The Impact of lncRNAs and miRNAs on Apoptosis in Lung Cancer

Apoptosis is a coordinated cellular process that occurs in several physiological situations. Dysregulation of apoptosis has been documented in numerous pathological situations, particularly cancer. Non-coding RNAs regulate apoptosis via different mechanisms. Lung cancer is among neoplastic conditions in which the role of non-coding RNAs in the regulation of apoptosis has been investigated. Non-coding RNAs that regulate apoptosis in lung cancer have functional interactions with PI3K/Akt, PTEN, GSK-3β, NF-κB, Bcl-2, Bax, p53, mTOR and other important cancer-related pathways. Globally, over-expression of apoptosis-blocking non-coding RNAs has been associated with poor prognosis of patients, while apoptosis-promoting ones have the opposite effect. In the current paper, we describe the impact of lncRNAs and miRNAs on cell apoptosis in lung cancer.


INTRODUCTION
Apoptosis is a well-organized and coordinated cellular process that happens in several physiological situations. Aberrant regulation of apoptosis has also been documented in numerous pathological situations, particularly cancer. In fact, cancer is one of the circumstances where this process is reduced, leading to evolution of malignant cells that will not perish. Apoptosis is regulated by a complex mechanism involving numerous pathways. Deficiencies in apoptotic pathways lead to malignant transformation of cells, enhancement of metastasis and induction of resistance to chemotherapy/ radiotherapy. Meanwhile, apoptosis has been considered as a target of several anticancer modalities (1). Both intracellular and extracellular stimuli can regulate apoptosis. This process is described by morphological alterations in the cells including fragmentation and condensation of the nuclear compartment, permeabilization of the outer membrane of mitochondria, membrane blebbing, cell shrinkage and finally formation of apoptotic bodies (2). Two extrinsic and intrinsic pathways are involved in the induction of cell apoptosis. While the extrinsic pathway is stimulated by death receptors, namely Fas, TNF receptors and TRAILs, the intrinsic pathway is initiated by DNA damage, energy starvation and hypoxia, which can dephosphorylate and cleave pro-apoptotic proteins, resulting in their recruitment in the mitochondria (3). Both pro-apoptotic and anti-apoptotic members of the Bcl-2 family proteins regulate intrinsic apoptotic pathway (4).
Recent studies have shown that non-coding RNAs (ncRNAs) have an important regulatory role on induction of apoptosis. In fact, regulation of cell apoptosis is the main route of function of many of these transcripts in the carcinogenic events (5). This group of transcripts has several types, two of them i.e. long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have attained more attention in cancer biology. LncRNAs have typically sizes more than 200 nucleotides and are transcribed by RNA polymerase II, except for few cases do not harbor open reading frame and translation-termination region, yet, lncRNAs can be spliced, 5'-capped and get polyadenylated tails. Their specific three-dimensional conformation permits them to interact with several classes of biomolecules including proteins, DNA or RNA. These interactions are framed through base pairing or construction of network (6). LncRNAs partake in regulation of gene expression, differentiation of cells and alteration of chromatin structure (6).
miRNAs have been shown to regulate expression of a high proportion of human genes. They mainly target 3' UTR of genes to suppress their expression or degrade the corresponding RNAs. Several aspects of cell functioning including apoptosis is regulated by miRNAs (7). Figure 1 illustrates that aberrant expression of various ncRNAs could contribute in modulation FIGURE 1 | A schematic representation of the role of non-coding RNAs in triggering the mitochondrial pathway of apoptosis in human lung cancer. The Bcl-2 family of proteins could play an effective role in modulating apoptosis via regulating mitochondrial cascade. The anti-apoptotic proteins Bcl-2 and Bcl-xL are located in the exterior part of mitochondrial wall and can suppress cytochrome c release. The pro-apoptotic Bcl-2 proteins Bax, Bad, Bim, and Bid could be located in the cytosol but may be transferred to mitochondria following induction of death signaling pathway, where they could elevate the release of cytochrome c (8,9). The mitochondrial cascade of apoptosis could be considered as the most commonly deregulated form of cell death in a variety of human cancers. Furthermore, aberrant expression of various non-coding RNAs could have a crucial part in dysregulating the mitochondrial pathway of apoptosis in lung cancer. of the mitochondrial pathway of apoptosis in the context of lung cancer.
In the current paper, we describe the impact of lncRNAs and miRNAs on cell apoptosis in lung cancer.

MIRNAS AND APOPTOSIS IN LUNG CANCER
Suppression of PI3K/AKT pathway in EGFR mutant lung cancer cells has led to dysregulation of 17 miRNAs among them have been members of the miR-17~92 cluster. These miRNAs function in a coordinated manner to increase the activity of the EGFR cascade. Suppression of miR-19b expression in EGFR mutant lung cancer cells has led to re phosphorylation of ERK, AKT and STAT and effector proteins. Consistently, it has resulted in enhancement of apoptosis, while reduction of cell cycle progression, colony formation and migration. Administration of gefitinib along with miR-19b antagonism has decreased migration and colony formation in a synergistic manner implying the cooperation between EGFR and miR-19b in the regulation of oncogenesis. PPP2R5E and BCL2L11 have been recognized as main targets of miR-19b, through their inhibition, miR-19b regulates cell proliferation and resistance to apoptosis, respectively (10). miR-21 is another miRNA that regulates apoptosis of lung cancer cells via influencing the PI3K/ Akt/NF-kB signaling pathway. Inhibition of miR-21 has enhanced apoptosis via this route. ASPP2 has been recognized as the target of miR-21 in NSCLC cells. miR-21 silencing has also inhibited migration, invasion, and epithelial-mesenchymal transition (EMT). Besides, miR-21 inhibition has stimulated cell apoptosis through caspase dependent route. Taken together, miR-21 silencing can induce cell apoptosis via reducing activity of the PI3K/Akt/NF-kB signaling (11). miR-24 is another oncogenic miRNA which is up-regulated in lung cancer tissues, particularly in high grade and large-sized tumors. Consistently, higher expression of miR-21 predicts lower overall survival (OS) of patients. Functionally, miR-24 enhances the viability, proliferation and cell cycle transition, while inhibiting cell apoptosis through binding with MAPK7 (12). miR-26 is a down-regulated miRNA in lung cancer cells. Forded overexpression of miR-26 induces cell apoptosis and enhances activity of caspase-3 and caspase-9. On the other hand, miR-26 FIGURE 2 | A schematic summary of the role of various non-coding RNAs in modulating the process of autophagy in human lung cancer. Several non-coding RNAs affect lung cancer progression through modulating autophagy and apoptosis cascades in human lung cancer cells. As an illustration, overexpression of lncRNA PANDAR as a tumor suppressor via directly targeting Beclin-1, LC3-I and LC3-II could activate both autophagy and apoptosis cascades, and thereby suppressing progression of lung cancer (14). In addition, lncRNA CASC2 could suppress autophagy and enhance apoptosis pathway in non-small cell lung cancer cells through modulating the miR-214/TRIM16 axis. Moreover, p62 expression level was significantly elevated but Atg-5 expression and the ratio of LC3-II/LC3-I were considerably reduced in the CASC2-overexpressing cells (15). silencing has increased levels of LC3 protein and the autophagyassociated genes in lung cancer cells. Besides, miR-26 has been shown to influence apoptosis and autophagy through suppressing expression of TGF-b in a JNK dependent route. Besides, miR-26 has been reported to affect the endoplasmic reticulum stress (ERS) signaling pathway (13). Figure 2 represents the role of several ncRNAs in regulating autophagy cascade in human lung cancer. Table 1 shows the list of miRNAs that regulate apoptosis in lung cancer.
Apoptosis-related miRNAs have been shown to influence survival of lung cancer patients. For instance, expression of miR-21 predicts lower OS of patients with NSCLC (12). Moreover, over-expression of miR-125b has been associated with poor prognosis in NSCLC (24).

LNCRNAS AND APOPTOSIS IN LUNG CANCER
Expression of FER1L4 has been remarkably decreased in plasma and tissue samples of patients with NSCLC as well as related cell lines. Forced over-expression of this lncRNA has reduced cell proliferation, migratory aptitude and invasiveness. FER1L4 has been shown to up-regulate PTEN and p53 expressions, suppress AKT phosphorylation expression, therefore enhancing the fraction of apoptotic cells. Functionally, these effects are mediated through the PTEN/AKT/p53 pathway (58). On the other hand, expression of PCAT1 has been increased in NSCLC tissues and cell lines. In vitro studies have shown that PCAT1 stimulates cell proliferation and invasion while suppressing cell apoptosis. In addition, PCAT1 has been shown to interact with the RNA-binding protein DKC1. PCAT1 and DKC1 exert synergistic effects in NSCLC. They enhance activity of VEGF/ AKT/Bcl-2/caspase9 pathway in these cells (59). WT1-AS is a down-regulated lncRNA in NSCLC cell lines which is shown to sponge miR-494-3p. Up-regulation of WT1-AS has increased apoptosis of lung cancer cells and attenuated progression of NSCLC through up-regulation of PTEN and subsequent inactivation of PI3K/AKT pathway (60). GACAT1 is another regulator of apoptosis which has been found to be up-regulated in NSCLC tissues in association with poor survival of patients. Functionally, GACAT1 enhances proliferation and cell cycle progression and inhibits apoptosis through sponging miR-422a and increasing expression of YY1 transcription factor (61). HOXC-AS2 is another up-regulated in NSCLC samples which increases proliferation, migration, and EMT, while suppressing apoptosis. HOXC13 has been identified as functional target of HOXC-AS2. Notably, HOXC-AS2 and HOXC13 can enhance expression of each other (62). Expression of SNHG1 has been found to be increased in NSCLC parallel with up-regulation of FRAT1. SNHG1 knock down has suppressed proliferation, increased cell apoptosis and precluded migration and invasiveness of these cells. Mechanistically, SNHG1 sponges miR-361-3p and to release FRAT1 from inhibitory effects of this miRNA (63). Table 2 shows the role of lncRNAs in regulation of apoptosis in lung cancer.

NCRNAS, CELL APOPTOSIS AND IMMUNOTHERAPY
Since immunotherapy has an emerging role in the treatment of lung cancer (98), identification of the role of ncRNAs in immune regulation and response of lung cancer to immunotherapy is important. A number of apoptosis-regulating ncRNAs have essential roles in this regard. For instance, miR-155 and miR-17~92 are involved in differentiation regulatory T cells (Tregs) and their function (99). miR-21 and miR-26 through downregulation of TAP1 and reduction in expression of HLA class I antigens affect response to immunotherapies (100). miR-138, miR-155, miR-34 and miR-146a have been found to affect immune checkpoints (101). MALAT1 is an lncRNA which is possibly involved in the immunotherapy resistance through induction of immunosuppressive phenotypes in stem cells (102). NEAT1 can affect response to immunotherapy through modulation of miR-155/Tim-3 (103). The exact roles of these ncRNAs in conferring resistance to immunotherapeutic approaches have not been elucidated in lung cancer; yet based on the results obtained from similar studies in other cancer types, these ncRNAs are expected to simultaneously affect apoptosis and response to immunotherapy in lung cancer.

DISCUSSION
Cell apoptosis, as one of the major dysregulated processes in the carcinogenesis of lung cancer has been shown to be regulated by ncRNAs. In the current review, we have explained the impact of miRNAs and lncRNAs on apoptosis in lung cancer. These ncRNAs interact with PI3K/Akt, NF-kB, Wnt/b-catenin, EGFR, TGF-b and other cancer-related pathways. Therefore, they not only regulate apoptosis, but also influence other aspects of lung carcinogenesis. Figure 3 depicts the role of ncRNAs in modulating apoptosis through Wnt/b-catenin cascade in human lung cancer.
Manipulation of expression of apoptosis-regulating lncRNAs and miRNAs represent a strategy for combating carcinogenesis as well as resistance to chemo/radiotherapy. Some of the apoptosis-regulating miRNAs/lncRNAs have been shown to influence prognosis of lung cancer. The observed correlation between their expression and patients' survival is due to their impact on disease progression as well as response of patients to EGFR inhibitors and chemotherapeutic agents. EMT is another important feature of lung cancer cells which is regulated by a number of apoptosis-regulating miRNAs/lncRNAs indicating the intercalation between cancer-related processes.
An acknowledged route of function of lncRNAs in the regulation of apoptosis in lung cancer is their impact on expression of miRNAs. Based on the importance of apoptotic pathways in determination of response of lung cancer patients to conventional as well as targeted therapies, identification of the impacts of lncRNAs/miRNAs on apoptosis and prior profiling of these ncRNAs in clinical samples would help in prediction of response of patients to each therapeutic regimen and design of personalized treatment strategies. The advent of high    Higher expression of HULC was associated with poor prognosis of NSCLC (85) 100 lung cancer tissues and their adjacent non-cancerous tissues from patients (67 males and 33 females; mean age, 62; age range,  Higher expression of ZEB2-AS1was associated with higher proliferation of NSCLC. (96) 56 NSCLC and adjacent tissues Lower levels of EPB41L4A-AS2 was associated with poor prognosis in NSCLC (86) 60 NSCLC patients Higher expression of TRPM2-AS was associated with poor prognosis in NSCLC (92) throughput sequencing strategies has facilitated conduction of this approach in the clinical settings. Finally, the possibility of lncRNAs/miRNAs tracing in the peripheral blood of patients has opened a new opportunity for early detection of emergence of resistance to conventional or targeted therapies and modulation of therapeutic regimens to enhance the survival of affected individuals.

AUTHOR CONTRIBUTIONS
MT and SG-F wrote the draft and revised it. HS, AA, JM, and MM collected the data, designed the tables and figures. All authors contributed to the article and approved the submitted version. FIGURE 3 | A schematic summary of the role of miRNAs and lncRNAs in regulating apoptosis cascade in lung cancer via Wnt/b-catenin pathway. Accumulating evidence has delineated that apoptotic cells are negative for b-catenin. This indicates that the Wnt/b-catenin signaling cascade could be inactive in apoptotic cells. Whilst, b-catenin is expressed in the membrane, cytoplasm, and nucleus of non-apoptotic epithelial cells around these apoptotic cells. Therefore, Wnt/b-catenin signaling cascade could be activated in non-apoptotic epithelial cells via apoptotic cells (104). As an illustration, downregulation of miR-125b could play an effective role in inhibiting expression of p-Akt, p-GSK3b, Wnt, and b-catenin, and could promote caspase-3 activity and Bax protein expression in human non-small cell lung cancer. Thereby, this could lead to suppressing the proliferation and triggering the apoptosis of tumor cells (24). Furthermore, another study have illustrated that upregulation of lncRNA SNHG20 could have a crucial part in elevating the proliferation and suppressing the apoptosis of NSCLC cells through targeting miR-197 via regulating the Wnt/b-catenin signaling cascade. Downregulation of this lncRNA could result in remarkable reduction of TCF and LEF1 expression in the Wnt/bcatenin pathway (75).