Jafari Naser, Dogaheh Hadi Peeri, Bohlooli Shahab, Oyong Glenn G, Shirzad Zohreh, Alibeiki Fatemeh, Asl Saeid Hosseini, Zargar Seyed Jalal
Department of Biochemistry, School of Medicine, Ardabil University of Medical Science Ardabil 56197, Iran ; Department of Cell & Molecular Biology, School of Biology, College of Science, University of Tehran Tehran, Iran.
Int J Clin Exp Med. 2013 Apr 12;6(4):269-74. Print 2013.
MicroRNAs (miRNAs) have recently been shown to play fundamental roles in diverse cellular processes and linked to variety of cancers. Dicer and Drosha are two major enzymes in the miRNA maturation process. DGCR8 is the assistant of Drosha in the microprocessor complex. In this study, we evaluated the mRNA expression profiles of major miRNA processing machinery Drosha, Dicer, and DGCR8 in human gastrointestinal (AGS, KYSE30 and HepG2) cancer cell lines.
The cells were cultured and harvested, and total cellular RNA was isolated from cells. Then, first-strand cDNA was synthesized from the RNA of cells. Afterward, Quantitative analysis was performed by real-time RT-PCR using the PowerSYBR Green PCR Master Mix.
Expression levels of Drosha in AGS and HepG2 cells were higher than the controls, whereas, Drosha's expression level in KYSE-30 cell line was lower. The Dicer expression levels in AGS and HepG2 cells were higher, while, its expression level in KYSE-30 cell was lower. The DGCR8 expression levels in all three cell lines were significantly higher than the control samples.
Expression levels of the two most important enzymes of the miRNA machinery, Drosha and Dicer, and microprocessor complex component, DGCR8 were noticeably dysregulated when compared to healthy controls.
微小RNA(miRNA)最近被证明在多种细胞过程中发挥着重要作用,并与多种癌症相关。Dicer和Drosha是miRNA成熟过程中的两种主要酶。DGCR8是微处理器复合物中Drosha的辅助因子。在本研究中,我们评估了主要miRNA加工机制Drosha、Dicer和DGCR8在人胃肠道(AGS、KYSE30和HepG2)癌细胞系中的mRNA表达谱。
培养并收获细胞,从细胞中分离总细胞RNA。然后,从细胞RNA合成第一链cDNA。之后,使用PowerSYBR Green PCR Master Mix通过实时RT-PCR进行定量分析。
Drosha在AGS和HepG2细胞中的表达水平高于对照,而在KYSE-30细胞系中Drosha的表达水平较低。Dicer在AGS和HepG2细胞中的表达水平较高,而在KYSE-30细胞中的表达水平较低。所有三种细胞系中DGCR8的表达水平均显著高于对照样本。
与健康对照相比,miRNA机制中两种最重要的酶Drosha和Dicer以及微处理器复合物成分DGCR8的表达水平明显失调。