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The interaction of miRNAs with mRNAs of the cell cycle genes in lung cancer R.Y. Niyazova, O.A. Berillo, S.A. Atambayeva, A.T. Ivashchenko
National Nanotechnology Laboratory, al-Farabi KazNU, 050038, Kazakhstan, raiguln@mail.ru

One of the specific features of carcinogenesis is increased cell proliferation caused by changes in the rate of the cell cycle and apoptosis [1, 2]. It is therefore important to examine the influence of miRNAs on these processes. Hundreds of genes and miRNAs involved in the development of malignant neoplasms serve as biomarkers of lung cancer. Identifying the association between miRNAs and their target genes is therefore critical for characterising the features of various tumours and their subtypes. In this study, the interaction of miRNAs with the mRNAs of genes involved in the cell cycle was assessed based on the Kyoto Encyclopaedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/). Materials and Methods. Human mRNAs were obtained from GenBank (http://www.ncbi.nlm.nih.gov). The nucleotide sequences of human mature miRNAs were downloaded from the miRBase database (http://mirbase.org). Target genes for miRNAs were determined using the MirTarget program [3]. This program defines the following features of binding sites: the start position of an miRNA binding site with respect to the mRNA sequence, the localisation of miRNA binding sites in the 5UTRs, CDSs and 3'UTRs of genes, the free energy of hybridisation (G, kJ/mole), and the schemes of nucleotide interactions between miRNAs and mRNAs. The G/Gm (%) ratio was estimated for each binding site, where G is equal to the free energy value of miRNA binding to its perfect complementary nucleotide sequence. The miRNA binding sites identified had G/Gm more than 90%. Results and Discussion. The miRNA binding sites in mRNAs of human cell cycle genes were identified, and are characterised in Table 1. We hypothesized that the expression of most cell cycle genes could be regulated by various miRNAs. Some mRNAs have binding sites for several miRNAs, which control their expression. For example, the DP-2 mRNA has eight binding sites. Five miRNAs have binding sites in GSK3, TGF, h300, SMAD4, h53, ATMATR, PTTG mRNAs, and four miRNAs have three binding sites in CDH1, MEN1, CDC6, CDK4, E2F1 mRNAs. Each mRNA of the SMAD3, RAD21, p57, ESP1, AB1, E2F1, E2F5, E2F4, CHK1 genes has three binding sites.


Table 1. The characteristics of miRNA binding sites in mRNAs of the cell cycle genes
Genes ABL1 ATM Characteristics of miRNA binding sites miR-149-3p, 3425, 90; miR-3685, 5136·, 91; miR-383-3p, 4007·, 90; miR-4519, 3648, 90. miR-1273a, 11053·, 90; miR-1273e, 11118·, 93; miR-1273g-3p, 11075·, 96; miR-5585-5p, 11155·, 91; miR-619-5p, 9792·, 98; miR-6507-5p, 2162, 90; miR-6829-3p, 266, 91. CDC25A miR-6749-3p, 1879, 90; miR-6809-3p, 1885, 94. CDC25B miR-4463, 457, 96; miR-4487, 756, 90; miR-6124, 664, 94; 668, 90. CDC6 miR-1273g-3p, 2286·, 93; miR-566, 2376·, 90; miR-5684, 2280·, 92; miR-6833-3p, 102, 90. CDC7 miR-4486, 1281, 91; miR-765, 81, 91. CDH1 miR-1273c, 3250·, 91; miR-1273g-3p, 3270·, 93; miR-1273h-5p, 3304·, 96; miR-3656, 486, 90; miR-4430, 51, 94; miR-7160-3p, 185, 91. CDK4 miR-1285-5p, 1940·, 92; miR-5095, 1694·, 91; miR-5096, 1774·, 96; miR-619-5p, 1700·, 95. CDKN1C miR-3714, 561, 90; miR-4463, 810, 91; 864, 94; 870, 94; 882, 91; 888, 91; 900, 91; miR4505, 904, 90; miR-762, 739, 92; 745, 91; 805, 94; 811, 94; 817, 91; 901, 92. CDKN2D miR-3940-3p, 133, 90; miR-4274, 115, 92; miR-6769a-3p, 734, 91. CHEK1 miR-4271, 63, 90; miR-5585-3p, 2574·, 93; miR-619-5p, 2567·, 95. E2 F1 miR-1913, 29, 90; miR-3960, 89, 92; miR-4749-3p, 2322·, 91; miR-6511a-3p, 2327·, 91; miR6511b-3p, 2326·, 93; miR-6786-5p, 268, 90; miR-6813-3p, 2537·, 91. E2 F2 miR-1273f, 4160·, 92; miR-1273g-3p, 4127·, 96; miR-4534, 39, 96; miR-4539, 1407, 90; miR548m, 2091·, 90; miR-5684, 4121·, 92; miR-760, 625, 93. E2 F4 miR-4265, 852, 90; miR-6791-3p, 160, 91; miR-7704, 80, 93. E2 F5 miR-1268a, 143, 90; miR-6068, 104, 90; miR-6791-3p, 233, 91. ELL miR-4800-5p, 2827·, 91; miR-6777-3p, 2964·, 93; miR-6817-3p, 2923·, 92. EP3 0 0 miR-1908-3p, 155, 90; miR-2682-3p, 298, 90; miR-3960, 52, 90; miR-574-5p, 8795·, 93; 8801·, 93; 8803·, 93; 8805·, 93; 8807·, 93; 8809·, 93; 8811·, 93; 8813·, 93. ESPL1 miR-6505-3p, 576, 90; miR-6735-3p, 1878, 95; miR-6815-3p, 3126, 91. GSK3B miR-1268a, 361, 92; miR-1268b, 359, 91; miR-3960, 9, 92; 12, 92; miR-466, 4712·, 91. MAD1L1 miR-4489, 2505·, 91; miR-6078, 1943, 98; miR-6132, 2467·, 90; MDM2 miR-1273e, 2520·, 93; miR-1273f, 6771·, 92; miR-1273g-3p, 2116·, 96; 2485·, 91; 6738·, 96; miR-1285-3p, 3217·, 91; miR-3929, 3012·, 93; miR-5684, 2479·, 90; 6732·, 90. MYC miR-1227-5p, 28, 94; miR-6761-5p, 989, 91. RAD21 miR-1322, 1575, 92; miR-3656, 186, 90; miR-4762-5p, 320, 90; miR-6124, 191, 92. RB1 miR-3960, 224, 92; miR-4736, 277, 90. RBL1 miR-5095, 3528·, 93; miR-5096, 3608·, 96; miR-619-5p, 3534·, 93; 3668·, 96. REEP5 miR-574-5p, 1477·, 93; 1479·, 93; 1485·, 93; 1487·, 93; 1489·, 93; 1491·, 93; 1493·, 93. SMAD2 miR-1273f, 6124·, 90; miR-566, 6181·, 92. SMAD3 miR-1227-5p, 4, 90; miR-4492, 107, 94; miR-4507, 2065·, 91; miR-4508, 110, 94; 242, 90; miR-4690-5p, 2065·, 92; miR-6089, 2077·, 91. SMAD4 miR-1273f, 4344·, 92; miR-1273g-3p, 4311·, 95; miR-1972, 4551·, 90; miR-5579-5p, 5307·, 90; miR-574-5p, 7741·, 91; 7743·, 93; 7745·, 93; 7747·, 93; 7749·, 93; 7751·, 93; 7755·, 91. SMC1A miR-1282, 2128, 90; miR-3119, 3496, 90. TFDP2 miR-1273f, 5323·, 90; 5858·, 92; 7373·, 92; miR-1273g-3p, 5292·, 98; 5824·, 91; 7341·, 96; miR-1285-5p, 9171·, 91; miR-1303, 4500·, 93; miR-5096, 9004·, 96; miR-5585-3p, 4393·, 91; miR-5684, 5286·, 92; 7335·, 92; miR-619-5p, 6779·, 95; 8930·, 96; 9064·, 96. TGFB1 miR-1234-5p, 2089·, 90; miR-3141, 873, 90; miR-4274, 254, 90; miR-4508, 2060·, 90; miR4530, 218, 92; miR-4651, 2086·, 95; miR-6089, 2064·, 91; miR-6125, 1, 91; miR-6742-5p, 2047, 90; miR-6824-5p, 707, 90; miR-6877-5p, 4, 90; miR-877-3p, 232, 93. TP53 miR-1273c, 2296·, 91; miR-1273g-3p, 2316·, 91; miR-1273h-5p, 2350·, 91. Notes: The first number after miRNA is the binding site position in mRNA (nucleotides); the second number is the ratio G/Gm (%); the symbols "", "·", "" indicate to binding sites in CDS, 3'UTR and 5 'UTR.


The mRNA of target genes with revealed miRNA binding sites can significantly alter the rate of the cell cycle. Unique miRNAs like miR-619-5p, miR-1273f, miR-1273g-3p, miR-574-5p, miR-3960, miR-619-5p, miR-1273e, miR-5096 and miR-5095 [4, 5] have binding sites in mRNAs of several genes. Consequently, there is a high probability that these miRNAs have important functions in tumourigenesis. It is necessary to note that some miRNAs may function as oncogenes or tumour suppressors. This bidirectional action of miRNAs complicates the unambiguous interpretation of their action; however, changes in their expression may impact the rate of the cell cycle and mediate tumorigenesis. Some of the genes influenced by miRNAs are transcription factors that can alter the expression of oncogenes and tumour suppressors. ATM, MDM2, CDH1, CDC6, EF2, SMAD1, SMAD4, TFTP2 and TP53 genes participate in regulation of the cell cycle, and its mRNA is a target for miRNAs of the miR-1273 family. The studied genes are involved in the development of cancer at various locations. For example, MDM2, an oncogene-encoded cellular phosphoprotein, negatively regulates p53 by blocking p53-mediated transactivation. This gene plays a significant role in human sarcomas, where the p53 wild-type allele is preserved. Therefore, it was proposed that MDM2 neutralizes the function of p53 in oncogenesis [6]. The MDM2 gene is a target for three miRNAs of the miR-1273 family (miR-1273e, miR1273f, miR-1273g-3p). miR-1273g-3p has multiple binding sites on the CDH1 mRNA, and CDH1 gene mutations correlate with tumorigenesis in different tissues, including the development of non-small cell lung cancer. The loss of function of its encoded protein leads to tumour progression via increased proliferation, invasion, and metastasis [7]. The CDH1 mRNA is a target for three miRNAs of the miR-1273 family (miR-1273c, miR-1273g-3p, miR-1273h-5p), and the CDH1 gene is specifically related to the development of large-cell lung carcinoma. The majority of miR-3960 binding sites are located in 5 UTRs and CDSs. The E2F1 gene is involved in cell cycle regulation, and its mRNA is a target for miR -3960 and miR-574-5p. The E2F protein family plays a key role in cell cycle control and in the function of tumour suppressor proteins [8]. E2F1 as well as E2F2, MYC, SMAD4 and TP53 genes are responsible for the development of small cell lung cancer [9, 10]. The protein encoded by the RB1 gene inhibits the cell cycle. The EP300 protein acts as a tumour suppressor in lung cancer neoplasm and its mRNA has multiple miR-574-5p binding sites. It


is known that miR-574-5p is highly expressed in lung cancer. miR-619-5p, miR-5096 and miR-5095 have binding sites on the ATM, CDK4, CHEK1, RBL1 and TFDP2 genes mRNAs. Increased expression of the CDK4 gene is an indicator of the aggressiveness of the tumour [11]. CHEK1 gene expression correlates with poor survival of patients with primary lung adenocarcinomas [12]. These results demonstrate that unique miRNAs of defined concentrations can strongly influence the expression of genes involved in cell cycle regulation. Increased levels of miR-1273f, miR-1273g-3p, miR-1273e, miR-574-5p, miR3960, miR-619-5p, miR-5096 and miR-5095 serve as an indication of carcinogenesis. Based on the results of the interactions of miRNAs and target genes, we propose that miR-619-5p, miR-1273f, miR-1273g-3p, miR-574-5p, miR-3960, miR-619-5p, miR-1273e, miR-5096 and miR-5095 be used as a marker of carcinogenesis. mRNAs of the DP-2, GSK3, TGF, h300, SMAD4, h53, ATMATR, PTTG, CDH1, MEN1, CDC6, CDK4, E2F1, SMAD3, RAD21, p57, ESP1, AB1, E2F1, E2F5, E2F4 and CHK1 genes are targeted by three and more studied miRNAs; therefore, this gene can also serve as a tumour marker since its expression is decreased in the presence of the predicted miRNAs. 1. J. Jin et al. (2014) Life Sciences, 108:48-53. 2. F. Kopp et al. (2014) Oncotarget, 5:85-195. 3. A. Ivashchenko et al. (2014) Bioinformation, 10:423-427. 4. A. Ivashchenko et al. (2014) BioMed Research International, 2014:620530. 5. A. Ivashchenko et al. (2014) BioMed Research International, 2014:720715. 6. Y. Zhao et al. (2014) Acta Biochimica et Biophysica Sinica, 2014:180-189. 7. E.Y. Kim et al. (2014) Respiratory Research, 15:26-32. 8. B.P. Coe et al. (2013) Plos One, 8(8):e71670. 9. C. Wu et al. (2013) BMC Bioinformatics, 17:365-372. 10. M. Taniwaki et al. (2006) International Journal of Oncology, 29:567­575. 11. Q. Li et al. (2012) Plos One, 7:e48278. 12. T.J. Chen et al. (2014) Tumour Biology, 35:7209-7216.