucleolar RNAs (snoRNAs) are a class of noncoding RNAs that guide the chemical modifications of ribosomal RNAs, transfer RNAs and small nuclear RNAs. SnoRNAs are located in the region of introns of genes that code for proteins or lncRNAs [84]. The snoRNAs also known to be a source of piRNAs (piR30840) [103]. The analysis of The Cancer Genome Atlas (TCGA) data has revealed that snoRNAs U60, U51, U28, U63, U104, HBI-100, U59B, HBII-419, HBII-142, and U30 are overexpressed, and HBII-420 is under-expressed, in lung cancer. Interestingly, SNORD15A was found to be significantly downregulated in non-smoker lung tissue with an overall difference pattern better distinguished in non-smokers compared to smokers [104]. The fact that SNORD78 is overexpressed in vitro, leads to cell proliferation, promotes EMT and consequently leads to enhanced invasion capacity, adds to the critical role snoRNAs play in NSCLC development [105]. Additionally, SNORD116–26 was found to be downregulated in tumor-initiating cells, while SNRA42 and SNORA3 were upregulated in the same cell type. Silencing of SNORA42 in cancer stem cells (CSCs) results in lower levels of tumorigenesis in lung cancer cells, while decreased expression of SNORA3 and SNORA42 in patients with lung tumors is associated with improved overall survival rate [106].

2.1.5 PIWI-interacting RNAs

The P-Element induced wimpy testis (PIWI) are a type of proteins highly conserved in plants and animals which are responsible for stem cell and germ cell differentiation [107]. The PIWI-interacting RNAs (piRNAs) make up the largest group of noncoding RNAs in animal cells and they originate from transposable elements (TE), mRNA and lncRNA (Table 3) [84, 113, 114, 115].

Type of ncRNA	Transcript	Expression in tumor vs. control	Biological function	Reference
piRNA	piR-34,871, piR-52,200	↑	↑ apoptosis; ↑ cell proliferation	[108]
	piR-35,127, piR-46,545	↓	None	[108]
	piR-L-163	↓	↓ cell migration; ↓ cell cycle progression	[109]
tRNA fragments	ts-46, ts-47, ts-101 and ts-53	↓	↓ cell proliferation	[101]
	tRF-Leu-CAG1	↑	↑ cell cycle progression; ↑ cell proliferation	[102]
	tRF-Leu-CAG2
YRNA	hY4 RNA	↑	↑ cell proliferation	[110]
SNORD	SNORA42	↑	Preserve tumor initiating make-up of cancer cells.	[106]
	U60,; U63,; U28,; U51,; U104,; HBII-419,; U59B,; HBII-142,; HBI-100, U30	↑	None	[104]
	HBII-420	↓
	SNORD78	↑	↑ in vivo tumorigenesis↑ lung tumor cell proliferation	[105]
	SNORA47, SNORA68, SNORA78, SNORA21, SNORD28 SNORD66	↑	None	[111]
	SNORD33, SNORD66 SNORD76	↑	None	[112]
NAT	NKX2–1-AS1	↑	↑ lung malignant cell proliferation.	[73]
	WRAP53		↑ lung malignant cell proliferation	[74]
	FAM83A		↑ tumor progression	[75]
	AFAP1-AS1		↑ cell invasion and metastasis	[76]
Pseudogene transcripts	SFTA1P	↓	↓ cell migration and invasion	[79]
	DUXAP8	↑	↑ malignant cell survival, proliferation, tumorigenesis	[77]
	DUXAP10		↑ malignant cell survival, proliferation, migration, tumorigenesis	[78]
T-UCR	Uc.338	↑	↑ malignant cell cycle progression, invasion, and migration.	[80]
	Uc.339	↑	↑ malignant cell cycle progression, and migration.	[81]
	Uc.454	↓	↓ malignant cell cycle progression, invasion, and migration	[82]

Table 3.

Examples of ncRNA involved in lung cancer and their biological effects (adapted from Braicu et al. [84]).

2.2 Role of ncRNAs in NSCLC diagnosis and prognosis

2.2.1 MicroRNAs

When diagnosing a lung cancer, several miRNAs can be used to determine the cancer subtype. As an example, miR-205 can be utilized to distinguish SCC from other lung cancer NSCLC subtypes, whereas miR-124a is known for its specificity to LAC [116, 117]. In addition, four upregulated miRNAs (miR-93, miR-221 miR-30e, and miR-205,) show specificity to SCC, while another five upregulated miRNAs (miR-100, let-7e, miR-125a-5p, miR-29b and miR-29c) are specific to LAC [118]. Being able to distinguish primary lung tumor from metastatic tumor plays an important role for diagnosis and prognosis. The high levels of miR-182 were reported to be associated with primary lung tumors, while overexpression of miR-126 points to tumors of metastatic nature [119]. In a similar fashion, overexpression of miR-552 and miR-592 helps to distinguish primary LAC from metastatic colorectal adenocarcinoma [120].

The possibility of performing diagnosis based on miRNA expression in bodily fluids (blood, plasma, sputum) remains a topic of high interest for researchers due to feasibility and high potential as a diagnostic tool. One of the suggestions is utilizing a set of miRNAs, a miR-test kit comprising of 13 miRNAs which include miR-140-5p miR30b-5p, miR148a-3p, let-7d-5p, miR-191-5p, miR-30c-5p, miR-328-3p, miR-331-3p, miR-374a-5p, miR-29a-3p, miR-484, miR-223-3p, and miR-92a-3p [121]. A commercially available kit named microRNA signature classifier (MSC) uses the expression ratio among 24 miRNAs in lung cancer as a diagnostic tool [122].

MiRNAs play an important role as prognosis tools as well. For example, under-expression [123] of let-7 miRNA is associated with shortened postoperative survival. Another study reported that miR-21 and miR-155 were associated with poor recurrence-free survival for NSCLC patients [124].

2.2.2 Long noncoding RNAs

Regarding lncRNAs in the context of lung cancer diagnosis and prognosis, a number of lncRNAs were found to be of use. For example, the overexpression of HOTAIR promotes metastasis and indicates a poor prognosis for NSLC patients [71]. Similarly, MALAT1 in conjunction with protein thymosin beta 4 can be used to predict metastasis and survival in early-stage NSCLC determined using early stage and metastasized tumor tissue removed with surgery [125]. The lncRNA CDKN2B-AS1 with a more common name as antisense noncoding RNA in the INK4 locus (ANRIL) is associated with advanced lymph node metastasis and poor overall survival. A high expression of large intergenic RNA (lincRNA) PVT1 was shown to be associated with tumor advanced stage and advanced stage of tumor-node-metastasis (TNM) in NSCLC tissues, with consequent poor prognosis [126]. Other lncRNAs associated with poor NSCLC prognosis are listed in the Table 4.

LncRNA	Expression in NSCLC (vs. normal tissue)	Clinical association	Reference
MALAT1	↑	Poor prognosis	[125]
HOTAIR	↑	Poor survival and prognosis	[71]
SPRY4-IT1	↓	Poor prognosis	[128]
CDKN2B-AS1	↑	Poor survival	[129]
CCAT2	↑	Poor prognosis	[130]
PVT1	↑	Poor prognosis	[131]
IRAIN	↓	Poor prognosis	[132]
LCAL1	↑	Poor prognosis	[133]
SOX2OT	↑	Poor survival and prognosis	[134]
AFAP1-AS1	↑	Poor prognosis	[135]
TATDN1	↑	Poor prognosis	[136]
FOXD2-AS1	↑	Poor prognosis	[137]
SNHG1	↑	Poor survival and prognosis	[138]
ARHGAP27P1	↑	Aggressive tumorigenesis and poor prognosis	[139]
UCA1	↑	Poor prognosis	[140, 141]
HOXA11-AS	↑	Poor prognosis	[142]
LINC00473	↑	Poor prognosis	[143]
LINC00673	↑	Poor prognosis	[144]
BC087858	↓	Poor prognosis	[145]

Table 4.

LncRNAs associated with poor NSCLC prognosis (adapted from Osielska et al. [127]).

2.2.3 YRNAs

YRNAs and YRNA-derived small RNAs (YsRNAs) are also found in NSCLC patients, and they are gaining traction as potential lung cancer biomarkers. When assessed in A549 NSCLC cell lines, Ys4RNA was significantly downregulated. On the other hand, a deep sequencing expression analysis of small RNAs in plasma extracellular vesicles from LAC patients, SCC patients and healthy individuals showed upregulation of Ys4RNA [110]. The finding shows it may be used as a potential circulating biomarker for NSCLC diagnosis.

2.3 Role of ncRNAs in NSCLC therapy

The last two decades have seen a significant change in lung cancer management and treatment. Achievements in tumor genotyping and other molecular approaches helped to develop tools for personalized medicine adding targeted therapy and immunotherapy to traditional chemotherapy and radiation therapy. Investigation of PD-L1 status and genetic mutations within tumor helps to predict the most appropriate targeted therapy or immune checkpoint blockers (ICBs). The combination of platinum-based doublet therapy has served as a standard for advanced stage NSCLC patients [146]. Surgical resection remains as the most effective therapy for NSCLC in stages I and II, however, there is a high percentage of tumor recurrence, with varying 5-year overall survival rate depending on the NSCLC stage [147, 148]. In this section, we will discuss the roles of noncoding RNAs in NSCLC therapy.

2.3.1 MicroRNAs

Owing to their role that they play in gene regulation, miRNAs do also impact the response to cancer radiotherapy, chemotherapy, and targeted therapy [149]. MiRNAs have been shown to modify the sensitivity and resistance to the most common platinum-based therapy. For example, cisplatin sensitivity in NSCLC in vitro was reported to increase as a result of miR-106b overexpression, its further suppression of polycystin 2 (PKD2) levels, and consequent downregulation of P-glycoprotein [150]. Another study reported how miR-503 blocked drug efflux mechanism and suppressed a number of proteins associated with drug resistance (MRP1, MDR1, survivin, Bcl-2 and ERCC1), consequently improving cisplatin sensitivity [151]. Interestingly, the upregulation of miR-196 had an opposite effect on the same proteins, thus promoting drug efflux and cisplatin resistance [152]. When it comes to radiation therapy, miR-200c was shown to have a positive impact on radiotherapy by suppressing oxidative response genes and inhibiting DNA repair [153]. The upregulation of miRNA-138 was reported to induce radio-sensitization in NSCLC cells [154]. Similarly, miR-25 modulated the radio-sensitivity in lung cancer cells by directly inhibiting BTG2 expression [155].

In the context of molecular targeted therapy, miR-21 were reported to induce resistance to gefitinib through activation of ALK and ERK and inhibiting PTEN in lung cancer [156, 157]. The ALK resistance is another aspect where miRNAs play a role, and it was reported that histone H3 lysine 27 acetylation (H3K27ac) loss and inhibition of miR-34a is one the ways how ALK-positive lung cancer acquire ALK inhibitor resistance [158]. Another study observed a fingerprint of 7 circulating miRNAs (miR-493-5p, miR-411-3p, miR-494-3p, miR-215-5p, miR-495-3p, miR-93-3p and miR-548j-5p) to have a strong association with improved survival in lung cancer patients following the nivolumab treatment.

Currently, the most studied therapeutic miRNAs are let-7, miR-150, miR-29b, miR-200c, miR-34, and miR-145. The miR-34a alters the p53/mir-34/PD-L1 pathway by targeting PD-L1 and liposomal mimic MRX34 is a first miRNA that was used in phase I clinical trials initially showing promising antitumor activity [159]. However, the trials had to be terminated due to adverse effects [18]. Table 5 summarizes the list of miRNAs associated with drug therapy and radiation sensitivity and resistance.

miRNA	Target/Pathway	Reference	miRNA	Target/Pathway	Reference
Chemo-sensitive			Chemo-resistant
let-7	LIN28	[160]	miR-15b	PEBP4	[161]
miR-7	EGFR	[162]	miR-21	PTEN, SMAD7	[163, 164]
miR-17, 20a, 20b	TGFβR2	[165]
miR-17 and miR-92 family	CDKN1A, RAD21	[166]	miR-27a	RKIP	[167]
miR-34a	PEBP4	[168]	miR-92a	PTEN	[169]
miR-101	ROCK2	[170]	miR-106a	Unknown	[171]
miR-106b	PKD2	[150]
miR-135b	FZD1	[172]	miR-181c	WIF1, Wnt/β-catenin pathway	[173]
miR-137	NUCKS1	[174]
miR-138	ZEB2	[175]	miR-196	MDR1, MRP1, ERCC1,	[152]
miR-146a	CCNJ	[176]		Survivin and Bcl-2
miR-181b	TGFβR1/Smad signaling pathway	[177]	miR-205	PTEN, Mcl-1 and Survivin	[178, 179]
miR-184	Bcl-2	[180]	miR-488	eIF3a-mediated NER signaling pathway	[181]
miR-218	RUNX2	[182]
mir-296	CX3CR1	[183]
miR-379	EIF4G2	[184]
miR-451	c-Myc-survivin/rad-51 signaling	[185]
miR-503	Down-regulation of MDR1, MRP1, ERCC1,	[151]
	Survivin and Bcl-2
miR-9600	STAT3	[186]
Radio-sensitive			Radio-resistant
miR-29c	Bcl-2, Mcl-1	[187]	miR-21	PTEN, HIF1α, PDCD4	[163, 188]
miR-138	SENP1	[154]	miR-25	BTG2	[155]
miR-200c	PRDX2, GAPB/Nrf2, and SESN1	[153]	miR-210	HIF1	[189]
miR-328	γ-H2AX	[190]	miR-1323	PRKDC	[191]
miR-449a	Unknown	[192]
miR-451	c-Myc-survivin/rad-51 signaling	[185]
EGFR TKI-sensitive			EGFR TKI-resistant
miR-126	Akt and ERK pathways	[193]	miR-21	PTEN, PDCD4	[194]
miR-134/miR-487b/miR-655 cluster	MAGI2	[195]	miR-23a	PTEN/PI3K/Akt pathway	[196]
miR-200c	PI3K/Akt pathway	[197]	miR-30 family	PI3K-SIAH2	[198]
miR-223	IGF1R/PI3K/Akt pathway	[199]	miR-214	PTEN/AKT pathway	[200]
miR-483-3p	integrin β3/ FAK/Erk pathway	[201]
ALK TKI-sensitive
miR-200c	ZEB1	[202]

Table 5.

miRNAs associated with lung cancer therapy and radiation sensitivity or resistance (adapted from Wu et al.) [15].

2.3.2 Long noncoding RNAs

The MALAT1 is the first lncRNA used in targeted therapy studies with lncRNAs molecules. The targeted therapy decreased the amount of MALAT1 using antisense oligonucleotides (ASOs), consequently reducing lung cancer metastasis in a murine model [203]. The protein STAT3 can impact the MRP1 transcription by binding upstream, thus STAT3 activation is associated with MRP1 and MDR1 upregulation, which in turn enhances lung tumor cells resistance to cisplatin [204, 205]. In similar manner, the lncRNA KCNQ1OT1 expression was shown to be positively correlated with MDR1 expression, and KCNQ1OT1 knockdown can improve paclitaxel sensitivity in LAC cells [206]. A different study has demonstrated the ability of the lncRNA XIST to act as a competing endogenous RNA by sponging miR-144-3p, and thus regulating the expression of MRP1 [207]. In another study, the lncRNA SNHG14 was found to regulate the DDP-resistance of NSCLC cell through miR-133a/HOXB12 pathway [208].

The EGFR-tyrosine kinase inhibitors (EGFR-TKIs) prevent the lung cancer cells growth by inhibiting EGFR activity. The lncRNA LINC00460 eliminates miR-769-5p, which in turn promotes EGFR expression and therefore enhances NSCLC cells resistance to Gefitinib. The association between expression of LINC00460 and expression of proteins with multidrug-resistance, such as BCRP, P-gp, and MRP1 was reported [209]. The lncRNA colon cancer-associated transcript-1 (CCAT1), targets the miR-130a-3p/SOX4 axis and promotes cisplatin resistance in NSCLC in vitro [210]. Another lncRNA that induces cisplatin resistance in NSCLC is SNHG7, and it does it by upregulating MRD1 and BCRP via PI3K/AKT pathway [211]. A larger list of lncRNAs involved in drug resistance in NSCLC can be found in Table 6.

Drug	lncRNA	Effect on NSCLC	miRNA Target	Expression in tumor vs. control	Reference
Gefitinib	LINC00460	Promote	miR-769-5p	↑	[205]
	LINC00665		EZH2		[213]
	RHN1-AS1	Inhibit	miR-299-3p	↓	[214]
Cisplatin	MALAT1	Promote	STAT3	↑	[215]
	CCAT1		miR-130a-3p		[207]
	SNHG7		PI3K/AKT		[211]
	ROR		Akt/mTOR		[216]
	LINC00485		miR-195		[217]
	BLACAT		miR-17		[218]
	XIST		miR-17		[219]
	HOXA-AS3		HOXA3		[220]
	PAX6		PI3K/AKT		[221]
	PVT1		miR-216b		[222]
	NBAT1	Inhibit	PSMD10	↓	[223]
	TUG1		miR-221		[224]
	MEG3		p53, Bcl-xl		[225]
Vincristine	MEG3	Inhibit	miR-650	↓	[226]
Paclitaxel	KCNQ1OT1	Promote	Not known	↑	[227]
	NEAT1		Akt/mTOR		[228]
Multiple	DGCr5	Promote	miR-330-5p	↑	[229]
	ITGB1		Snail		[230]
	SNHG12		miR-299-3p		[231]
	SOX21-AS1		p57		[232]
	TUSC-7	Inhibit	miR-146	↓	[233]
	FENDRR		HuR		[234]
	MBNL1-AS1		miR-301b-3p		[235]
Crizotinib	HOTAIR	Promote	ULK1	↑	[236]
	ROR		ALK		[237]
EGFR-TKIs	UCA1	Promote	Akt/mTOR	↑	[238]
	LINC00460		miR-149-5p		[239]

Table 6.

LncRNAs related to drug resistance in NSCLC (adapted from Zhou et al. [212]).

3. The role of ncRNAs in small cell lung cancer

Small-cell lung cancer makes up 15% of all lung cancer incidences with 250,000 new cases and at least 200,000 annual death rates globally [240]. Carcinogens present in tobacco are responsible for the initiation of SCLC with accompanying inactivation of tumor suppressors p53 and RB in the majority of SCLC patients [241]. Amplification of MYC family members and frequent mutations of chromatin modeling proteins (EP300, CREBBP and MLL2) and Notch family members were also observed in SCLC patients [241, 242]. The ability of ncRNAs to modulate gene expression at transcriptional, post-transcriptional and epigenetic levels makes them significant factors, with clinical implications and functional roles in SCLC.

3.1 Role of miRNAs in SCLC

The miR-335 is downregulated in multi-drug resistant human SCLC cell lines. When overexpressed, miR-335 leads to the sensitization of the human SCLC cell lines to chemotherapy and radiotherapy by targeting PARP-1 (Poly [ADP-ribose] polymerase 1) gene. On top of that, overexpression of miR-335 promoted cell apoptosis, inhibited cell migration ability in vitro, and inhibited tumor growth in vivo [243]. In a similar fashion, the noncoding miR-22 was found to enhance the radiosensitivity in human SCLC cell line by targeting WRNIP1, promoting apoptosis and inhibiting cell migration when overexpressed [244]. Another noteworthy observation is that methylation in SCLC cell lines reduced the expression of miR-34 family members, which are known tumor suppressive miRNAS. The transfection of miR-34b/c to SCLC cell lines (H1048 and SBC5) resulted in significantly decreased cell growth, migration, and invasion [245]. The circulating miR-141 from plasm exosomes and serum of SCLC patients was found to be upregulated when compared to healthy volunteers. The ability of miR-141 to promote angiogenesis by targeting KLF12 (Kruppel-like factor 12) was demonstrated [246]. Earlier, miR-126 was found to inhibit SCLC cells proliferation by targeting SLC7A5, whereas the miR-217 was reported to inhibit proliferation and to promote apoptosis in SCLC cells by targeting PCDH8 [247, 248]. The miR-195 promoted SCLC apoptosis by inhibiting Rap2C protein-dependent MAPK signal transduction [249]. Similarly, the miR-26b was reported to promote apoptosis and suppress tumorigenicity by targeting myeloid cell leukemia 1 protein [250]. In another study, miR-485-5p was found to suppress the proliferation, migration, and invasion of SCLC cells by targeting flotillin-2 [251].

3.2 Role of lncRNAs in SCLC

There are five lncRNAs that are known to be involved in SCLC: HOTTIP, HOTAIR, TUG1, CCAT2 and PVT1 [252]. The lncRNA HOTTIP is upregulated in SCLC, is associated with chemoresistance and shorter survival. The HOTTIP acts through HOTTIP/miR-574-5p/EZH1 and HOTTIP/miR-216a/BCL-2 axes [253, 254]. The lncRNA HOTAIR, same as in NSCLC, is upregulated in SCLC, and is associated with lymphatic invasion and chemoresistance in SCLC [255, 256]. The ncRNAs TUG1 and CCAT2 are also upregulated in SCLC, where TUG1 is associated with chemoresistance, clinical stage and shorter survival, whereas CCAT2 is associated with malignant status and poor prognosis [257, 258]. Similarly, the lncRNA PVT1 is upregulated in SCLC patients and is associated with lymph node metastasis and clinical stage [259]. Recently, Linc00173 was reported to modulate glucose metabolism and multidrug chemoresistance in SCLC [260].

4. Conclusion

Lung cancer remains to be a leading cause of cancer death. The past two decades have witnessed an unprecedented growth in cancer research, and new findings have continuously supported and expanded the notion that ncRNAs play a significant part in all aspects of lung cancer. Future advancements in molecular technology will help researchers answer more complicated questions about this disease, including uncovering the additional ncRNAs and the mechanisms by which these regulated lung cancer development, progression, and response to therapy. The heterogeneity of lung cancer subtypes, variation in mutational burden, sex differences, and different stages of cancer further necessitate a combination of individualized therapies, such as platinum-based doublet chemotherapy, immunotherapy, and targeted therapy to combat lung cancer effects on human health. The relative success of combined therapies underlines the need to further investigate the roles and functions of ncRNAs within cancer processes. A better understanding of tumorigenesis mechanisms and identification of ncRNAs and other participants will play an important role in improving lung cancer diagnosis, prognosis, prevention, and therapy.

Acknowledgments

This work was supported by NIH grant HL159764 (PS).

Conflict of interest

The authors declare no conflict of interest.