miR-30a-5p together with miR-210-3p as a promising biomarker for non-small cell lung cancer: A preliminary study

1. Introduction

Lung cancer incessantly remains not only the most common (around 13% of cancer incidences) but also the most deadly type of cancer in the world. It is responsible for more than 1.5 million deaths per year [50]. Most frequently, diagnosis of lung cancer is done at the advance stage of tumour development, when metastasis have already occurred and when surgical intervention cannot be done. Around 70% of lung cancers are unresectable, that is why small biopsy and cytology specimens are the primary method of diagnosis [51]. Late symptoms of lung cancer are the main reason of the overdue diagnosis and it is known that the advance stage of cancer is correlated with unfavourable prognosis and with very poor chance for recovery. Development of an early detection program is crucial for effective fighting with this disease and will allow to apply and perform treatment at the time when it can bring the biggest benefits. It may critically improve overall five-year survival rate which for NSCLC patients remains less than 15% [43, 8].

NSCLC is heterogeneous group comprised of subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (AC) and a lesser extent large cell carcinoma (LCC). Rising number of data confirm the highly complex histological nature and intricate molecular pattern of lung cancer which can differ even within the same histological type [61].

With an advent of personalized therapies, histological subclassification is becoming essential to provide effective treatment for patients with NSCLC. The substantial differences in clinical tumor behaviour and management, depends on different histology and molecular pattern of the tumour. For example patients with SCC should not be treated with bevacizumab because of an increased risk of life-threatening haemorrhage [51]. Therefore, finding a reliable diagnostic tumour marker, not only would reduce the histologic misclassification of small biopsy specimens but also would significantly contribute to lowering lung cancer mortality.

In the diagnostic medicine, a novel and very promising class of RNA molecules called microRNA (miRNA) turn out. These single-stranded, 22–24 nucleotides in length molecules are non-coding, fundamental regulators of gene expression. They act by binding to the 3’-UTR (untranslated region) of target transcripts and initiate translational repression or mRNA degradation [14]. The increasing body of evidences have indicated that dysregulation of miRNA expression may be a key factor underlying tumorigenesis [35]. miRNAs can exert impact on signalling, differentiation, growth, transformation and other cellular processes [12, 27, 55]. Genes coding miRNA are located within introns or exons of protein-coding genes and in intergenic regions. More than 50% of genes of the known miRNAs are placed in areas of genome which are common breakpoints, highly associated with cancer like: minimal regions of loss of heterozygosity, fragile sites or close to it and regions of amplification [5]. For example miR-30a-5p is located on the chromosome 6q13, which has been reported to be a genomic fragile region, linked to the risk of lung cancer development [19], miR-205 associated with the 1q32.2 regions is frequently amplified in AC lung cancer, whereas miR-126 with the 9q34.3 region commonly deleted in lung cancer [3]. The dysregulation of miRNAs can be implicated in the initiation and progression of cancer. Moreover, accumulating evidences are suggesting that microRNAs can play an important role as biomarkers for various cancers [17, 18, 58], including NSCLC also at the early stage of the disease [9, 22, 45].

Researchers are incessantly reporting about abnormalities concerning various miRNAs also ones abovementioned, but diagnostic value of miRNAs in NSCLC still remains not consistent and currently no single miRNA or panel of miRNAs can be applied in screening tests. The purpose of this preliminary study was to investigate the expression profile of six chosen miRNA: miR-21-5p, miR-30a-5p, miR-126-3p, miR-205-5p, miR-210-3p and miR-486-5p in NSCLC tissues and to test their diagnostic abilities.

2. Materials and methods

3. Results

4. Discussion

Six highly implicated in lung cancer genesis and development miRNAs were selected for testing in this preliminary study and RT-qPCR was employed to evaluate their expression in NSCLC tissues. Performed discriminant analysis revealed that miR-30a-5p together with miR-210-3p can distinguish cancer from normal adjacent tissue. Moreover, ROC analysis revealed the high diagnostic value of combination of these two miRNAs. Several research groups have reported about the altered expression of miR-30a-5p [16, 25, 64] and miR-210-3p in NSCLC [38], but never before, according to our knowledge, the combination of these two miRNAs was mentioned as an independent biomarker. Interestingly, careful insight into the discriminant model determined in this study revealed that miR-30a-5p and miR-210-3p are almost perfectly correlated in non-cancerous lung tissue (r = 0.95), but not so much in cancerous tissue (r = 0.61). This may indicate that potential common pathway of regulation of expression of these two miRNAs is involved, which warrants further research.

Kumarswamy et al. [25] found significant differences in miR-30a-5p expression between cancer and their adjacent non-cancer lung tissues ( n = 64). They revealed that expression of miR-30a-5p is inversely correlated to invasive capabilities. They indicted that miR-30a-5p may act as a tumour suppressor and has ability to modulate an epithelial mesenchymal transduction and to inhibit invasion and metastasis through targeting Snail1 gene (a known transcriptional repressor of E-cadherin). In studies conducted by Jiang et al. [23] miR-30a-5p was downregulated 3.9 fold times in NSCLC tissue compared to adjacent normal tissue ( n = 22). Moreover, authors presented a mechanism of  the  inhibition  of  BCL11A  protein  expression  by

miR-30a in in vitro studies. BCL11A protein is specifically upregulated in NSCLC tissues what correlates with disease-free survival and OS in early stage SCC patients. Xu et al. [56] found that miR-30a-5p expression is negatively correlated with expression of BCL-2 protein, a regulator of apoptosis, involved in tumorigenesis and chemoresistance in NSCLC tissues. Zhu et al. [64] found that CD73/NT5E is a direct target of miR-30a-5p, they reveal that expression level of this miRNA influence the cancer cells ability to proliferate. Moreover, according to Meng et al. [33] miR-30a-5p may regulates cell apoptosis and cell invasion with migration properties through controlling the PI3K/AKT signalling pathways. Results of research conducted by Tang et al. [49] on a bigger set of samples ( n = 125) are in line with ours and indicate downregulation of miR-30a-5p in tumour tissue. Moreover, expression of miR-30a in their studies turned out to be negatively correlated with tumor size, lymphatic metastasis, clinical TNM stage and OS.

Researchers reported several times about increased miR-210-3p expression is different cancers [6, 32, 39]. Hypoxia, which is a hallmark of many solid tumors, is inducing the gene expression of miR-210 via hypoxia-inducible factors HIF-1a and HIF-2a [6, 24, 32]. Moreover, this miRNA targets many genes which are involved in carcinogenesis and are implicated in processes regulating apoptosis, angiogenesis and tumor growth [20]. miR-210 inhibits expression of transcription factors MNT, an antagonist of MYC and cell-cycle regulator E2F3 what results in the induction of cell proliferation [10, 60]. miR-210-3p has been reported to be increased in lung tumor tissues and sputum from NSCLC patients and to be correlated with patient’s survival [13, 44]. Recent research conducted by Daugaard et al. revealed that miR-210-3p is upregulated in AC tissues of patients showing distant metastasis and indicates the high potential of this miRNA to become a biomarker for formation of distant metastasis ( n = 52) [11].

This preliminary study also confirmed the different expression of miR-126-3p, miR-486-5p and miR-205-5p in NSCLC tissue compared to non-cancerous adjacent tissue. Shao et al. [42] reported about downregulated level of miR-486-5p in NSCLC tissues ( n = 38). This miRNA is known from acting as a tumor suppressor by targeting CDK4, an oncogene regulating cell cycle G1/S phase progression, IGF1R responsible for controlling of cell survival and proliferation [37] and ARGAP5, a protumorigenic gene [54]. miR-126-3p is reported to act as a tumor suppressor through targeting VEGF-A and EGFL7 involved in angiogenesis and metastasis development [28, 47]. Recently Song et al. [46] have also highlighted that miR-126 is exerting an impact on cells ability to proliferate, migrate and invasion. In studies on cell line A549 they show that miR-126 through targeting PIK3R2 can influence the PTEN/PI3K/AKT signalling pathway of multiple biological processes related to metabolism, cell growth and proliferation.

Role of miR-205 is carcinogenesis is not clear, it depends on cancer type and can be downregulated [21] or upregulated [7]. In NSCLC it is observed to be overexpress in tumor tissue [2, 4, 31]. Lebanony et al. [26] ( n = 122) and Patnaik et al. [36] ( n = 77) are indicating that miR-205 may become a specific biomarker for distinguishing SCC from AD. In vitro studies conducted by Bai et al. [2] presented that miR-205 may act as a tumor suppressor through inhibiting proliferation of cells by targeting PTEN. However Cai et al. [4] report that miR-205 through targeting PHLPP2 and PTEN leads to activation of both AKT/FOXO3a and AKT/mTOR pathways, consequently resulting in accelerated proliferation and enhanced angiogenesis in NSCLC.

Some studies indicate panels of miRNAs which can serve as biomarkers of NSCLC. Tan et al. [48] reported about combination of 5 miRNAs (miR-210, miR-182, miR-486-5p, miR-30a, and miR-140-3p) which contains three of tested by us miRNAs, that can distinguish cancerous SCC lesions from adjacent normal tissues ( n = 148). Zhu et al. [65] noted the downregulated levels of miR-486-5p and miR-30a ( n = 80). Moreover, meta-analysis conducted by Vosa et al. [52], covers highly deregulated miRNAs in NSCLC across 20 different studies and is highlighting diagnostic validity of investigated in this study miRNAs.

Nevertheless, the present preliminary study has several limitations, mostly resulting from the limited number of samples. This survey did not cover patients with metastasis, thus the correlation of miRNA-30a-5p or miR-210-3p (which are reported to be involved in process of metastasis formation [11, 25, 49]) with this variable could not be determined. Additionally the analysis of OS was performed on modest sample size ( n = 33), what might be the main reason why none of the selected miRNAs were associated with OS. An attempt to determine the role of selected miRNAs in subclassification of NSCLC did not succeed, most probably because of the poor sample representation of each subtype.

miR-21 is reported to be overexpressed in NSCLC patients tissue, moreover, enhanced expression of this miRNA is reported to affect the bad prognosis and shorter survival of the patients [16, 31, 40, 41, 53, 57, 59]. miR-21 targets many genes, it is regulating EGFR-Akt pathway activity, the expression of tumour suppressors genes PDCD4, TPM1 and apoptosis-related proteins, and thus has impact on proliferation and metastasis of cancer cells [1, 15, 30, 62, 63]. Interestingly, in our research miR-21-5p showed only tendency to increase in expression in tumor tissue (1.36 fold) but, this value did not reach a statistical significance ( P = 0.072). Additionally this miRNA showed tendency to be downregulated in cancer tissue with increased tumour size comparing to non-cancerous tissue ( P = 0.025), but this result after correction for testing multiple hypothesis also did not reach statistical significance.

Carcinogenesis is a complex process in which miRNAs undoubtedly plays an important role. miRNAs are proved to be more stable and reproducible than mRNAs [34], what gives them opportunity to become a perfect biomarkers. Assessment of the expression of indicated in this preliminary study miRNAs in blood samples form NSCLC patients may provide additional evidence supporting the utility of these miRNAs as reliable diagnostic biomarkers. Additionally, further investigation of the role of these miRNAs in cancer and their mechanisms of action is substantiated and would increase knowledge concerning cancer biology.

In conclusion, we have consolidated the claim that miRNAs selected to this preliminary study have potential to become diagnostic biomarkers for NSCLC, especially miR-30a-5p together with miR-210-3p due to a high sensitivity in distinguishing cancer from non-cancerous adjacent tissue. Nevertheless because of the small sample size these preliminary results require confirmation by a further study.