EXPRESSION ANALYSIS OF MFN2, MT-ATP6, MIDN AND KPNB1 GENES ISOLATED FROM HEART-SPECIFIC SUBTRACTIVE HYBRIDIZATION CDNA LIBRARY IN ADULT, NEONATAL AND EMBRYONIC TISSUES

Yıl 2017, Cilt: 7 Sayı: 14, 21 - 33, 14.12.2017

Bilge Ozsait Selcuk , Evrim Komurcu Bayrak , Muhammed Abdulvahid Kalkan , Gizem Çelebi Nihan Erginel Ünaltuna

Öz

Objective: Identification of
differential gene expression and determination of co-expressed genes is
important in the characterization of tissue specific molecular pathways. In our
previous studies, we constructed a heart-specific subtractive hybridization
cDNA library (SHL) and conducted several functional studies in order to analyze
the role of the isolated transcripts in heart tissue. In this study, our aim
was to investigate the expression patterns of four selected transcripts from
SHL in multiple tissues and, total embryos tissues of different stages.

Materials
and Methods:
Total RNA isolation and cDNA synthesis were performed from 16 different
tissues, 5 different stages of total embryos and neonatal heart and skeletal
muscle of BALB/c mice. The expression profiles of SHL isolated four genes,
mitofusin-2 (Mfn2), mitochondrial ATP
synthase 6 (mt-Atp6), midnolin (Midn) and karyopherin (importin) beta 1
(Kpnb1) were analyzed by quantitative
real time PCR. Additionally, Northern blot analysis of mt-Atp6 in multiple tissues was performed.

Results: Gene expression levels
of the four transcripts were higher in neonatal heart than in neonatal skeletal
muscle. Similar pattern was observed in adult heart and skeletal muscle.
However, mt-Atp6 and Mfn2 genes were over-expressed in heart
tissue compared to all other tissues and embryonic stages.

Conclusion: Differential expression
of the transcripts among different tissues as well as in different embryonic
stages suggests that these four genes have crucial roles in cellular physiology.
Demonstration of the gene expression levels of the transcripts in the SHL in
healthy tissues and embryonic development will contribute to the identification
of the function of the genes in related metabolic pathways.

Anahtar Kelimeler

subtractive hybridization, qRT-PCR, Mfn2, mt-Atp6, Midn, Kpnb1

KALBE ÖZGÜ “SUBTRACTIVE” HIBRIDIZASYON CDNA KÜTÜPHANESINDEN ELDE EDILEN MFN2, MT-ATP6, MIDN VE KPNB1 GENLERININ YETIŞKIN, NEONATAL VE EMBRIYONIK DOKULARDAKI EKSPRESYON ANALIZI

Öz

 

Amaç: Eş zamanlı eksprese olan genlerin belirlenmesinde ve
farklılaşmış gen ekspresyonunun tanımlanmasında dokuya özgü moleküler
yolakların belirlenmesi önem taşımaktadır. Daha önceki çalışmalarımızda, “subtractive”
hibridizasyon cDNA kütüphanesi (SHL) oluşturulmuş ve ardından bu
transkriptlerin kalp dokusundaki rollerinin tanımlanabilmesi amacı ile çok
sayıda fonksiyonel çalışma yürütülmüştür. Bu çalışmadaki amacımız, SHL’den
seçilen dört transkriptin çoklu doku ve farklı evrelerdeki total embriyo
dokularındaki ekspresyon özelliklerinin araştırılmasıdır.

Gereç ve Yöntem: Toplam 16
farklı yetişkin BALB/c fare dokusu, neonatal kalp ve iskelet kası dokusu ve 5
farklı embriyonik döneme ait dokularda total RNA izolasyonu ve cDNA sentezi
gerçekleştirildi. SHL’den elde edilen dört genin, mitofusin-2 (Mfn2), mitokondriyal ATP sentaz 6 (mt-Atp6), midnolin (Midn) ve karyoferin (importin) beta 1 (Kpnb1) genlerinin qRT-PCR yöntemi ile gen ekspresyon analizi
yapıldı. Ek olarak, mt-Atp6 geninin
ekspresyonu çoklu doku Northern blot analizi ile araştırıldı.

Bulgular: Araştırılan dört transkriptin
neonatal dönemde iskelet kasına oranla kalp dokusundaki ekspresyon seviyelerinin
daha fazla olduğu tespit edildi. Yetişkin kalp ve iskelet dokularında da benzer
yapı gözlendi. Diğer yandan, mt-Atp6 ve Mfn2 genlerinin diğer tüm dokular ile
karşılaştırıldığında kalp dokusunda daha fazla ekspresyonunun olduğu
belirlendi.

Sonuç: Farklı dokular ve embriyonik dönemler arasında farklılaşmış
gen ekspresyonlarının olması, bu dört transkriptin hücre fizyolojisinde önemli
bir role sahip olduğunu düşündürmektedir. SHL’den izole edilen bu
transkriptlerin seviyelerinin sağlıklı dokularda ve embriyonik gelişim dönemlerinde
gösterilmesi, kalp fizyolojisi ile ilişkili metabolik yolaklardaki işlevlerinin
tanımlanması için önem taşımaktadır.

Anahtar Kelimeler

“subtractive” hibridizasyon, qRT-PCR, Mfn2, mt-Atp6, Midn, Kpn1b

Kaynakça

• Calore M, De Windt LJ, Rampazzo A. Genetics meets epigenetics: genetic variants that modulate noncoding RNA in cardiovascular diseases. J Mol Cell Cardiol 2015;89: 27-34.
• Elia L, Condorelli G. RNA (Epi)genetics in cardiovascular diseases. J Mol Cell Cardiol 2015;89: 11-16.
• Stanton L.W. Methods to Profile Gene Expression. Trends Cardiovasc Med 2001;11:49–54.
• Diatchenko L., Lau Y.F., Campbell A.P., Chenchik A., Moqadam F., Huang B., Lukyanov S., Lukyanov K., Gurskaya N., Sverdlov E.D., Siebert P.D. Suppression subtractive hybridization: a method for generating differentially regulated or tissuespecific cDNA probes and libraries 1996; PNAS USA 93:6025-6030.
• Komurcu-Bayrak E, Ozsait B, Erginel-Unaltuna N. Isolation and analysis of genes mainly expressed in adult mouse heart using subtractive hybridization cDNA library. Mol Biol Rep 2012;39:8065-8074.
• Hara E, Yamaguchi T, Tahara H, Tsuyama N, Tsurui H, Ide T, Oda K. DNA–DNA subtractive cDNA cloning using oligo(dT)30-Latex and PCR: identification of cellular genes which are overexpressed in senescent human diploid fibroblasts. Anal Biochem 1993;214:58–64.
• von Stein OD, Thies WG, Hofmann M. A high throughput screening for rarely transcribed differentially expressed genes. Nucleic Acids Res 1997;25:2598–2602.
• Zhang H, Zhou L, Yang R, Sheng Y, Sun W, Kong X, Cao K. Identification of differentially expressed genes in human heart with ventricular septal defect using suppression subtractive hybridization. Biochem Biophys Res Commun 2006;342:135–144.
• Özsait Selçuk B. “Subtractive” hibridizasyon kütüphanesinden izole edilen ve kalp gelişiminde rolü olduğu düşünülen genlerin analizi, Yüksek Lisans Tezi, İstanbul Üniversitesi, İstanbul, 2003.
• Özsait Selçuk B, Kömürcü Bayrak E, Erginal Ünaltuna N. Higher Expression level of Bat3 is associated with silencing of Midn gene in primary 2 mouse cardiomyocytes. Turk J Biol 2016;40: 1295-1302.
• Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, Zhang J, Soden R, Hayakawa M, Kreiman G, Cooke MP, Walker JR, Hogenesch JB. A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci U S A. 2004;101(16):6062-7
• Hwang DM, Dempsey AA, Wang RX, Rezvani M, Barrans JD, Dai KS, Wang HY, Ma H, Cukerman E, Liu YQ, Gu JR, Zhang JH, Tsui SK, Waye MM, Fung KP, Lee CY, Liew CC. A genome-based resource for molecular cardiovascular medicine: toward a compendium of cardiovascular genes. Circulation. 1997;96(12):4146-203.
• Ware, S. M., El-Hassan, N., Kahler, S. G., Zhang, Q., Ma, Y.-W., Miller, E., Wong, B., Spicer, R. L., Craigen, W. J., Kozel, B. A., Grange, D. K., Wong, L.-J. Infantile cardiomyopathy caused by a mutation in the overlapping region of mitochondrial ATPase 6 and 8 genes. J. Med. Genet. 2009;46: 308-314.
• Santel A, Fuller MT. Control of mitochondrial morphology by a human mitofusin.J Cell Sci. 2001 Mar;114(Pt 5):867-74
• Yang C, Aye CC, Li X, Diaz Ramos A, Zorzano A, Mora S. Mitochondrial dysfunction in insulin resistance: differential contributions of chronic insulin and saturated fatty acid exposure in muscle cells. Biosci Rep. 2012 Oct;32(5):465-78.
• Zorzano A, Hernández-Alvarez MI, Sebastián D, Muñoz JP. Mitofusin 2 as a driver that controls energy metabolism and insulin signaling. Antioxid Redox Signal. 2015 Apr 20;22(12):1020-31.
• Givvimani S, Pushpakumar S, Veeranki S, Tyagi SC.Dysregulation of Mfn2 and Drp-1 proteins in heart failure. Can J Physiol Pharmacol. 2014 Jul;92(7):583-91
• Zuchner S, Mersiyanova IV, Muglia M, Bissar-Tadmouri N, Rochelle J, Dadali E L, Zappia M, Nelis E, Patitucci A, Senderek J, Parman Y, Evgrafov O, Jonghe PD, Takahashi Y, Tsuji S, Pericak-Vance MA, Quattrone A, Battaloglu E, Polyakov AV, Timmerman V, Schröder JM, Vance JM. Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nature Genet. 36: 449-451, 2004.
• Tsukahara M, Suemori H, Noguchi S, Ji ZS, Tsunoo H . Novel nucleolar protein, midnolin, is expressed in the mesencephalon during mouse development. Gene. 2000; 254: 45-55.
• Quan Y, Ji ZL, Wang X, Tartakoff AM, Tao T. Evolutionary and transcriptional analysis of karyopherin beta superfamily proteins. Mol Cell Proteomics. 2008;7(7):1254-69.
• Loveland K L, Hogarth C, Szczepny A, Prabhu S M, Jans DA. Expression of nuclear transport importins β 1 and β 3 is regulated during rodent spermatogenesis. Biol. Reprod. 2006;74, 67–74.
• Nachury MV, Maresca TJ, Salmon WC, Waterman-Storer CM, Heald R, Weis K. Importin β is a mitotic target of the small GTPase Ran in spindle assembly. Cell. 2001;104:95-106.