Review of miRNA Technology

Zhang Tangyao(University of Melbourne,Australia)

MicroRNAs (abbr. miRNAs) are RNA molecules widely exist in eukaryotic with 22 nucleotides, and they can regulate some transcriptional and post-transcriptional genes expression by specific binding within target mRNA molecules.(Chen and Rajewsky, 2007) MiRNAs are encoded by nuclear DNA, however, they cannot be translated into proteins thus they are non-coding RNA. In animals, a miRNA can usually regulate multiple genes and influence cell development,differentiation, proliferation and apoptosis. MiRNAs not encode any proteins, nevertheless, many miRNAs have been found to have an important physiological function in human health.(Van Roosbroeck et al., 2013) Because miRNAs are involved in regulation of genes expression widely in eukaryotic cells,therefore the abnormal expression of miRNAs are also lead to some disease. This essay will describe how analysing the profile of miRNA expression could be used to diagnose disease and the real-time polymerase chain reaction technique which can be applied to detect the changes in miRNA expression.

The miRNA is generated from a primary transcript which is also called a pri-miRNA transcribed by RNA polymerase II, then become a pre-miRNA with hairpin loop and export from the nuclear by nucleocytoplasmic shuttle exp-5 (Fig.1). Next, RNase III enzyme Dicer modified the pre-miRNA hairpin in cytoplasm, then generate an uncompleted miRNA:miRNA* duplex. The duplex could sometimes function as miRNA, however one strand is usually combined with the RNA induced silencing complex (RISC) where the miRNA interact with the particular mRNA target.

Fig.1 miRNA Biogenesis (Okamura et al., 2007)

The expression of miRNA involved many human disease such as cancer and heart disease. Chronic lymphocytic leukemia (CLL) was the first human disease identified to be associated with miRNA deregulation.(Mraz and Pospisilova,2012) Protein 53, encoded by the TP53 gene, is known as the guardian of the genome because of it can conserve stability by avoid genome mutation. Yamakuchi shows that the target of mir-34 microRNA precursor family is silent information regulator 1 (SIRT1) gene and it resulting the influence on p53.(Yamakuchi and Lowenstein, 2009) Marek Mraz and Sarka Pospisilova(Mraz and Pospisilova, 2012)address that early diagnose of p53 abnormal would be helpful to CLL patients, and miR-34a deregulation can be used as a sensor for identification of p53 abnormality during the progress of CLL.

An oncomiR is a miRNA such as miR-34a which associated with cancer like CLL. Like miR-34a, oncomiRs result in cancer by down-regulating genes by both repressing gene translation and destabilising mRNA mechanisms. These downregulated genes may code for proteins like p53 that control the cell cycle. In cancerous tissue, oncomiRs may be increasing or decreasing. If the target gene for an oncomiR is a tumor suppressor gene or apoptosis gene, the observed activity of oncomiRs will theoretically increase in cancerous tissue. On the other hand, if the target is a gene involved cell proliferation,the under-expressed oncomiR will lead to unlimited cell proliferation.

The first miRNA to be identified as an oncomiR is MiR-21 family. After the discovery of first oncomiR, approximately 44 families of miRNA have been identified associated with cancer include leukemia, breast cancer, lung cancer,cholangiocarcinoma, colorectal cancer, glioblastoma etc.(Cho,2007) Lu et al. analyzed 217 animal miRNAs from 334 samples include cancer cells such as liver cancer, colon cancer,stomach cancer, pancreatic cancer, by using bead-based flow cytometric miRNA expression profiling method. They successfully use miRNA expression profiles to categorize poorly differentiated cancer, and their research reveals the important value of miRNA expression level in cancer diagnosis.(Lu et al., 2005) Lee et al. applied real-time PCR techniques to profiling of over 200 miRNA precursors derived from specimens of human pancreatic adenocarcinoma, paired benign tissue, normal pancreas, chronic pancreatitis and nine pancreatic cancer cell lines. They find that the abnormal expression of miR-221, miR-301 and miR-376a were only founded in pancreatic cancer cell lines.(Lee et al., 2007).

The main methods to analyzing the profile of miRNA expression for diagnosing cancer are microarray test and real-time PCR. Bottoni et al. analyzed 32 pituitary adenomas and 6 normal pituitary samples by using microarray and by real-time PCR.(Bottoni et al., 2007) The results of profiling entire miRNAome of all samples show that miRNA expression level can distinguish between micro-adenomas and macro-adenomas, and they also identified some miRNAs are act functionally in cell propagation and apoptosis.Realtime PCR integrated with reverse transcription can be used to quantify miRNA (mRNA and non-coding RNA) in cells or tissues as well as diagnose such as Alzheimer's disease.(Tian et al., 2013) Different from normal PCR, fluorescent dyes(non-specific fluorescent dyes or sequence-specific fluorescent reporters) are used in real-time PCR. By using fluorescent dyes, real-time PCR has more advantages: fast ,sensitive and do not need to detect after end-point.

Armstrong et al. suggest a method to detect expression of miRNA changes using real-time PCR.(Armstrong et al.,2012) TaqMan Array Human MicroRNA A+B Cards Set v3.0 was employed in this research, which is a product of Life Technologies Corporation.It includes A and B arrays and each of them have 384 wells contain different kinds of oligoinucleotides, therefore this set could quantify 754 human miRNAs in 5 hours (Fig.2). It is a proven and commercialized real-time PCR technique which can detect look the changes in miRNA expression by simply run several times of PCR reactions.

In conclusion, the phenomena of RNA interference have new biomedical significance after hounders of miRNAs were discovered. MiRNAs, which are short non-coding RNA and have genes regulation function, involved in many pathogenesis of human diseases especially cancers. The monitoring of cancer related miRNAs, oncomiRs, will help diagnose caners.Furthermore, the studies on miRomics will bring revolutionary breakthrough to medicine and carcinobiology.

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