MicroRNAs are small, single-stranded, non-coding, 16–29 nucleotide RNA molecules (approximately 22 nucleotides in length) comprising of an evolutionarily conserved class of endogenous ribo-regulators that regulate (modulate) gene expression (Wiemer, 2007, p.1529; Zhang et al., 2006, p.9136; Garzon et al., 2009, p.168). They were discovered in 1993 in the nematode Caenorhabditis elegans (Espinosa & Slack, 2006, p.131). Currently, 4167 miRNAs are listed in the miRNA registry and 474 human miRNAs have been identified (Wiemer, 2007, p.1530).
The miRNA genes are dispersed across the genome in gene clusters or as single genes. At least 50% of the miRNA genes are in defined transcription units, however, some are located in intergenic regions (Wiemer, 2007, p.1530).
MiRNAs are transcribed by the RNA Polymerase II into large precursor RNAs, called primary miRNAs or pri-miRNAs (Espinosa & Slack, 2006, p.131-2; Wiemer, 2007, p.1530; Garzon et al., 2009, p.168) with several kilobases in length. In the nucleus, the pri-miRNAs are capped and polyadenylated before being processed by the Drosha (a member of the RNase III enzyme family), together with DGCR8/Pasha (a double-stranded RNA-binding protein) (Espinosa & Slack, 2006, p.132). The processing step produces segments of about 60-100 nucleotides long, which fold into pre-miRNAs (stem-loop structures) (Wiemer, 2007, p.1530). The pre-miRNAs are exported from the nucleus by exportin 5 in a GTP-dependent fashion and are subjected to additional processing by Dicer, another RNase III enzyme. The product of this step is a double-stranded RNA duplex, approximately 22 nucleotides in length, which is then incorporated into the miRISC complex in a fashion comparable to that in RNA interference (RNAi). In the miRISC complex, the mature miRNA strand is retained, making the complex capable to regulate its target genes (Espinosa & Slack, 2006, p.132).
How to identify miRNA targets
One of the greatest challenges today is the identification of miRNA target transcripts (Espinosa & Slack, 2006, p.132; Wiemer, 2007, p.1532). This is due to the fact that, in animals, most of the miRNAs have a limited complementarity to the target sequences, which makes it difficult to identify the potential target mRNAs based on computational analyses. Only the 6-7 nucleotides (nucleotides 2–8, also called “seed sequence”) of the 21 nucleotides comprising a miRNA, have been identified as critical and sufficient for target silencing (Wiemer, 2007, p.1532).
Most miRNAs have multiple targets, and target mRNAs may bind multiple miRNAs (Wiemer, 2007, p.1532). Be that as it may, various computer programs (algorithms) have been developed to identify the putative target genes for miRNAs. For vertebrate miRNAs, the online programs include miRanda, Target-Scan/TargetScanS, PicTar, and mirBase. Wiemer (2007, p.1532) suggests that additional experimental verification is needed before an mRNA can be considered as a genuine target for a given miRNA.
Function of miRNAs
MiRNAs are crucial for the development of vertebrates due to the fact that depletion of the components of miRNA processing pathway, interfering with maturation of miRNAs, is not compatible with life (Wiemer, 2007, p.1532). The deletion of Dicer due to knockout approaches makes embryogenesis to stall during the early developmental phase. This results in lethality. Recently, it has been shown that miRNAs are involved in a variety of cellular process, especially the metabolic and developmental processes such as cell proliferation and differentiation, haematopoietic lineage differentiation, fat metabolism, developmental timing, insulin secretion, apoptosis, neuronal patterning, stem cell maintenance, and flower development in plants (Wiemer, 2007, p.1532).
An altered miRNA expression or a perturbed miRNA function can disorganize cellular processes and contribute to or cause disease (Wiemer, 2007, p.1532). Some evidence also links miRNA processing machinery to cancer (Wiemer, 2007, p.1534). In addition, poor prognosis in non-small cell lung carcinomas is associated with reduced expression of Dicer, besides Dicer upregulation being shown in prostate adenocarcinoma. Abnormalities of Dicer1 and Ago2 also results in breast cancer and ovarian cancer (Wiemer, 2007, p.1534).
Tumorigenesis
Tumorigenesis is the process of production or formation of tumors (mass of body cells or uncontrolled growths with no physiological function, and are either malignant or benign). This paper focuses on the roles of miRNAs in tumorigenesis. The study reveals that miRNAs function as tumor suppressors or as oncogenes, which makes it possible to regulate miRNA expression or inject miRNAs to regulate the formation of cancer.
MiRNAs and Cancer
All the types of cancer have several characteristics in common. These include: loss of cellular identity, increase in the ability to grow and proliferate and the alterations in systems controlling cell death (Espinosa & Slack, 2006, p.132). Various studies reveal that miRNAs have the ability to regulate these aforementioned cellular processes, suggesting that miRNAs could be involved in cancer. More so, miRNA profiling experiments revealed that many miRNAs are abnormally expressed in clinical cancer samples (Wiemer, 2007, p.1534).
Tumor formation may be promoted by the alterations in the miRNA expressions through modulating functional expression of critical genes that are involved in the tumor cell proliferation or survival. This, however, doesn’t mean that miRNAs whose expression is perturbed are all involved directly in tumorigenesis or cancer progression (Wiemer, 2007, p.1534).
MicroRNAs with tumor suppressing activity
Studies reveal that miR-15a and miR-16-1 play a role in chronic lymphocytic leukaemia (CLL). From deletion analysis, clustered miR-15a and -16-1 are the only genes lost in CLL patients. This suggests that the two miRNAs play a causative role in the pathogenesis of CLL. These miRNAs act as tumor suppressors and their loss of function may contribute to malignant transformation, thereby preventing apoptosis (Wiemer, 2007, p.1534).
The let-7 family plays a crucial role in pathogenesis of lung cancer and studies reveal that its expression is reduced in nonsmall cell lung cancer (NSCLC) (Wiemer, 2007, p.1534). Considering most studies, let-7 family regulates the oncogene RAS thus seems to operate as a tumor suppressor in lung cancer (Wiemer, 2007, p.1537).
MicroRNAs with oncogenic potential
Recent studies reveal that miR-372 and miR-373 are oncogenes in the human testicular germ cell tumors. They have the ability to overcome cellular senescence induced by oncogenic Ras (Wiemer, 2007, p.1537).
MiR-21 is highly expressed in numerous cancers such as breast cancer, pancreatic cancer, and glioblastoma. Its downregulation by transfection of breast cancer cell lines with anti-sense miR-21 leads to a reduced proliferation rate and an increase in apoptosis. It inhibits pro-apoptotic genes, thus functions as an oncogene (Wiemer, 2007, p.1537).
MiR-17-92 cluster also displays tumor suppressing activity by decreasing transcription factor levels of E2F1, thereby tightly regulating c-Myc mediated cellular proliferation. MiR-155 also has the oncogenic potential (Wiemer, 2007, p.1537).
The following section highlights the various studies on the role of miRNA in tumorigenesis and the findings.
Research findings from similar studies
Highlighted herein are the various studies conducted on the role of miRNA in tumorigenesis and the findings of these studies. Of specific interest is the breast tumorigenesis (breast cancer).
Leaderer et al. (2011) performed genetic and association studies of XPO5 in a case control study of breast cancer to investigate the roles of the microRNA biogenesis gene. In the study, two missense SNPs in XPO5, rs34324334 (S241N) and rs11544382 (M1115T) were genotyped, and methylation levels analyzed in XPO5 promoter region for the blood DNA samples. The results showed that the variant genotypes of rs11544382 were associated with the breast cancer risk as compared to the homozygous common genotype. Through menopausal status stratification, it was found that the variant alleles of both rs11544382 and rs34324334 were associated with breast cancer risk. Methylation analysis revealed that high or middle tertiles of methylation index are associated with reduced risk of breast cancer. The results were confirmed by the data from tissue array which depicted lower levels of XPO5 expression in the healthy controls as compared to adjacent tissues from breast cancer patients (tumor tissues). The tumor tissues exhibited the highest expression levels. The findings led to a conclusion that, genetic and epigenetic variations in the miRNA biogenesis gene exportin-5 significantly influence the susceptibility of breast cancer.
A study by Zhang et al. (2006) revealed that miRNAs exhibit high frequency genomic alterations in human cancer. The study involved an analysis of 283 known human miRNA genes by high-resolution arraybased comparative genomic hybridization in breast cancer, melanoma, and ovarian cancer specimens. DNA copy number alterations were exhibited in genomic loci containing miRNA genes; breast cancer (72.8%), melanoma (85.9%), and ovarian cancer (37.1%). The study showed that miRNA copy changes correlate with the miRNA expression. Also, high frequency copy number abnormalities of Dicer1, Argonaute2, were identified. The findings led to a conclusion that in cancer, there is high prevalence of the copy number alterations of miRNAs and their regulatory genes, partly accounting for frequent miRNA gene deregulation observed in various types of tumor.
Another study to explore the role of microRNAs in mediating cancer metastasis was conducted by Ma et al. (2007). The study was aimed at investigating tumor invasion and metastasis initiated by microRNA-10b in breast cancer. The study showed, using a combination of human and mouse cells, that miR-10b (microRNA-10b) is highly expressed in the metastatic breast cancer cells, and regulates positively cell invasion and migration. In the non-metastatic breast tumors, overexpression of miR-10b initiated robust invasion and metastasis. The study also showed that the expression of miR-10b is normally induced by transcription factor Twist, which directly binds to mir-10b’s putative promoter (MIRN10B). After the induction, the miR-10b proceeds to inhibit the translation of the messenger RNA encoding homeobox D10, which then results in an increase in the expression of RHOC, a well-characterized pro-metastatic gene. The level of miR-10b expression in the primary breast carcinomas significantly correlates with the clinical progression. The findings of this study suggest the workings of undescribed regulatory pathway, where expression of a specific microRNA is induced by a pleiotropic transcription factor. The microRNA’s direct target is then suppressed and pro-metastatic gene activated. This eventually leads to tumor cell invasion and metastasis.
Other studies, though not related to breast cancer, but have similar findings include El-Murr et al (2012) and Kota et al (2009). The first study was aimed at investigating the role of miRNA genes in Colorectal Cancer. The results of the study depicted that DNA repeats in human miRNA genes are rare and not involved in mutations because of MSI in MMR-deficient cancer cells. It was therefore concluded that miRNA genes constitute new targets for microsatellite instability in colorectal cancer. The latter study was aimed at investigating the role of miRNA in tumorigenesis in a Murine Liver Cancer Model, through the replacement therapy. The findings demonstrated that hepatocellular carcinoma cells (HCC) exhibit reduced expression of the miR-26a. Normally, miR-26a is expressed at high levels in the diverse tissues. The findings suggested that the delivery of miRNAs which are highly expressed and tolerated in the normal tissues but lost in the disease cells provide a strategy for the miRNA replacement therapies.
Potential use of miRNAs in clinics
Based on the various properties of miRNAs, they can be used to classify tumors in accordance to their differentiation state and the developmental origin. Prognostic miRNA expression signatures can be identified within tumor groups that predict conditions such as high progression risk, poor survival or presence of metastases. MiRNAs may also be targets or means for therapeutic intervention or cancer prevention (Wiemer, 2007, p.1538).
If induced with let-7 expression or re-expressed with miR-15a and -16-1, one may prevent formation or progression of these types of cancers (Wiemer, 2007, p.1538).
Conclusion
MicroRNAs are involved in several cellular processes like the metabolic and developmental processes and their roles in these processes are significant. For the case of an altered miRNA expression or a perturbed miRNA function, the cellular processes can be disorganized and this can contribute to or cause disease. The research herein also links miRNA processing machinery to cancer.
References
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Kota J, Chivukula RR, O’Donnell KA, Wentzel EA, Montgomery CL, Hwang H, Chang T, Vivekanandan P, Torbenson M, Clark KR, Mendell JR, Mendell JT (2009) Therapeutic microRNA Delivery Suppresses Tumorigenesis in a Murine Liver Cancer Model. Cell, 137:1005–1017.
Leaderer D, Hoffman AE, Zheng T, Fu A, Weidhaas J, Paranjape T, Zhu Y (2011) Genetic and epigenetic association studies suggest a role of microRNA biogenesis gene exportin-5 (XPO5) in breast tumorigenesis. International Journal of Molecular Epidemiology and Genetics, 2(1):9-18.
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