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Cancer Biology and MicroRNAs Essay
Cancer Biology and MicroRNAs Essay
Cancer Biology and MicroRNAs
Just like the DNA, the RNA is made up of coding and non-coding regions. The coding regions are responsible for gene expression. Looking at the central dogma and the transfer of a characteristic from the gene to the final protein, the genetic material is stored in the DNA which is then transferred to the RNA by transcription. These various RNAs then decide how the amino acids will be coded to make the final protein chain and hence the expression of the genes. MicroRNAs however, are non-coding which means that they are not involved in the transcription and the translation process in any way. They are small molecules and they contain about twenty to twenty five nucleotides. Cancer Biology and MicroRNAs Essay.
The role of the micro RNAs is to regulate gene expression as the work of the coding RNA needs to be streamlined to avoid abnormal or wrong coding. They are also very important in RNA silencing which involves suppressing the effect of a gene or more than one gene after coding has taken place (Dutta & Lee, 2010). This can also be important in the elimination of certain unwanted genes form the body. RNA silencing involves the use of antisense RNA which is used in the gene silencing. This work will look at the role of the microRNAs in cancer. Regulation of genes is important in gene expression. Studies have shown that micro RNAs are important in cancer developmental stages such as proliferation, apoptosis and invasion. This essay will address these roles in cancer development.
Understanding miRNA
The identification of the two were among the greatest works done in this field in the last century. Of more importance is the realization that miRNA were significant players in the etiology of cancer. Further studies have shown that the miRNAs can be used in cancer therapy and so understanding their nature existence and roles is of great importance to molecular biology. miRNAs are often expressed differently in various stages of development in the organism an aspect that made them to be called small temporal RNA due to their temporary expression (Dutta & Lee, 2010). The techniques used to measure their expression include Northern blotting and primer extension assay. Cancer Biology and MicroRNAs Essay. This have been developed to help in understanding the small aspects of DNA and RNA that scientists could have ignored or bypassed in the past.
Post Transcriptional Suppression
As highlighted earlier, micro RNAs play a role in suppressing target messenger RNA expression. This is a complex interaction which includes interacting with 3’UTR in the mRNA. What has been noted is that there are many mismatches in the micro RNA target sites a situation that is still being probed to establish the cause and effects in gene mRNA expression. miRNA is also known to inhibit or decrease the target levels in the respective mRNA and so this can affect the organism adversely or serve as a predisposing factor for cancer (Dutta & Lee, 2010).
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Cited Work
Dutta, Anindya. & Lee, S. Yong. MicroRNAs in Cancer. US National Library of Medicine. 2010. Available at.
Epigenetics is defined as the heritable chances that affect gene expression without changing the DNA sequence. Epigenetic regulation of gene expression can be through different mechanisms such as DNA methylation, histone modifications and nucleosome positioning. MicroRNAs are short RNA molecules which do not code for a protein but have a role in post-transcriptional silencing of multiple target genes by binding to their 3′ UTRs (untranslated regions).Cancer Biology and MicroRNAs Essay. Both epigenetic mechanisms, such as DNA methylation and histone modifications, and the microRNAs are crucial for normal differentiation, development and maintenance of tissue-specific gene expression. These mechanisms also explain how cells with the same DNA content can differentiate into cells with different functions. Changes in epigenetic processes can lead to changes in gene function, cancer formation and progression, as well as other diseases. In the present chapter we will mainly focus on microRNAs and methylation and their implications in human disease, mainly in cancer.
Mature microRNAs (miRNAs) are single-stranded RNA molecules of 20–23-nucleotide (nt) length that control gene expression in many cellular processes. These molecules typically reduce the translation and stability of mRNAs, including those of genes that mediate processes in tumorigenesis, such as inflammation, cell cycle regulation, stress response, differentiation, apoptosis, and invasion. miRNA targeting is initiated through specific base-pairing interactions between the 5′ end (“seed” region) of the miRNA and sites within coding and untranslated regions (UTRs) of mRNAs; target sites in the 3′ UTR lead to more effective mRNA destabilization. Since miRNAs frequently target hundreds of mRNAs, miRNA regulatory pathways are complex. To provide a critical overview of miRNA dysregulation in cancer, we first discuss the methods currently available for studying the role of miRNAs in cancer and then review miRNA genomic organization, biogenesis, and mechanism of target recognition, examining how these processes are altered in tumorigenesis. Given the critical role miRNAs play in tumorigenesis processes and their disease specific expression, they hold potential as therapeutic targets and novel biomarkers. Cancer Biology and MicroRNAs Essay.
1.1 miRNA Overview
miRNAs were originally shown to be important in timing of larval development in C. elegans, leading to the identification of the miRNAs lin-4 and let-7 [1, 2]. Our initial understanding of miRNA-mRNA target recognition came from observations of sequence complementarity of the lin-4 RNA to multiple conserved sites within the lin-14 3′ UTR [1, 3]; molecular genetic analysis had shown that this complementarity was required for the repression of lin-14 by lin-4 [4]. Homologues of let-7 or lin-4/mir-125 were thereafter shown to have temporal expression patterns in other organisms, including mammals, and to regulate mammalian development [5–8]. Given their integral role in development, it was no surprise that miRNAs were soon found to be important in tumorigenesis, and since their discovery close to 5,000 publications associate miRNAs to cancer, including over 1,000 reviews (recent examples include [9–11]). miRNAs were initially linked to tumorigenesis due to their apparent proximity to chromosomal breakpoints [12] and their dysregulated expression levels in many malignancies [13, 14].
Given the wealth of rapidly accumulating information implicating miRNAs in cancer, to allow the reader to critically assess the reports exploring the function of miRNAs in malignancies, we first review the methods used to study the expression and role of miRNAs in tumors, and then review the evidence that relates miRNA genomic organization, biogenesis, target recognition and function to tumorigenesis. Cancer Biology and MicroRNAs Essay. An overview of miRNA cistronic expression and sequence similarity allows a better understanding of the regulation of miRNA expression and the factors contributing to technical limitations in accuracy of miRNA detection. Understanding the regulatory potential of miRNAs based on sequence similarity families and miRNA abundance allows evaluation of which miRNAs are important regulators of tumorigenesis pathways.
1.2 Methods for Studying miRNA Genetics and Expression
1.2.1 miRNA Profiling
The main methods currently used for miRNA profiling are sequencing, microarray and real-time RT-PCR based approaches (reviewed in [15–17]). The input material initially used for these studies comprised high quality preserved fresh frozen samples, but recently it has been possible to obtain reproducible and comparable profiles using formalin-fixed paraffin-embedded tissues (FFPE), making these archived tumor collections accessible for study [18–20]. Microarrays generally provide fold-changes in miRNA expression between samples, with members of miRNA sequence families prone to cross-hybridization [21–24]. More recently, calibration cocktails of synthetic miRNAs were used in array experiments to derive absolute abundance of miRNAs [25]. RT-PCR methods are lower throughput and require normalization (i.e. candidate reference genes including other small noncoding RNAs [26, 27]). Mean expression normalization has been suggested as an alternative RT-PCR normalization method for reduction of technical variation to allow appreciation of biological changes [28]. If external miRNA standards are used for quantification (i.e. [29, 30]), the most abundant miRNA, which may vary in length due to 3′ end heterogeneity, should be used as a calibration standard. Sequencing methods, besides their obvious potential to identify new miRNAs, editing and mutation events, estimate miRNA abundance based on frequency of sequence reads (e.g. [5, 7, 8, 31–34]). Given the dramatic increase in sequencing power, bar-coding samples can allow multiple specimens to be processed at the same time, reducing the cost and effort of profiling, and paving the way for large specimen studies [34–36]. Cancer Biology and MicroRNAs Essay. Ligation biases between miRNAs and 5′ and 3′ adapters for RT-PCR amplification exist in sequencing methods, and miRNA read frequencies may not always reflect the absolute expression levels, but these variations are irrelevant when monitoring fold-changes between samples. A study with a synthetic pool of 770 miRNA sequences showed that overall, these biases do not prevent identification of miRNAs, and allowed estimation of these biases [36]. For example, certain miRNAs could be over-represented due to higher ligation efficiency (such as miR-21, which was ~2-fold over-represented), while other miRNAs could be under-represented (such as miR-31, which was > 5-fold under-represented). However, given the increasing depth of sequencing, most under-represented miRNAs are identified with sufficient sequence reads to allow for a statistically significant comparison across parallel processed samples.
Recent studies have compared the results obtained using multiple platforms [37]. A study of miRNA expression in liposarcoma revealed excellent agreement between bar-coded next generation sequencing and microarray profiles [38], while another study of miRNA expression in breast cancer showed good agreement between bar-coded sequencing and another hybridization-based method, Northern blotting [39].
Finally, choosing the appropriate statistical analysis to evaluate the data depends on the methodology used to obtain the profiles, ranging from established SAM analysis for microarray data [40], to newly developed techniques for sequencing data [34, 41, 42]. Recent in situ hybridization (ISH) advances allowed sensitive detection of miRNAs in heterogeneous tissues, defining miRNA cellular localization [43–45]. The potential of miRNA localization to suggest function for a subpopulation of cells was demonstrated early on, as in the case of lsy-6 expressed in less than ten neurons in C. eleganscontrolling left/right asymmetry [46]. Cancer Biology and MicroRNAs Essay.
1.2.2 miRNA Databases and Validation
It is critical to know which miRNAs are validated and have the potential to regulate cellular functions, especially given the frequent revisions of the miRNA database, miRBase (www.mirbase.org) [47], and the dramatic increase in the number of novel and re-annotated miRNAs through the use of deep-sequencing technologies. It is extremely challenging to establish the validity of novel miRNAs, particularly when their definition is based on a handful of sequence reads. The latest release of miRBase (version 17) includes 1,424 human miRNA precursors. Compared to version 16, version 17 includes 385 novel human miRNA precursors, 45 name changes, 1 sequence revision, and the removal of 2 precursors. Given the recent explosion in acquisition of next generation sequencing profiles, miRBase has now added features to allow evaluation of microRNA annotation [48]. The database mapped reads from short RNA deep-sequencing experiments to miRNAs and developed web interfaces to view these mappings. This is an important step in characterizing the newly identified miRNAs as prototypical miRNAs (consisting of a hairpin structure and processing sites consistent with RNase III cleavage steps).
The challenge of constantly revising and curating existing databases based on newly acquired sequencing data is illustrated in two recent studies re-evaluating mouse and human miRNAs. A recent study of 60 million small RNA sequence reads generated from a variety of adult and embryonic mouse tissues confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes but was unable to find sequencing evidence for 150 previously annotated mouse miRNAs. Ectopic expression of the confirmed and newly identified miRNA hairpin sequences yielded small RNAs with the classical miRNA features but failed to support other previously annotated sequences (of the 17 tested miRNAs with no read evidence, only one yielded a single sequence read, while of 28 tested miRNAs with insufficient number of reads, only 4 were verified) [49]. A more recent study has reannotated human miRNAs based on read evidence from over 1,000 human samples [39] miRNAs were curated both on the basis of read counts, as well as patterns compatible with traditional miRNA processing, re-defining prototypical miRNAs (557 precursors, corresponding to 1,112 mature and star sequences (miRNA*, described in the following section), miR-451 and miR-618 being the only miRNAs without a star sequence).Cancer Biology and MicroRNAs Essay. 269 not yet reported star sequences were added (compared to miRBase 16), putative miRNAs from miRBase, for which read evidence was not obtained, were ignored, and specific miRNAs were renamed according to the read ratio between mature and star sequences. The importance of curated miRNA databases is especially evident in assessing the statistical significance of differentially expressed miRNAs to identify potential biomarkers based on microarray studies. Including miRNAs without strong read evidence in such comparisons could skew the results.
1.3 Mechanisms of Alteration of miRNA Levels in Malignancy
We review miRNA biogenesis (Fig. 1.1) and illustrate which steps of the biogenesis pathway are linked to malignancy, starting from miRNA genomic localization, transcriptional regulation, processing steps and post-transcriptional modification. There is evidence supporting the association of the first three processes and/or the factors that control them with tumorigenesis, whereas evidence relating post-transcriptional miRNA modifications to cancer is not clear-cut.
miRNA biogenesis pathway. miRNAs are transcribed by RNAPII to produce pri-miRNAs. Canonical miRNAs are processed by the endoribonuclease Drosha in partnership with its RBP partner DGCR8; mirtrons are instead processed by the spliceosome. The processed premiRNA is transported to the cytoplasm through an export complex consisting of exportin 5.Cancer Biology and MicroRNAs Essay. The pre-miRNA is subsequently processed in the cytoplasm by another endoribonuclease Dicer in partnership with its RBP partner TRBP to form the final 21–23 nucleotide miRNA product. miR-451 is not processed by Dicer, but is rather cleaved by AGO2. Mature miRNAs (indicated in red) are then incorporated into AGO 1 through 4, forming miRNPs, also known as miRISC. miRNPs also incorporate other proteins, such as GW182. miRNPs are thought to direct miRNA mediated destabilization (i.e. through interaction with CCR4) or miRNA mediated translational repression (i.e. through interaction with ribosomes) of miRNAs without perfectly complementary mRNA targets. miRISC is thought to direct AGO2 catalyzed target mRNA cleavage of miRNA fully or nearly fully complementary mRNA targets
1.3.1 General Principles of miRNA Genomic Organization
miRNAs are frequently expressed as polycistronic transcripts. To date, 1,424 human miRNA precursor sequences have been deposited in miRBase [47]. Approximately one-third (497) of these miRNAs are located in 156 clusters, each measuring ≤51 kb in the human genome (51 kb being the longest distance between miRNAs belonging to the same cluster, Fig. 1.2). These miRNA clusters are co-expressed based on evidence from miRNA profiling data from a variety of tissues and cell lines [22, 33, 34, 49]. The genomic organization of representative oncogenic (miR-17 and miR-21) and tumor suppressor (let-7 and miR-141) sequence families (described in following section) is illustrated in Fig. 1.2. Presentation of miRNA profiles in the form of expression clusters provides a readily interpretable summary of expression data and stresses the importance of cistronic expression regulation; dysregulation of one member of the cluster should be accompanied by similar dysregulation of other cluster members [39]. Since miRNA genes are frequently multi-copy, determining the relative contribution of each genomic location to mature miRNA expression is challenging.Cancer Biology and MicroRNAs Essay.
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miRNA genomic and functional organization. The genomic and functional organization of four miRNA clusters is clarified: (a) let-7/mir-98 cluster, (b) mir-141/mir-200a cluster, (c) mir-21 cluster and (d) mir-17-92 cluster. The genomic locations for each of the miRNA members are defined. Grey lines denote intronic regions. miRNA mature sequences are color coded according to the sequence family they belong to (i.e. in the let-7/mir-98cluster red signifies the let-7 sequence family). The star sequence is defined with a greybar. The sequence families are depicted as sequence alignments compared to the most highly expressed miRNA family member shown on top, based on profiles of over 1,000 human specimens [39]. Shaded residues denote differences from the most highly expressed miRNA family member
1.3.2 Alterations in Genomic miRNA Copy Numbers and Location
Changes in miRNA expression between normal and tumor specimens are often attributed to the location of miRNAs in regions of chromosomal instability (amplification, translocation or deletion), or nearby chromosomal breakpoints, initially locating 52.5% of miRNA genes in cancer-associated regions or fragile sites [12]. Cancer Biology and MicroRNAs Essay. The miRNA cluster mir-15a/16-1 is located in a frequently deleted genomic locus containing a putative tumor suppressor-containing region in chronic B-cell lymphocytic leukemia (B-CLL) [50]. Other examples include deletion of let-7g/mir-135-1 in a variety of human malignancies [12], amplification of mir-17-92 cluster in lymphoma [51], translocation of mir-17-92 in T-cell acute lymphoblastic leukemia (T-ALL) [52], and amplification of mir-26a in glioblastoma [53].
1.3.3 Alterations in miRNA Transcriptional Regulation
Some autonomously expressed miRNA genes have promoter regions that allow miRNAs to be highly expressed in a cell-type-specific manner, and can even drive high levels of oncogenes in cases of chromosomal translocation. The mir-142 gene, strongly expressed in hematopoietic cells, is located on chromosome 17 and was found at the breakpoint junction of a t(8;17) translocation to MYC, which causes an aggressive B-cell leukemia [54]. The translocated MYC gene, which was also truncated at the first exon, was located only four nucleotides from the 3′ end of the mir-142 precursor, placing it under the control of the upstream mir-142 promoter. In an animal model for Hepatocellular Carcinoma (HCC), a similar event placed C-MYC downstream of the mir-122a promoter which is active only in hepatocytes [55].
Many transcription factors regulate miRNA expression in a tissue-specific and disease state-specific fashion, and some miRNAs are regulated by well-established tumor suppressor or oncogene pathways such as TP53, MYC, and RAS (reviewed in [56]). The miRNA and its transcriptional regulators can participate in complex feedback regulation loops. Examples include the TP53 regulated mir-34a [57, 58], the RAS regulated mir-21[33, 59, 60] and the MYC regulated mir-17-92 gene cluster [61, 62].
miRNA dysregulation has also been linked to changes in epigenetic regulation, such as the methylation status of miRNA genes, which results in alterations in their expression levels [63, 64]. Examples of methylated miRNA genes include mir-127 in bladder cancer cells [65] and mir-9-1 in breast cancer [66]. Cancer Biology and MicroRNAs Essay.
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