Overview upon miR-21 in lung cancer: focus on NSCLC

Abstract

Considering the high mortality rate encountered in lung cancer, there is a strong need to explore new biomarkers for early diagnosis and also improved therapeutic targets to overcome this issue. The implementation of microRNAs as important regulators in cancer and other pathologies expanded the possibilities of lung cancer management and not only. MiR-21 represents an intensively studied microRNA in many types of cancer, including non-small cell lung cancer (NSCLC). Its role as an oncogene is underlined in multiple studies reporting the upregulated expression of this sequence in patients diagnosed with this malignancy; moreover, several studies associated this increased expression of miR-21 with a worse outcome within NSCLC patients. The same pattern is supported by the data existent in the Cancer Genome Atlas (TCGA). The carcinogenic advantage generated by miR-21 in NSCLC resides in the target genes involved in multiple pathways such as cell growth and proliferation, angiogenesis, invasion and metastasis, but also chemo- and radioresistance. Therapeutic modulation of miR-21 by use of antisense sequences entrapped in different delivery systems has shown promising results in impairment of NSCLC. Hereby, we review the mechanisms of action of miR-21 in cancer and the associated changes upon tumor cells together a focused perspective on NSCLC signaling, prognosis and therapy.

Electronic supplementary material

The online version of this article (10.1007/s00018-018-2877-x) contains supplementary material, which is available to authorized users.

Introduction

Data collected into GLOBOCAN 2012 revealed that lung cancer classifies as the third most diagnosed cancer having also a high rate of mortality; the fact that makes this malignancy a major concern worldwide [1]. Non-small cell lung cancer (NSCLC) represents the most common subtype of lung cancer, with a higher incidence in developed countries [2]. The decreased survival rate is partially determined by late diagnosis and challenges in prevention, such as the poor impact of anti-smoking commercials [3]. The molecular alterations that characterize NSCLC are multiple and they work in networks leading to a molecular imbalance that generates a malignant phenotype. These modifications include changes in gene expression, copy number alterations, different methylation patterns, but also changes in the expression of non-coding RNAs (ncRNAs) [4]. It has been proved that the expression pattern of particular microRNAs (miRNAs) can be used as biomarkers for the histotype classification of NSCLC [5]. Therefore, the discovery of new biomarkers for early diagnosis and putative therapeutic targets for personalized medicine could significantly improve the clinical care and survival rate of lung cancer patients.

Once considered ‘junk DNA’, the non-coding part of the genome has proven to be of crucial importance, as the resulted transcripts of these sites exert modulatory functions upon target genes/molecules that further affect important signaling pathways [6]. These DNA regions are represented by different ncRNAs, and one major class is delineated by miRNAs. These small sequences constitute a class of ncRNAs encountered all over the genome [7], with nearly 22 nucleotides in final length after the process of maturation, form in which they can complementary bind to a messenger RNA (mRNA) of different genes [8–11]. The essential role of miRNAs results from the fact that one single molecule is able to target multiple mRNAs, thus influencing the expression of hundred of genes simultaneously [12–14]. The report is that about 30% of the genes are targeted by different miRNAs in humans [15].

The association of miRNAs with different types of cancer was first reported upon the illustration that downregulation of miR-15 and miR-16 was positively correlated with the presence of chronic lymphocytic leukemia phenotype in patients [6]. The involvement of miRNAs in several cancers was emphasized in a comprehensive review that underlines the implications of miRNAs in all hallmarks of cancer [16]. Besides, several other comprehensive studies are taking in discussion the importance of miRNAs in various types of cancer [17–22].

The present paper is focused on the role of miR-21 in lung cancer, more specifically in NSCLC. As this sequence was repeatedly reported as upregulated in this malignancy with further implications upon cancer progression and also patients’ prognosis, we are taking in discussion the possible utilization of this miRNA as early diagnosis tool and also therapeutic target. Moreover, specific mechanisms of action of miR-21 within the malignant environment are presented.

MiR-21 functions as an oncogene in several cancers

One of the first characterizations of pri-mir-21 appeared in 2004, being also one of the first miRNAs to be described with its processing steps [23]. Since then, deregulated expression of this miRNA together with its role has been reported in a wide range of malignancies (Table 1) [24, 25]. The aberrant expression appears because of altered molecular networks that may involve proteins such as signal transducer and activator of transcription 3 (STAT3), epidermal growth factor receptor (EGFR), transforming growth factor-β (TGF-β) [24, 26]. Moreover, it seems that miR-21 is also able to influence the p53 pathway [27], a well-known gene with decisive roles in cancer [28–30]. One of the first indications refers to the role of miR-21 in the apoptosis of glioblastoma cells. Reporting upregulated expression of miR-21 in tissue samples from glioblastoma patients, the researchers further demonstrated the association of high levels of miR-21 with the inhibition of apoptosis in several glioblastoma cell lines [31]. Abnormal expression of miR-21 in glioblastoma patients was confirmed within a further study, as it appears to be upregulated in tumor tissue when compared with non-tumor brain samples [32]. The possible use of miR-21 as a circulating biomarker for minimally invasive diagnosis approaches, can apply to glioblastoma patients, as its levels in plasma samples are significantly increased than in those with other neurological disorders [33]. In breast cancer patients, the expression of miR-21 appears to be upregulated in tumor compared with normal breast tissue [34]. Moreover, the expression of miR-21/miR-155/miR-365 in serum samples could help discriminate between breast cancer patients and healthy controls [35]. Another study found that in vitro and in vivo breast cancer models, as well as sera of actual patients treated with metformin exhibit a reduction in miR-21; this inhibition was further examined in vitro where SESN1 and CAB39L (direct target genes of miR-21) were found as increased, with further effects upon AMPK activation, mTOR reduction and also impairment of cell invasion and migration [36]. In the case of young gastric cancer patients, miR-21-5p was found as upregulated in tissue samples and the expression was correlated with recurrence in these individuals [37]. Hematological malignancies are also characterized by deregulated miR-21 profiles, as this miRNA was reported as overexpressed in patients with nucleophosmin 1 gene (NPM1)—mutant acute myeloid leukemia [38]. Another study refers to the elevated expression of miR-21 in colon cancer patients, expression that was also associated with high mortality rates in these patients [39]. Using in situ hybridization (ISH) and microarray analyses on tissue samples from patients with prostate cancer, Li et al. [40] showed that higher grades were associated with upregulated expression of miR-21 and its overexpression predicts poor biochemical recurrence-free survival. Likewise, using peripheral blood mononuclear cell samples for evaluation of mi-21 expression in prostate cancer patients, it was assessed that high expression level could be a useful predictor of the presence, recurrence and metastasis [41]. The high expression of miR-21 in hepatocellular carcinoma tissues obtained from patient samples is a measure for shorter disease-free survival (DFS) and overall survival (OS) in an Asian population [42]. In addition, the levels of miR-21 in serum samples from patients with the same malignancy can separate healthy volunteers from hepatocellular carcinoma patients, being also coupled with the clinical stage and the presence of metastases [43].

Table 1.

MiR-21 target genes and altered pathways in different types of cancer

Cell line/animal model	Target genes	Pathway	References
Trp53 (–/–) miR-21 (–/–) mice	PTEN	TP53-associated apoptosis and senescence	[27]
MCF10A (ER-Src cells)	PTEN	PTEN/Akt/NF-κB	[26]
SUM159PT	SESN1 and CAB39L	Invasion and migration	[36]
CCR6+ Treg (from murine breast cancer)	PTEN	Cell proliferation	[47]
HeLa, HT-3	RASA1	EMT and cell migration	[48]
DU145	PTEN	Angiogenesis	[49]

PTEN phosphatase and tensin homolog, SESN1 sestrin 1, CAB39L calcium binding protein 39 like, RASA1 RAS P21 protein activator 1

The importance of miR-21 in regulating metastasis in different types of cancer is already known, as it was stated in several publications [44, 45]. In triple negative breast cancer (TNBC) cells, miR-21 modulates the invasive and metastatic potential of the cells, as part of the LPA₁/Pi3 K/ZEB1/miR-21pathway that further regulates the lysophosphatidic acid (LPA) activity [46]. The aberrantly expressed miR-21 also mediates the activity of CCR6⁺Treg cells, through PTEN/Akt pathway, controlling their capacity to proliferate in breast cancer tumors [47]. MiR-21 contributes to epithelial-to-mesenchymal transition (EMT) in cervical cancer by modulating the expression of Rasa1 gene (RAS p21 protein activator 1). Therefore, by indirectly influencing the activity of Ras, miR-21 contributes to the migration potential of these cells [48]. Besides, miR-21 is known to modulate angiogenesis in prostate cancer cells; effect enabled by the indirect modulatory action upon HIF1α and VEGF [49].

All these investigations suggest that miR-21 functions as an oncogene in several types of cancer, making this molecule an important element that should be considered for early diagnosis, but also as therapeutic target for new precision medicine approaches (Fig. 1).

Fig. 1.

Mir-21 is upregulated in several types of cancer, contributing to the malignant transformation. The ubiquitous presence of upregulated miR-21 in many cancers reveals the importance of this miRNA as diagnosis/prognostic marker and therapeutic target for precision medicine

In NSCLC, it was stated that NF-kB influences the expression of miR-21 through a mechanism that involves the binding of NF-kB to the promoter region of miR-21, leading to its upregulation [50]. As reminded, the mechanism of action of miR-21 as an oncogene in different types of cancer involves the modulation of several genes (Supplemental Fig. 1—miR-21 target genes based on strong experimental evidences) that are part of important signaling pathways, such as PDCD4/NF-κB/TNF-α [51], PTEN/PI3K/AKT [52], or PTEN/Akt/HIF-1α pathway [53]. In NSCLC, the publications in which the target genes of miR-21 were evaluated in cell lines, identified PTEN [50, 54, 55], PDCD4 [56], HIF1α and hMSH2 [56] as direct targets (Table 2). PTEN is a well-known tumor suppressor gene, frequently mutated or inactivated in NSCLC cell lines [57–59]. By targeting PTEN transcript in NSCLC, miR-21 influences the growth and invasion of these cells [54].

Table 2.

Validated target genes for miR-21 in NSCLC cell lines

Cell line	Target genes	Pathway	References
A549	PTEN	Cell growth, invasion	[54]
NCI-H446, A549, NCI-H460	hMSH2	Cell proliferation	[60]
A549	SMAD7	Cell invasion/chemosensitivity	[61]
A549	PTEN	Cell growth, metastasis, chemo-/radioresistance	[55]
A549	PTEN	Cell viability/cisplatin sensitivity	[50]
A549	HIF1α	Radioresistance	[62]
A549	PDCD4	Radioresistance	[56]

hMSH2 human MutS protein homolog 2, PTEN phosphatase and tensin homolog, HIF1α hypoxia-inducible factors 1 alpha, PDCD4 programmed cell death protein 4, SMAD7 mothers against decapentaplegic homolog 7

Reports have shown that miR-21 intervenes in EMT by modulating the expression of SMAD7 in NSCLC cells. Besides, as SMAD7 represents the target for carboplatin therapy, miR-21 expression could serve as an important biomarker for carboplatin chemosensitivity screening of patients with NSCLC [61]. The signaling pathways disturbed according to the validated target genes of miR-21 in NSCLC cell lines, and their mechanism of action are illustrated in Fig. 2.

Fig. 2.

Processes influenced by the action of miR-21 in NSCLC, according to the published literature. By inhibiting the expression of Akt via PTEN, miR-21 increases the rate of growth and proliferation. The inhibition of PTEN also results in impairment of migration and invasion and in sensitivity to chemotherapy. Sensitivity to radiotherapy is controlled via inhibition of PDCD4 or activation of HIF-1α gene and PI3 K/Akt/mTOR pathway by miR-21. Increased glycolysis appears as a result of stimulated expression of HIF-1α by miR-21. The action of stimulating the migration and invasion is reflected in the inhibition of SMAD7 that modulates the expression of TGF-β

Mechanisms and regulation of miR-21 biogenesis and dysregulation in cancer

MiR-21 is localized in the 17q23.2 chromosome region within an intron of vacuole membrane protein 1 (VMP1) coding region—its human homologue being TMEM49. The same publication revealed the fact that AP-1 promotes the transcription of miR-21, process dependent on chromatin remodeling complexes [63]. Post-transcriptional regulation of miR-21 is also backed by the fact that when pre-miR-21 of exogenous origins is expressed in A549 and MCF-7 cells, mature miR-21 is not produced [64].

The expression of miR-21 appears to be regulated by different factors. In prostate cancer, it has been reported that reactive oxygen species (ROS) play an important role in regulating its expression. Within this context, it was previously described that in prostate cancer, ROS resulting from NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activity, is necessary for miR-21 expression. The enhanced expression of NADPH and miR-21 contributes to the invasive potential of prostate cancer cells [65]. Mir-21 expression in also indirectly regulated by CD24, which activates Src, leading to the upregulation of miR-21 in RKO and Geo colorectal cell lines [66].

In glioma cells, the biogenesis of miR-21 is modulated by DDX23 (DEAD-box RNA helicase), a gene with implications in mRNA splicing [67]. DDX23 influences the expression of miR-21 during the processing steps of pri-miR-21 to the precursor one, by intervening at the level of Drosha protein [68]. In glioblastoma cell lines, miR-21 appears to be regulated by MPS1 (Monopolar spindle 1), a key protein whose action is necessary to ensure spindle stability [69], which in turn, modulates the expression of PDCD4 and MSH2 (MutS protein homolog 2), with further effects on cell proliferation [70]. Multiple myeloma represents another pathology characterized by miR-21 deregulation. In this malignancy, the STAT3 pathway through IL-6 (Interleukin-6) is responsible for the upregulation of miR-21 [71].

Another mechanism involved in the regulation of miR-21 refers to the epigenetic changes that disturb the expression of this miRNA. Therefore, in colorectal cancer, it was demonstrated that miR-21 is under epigenetic regulation, the promoter of miR-21 being activated or repressed upon the action of histone modifications [72]. In addition, the mechanism of miR-21 modulation by retinoblastoma binding protein 2 (RBP2) in chronic myeloid leukemia cells implies also epigenetic changes, such as decreased H3K4 trimethylation in its promoter [73].

Deregulated expression of miR-21 in lung cancer

Regarding the expression of miR-21 in lung cancer, specifically NSCLC, the studies published up to date have reported upregulated levels of this sequence in diagnosed patients (Supplemental Table 1). One of the first publication that mentions the possible role of miR-21 expression profile in NSCLC patients as prognostic factor was in 2008, when Markou et al. [74] measured the levels of the miRNA in tissue samples from NSCLC patients and reported the upregulated levels in tumor tissue compared with its paired normal controls. The investigation went further, as it was observed that overexpression of miR-21 resulted in poor overall survival within these patients. The results were confirmed by the work of Cho et al. [75], which validated the upregulation of miR-21 in tumor tissue vs. non-tumor ones through microarray analysis and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The feasible value of miR-21 as a non-invasive biomarker for NSCLC diagnosis, assessed from sputum samples, was confirmed with high specificity and sensitivity, as its expression was significantly higher than in sputum samples from cancer-free individuals [76]. The same observation was made on plasma samples. In this sense, upregulated expression of miR-21 could stand as a biomarker for distinguishing between NSCLC patients and healthy plasma donors [77]. Another important aspect concerning the deregulated expression of miR-21 in NSCLC refers to its relevance in gefitinib resistance/sensitivity. This feature of miR-21 was assessed in pc-9 and pc-9/GR (Gefitinib-resistant) cells, where upregulation of miR-21 correlated with downregulation of PTEN, further inducing low-gefitinib sensitivity and decreasing the overall survival of these patients [78].

Another aspect worth mentioning is the fact that miR-21 is often part of panels of genes/miRNAs proposed as biomarkers for NSCLC early detection. In this concern, along with miR-221, miR-145, miR-20a, and miR-223, miR-21 is present as a member of the 5 miRNAs signature from plasma samples with high potential as minimally invasive method for early diagnosis of NSCLC patients [79]. Similarly, another panel of miRNAs (miR-20a, miR-21, miR-223, and miR-145) detected in plasma samples that includes also miR-21, could stand as a potential tool for early diagnosis, as their elevated levels could discriminate between NSCLC patients and healthy controls [80]. Upregulated expression of miR-21 is also encountered in serum samples of NSCLC patients together with downregulated expression of tumor suppressor miRNAs: miR-148a/b and miR-152, profile that could also serve as a biomarker tool for NSCLC screening [81].

Exosomal miR-21 expression in lung cancer

MiRNAs can exert their functions not only within the cell in which they are expressed but also in other parts of the body, their small size enabling them to travel together with the biological fluids—including through small nanoparticles released by cells [82, 83]. Exosomes are vesicles derived from donor cells with a size ranging between 20 and 120 nm [84, 85]. Their composition includes the presence of proteins usually localized in the cell at the cytosol level, in the membranes of endocytic compartments and also plasma membrane [86, 87], suggesting that exosomes are derived from lipid structures called intraluminal vesicles and are extravasated from the cell in a process that involves the fusion with the plasma membrane [88]. Their role mainly consist in the ensuring of intracellular communication through transportation of different regulatory small molecules [89]. The exosomal cargo contains also miRNAs and mRNAs, thus underlining the importance of miRNAs in processes of cell communication [90]. Exosomes have been reported to play key roles in cancer via internalization and transport of regulatory molecular cargos with further direct consequences upon the target cell and also tumor development [91]. MiRNAs that are exported via exosomes, with their oncogenic/tumor suppressor potential, contribute also to the involvement of exosomes in cancer progression [92, 93], but can be also exploited as potential biomarkers for diagnosis/prognosis due to the presence in liquid samples (Fig. 3).

Fig. 3.

Upregulation of miR-21 in NSCLC cells and exosomes released from malignant transformed cells. The expression of miR-21 in exosomes harvested from NSCLC patients could be used as minimal invasive biomarkers for early diagnosis/prognosis

In NSCLC, several studies investigated the association between the levels of exosomal miR-21 and the prognostic of patients affected by this pathology. Therefore, the upregulation of miR-21-5p in exosomes isolated from plasma was confirmed, indicating that exosomal miR-21 could stand as a possible biomarker for minimally invasive diagnosis of NSCLC [94]. As it can be observed in Table 3, miR-21 present in exosomes can offer valuable information concerning the prognosis of patients with NSCLC. Accordingly, it was stated that using Cox proportional hazard model, high miR-21-5p levels in plasma exosomes of these patients determined a poor overall survival (OS) [95].

Table 3.

List of publications evaluating the correlation between the expression levels of miR-21 and the outcome of patients with NSCLC

Expression of miR-21	Outcome of NSCLC patients	Sample size/type	Statistical method	References
↑	Shorter RFS (p = 0.042) and OS (p = 0.043)	210 patients/tissue	Log-rank test	[96]
↑	Poor prognosis for OS (p = 0.011)	423 patients (TCGA data)	Cox regression analysis	[101]
↑	Poor OS (p = 0.003)	10 patients/plasma exosomes	Cox proportional hazard analysis	[95]
↑	Poor DFS (p = 0.016)	195 patients/plasma exosomes	Log-rank test/multivariate Cox analysis	[106]
↑	Worse cancer-specific survival (p = 0.018/0.014) and RFS (p = 0.0005)	317 patients/tissue	Univariate Cox analysis	[97]
↑	Reduced OS (p = 0.027)	48 patients/tissue	Log-rank test	[74]
↓	Worse DSS (p = 0.027)	95 lymph-node positive patients/tumor cells	Multivariate analysis	[105]
↑	Lower DFI (p = 0.022—tissue/p  = 0.045—plasma) and OS (p = 0.037—tissue/p = 0.065—plasma)	40 patients/tissue 37 patients/plasma	Log-rank test	[107]
↑	Worse OS (p = 0.0045)	261 patients (gefitinib treatment)/plasma	Univariate and multivariate analysis	[102]
↑	Shorter DFS (p = 0.008)	58 patients/tissue	Cox proportional hazard regression model	[98]
↑	Poor OS (p = 0.015)	88 patients/serum	Univariate and multivariate analysis	[103]
↑	Shorter OS and PFS (p < 0.001)	204 patients/tissue	Multivariate logistic regression	[99]
↑	Shorter OS (p = 0.0065—tissue) Poor prognosis (p = 0.019—serum)	70 patients/serum and tissue	Log-rank test Univariate cox analysis	[108]
↑	Lower survival time (p = 0.001)	80 patients/serum	Pearson correlation	[104]
↑	Lower PFS (p < 0.001)	108 patients/plasma	Kaplan–Meier analysis	[109]
↑	Shorter DFS	46 patients/tissue	Kaplan–Meier analysis	[78]
↑	Lower PFS (p = 0.0003) and OS (p = 0.0045)	80 patients/tissue	Kaplan–Meier analysis	[100]

NSCLC non-small cell lung cancer, RFS relapse/recurrence-free survival, OS overall survival, PFS progression-free survival, DFS disease-free survival, DSS disease-specific survival, DFI disease-free interval

MiR-21 prognostic value in lung cancer

The large number of studies that assessed the expression of miR-21 in patients with NSCLC and investigated the association with survival, indicate that this molecule is actively involved in the modulation of malignant transformation and progression. Table 3 illustrates the studies that reported the connection of high miR-21 expression and its prognostic significance. The majority of the results indicate that independent of the sample type, upregulation of miR-21 is correlated with negative outcome in tissue [96–100], plasma [95, 101, 102] or serum samples [103, 104]. One particular case is represented by the work of Stenvold et al., where they used ISH for the evaluation of miR-21 independent expression on tumor cells and stromal cells. Therefore, it was reported that the upregulation of miR-21 in tumor cells positively correlates with better prognosis, these results being limited only to lymph-node positive NSCLC patients. When the expression was evaluated in the stroma, overexpression of miR-21 appeared to negatively correlate with the prognosis of patients with lymph-node negative status [105].

MiR-21 turned out to be an important biomarker in predicting brain metastases in patients with NSCLC. In this regard, a recent publication reported that when dividing the patients with NSCLC according to their brain metastasis status (present/absent), the expression of miR-21 is higher in those with positive for brain metastases [110].

Data from the Cancer Genome Atlas (TCGA) strengthen the importance of miR-21 in the context of the present article. Thus, to achieve a more comprehensive view upon the effects of hsa-miR-21 expression in lung cancer, TCGA miRNA-seq data were downloaded from the UCSC (University of California Santa Cruz) Cancer Browser in the form of a data matrix containing the normalized, log2 transformed miRNA expression levels for 515 tumor tissues and 46 peritumoral normal tissues, together with the corresponding clinical parameters. After removing the normal tissues and selecting the samples with complete survival information, Kaplan–Meier survival analysis was performed for 486 samples for which miR-21 expression levels were separated into high-expression and low-expression according to the median. The test presented statistical significance, with a p value of 0.047, as seen in Fig. 3, meaning that the expression of miR-21 in lung adenocarcinoma patients can be correlated with the overall survival of these patients. Precisely, upregulated expression of miR-21 is associated with poor overall survival of patients with LUAD (Fig. 4).

Fig. 4.

MiR-21 expression associated with cancer-specific mortality in lung adenocarcinoma patients. Kaplan–Meier curve on TCGA data from patients with LUAD (n = 486). Patients were separated in low- and high-expression based on the median value; TCGA The Cancer Genome Atlas, LUAD lung adenocarcinoma; *p < 0.05

Association with long non-coding RNAs in NSCLC

Long non-coding RNAs (lncRNAs) represent a class of ncRNAs, along with miRNAs, with gene sequences that are not transcribed into proteins, but unlike miRNAs, this class contain transcripts that have more than 200 nucleotides in length and their post-transcription modifications include alternative splicing and 5′ capping [111, 112]. LncRNAs role in cancer is now an established fact, as their involvement in modulation of mRNAs stability, splicing and translation [113] has indirect repercussions on the disruption of signaling pathways that contribute to neoplastic transformations [114].

As it was formerly stated, upregulated miR-21 expression correlates with the presence of NSCLC. Contrarily, GAS5 (Growth Arrest Specific 5) lncRNA is found with as poorly expressed in NSCLC, possibly indicating an association between this lncRNA and miR-21. Therefore, the link was explored, and the obtained data suggested that miR-21 affects the sensitivity of H157 and H460 cells to cisplatin by regulating the expression of GAS5 through interfering with PTEN pathway [115]. H19, an lncRNA whose role was assessed in extensive studies, was studied together with miR-21 in the context of NSCLC. It was revealed that upregulated expression of both mir-21 and H19 appear in patients with NSCLC and that their expression, taken together, has prognostic value for these individuals. These results indicate the fact that a panel consisting of miR-21 and H19 could stand as a biomarker of diagnosis/prognosis [116]. Supplemental Table 2 presents the putative mir21—lncRNAs interactions retrieved from miRWalk database and sorted according to the seed length, demonstrating other possible regulatory mechanisms involving miR-21 and lncRNAs that are not yet backed up by research data.

MiR-21 delivery systems for targeted therapy

Concerning the connection between miR-21 expression and other features that characterize malignant cells, such as resistance to therapy, there are several publications that assessed the implication of miR-21 in blocking the response to therapy. It was underlined fact that upregulation of miR-21 in various types of cancer is correlated with resistance to different categories of chemotherapeutics, such as cisplatin, gemcitabine, and teniposide [117]. The association between plasma levels of miR-21 and the acquired resistance to the treatment consisting of epidermal growth factor receptor (EGFR) and tyrosine kinase inhibitors (TKIs) was assessed in patients that followed the above-mentioned therapies and the disease continued to progress after the treatment. The conclusion was that in these patients, miR-21 levels were more increased than at baseline [118]. The radiosensitivity mediated by PI3K/Akt pathway represents also an aspect controlled by miR-21. Accordingly, it was reported that in A549 cells, the inhibition of miR-21 improved the sensitivity to radiotherapy, followed by increased apoptosis and decreased cell proliferation [119]. Furthermore, the action of miR-21 in mediating radiation-induced bystander effects through TGF-β1-miR-21-ROS pathway was previously described. Therefore, upregulated expression of miR-21 in bystander H1299 cells determined increased ROS levels and also DNA damage. As expected, inhibited expression of miR-21 significantly reduced cell proliferation [120]. In plasma samples, the high levels of miR-21 in NSCLC patients divided based on their reaction to cisplatin-based therapy, respectively, responders and non-responders, could be a useful predictor of the therapeutic response, and constructive, worse outcome [109].

The use of different delivery systems for miRNAs with tumor suppressor properties could represent a major improvement for personalized treatment in cancer patients. The limitations of this treatment option are imputed to the deficient stability of these molecules, but also to the faulty delivery systems used [121–123].

Given the ubiquitous character of miR-21 as an oncogene in a large number of cancers, several publications evaluated the effect of miR-21 or antisense miR-21 (anti-miR-21) in different cancer cell lines. In this interest, using poly[lactic-co-glycolic]acid (PLGA) nanoparticles as carriers for antimiR-21 in combination with temozolomide in glioma cell lines (miR-21 is overexpressed in glioblastoma), it was demonstrated that PLGA is an efficient delivery system for miRNAs, leading to the downregulation of the endogenous sequence and further reducing the number of viable cells [124]. An efficient delivery system for antisense-oligodeoxynucleotide (antisense-ODN) against miR-21 in glioblastoma cells was also developed by Song et al., using a R3V6 complex stable enough to deliver anti-miR-21 and inhibit its effects on cells. Therefore, anti-miR-21 administrated in R3V6 reduced the survival of glioma cells showing also less cytotoxicity than PEI25k (polyethylenimine) system [125]. It was previously shown that combination therapies could have a significant impact on glioblastoma cells [126], strategy that could work also with miR-21 inhibitors combined with other targeted agents (e.g., metformin, sorafenib). Lentiviral vectors are also used as delivery systems for miR-21 inhibitors, precisely for multiple myeloma cells using in vitro and in vivo models with anti-proliferative effects [127].

Anti-miR-21 was also used in mice models, and orthotropic injection of 3WJ-EGFRapt nanoparticles carrying anti-miR-21 appeared to specifically target the TNBC tumor and release the miRNA within these cells. The inactivation of miR-21 was observed upon the effect on its target genes, PDCD4 and PTEN, their expression being upregulated in the targeted TNBC cells [128]. In solid cancer tumors, graphene represents a valuable biomaterial for targeted therapy due to its characteristics, such as biocompatibility and the possibility to be functionalized [129]. A complex that incorporated graphene was utilized for in vitro studies of breast cancer tumors as carrier for miR-21 to overcome multi-drug resistance (MDR). Precisely, the researchers used polyethylenimine (PEI)/poly (sodium 4-) (PSS)/graphene oxide (GO), a complex easy to be internalized by target cells, as a delivery system for adriamycin and anti-miR-21; their results showed that this complex managed to keep its properties and also efficiently released the anti-miR-21 sequence [130]. In pancreatic cancer, nanoparticles consisting of PEG-PEI-IONPs (polyethylene glycol–polyethylenimine–magnetic iron oxide nanoparticles) were used to co-deliver gemcitabine and anti-miR-21 in pancreatic cancer cells, where the inhibition upon migration and proliferation of these cells confirmed the appropriate delivery and release of the therapeutic agents. The overexpressed PDCD4 and PTEN and underexpressed E-cadherin and Vimentin validated the inhibition of miR-21 [131]. Nanocapsules containing anti-miR-21 trapped inside the shell through in situ polymerization were used upon glioblastoma and breast cancer cell lines, and the results evidenced the fact that nanocapsules carrying anti-miR-21 are more efficient than lipofectamine. The same therapeutic strategy was used on murine models, where the tumor growth was suppressed as a result of angiogenesis inhibition in mice injected with U87 tumor cells [132].

Natural compounds delivered within cancer cells were also associated with modulatory capacity upon miR-21 levels—another perspective upon modifications of miRNAs level [133, 134]. Besides the suppressive action upon miRNAs, these compounds have additional inhibitory effects on cancer progression and can be also delivered inside targeted nanostructures [9, 135, 136].

The use of nanoparticles to deliver anti-miR-21 in NSCLC studies appears to be a domain insufficiently explored, as there are limited publications of this type; one study used calcium phosphate nanoparticles as carriers for anti-miR-21 in NSCLC cell lines. The anti-miR-21 was used to overcome radioresistance, determined and maintained by the presence of ALDH1⁺ and CD133⁺ cancer stem cells. By targeting these cells and releasing miR-21 and miR-95 inhibitors within them, subsequently inhibition of miR-21 and miR-95 was achieved, and to the lung cancer tumors were sensitized to radiation therapy [137].

Conclusions

MiR-21 is one of the most intensively studied miRNA, and its role as an oncogene was proven in many types of cancer with an upregulated expression in patients affected by these malignancies. The deregulated expression of miR-21 affects pathways that control cell proliferation, migration, survival, but also angiogenesis. The already mentioned publications suggest a possible role of miR-21 as a valuable target for personalized treatment of cancer patients. The limited number of target genes for miR-21 in NSCLC validated up to date, represents a major advantage, as its inhibition would only disrupt a restrained amount of signaling pathways, and its action would be specifically targeted and easily controlled. The advancements made within biomaterials development and nanodelivery systems are encouraging for the future use of miR-21 as part of the standard treatment for different types of cancers, and particularly for NSCLC. The use of anti-miR-21 agents encapsulated in different functionalized nanoparticles—to prevent their degradation, minimize their interaction with cell structures and maximize their uptake—brings new insights into the management of NSCLC.

Electronic supplementary material

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Abbreviations

NSCLC

Non-small cell lung cancer

PTEN

Phosphatase and tensin homolog

LUAD

Lung adenocarcinoma

TCGA

The Cancer Genome Atlas

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.