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A NOVEL Cerna ANALYSIS for LMNA and ZMPSTE24 RELATED to MECHANISMS of PROGEROID LAMINOPATHIES

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A NOVEL Cerna ANALYSIS for LMNA and ZMPSTE24 RELATED to MECHANISMS of PROGEROID LAMINOPATHIES EJONS International Journal on Mathematic, Engineering and Natural Sciences ISSN 2602 - 4136 Article Arrival Date Article Type Article Published Date 18.05.2020 Research Article 12.06.2020 Doi Number: http://dx.doi.org/10.38063/ejons.251 A NOVEL ceRNA ANALYSIS FOR LMNA AND ZMPSTE24 RELATED TO MECHANISMS OF PROGEROID LAMINOPATHIES Nazlı Irmak GİRİTLİOĞLU Molecular Biologist and Bioengineering Specialist, Istanbul/Turkey [email protected], https://orcid.org/0000-0001-5034-0984 ABSTRACT Progeroid syndromes are diseases that display old-age features such as skin aging (loss of elastin and skin thinning), cardiovascular problems, alopecia at young ages. One of the most known progeroid syndromes is Hutchinson-Gilford Progeria Syndrome (HGPS). HGPS is caused by LMNA de novo point mutation and as a result, a toxic protein that accumulates in the inner nuclear membrane called progerin is produced. Zinc Metallopeptidase STE24 (ZMPSTE24, also known as FACE-1 in humans) is an enzyme that has important functions in post-translational modifications. ZMPSTE24 mutations also can cause progeroid syndromes. Competing endogenous RNA (ceRNA) is a relatively new hypothesis that claims RNAs communicate with each other and regulate each other’s expressions via shared microRNA (miRNA)s. In this study, a ceRNA analysis was performed for mRNAs of LMNA and ZMPSTE24. Firstly, related miRNAs of LMNA and ZMPSTE24 mRNAs were found in miRTarBase, and the results with both strong and less strong evidence were considered. Then, other genes related to these miRNAs that have only strong evidence in miRTarBase were chosen and hit scores of these genes were calculated. The Gene Ontology (GO) Resource GO Enrichment analysis 359 algorithm was used for P values (<0.05) of molecular functions of these genes which have the highest hit scores. The highest hit score of transcripts associated with LMNA mRNAs is 3 (BCL2, CCND1, CCND2, CDK6, EZH2) and the highest hit score of transcripts associated with ZMPSTE24 mRNAs is 2 (CBL, CCND2, CLOCK, EZH2, MAPK14, SERP1). The most important result in this study is EZH2 interaction with both LMNA and ZMPSTE24 networks and its epigenetic involvement might be key for treatments of some progeroid syndromes. It was known that downregulated EZH2 with loss of the H3K27me3 marks causes the weaker heterochromatin-lamina associations. The study provides a novel analysis for connected ceRNAs of LMNA and ZMPSTE24 mRNAs. Keywords: ceRNA, miRNA, Progeroid, LMNA, ZMPSTE24 1. INTRODUCTION Nuclear lamins (type V intermediate filament proteins) are important, structural components of the proteinaceous meshwork called nuclear lamina (NL), and directly associated with the nuclear genome. NL provides a proper morphology to cells and has regulatory functions on epigenetic mechanisms (Hah & Kim, 2019). Lamins are capable of binding to chromatin, DNA, and histones (Shevelyov & Ulianov, 2019) so they control gene expressions. Vertebrates have two A type of lamins (lamins A and C) that are encoded by LMNA, also there are less abundant isoforms (lamin A10 ve C2) (Dechat, Adam, Taimen, Shimi, & Goldman, 2010). A-type lamins are expressed in most somatic cell lines but not in undifferentiated pluripotent stem cells (Chojnowski, Ong, & Dreesen, 2015). Accelerated aging syndromes comprise of the many types of diseases which include classical/non- classical HGPS (both of them progerin producing type), non-progerin producing progeroid laminopathies (in consequence of heterozygous LMNA pathogenic variant or pathogenic variants in other genes such as ZMPSTE24), non-progeroid laminopathies, and non-laminopathy progeroid Year 4 (2020) Vol:14 Issued in JUNE, 2020 www.ejons.co.uk EJONS International Journal on Mathematic, Engineering and Natural Sciences ISSN 2602 - 4136 syndromes (Gordon, Brown, & Collins, 2003). Laminopathies are caused by the genes (LMNA, LMNB1, and LMNB2) which express the lamin proteins and LMNA mutations are the most involved type of these diseases such as muscular dystrophy, neuropathy, cardiomyopathy, metabolic disorders, and accelerated aging and their combinations (Ho & Hegele, 2019). HGPS is one of the most known type of progeroid syndromes and caused by an aberrant process in post-translational modification of prelamin A. Cytosine (C) changes to thymine (T) in LMNA exon 11 (c.1824C>T, G608G). Prelamin A has CAAX (C=cysteine, A=aliphatic amino acid, and x=any amino acid) motif that goes through some processes at the C-terminus: 1) Farnesyltransferase adds a farnesyl group to Cysteine, 2) aaX aminoacids are cleavaged by zinc metalloproteinase (ZMPSTE24 or also known as FACE-1), 3) Prenylcysteine carboxy methyltransferase adds a carboxymethyl group to farnesylated cysteine, and 4) 15 terminal amino acid residues are cleavaged by ZMPSTE24. In HGPS, ZMPSTE24 does not recognize the second cleavage site because of the de novo LMNA mutation (then aberrant mRNA splicing) and a toxic protein is formed called progerin (Eriksson et al., 2003; Ramirez, Cadinanos, Varela, Freije, & Lopez-Otin, 2007). When the functional lamin A concentration is low, the cell would be more unstable, and external forces can easily affect it (Al- Saaidi & Bross, 2015). If the farnesylated prelamin A accumulates in the inner nuclear membrane, nuclear blebbing occurs (Yang et al., 2005). Mutations in ZMPSTE24 also cause progeroid syndromes. Denecke et al. report a patient with homozygous ZMPSTE24 null mutation and heterozygous LMNA mutation (Denecke et al., 2006). Shackleton et al. describe a 2 years old female patient that shows characteristics of HGPS, mandibuloacral dysplasia, and restrictive dermopathy with compound heterozygous ZMPSTE24 mutations (Shackleton et al., 2005). miRNAs are short (approximately 19-25 nucleotide), endogenous RNA molecules and basically, they have functions in gene silencing (Lu & Rothenberg, 2018). RNA-RNA interactions can comprise the regulations of RNA transcripts by miRNAs. In this context, ceRNA is a potent hypothesis that specifies the mRNAs of genes communicate between them and control each other’s expressions with 360 miRNAs (Tay, Rinn, & Pandolfi, 2014). There are some computational algorithms such as mirWalk (Dweep, Sticht, Pandey, & Gretz, 2011) or mirTaRBase (Huang et al., 2019) to show miRNAs that associate with mRNAs. Counts of shared miRNAs for mRNAs are indicated the connections between these mRNAs. In this study, ceRNAs of LMNA and ZMPSTE24 mRNAs were searched in bioinformatic tools and the potential ceRNAs were discussed. 2. MATERIALS AND METHODS 2.1. Detection of related miRNAs of LMNA and ZMPSTE24 mRNAs based on strong and less strong evidence Related miRNAs of LMNA and ZMPSTE24 mRNAs were detected in miRTarBase which is a database that shows the validated miRNAs with strong and less strong experimental evidence (Huang et al., 2019) (Table 1 and Table 2). Year 4 (2020) Vol:14 Issued in JUNE, 2020 www.ejons.co.uk EJONS International Journal on Mathematic, Engineering and Natural Sciences ISSN 2602 - 4136 Table 1. LMNA mRNAs related miRNAs with strong and less strong valuation methods in miRTarBase (http://mirtarbase.cuhk.edu.cn/php/index.php) Target miRTarbase ID miRNA Sequence Valuation Methods Site MIRT019532 hsa-miR-340-5p UUAUAAAGCAAUGAGACUGAUU 3' UTR NGS MIRT021388 hsa-miR-9-5p UCUUUGGUUAUCUAGCUGUAUGA 3' UTR Microarray MIRT022394 hsa-miR-124-3p UAAGGCACGCGGUGAAUGCC 3' UTR Microarray and other MIRT038445 hsa-miR-296-3p GAGGGUUGGGUGGAGGCUCUCC 3' UTR NGS MIRT040169 hsa-miR-615-3p UCCGAGCCUGGGUCUCCCUCUU 3' UTR NGS MIRT051592 hsa-let-7e-5p UGAGGUAGGAGGUUGUAUAGUU 3' UTR NGS MIRT052409 hsa-let-7a-5p UGAGGUAGUAGGUUGUAUAGUU 3' UTR NGS Reporter assay and MIRT731197 hsa-miR-205-5p UCCUUCAUUCCACCGGAGUCUG 3' UTR microarray Table 2. ZMPSTE24 mRNAs related miRNAs with strong and less strong valuation methods in miRTarBase (http://mirtarbase.cuhk.edu.cn/php/index.php) Target miRTarbase ID miRNA Sequence Valuation Methods Site MIRT004942 hsa-miR-98-5p UGAGGUAGUAAGUUGUAUUGUU 3' UTR qPCR and other MIRT022576 hsa-miR-124-3p UAAGGCACGCGGUGAAUGCC 3' UTR Microarray MIRT438256 hsa-miR-141-3p UAACACUGUCUGGUAAAGAUGG 3' UTR Reporter assay 2.2. Determining the genes associated with LMNA and ZMPSTE24 miRNAs and calculating the hit scores of the potential ceRNAs All the miRNAs in Table 1 and Table 2 were searched in miRTarBase and the genes with only the strong evidence grade were selected (Table 3 and Table 4). 361 Hit score in this study means that the number of the genes according to their shared miRNAs. For example; BCL in Table 3 shares three miRNAs on the strong evidence grade with LMNA, so the hit score of BCL is 3. 2.3. GO molecular function analysis of the potential LMNA and ZMPSTE24 ceRNAs “Molecular function” term explains the functional activities of the gene products at the molecular level. The genes which have the highest hit scores were inserted into GO Enrichment Analysis (powered by PANTHER) algorithm (http://geneontology.org/) and “Molecular function” and “Homo sapiens” were selected. P values (<0.05) were ordered from the lowest to the highest (Table 7 and Table 8). Year 4 (2020) Vol:14 Issued in JUNE, 2020 www.ejons.co.uk EJONS International Journal on Mathematic, Engineering and Natural Sciences ISSN 2602 - 4136 Table 3. The genes that their expressions are regulated by the same miRNAs with LMNA miRNA Genes with strong evidence CCND1, CCND2, CDK6, HNRNPA2B1, KRAS, MECP2, MET, PTBP1, PUM1, hsa-miR-340-5p PUM2, RHOA, ROCK1, SKP2, SOX2 ACAT1, AP3B1, ATG5, BACE1, BCL2, BCL2L11, BCL6, CCND1, CCNG1, CD34, CDH1, CDX2, CHMP2B,
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