Expression of miR-143, miR-145, miR-192, Tumor Suppressor miRNAs using qPCR in Colon Cancer stage II

Yıl 2017, Sayı: 3 - IONCC 2017 Özel sayısı, 40 - 45, 30.12.2017

Çiğdem Güngörmez , Hatice Gumushan Aktas , Ersin Borazan

Öz

Background: In this study, it was aimed to determine
the expression levels of tumor suppressor microRNAs
(miRNA) including miR-143, miR-145 and miR-192
in tumor and normal colon / rectum tissues of Stage II
colorectal cancer (CRC) patients by using qPCR and to
compare the obtained data.
Materials and Methods: This study was performed on
tumor or normal (control-clean surgical margin) colon
/ rectum tissues of 9 patients diagnosed with colorectal
cancer as a result of clinical evaluation with laboratory
and pathological findings in Gaziantep University
Medical Faculty Research and Practice Hospital
General Surgery Department. miRNAs isolated with
miRNeasy mini kit were used for qPCR. Isolated
miRNAs were converted into cDNA by miScript RTPCR
commercial kit. The miRNA expression levels of
tissue samples were determined by using RT- SYBR
Green qPCR kit in Rotor Gene Q.
Result: The mean level of miRNA-143 expression in
9 patients diagnosed with colorectal cancer appears to
have decreased 0.096 fold compared to normal tissue
and tumor tissue, while miR-145, 1.52 fold. This
decrease was considered as statistically significant (p
<0.05). When the fold chance values of normal and
tumor tissue are compared, despite being tumor
supressor miRNA, miR-192 appears to have
increased 3.25 fold and was not considered
statistically significant (p>0.05).
Conclusion: According to the findings obtained in
our study, the expression levels of tumor supressor
miRNA miR-143 and miR-145 in tumor colon
tissues of patients with colorectal cancer have
decreased. miRNAs have attracted major interest as
a means to analyze the molecular pathways
involved in cancer development and progression.
In addition to their important cellular functions, it
is possible that secreted miRNAs may be diagnostic
biomarkers for cancer detection. 

Anahtar Kelimeler

miRNA, Tumor suppressor miRNA, Biomarker, Colorectal cancer

Stage II Kolon Kanserinde Tümör Süpressör miRNA olan miR-143, miR-145 ve miR-192’nin Ekspresyonu

Öz

Amaç: Bu çalışmada Stage II kolorektal kanserli
hastalardan alınmış tümörlü ve normal kolon/rektum
dokularında Tümör süpressör mikroRNA (miRNA)
olan miR-143, miR-145 ve miR-192’in ekspresyon
seviyelerinin qRT-PCR ile tespit edilmesi ve elde
edilen verilerin karşılaştırılması amaçlanmıştır.
Materyal ve Metod: Bu çalışma Gaziantep
Üniversitesi Tıp Fakültesi Araştırma ve Uygulama
Hastanesi Genel Cerrahi Anabilim Dalı’nda laboratuar ve patalojik bulgular ile klinik değerlendirme
sonucunda kolorektal kanser tanısı konmuş 9 hastanın
tümörlü ve normal (kontrol-temiz cerrahi sınırları)
kolon/rektum dokuları ile gerçekleştirilmiştir. qRTPCR
uygulaması için miRNeasy mini kit ile izole
edilen miRNA’lar kullanıldı. İzole edilen miRNA’lar
miScript RT-PCR ticari kit ile cDNA’ya dönüştürüldü.
Doku örneklerindeki miRNA ekspresyon seviyeleri
RT- SYBR Green qPCR kiti kullanılarak Rotor Gene
Q sistemi ile belirlendi.
Bulgular: Kolorektal kanser teşhisi konulmuş 9
hastanın ortalama miRNA-143 ekspresyon seviyesi
normal doku ve tümörlü doku karşılaştırıldığında
0.096 kat azaldığı, miR-145’in ise 1.52 kat azaldığı
tespit edilmiştir. Bu artış istatiksel olarak anlamlı
bulunmuştur (p˂0,05). miR-192 tümör baskılayıcı
miRNA olmasına rağmen ekpresyon miktarının
normal ve tümör dokusu fold chance değerleri
karşılaştırıldığında 3.25 kat arttığı tespit edilip
istatiksel olarak anlamlı bulunmamıştır (p˃0,05).
Sonuç: Çalışmamızdan elde ettiğimiz bulgulara göre
kolorektal kanserde tümör süpressör miRNA olan
miR-143 ve miR-145’in hastaların tümörlü kolon
dokularında ekspresyon seviyeleri azalmıştır.
miRNA'lar, kanser gelişimi ve ilerlemesindeki
moleküler yollarda önemli etkiler etmektedir.
miRNA’lar hücresel birçok işleve ek olarak, kanser
tanı ve tedavisinde biyobelirteç olarak
kullanılabileceği düşünülmektedir.

Anahtar Kelimeler

miRNA, Tümör baskılayıcı miRNA, Biyobelirteç, Kolon kanseri

Kaynakça

• 1) Atkin W.S, Edwards R, Kralj-Hans I, Wooldrage K, Hart AR, et al. ) Once-only flexible sigmoidoscopy screening in prevention of colorectal cancer: a multicentre randomised controlled trial. Lancet 2010; 375: 1624–1633. 2) Bretthauer M. Evidence for colorectal cancer screening. Best Pract Res Clin Gastroenterol 2010; 24: 417–425 3) Ogata-Kawata H, Izumiya M, Kurioka D, et al. Circulating Exosomal microRNAs as Biomarkers of Colon Cancer . PLoS ONE 2014; 9(4): e92921. 4) Lee R.C, Feinbaum R.L, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75(5):843 54. 5) Pancione M, Remo A, Colantuoni V. Genetic and epigenetic events generate multiple pathways in colorectal cancer progression. Patholog Res Int 2012; pp: 1-11. 6) Corte H, Manceau G, Blons H, Laurent-Puig P. MicroRNA and colorectal cancer. Dig Liver Dis 2012; 44: 195-200. 7) Lin S, Gregory R.I. MicroRNA biogenesis pathways in cancer. Nat Rev Cancer 2015; 15: 321-333. 8) Ren A, Dong Y, Tsoi H, Yu J. Detection of miRNA as non-invasive biomarkers of colorectal cancer. Int J Mol Sci 2015; 16: 2810-2823. 9) Chang K, Mestdagh P, Vandesompele J, Kerin J.M et al. MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer. BMC Cancer 2010; 10:173. 10) Aaron J. Schetter, Hirokazu Okayama, and Curtis C. Harris. The rol of microRNAs in Colorectal Cancer. Cancer J. 2012 ; 18(3): 244–252. 11) Zhang J.X, Song W, Chen Z.H, et al. Prognostic and predictive value of a microRNA signature in stage II colon cancer: a microRNA expression analysis. Lancet Oncol 2013; 14:1295-306. 12) Michael M.Z, O'Connor S.M, Nicholas G. Reduced Accumulation of Specific MicroRNAs in Colorectal Neoplasia. Mol Cancer Res 2003; 1: 882-891. 13) Hayes J, Peruzzi P.P and Lawler S. microRNAs in cancer: biomarkers, functions and therapy. Trends in Molecular Medicine 2014; 20(8). 14) Mazeh H, Mizrahi I, Ilyayev N, Halle D, Brücher B, et al. The Diagnostic and Prognostic Role of microRNA in Colorectal Cancer-a Comprehensive review. J Cancer 2013; 4: 281-295. 15) Shen J, Stass SA, Jiang F. MicroRNAs as potential biomarkers in human solid tumors. Cancer Lett 2013; 329: 125-136 16) Wang J.C, Zhou Z.G, Wang L, et al. Clinicopathological significance of microRNA-31, -143 and -145 expression in colorectal cancer. Disease Markers. 2009; 26(1):27–34. 17 ) Slaby O, Svoboda M, Fabian P, et al. Altered expression of miR-21, miR-31, miR-143 and miR-145 is related to clinicopathologic features of colorectal cancer. Oncology. 2008; 72(5-6):397–402. 18) Di Leva G, Garofalo M, Croce CM. MicroRNAs in cancer. Annu Rev Pathol 2014; 9: 287-314. 19) Hayes J, Peruzzi P.P, Lawler S (2014) MicroRNAs in cancer: biomarkers, functions and therapy. Trends Mol Med 2014; 20: 460-469. 20) Hamfjord J, Stangeland A.M, Hughes T, Skrede M.L, Tveit K.M, et al. Differential expression of miRNAs in colorectal cancer: comparison of paired tumor tissue and adjacent normal mucosa using high-throughput sequencing. PloS one 2012; 7: e34150 21) Ye J.J, Cao J. MicroRNAs in colorectal cancer as markers and targets: Recent advances. World J Gastroenterol 2014; 20: 4288-4299. 22) Volinia S, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006; 103(7):2257–2261. 23) Siegel R.L, Miller K.B, Jemal A. Cncer Statistic, 2017. Cancer Journal for Clinicians 2017; 67: 7-30