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Features of miR-574-5p and miR-574-3p binding sites in mRNA of target genes A.T. Ivashchenko, R.Y. Niyazova, O.A. Berillo, S.A. Atambayeva, A.Y. Pyrkova
Institute of Biology and Biotechnology Problems, al-Farabi KazNU, 050038, Kazakhstan, a_ivashchenko@mail.ru

miRNAs function as key regulators of developmental timing, and are highly conserved. They have specific nucleotide sequences and participate in post-transcriptional regulation of their target genes [1]. miRNAs with high affinity to mRNAs of several hundred genes were recently identified [2-4]. Their binding sites are located in 5 UTRs, CDSs, and 3 UTRs. These miRNAs have unique properties; for example, some miRNAs have specific binding site locations at a certain distance in mRNAs of different genes [5]. Other miRNAs have multiple binding site locations within several tens of nucleotides [2-4]. Such unique miRNAs have hundreds of target genes that regulate several key biological processes. Therefore, it is important to understand both the characteristics of the binding sites in the mRNAs of target genes as well as the biological processes they regulate. Specifically, miR -574-5p is a unique miRNA that has multiple binding sites in mRNAs of target genes, and their specific functions need to be extensively investigated. While there is some data regarding the function of miR 574-5p in different processes and pathologies, further research in this regard is warranted. The present work examined the interaction of miR-574 with the mRNAs of its target genes as well as analysed the functions of these genes. 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 [5]. 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 Gm 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. Pre-miR-574 consists of miR-574-3p and miR-574-5p, and is encoded in an intron of a family with sequence similarity 114 member A1 gene (FAM114A1) located on chromosome 4. ble 1 Characteristics of multiple miR-574-5p binding sites in 3'UTR of mRNA
Binding site position, nt. ACVR2B 10963-11013 (26) ADAT2 3307-3329 (12) ADRBK2 3880-3896 (8) AFF3 4208-4224 (7) AMOTL1 6023-6037 (8) ANKRD42 1888-1908 (10) ANO8 4031-4041 (6) ARHGAP35 7726-7740 (8) ARHGEF9 2537-2549 (7) ARRB1 6399-6417 (10) ATMIN 4182-4192 (6) BDH1 2611-2710 (35) BTBD9 5797-5825 (15) C10orf71 4716-4735 (10) C15orf57 862-904 (22) CAMK2N1 1850-1864 (8) CARNS1 3225-3235 (6) CD22 2733-2753 (9) CD40LG 1549-1579 (16) CD93 3489-3499 (6) CDH6 4747-4759 (7) CHRDL1 2247-2265 (9) CHST11 5219-5233 (8) CLIC6 2573-2587 (8) CNGA4 2098-2120 (12) CPPED1 5408-5422 (8) CREB3L2 6066-6082 (9) CYP4V2 2906-2916 (6) DNAJC15 1131-1141 (6) DNAJC6 5549-5563 (8) DOK6 7831-7851 (11) DPYSL5 2933-2949 (9) EHD3 2274-2284 (6) EOGT 2684-2694 (6) EP3 0 0 8566-8584 (10) FAM117B 2078-2104 (14) FAM163A 2108-2122 (8) FAM167A 3152-3178 (9) FAM83C 2511-2523 (7) FBRSL1 4180-4190 (6) FOXN3 2420-2446 (13) Note. In parentheses is shown the n Gene Binding site position, nt. GFRA1 8453-8481 (15) GLI2 6124-6148 (13) GLP1R 1546-1562 (9) GLRA2 2526-2568 (20) GLYR1 1965-1979 (8) GRIA3 3351-3363 (7) GRIA4 5084-5098 (8) HPS3 3253-3267 (8) HS6ST3 3847-3855 (5) IFFO2 4395-4407 (6) IGF1 4042-4062 (10) INHBA 2041-2053 (7) ITGA11 4598-4638 (20) KATNAL1 4198-4526 (30) KCNIP3 1007-1021 (8) KCNK10 6170-6178 (5) KCNQ3 3560-3576 (9) KCNQ5 5370-5412 (22) KIAA0895L 2882-2928 (23) KIAA1549L 9102-9118 (9) KIAA2018 9953-9965 (6) KIF1B 5813-5823 (6) LDB3 4421-4447 (14) LEPREL1 2947-2963 (9) LHFPL5 1461-1531 (13) LHFP 1397-1426 (15) LRRTM2 2949-2973 (12) LYRM7 5255-5279 (13) MAF 2106-2127 (10) MAP2 8797-8809 (7) MARCH4 3767-3785 (10) MCM8 2994-3020 (13) MNT 4275-4289 (7) NAV1 7555-7579 (13) NCDN 3557-3587 (15) NR2E1 2953-2981 (15) PCDHB16 198-212 (8) PPARA 9024-9036 (6) PPP2 R1 B 4561-4581 (11) PRKCB 7056-7087 (16) PTCHD1 2900-2922 (12) umber of repeated miR -574-5p bind Gene Gene RAB3 I P RAB7 A RABGAP1L RALGAPB REEP5 RPH3A RUNX1T1 SAMD9L SBK1 SDK2 SEPT6 SH3TC2 SHB SLC31A1 SLITRK3 SMAD4 SNAP29 SNX2 SPATA6 SRD5A3 S S X1 S S X2 B S S X2 S S X5 STXBP6 SYNPO TCTE1 TMEM130 TNS4 TREML2 TRIOBP UBLCP1 VAMP4 VSNL1 WNT4 XRCC1 ZEB1 ZFP92 ZNF618 ZRANB1 ZSWIM1 ing sites. Binding site position, nt. 4086-4100 (7) 1021-1031 (6) 34-66 (16) 7474-7488 (7) 1478-1496 (10) 2859-2869 (6) 3269-3301 (17) 6484-6508 (12) 2321-2362 (21) 7582-8030 (7) 4493-4521 (15) 20874-20898 (13) 3405-3481 (9) 867-879 (7) 4415-4437 (11) 7742-7756 (7) 1361-1385 (12) 1737-1753 (8) 3213-3231 (10) 1415-1433 (10) 1066-1080 (8) 1275-1306 (15) 1102-1129 (13) 1199-1217 (10) 2559-2573 (8) 3601-3623 (12) 2238-2266 (15) 2003-2026 (11) 3471-3511 (21) 2350-2380 (16) 7488-7575 (8) 1550-1574 (13) 1890-1900 (6) 1023-1045 (12) 1981-2001 (11) 2035-2051(9) 3587-3605 (10) 3289-3307 (10) 2954-2976 (11) 3797-3807 (6) 1627-1645 (9)


miR-574-5p and miR-574-3 have different properties despite their common origin. miR574-5p has 13 purines and 10 pyrimidines, while miR-574-3 has 9 purines and 13 pyrimidines. miR-574-5p has 245 target genes despite its longer length, whereas miR-574-3 has only six target genes with G/Gm ratios of 90% or more. miR-574-5p and miR-574-3 have binding sites in the mRNAs of 1764 and six target genes, respectively. The average number of binding sites for miR-574-5p and miR-574-3p in the mRNA of a target gene is 7.2 and 1.0, respectively (table 1). We therefore propose the following hypothesis based on the observed properties of miR-574-5p and miR-574-3. In addition, studies measuring the concentration of miR-574-3 without miR-574-5 observed that changes in biological liquids correlated with tumour development. Therefore, we hypothesize that it is possible that tumorigenesis is initiated by changes in the concurrent concentration of miR-574-3p and miR-574-5p, since they mature from a common precursor. This hypothesis is supported by the fact that miRNA-5p and miRNA-3p are coded by a single pre-miRNA. The effect of the concentration of miRNA-5p has been investigated without a control comparison with miRNA-3p. Therefore, the results of these studies are incomplete and only a few studies have described the co-expression of miRNA-5p and miRNA-3p. A similar pattern of regulation was observed for miR-574-3p and miR-574-5p, which are specifically downregulated in lymphoblastic cell lines. miR-574-5p binding sites are located in adeninecytosine repeats (AC-repeats) in mRNA UTRs. A significant positive correlation between the sequence length of 3 UTRs and the presence of tandem repeat sequences has been reported. Therefore, the majority of miR-574-5p sites are located in 3 UTRs. Regulation of human CBR1 gene expression by miR-574-5p depends upon the rs9024 genotype status. It was established that the concentration of serum miR-574-5p was significantly different and was associated with death from sepsis [6]. Moreover, varying miR-574 expression was demonstrated in different diseases and its possible gene targets were investigated [7-12]. miR-574 is overexpressed in myotonic dystrophy, oesophageal squamous cell carcinoma, and early-stage NSCLC. It was shown that TLR9 signaling was elevated due to the expression of miR-574-5p in human lung cancer cells [9]. Furthermore, miR-574-3p plays a significant role in cellular processes and might also be required for mesenchymal stem cell multipotency [10]; it has also been associated with neurodegenerative diseases and gastric cancer [11]. In


vitro transfection of mesenchymal stem cells with pre-miR-574-3p resulted in inhibition of chondrogenesis, indicating its role during the commitment of mesenchymal stem cells towards chondrocytes. Studies have shown that nucleotide repeats are connected to miRNA target gene regulation. Therefore, we studied CA-repeats of genes that are targets for miR574-5p. We determined that 162 miR-574-5p binding sites are located in 5 UTRs, two in CDSs and 1,606 in 3 UTRs with a G/Gm ratio of 90% or more. The target genes identified play important roles in various cell processes involving 36 transcription factors, 26 kinases (including 7 transcription factors), 31 cell cycle related genes, and 27 apoptotic genes. Some of these target genes participate in carcinogenesis. Specifically, 18 and 11 proteins of identified target genes are involved in breast and lung cancer development, respectively. All 11 target genes participating in the development of lung cancer are included in the group of 18 target genes that regulate breast cancer development. We established that the predicted target genes of miRNAs are transcription factors, kinases, and cell cycle and apoptosis related genes that participate in the development of various diseases such as breast cancer, lung cancer, and cardiovascular diseases, etc. 1. M. Lagos-Quintana et al. (2001) Science, 294(5543):853-8. 2. A. Ivashchenko et al. (2014) BioMed Research International, 2014:620530. 3. A. Ivashchenko et al. (2014) BioMed Research International, 2014:720715. 4. R. Niyazova et al. (2014) BioMed Research International, 2014:988786. 5. A. Ivashchenko et al. (2014) Bioinformation, 10:423-427. 6. H. Wang et al. (2012) Shock, 37:263-267. 7. M. Meyers-Needham et al. (2012) EMBO Mol Med, 4:78-92. 8. A. Perfetti et al. (2014) Neuromuscul Disord, 24:509-15. 9. Q. Li et al. (2012) PLoS One, 7:e48278. 10. D. GuИrit et al. (2013) PLoS One, 8:e62582. 11. Y. Su. (2012) Int Immunopharmacol, 13:468-475. 12. K.M. Foss et al. (2011) J Thorac Oncol, 6:482-488.