microRNA

and their role in cancer critical gene regulation

By: Chad Kenneth Cain Pacific Lutherin University Nov 28, 2012

microRNA (miRNA) is a single stranded polymer of 20-30 nucleotides that functions principally through post-transcriptional repression of messenger RNA. By regulating expression of transcriptional products destined to become proteins, aberrant levels of miRNA have been shown to affect oncogenesis in a variety of cancers due to the type of cancer-critical proteins regulated (oncogenes or tumor suppressors). Using patient donated cancerous tissues and cellular models research has indicated that oncogenes RAS, HMGA2, and BCL2 are sensitive to let-7, and miRNA-15a/16 down regulation. These examples are documented in Leukemia, lung, breast, and colon cancers, which show that decreased levels of certain miRNAs can result in elevated concentrations of their target mRNA when compared to normal adjacent tissues. Up regulated expression of miRNA-21, miRNA-221/222 and miRNA-155 targeting tumor-suppressors p53, and p27Kip1 has been documented in various lymphomas and prostate carcinomas. Due to the broad influence of microRNA on the expression of crucial cancer related genes, it has become a major focus of todays leading cancer researchers in the fight for discovering better possible treatments.

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Introduction: Ironically, with the 3 billion base pairs that make up the human genome sequenced, and the 21,000 protein coding genes identified, the Human Genome Project (HGP) produced more questions than it did answers following its completion in 2003. Since protein-coding genes, known as exomes, only accounted for roughly 1.5% of the total sequenced DNA much of the non-coding DNA was considered to be without biological function (Maher 2012). However, since 2005 90% of single-nucleotide polymorphisms documented by Genome Wide Association Studies (GWAS) that appear to be associated with diseases existed outside the previously mentioned 21,000 protein-coding regions uncovered by the HGP (Maher 2012). These findings contradicted the popular belief that these long non-coding regions were junk DNA accumulated over time. It wasnt until the US National Human Genomic Research Institute (NHGRI) launched the Encyclopedia of DNA Elements (ENCODE Project), a public research consortium charged with locating all the functional elements in the human genome, did these non-coding regions begin to be finally understood. By 2012, the preliminary results of the study were published in a series of 30 papers that collectively assigned biochemical functions to approximately 80% of the previously unidentified noncoding regions (Dunham et al. 2012). Many of the assigned regions included sites of transcription, transcription factor association, chromatin structure and histone modification, all of which suggest that these sections are frequently involved in regulating the expression of coding regions, earning them the title regulomes (Dunham et al. 2012). Of the transcriptional products derived from genomic regions investigated by the ENCODE Project, microRNA (miRNA) has received particular interest from cancer researchers due the scope of miRNAs involvement in the cell in addition to the availability

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of evidence showing that altered levels of these molecules are associated with a various forms of cancers. Improvements in researchers ability to detect loci on the exomes where miRNA interact have revealed that potentially 60% of all genes are under selective pressure to maintain their interaction with miRNA (Fredman et al. 2009). Of the 186 miRNA genes documented in 2004, 52.9% were found to be located at or within sites prone to deletion, rearrangement, amplification and/or breakage (Calin et al. 2004). Even though the number of documented mature miRNA is somewhere above 5922, its still important to note that many discovered at early stages of research were located at fragile sites especially when you take into account how important they are in regulating gene expression (Griffith-Johns et al. 2008).
Figure 1 Pathway and enzymes responsible for converting large hairpin pre-miRNA in the nucleus to mature functional miRNA in the cytoplasm capable of repressing mRNA translation. (www.nature.com/nbt/journal/v25/n6/fig_tab/nbt0607-631_F2.html)

Figure 1 outlines the pathway necessary for a mature miRNA to become functional in

the cytoplasm. Biologically active mature miRNA originate from a large multi-stem precursor that is transcribed by RNA Polymerase II. In many cases, each fabricated multi- stem precursor can contain multiple pre-miRNAs, which are then released by endonuclease Drosha/Pasha while still in the nucleus. Once cleaved the pre-miRNAs still take the shape

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of a hairpin loop and are not fully cleaved until they are exported out of the nucleus by nuclear membrane protein Exportin 5 into the cytosol (Croce 2009). While still labeled as pre-miRNA, they must undergo yet further enzymatic modification by Dicer, a protein responsible for unwinding/cleaving the hairpins before they are loaded onto the RNA Induced Silencing Complex (RISC). Once the mature miRNAs have been loaded onto the RISC and the stabilizing proteins, Argonaute, associate with the final structure, the miRNA is finally capable of binding to specific mRNA targets and repressing their translation into proteins. Successful gene repression is reliant on complementarity between sequences found on the miRNA and stretches of 8-15 nucleotides located at sites within 3 untranslated region (UTR) of target mRNA (Lewis et al. 2003). Once the miRNA has associated with its target, translation is prevented through one of two mechanisms that is dictated by the extent of complementarity between the two RNAs. If base pairing is high enough the bound stabilizing protein, Argonaute, cleaves the mRNAs poly-A tail exposing it to exonuclease degradation. If base pairing is mild, the miRNA simply remains bound to the mRNA target thus preventing any successful interaction with translational machinery. Since miRNAs that trigger degradation are capable of binding to multiple targets following their interaction, a small concentration can result in a high degree of regulation. In the case of miRNAs that function by preventing targets from associating with ribosomes, the magnitude of repression is dependent on the levels of both miRNA and mRNA present. The differences in the two mechanisms allows miRNA to exhibit behaviors similar to either a valve, damping the total amount of gene expression taking place, or a switch, shutting off expression entirely, if complementarity is extensive enough (Alberts et al. 2008).

While the principle mode of regulating gene expression occurs at the level of

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transcription through the actions of various transcription factors, miRNAs post- transcriptional regulation has several features that make it exceptionally useful in the cell. For instance, a single type of miRNA can be responsible for repressing multiple mRNA genes if they all contain the same complementary sequences in their 3UTRs. Another advantage is that miRNA repression can be combinatorial, so if the dosage of one type of miRNA is insufficient to alter expression levels a second set of miRNA may bind to further limit expression. Cumulatively, miRNAs post-transcriptional regulation exhibits behavior akin to a switch or in some cases a flow valve fine-tuning the dosage in which a gene is expressed (Alberts et al. 2008).
Figure 2 Due to miRNAs having different cancer related targets, certain types of gene mutation are needed or miRNA to induce cancer. (http://en.wikipedia.org/wiki/Gene_mutation) f

miRNAs association with cancer begins with the type of mutation its genes undergo

and the category of cancer-critical genes the miRNA is responsible for repressing. Figure 2 shows the categories of miRNA mutation and cancer-critical mRNA targets required for a cell to exhibit cancerous behavior. If miRNAs genes acquire point mutations, inversions, or deletions the result can decrease the amount of transcriptional output or have an effect on

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whether the product is functional. This when combined with mRNA targets that belong to the gain-of-function category of cancer-critical genes, called proto-oncogenes, can drive a cell to become overactive (Alberts et al. 2008). Normal levels of proto-oncogene expression are often dependent on a certain degree of miRNA repression at baseline. If miRNA repression is relieved, higher than normal levels of proto-oncogenes may result (as seen in proto-oncogenes: RAS, HMGA2, and Bcl2). At the other end of the spectrum lie translocations and duplications to miRNA genes. These mutations may contribute to elevated levels of miRNA transcription. If the miRNA targets belong to the loss-of-function category, tumor suppressors, the ability of the cell to express these mRNAs, when triggered to do so, may be limited or entirely repressed thereby leading to transformation (as seen in tumor suppressors p53 and p27kip1). miRNAs potential for regulating expression of both oncogenes and tumor suppressor genes, along with its susceptibility to mutations (i.e. location on or near fragile sites) are the reasons for its newly acquired spotlight in recent literature. Interestingly, at the time when miRNAs were first documented, post-transcriptional regulation from miRNAs were thought only to be a nuance of the model organism in which they were found. miRNAs were discovered in 1993 by Rosalind Lee, Victor Ambros, and Rhonda Feinbaum, in the course of exploring gene expression in developing Caenorhabditis elegans. The outcome of this study revealed the maturation and function of small class of RNA molecules, roughly 20-30 nucleotides in length, they coined as microRNA, as being crucial for regulating levels of certain proteins essential to early larval development (Lee et al. 1993).

Current literature on miRNAs capacity for regulating oncogenes and tumor

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suppressor genes share a similar routine for investigating miRNA. Typically researchers begin by drawing correlations between altered levels of cancer related proteins and atypical miRNA expression. These correlation studies are usually compared between unaffected tissues and cancerous tissues, however in some cases the correlations are made between low-grade and high-grade cancers. The next commonly used step is detecting if complementary sequences exists between the mRNA of the cancerous protein and suspicious miRNA through sequence analysis. This indicates whether the miRNA suspected repressing cancer related gene expression has the necessary sites to interact with the appropriate mRNA. Sequence analysis revealing similar homologies are typically followed by some form of luminescence repression study. This is used to indicate that the sequence similarities are significant enough to allow the two molecules to interact with each other. The final common strategy among prevailing research investigating miRNA involvement is transfection analysis. Here the expression of miRNA is purposely altered to reveal how cell behavior is either repaired or further disrupted. The shared trends in current literature investigating miRNA provided the necessary framework behind the formatting of this papers thesis. Using a similar paradigm for discussing miRNAs relation to cancer allows the reader to approach an understanding that reflects the same deductive pathway followed by researchers. This review argues that the aberrant levels of certain miRNAs being correlated with cancer gene expression, miRNAs successful interaction with cancer related mRNAs, and the evidence from purposely altering miRNA expression, all support miRNAs potential for exhibiting behaviors akin to tumor suppressors and oncogenes.

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miRNAs presence is inversely correlated to cancer-related protein expression: The inverse relationship seen in the expression of miRNAs and cancer related proteins offered the first piece of evidence suggesting miRNAs post-transcriptional regulation is capable of exhibiting behaviors akin to oncogenes and tumor suppressors. More specifically, when genetic mutations decrease the presence of certain miRNAs and the expression of characterized oncogenes are conversely elevated, these miRNAs are suspected of behaving comparably to tumor suppressors. This is an appropriate description because the evidence then suggests that the transformation of cells to a cancerous state can be attributed to the lack of miRNA gene activity. This parallels the defining feature of a tumor suppressors, loss of gene activity equals the promotion of cancerous cell behavior (Alberts et al. 2008).

Figure 3 (A) The expression of let-7 in breast, colon, and lung tumors against NAT by fluorescently labeling miRNA with specific probes and quantifying via microarray. Results indicate significant under expression of let-7 mainly in lung cancer tissue samples (B) Northern blot showing the inverse relationship between Ras protein expression (GAPDH reference) and the western blot of let-7 miRNA presence. (C) qRT-PCR of Ras mRNA between cancerous tissues and NAT showing no significant difference.

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Slack et al. (2005) showed that miRNA lethal-7 (let-7) was under expressed in 21 cases of lung, breast, and colon cancer, when compared to normal adjacent tissues (NAT) using fluorescently labeled RNA probes. Lung cancer tissues showed the greatest decrease in expression, with an average 50% reduction in let-7 presence (Johnson et al. 2005) (See Figure 3A). This degree of under expression prompted further investigation to see if the altered let-7 presence could be tied to lung cancers characteristically over expressed oncogene, Ras (Ahrendt et al. 2001). Ras oncogenic properties stem from its GTPase activity being capable of switching on/off various factors responsible for elevating rates of cellular division (McKay et al.1986; Pulciani et al. 1985). To investigate the relationship of let-7 with abnormal levels of Ras, Northern blot analysis and quantitative Real Time (qRT)- PCR of the RNA extracts from 3 different lung cancer cell lines were performed (Johnson et al. 2005). Let-7 and Ras mRNA were normalized to the concentrations of U6-RNA and glyceraldehyde 3-phosphate dehydrogenase mRNA (GAPDH) respectively (See Figure 3B). The resulting data were compared with the Western blot of Ras protein expression (normalized to NAT) in the same lung cancer cell lines (See Figure 3C). The data indicated that a 10-fold increase in RAS protein corresponded with an 8-fold decrease in let-7 expression in lung cancer while showing no correlation between let-7 and Ras mRNA expression. This illustrates one of miRNAs known modes of action of inhibiting translation without triggering degradation. The correlation studies of let-7 presence and Ras mRNA/protein offer supporting evidence for miRNAs role as a tumor suppressor by down regulating known oncogenes. Even though the traditional role of tumor suppressors isnt involved in regulating the expression of oncogenes, this description of miRNA still operates within the widely accepted definition of a tumor suppressors; loss of gene activity

 contributes to cancerous transformation.

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Let-7 is not the only miRNA suspected of behaving similarly to tumor suppressors due to its inversely correlated expression to known oncogenes. Cimmino et al. (2006) investigation of chronic lymphocytic leukemia (CLL) was able to provide another key example of how down-regulated miRNAs correlate to altered expression of oncoproteins, testifying miRNAs role as a tumor suppressor. In most cases of CLL, known oncogene Bcl-2 (anti-apoptotic B-Cell Lymphoma) protein is over expressed by margins of 60-80% (Kitada et al. 1998). This negatively impacts the cell because Bcl-2 associates with the mitochondrial membrane to prevent BH123 proteins from releasing pro-apoptotic factors like Cytochrome C (Cimmino et al. 2006). The anti-apoptotic influence of elevated Bcl-2 sheds light on why CLL is characterized as a malignant slow-dividing B-cell leukemia. If subsequent mutations occur, the inability of cells to undergo apoptosis results in the accumulation of large dysfunctional B-cells in the various capillaries and lymph systems. In time, this eventually leads to toxicity through the bodys natural inflammatory responses (Harris et al. 1999). In order to draw the necessary correlations between miRNA dysregulation and over-expression of Bcl2 Cimmino et al. (2005) performed a broad- spectrum analysis of 30 cases of CLL, using a microarray containing 368 miRNA probes, followed by a Western blot with antibodies targeting Bcl-2 protein (See Figure 4). The data revealed that miRNA-15a and miRNA-16 show inversely correlated coefficient of 94 and 95% with respect to Bcl2 protein levels normalized to -actin concentrations (Cimmino et al. 2006). Discoveries of CLLs shared pattern of dysregulation between miRNA-15a/16 and Bcl2, serve to corroborate existing evidence from Slack et al. (2005) that miRNAs can potentially serve as tumor suppressors through post-transcriptional

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Figure 4 368 miRNA probe microarray analysis and Western blot of Bcl-2 (-actin normalized) revealed miRNA-15a and 16-1 show inversely correlated expression in tested CLL tissues samples.

oncogene regulation. Many forms of cancer share an atypical over expression of certain miRNAs that relate to a significantly reduced level of key tumor suppressors deemed necessary for cancerous transformation. This suggests that a fraction of miRNA may also perform actions that relate to oncogenes in addition to the previously noted role as a tumor suppressor (Asangani et al. 2008). In normal cells the up regulation of programmed cell death protein 4 (Pdcd4) prevents cancerous invasion and metastasis by inhibiting transcription initiating factors eIF4A and eIF4g (Yang et al. 2004). In lung, breast, and colorectal cancer however, Pdcd4 can be down regulated and is frequently used by physicians as a reliable indicator of poor prognostic outcome (Chen et al. 2003, Zhu et al. 2008). Using the 652 nt-3UTR sequence of Pdcd4 mRNA and comparing it to a bioinformatics database of known miRNAs revealed a 100% match between an 11-nt region of Pdcd4 and miRNA-21 (Asangani et al. 2008). To identify if any corollaries existed between pdcd4 (mRNA and protein) expression and miRNA-21, Northern/Western blot analysis and qRT-PCR were performed across 10 different colorectal cancer cell lines. With a statistical significance value (p = 0.01) rejecting the null hypothesis, indicating the results were not a product of

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experimental error or coincidence, Pdcd4 protein was shown to have an inversely related expression to miRNA-21 in all 10 samples (See Figure 5). Pdcd4 mRNA and miRNA-21 levels, however, did not result in a statistical significance value capable of rejecting the null hypothesis (p = 0.1). This is a notable attribute of miRNA post-transcriptional regulation whose base pairing is significant enough to prevent translation but not trigger mRNA degradation. The evidence shows that the amount of functional Pdcd4 protein available to reduce tumorgenicity is likely to be reliant on sustaining low level of miRNA-21 intervention (Asangani et al. 2009).

Figure 5 Pdcd4 protein densitometric Western analysis (actin normalized) against miRNA-21 qRT- PCR revealing an inverse relationship in expression across all 10 cell lines.

Further evidence illustrating a putative role for miRNA as an oncogene can be drawn from miRNA-221 and 222s over-expression in aggressive forms of human prostate carcinoma (PC3) (Galardi et al. 2007). Galardi et al. (2007) sought to better understand dysregulation in the availability of cyclin-dependent kinase inhibitor 1B (p27Kip1) in various forms of human prostate carcinoma because of its reliability in predicting poor

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prognostic outcome. p27Kip1, a known tumor suppressor, regulates cell cycle progression at

Figure 6 (A) Cell cycle depicting the natural accumulation of cycle D that would otherwise lead to CKD complex formation and the subsequent activation of phosphorylation cascade. (B) starting from the top: miRNA-221 and 222 northern blot analysis for PC3, 22Rv1, and LCAP (snRNA U6 normalized) along with the western blot for p27Kip1 protein (actin normalized).

the G1/S checkpoint by inhibiting the cyclin dependant kinase (CDK) complex from initiating a phosphorylation cascade that would otherwise drive the cell towards growth and division. If left uninhibited by p27Kip1, elevated levels of cyclin D that naturally accumulate as G1 of interphase progresses are free to activate CKD complex (See Figure 6A). Western blot analysis revealed that the aggressive androgen insensitive PC3 expresses little to no p27Kip1 protein when compared to slower growing androgen responsive (22Rv1) and androgen dependent (LNCaP) cell lines (Galardi et al. 2007) (See Figure 6B). When the expression of miRNA-221 and/or 222 was knocked down through transfection with locked nucleic acid (LNA) oligonucleotides (which are modified

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nucleotides that form a duplex with mRNA strands causing them to be inaccessible to ribosomal translation), the subsequent Western blot revealed that p27Kip1 protein levels had doubled in all three trials (See Figure 7B). Concurrently, when LNCaP cell lines were transfected with miRNA-221 and/or 222 plasmid constructs all cell lines resulted in a roughly 50% reduction of p27Kip1 expression (See Figure 7A). Surprisingly, after p27Kip1 mRNA expression levels were assessed between PC3 and slow growing prostate carcinoma cell lines (LNCaP), they were determined to be relatively equal to each other (Galardi et al. 2007). This supports the theory that disruption in some component of post-transcriptional gene regulation is responsible for the varied expression of p27Kip1 protein between PC3 and LNCaP cell lines. P27Kip1 mRNA levels matching between PC3 and LNCaP cell lines along with the results of knocked down or upregulated miRNA-221/222 expression on p27Kip1 protein availability reinforce
Figure 7 (A) Western blot for testing for p27 in LNCaP transfected with miRNA constructs. p-221 and p-222 represents miRNA-221 and 222 insert respectively p-T contains both plasmids while c is an empty vector. (B) Western blot of PC3 cells transfected with LNA-oligos. LNA-contr (blank vectors), LNA-T (miRNA-221 and 222), LNA-221 (miRNA-221), and LNA-222 (miRNA-222)

Galardi et al. (2007) theories of miRNA-221/222 function in repressing translation of p27Kip1.

The earliest evidence supporting miRNAs role in oncogenesis was its common dysregulation in a multitude of cancers. This dysregulation is frequently coupled with changes to known oncogenes or tumor suppressors expression. Literature clearly supports miRNAs post-transcriptional regulating functions as the presumed cause for this trend. The underexpression of let-7 and miRNA-15a/16s in CLL, lung, colon, and breast cancers,

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along with the comparable decrease in their respective oncogene, implies miRNA has normal pre-cancer functions as a tumor suppressor (Slack et al. 2005; Cimmino et al. 2006). The overexpression of miRNA-21 in lung, breast and colorectal cancer and miRNA- 221/222s increased expression seen only in aggressive forms of prostate cancer when viewed with respect to the decreased presence of tumor suppressor, attests to miRNAs potential as an oncogene (Asangani et al. 2009; Galardi et al. 2007). The predicted alternate roles of miRNA as a tumor suppressor and oncogene are not dependant on miRNA functioning in a manner that is contrary to how these regulatory RNAs are historically understood to function. miRNA in both cases, as an oncogene or tumor suppressor, are still operating by post-transciptionally regulating mRNA expression. The literature available on cancer expression patterns indicates that the differences lie in the form of aberrant miRNA gene expression (amplification, deletion, rearrangement etc.) occurring and the category of genes being targeted (i.e. oncogene or tumor suppressor gene). These trends provide novel insight on what specific miRNAs may actually be at the root of the problem, which highlights potentially suitable targets for therapeutic intervention. Complementary sequences between miRNA & 3UTR of cancer related mRNA: When correlations have been documented between a miRNAs expression profile and cancer-risk mRNAs, genomic sequence analysis, bioinformatics, and luminescence repression studies are used to investigate whether the miRNA is interacting with that particular mRNA. This is necessary because simply relying on correlations between paralleled expression patterns is insufficient to establish actual causation. Based off of the correlation studies uncovering the relationship between let-7 and Ras expression, Johnson et al. (2005) performed a luminescence repression assay (LRA) to indicate whether the similar homologies (revealed by genomic sequence analysis) shared by let-7 and Ras mRNA

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Figure 8 Percent luminescence repression indicating the relative amount of direct interaction between tagged Ras mRNA and miRNA let-7

in response to ectopic levels of transfected let-7 miRNA (10nM or

30nM) indicating successful interaction (See Figure 8). Not only did this support a successful interaction between the two molecules but it also revealed that Ras mRNA exhibits a dosage dependant repression by let-7 (See Figure 8).

Figure 9 (A) Luciferase repression assays showing the influence of miRNA- Let-7 target pairing with mutated Ras mRNA luciferase constructs Luc-m (B) Design of the murine luciferase HMGA2 3UTR mRNA reporter constructs and the 3UTR sites that have been deleted

mRNAs from different genes can share complementarity between a single class of miRNAs, so it was not surprising that other targets of let-7 besides Ras might exist. Fueled by similar correlation studies used by Johnson et al. (2005), Mayr et al. (2008) sought to investigate whether oncogene high mobility group protein a2 (HMGA2) with its 3UTR

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containing multiple homologies to let-7 was capable of the interaction needed induce repression. The magnitude of luminescence repression was found to be directly proportional to the number of complementary sites left unmutated by researchers on the 3UTR of HMGA2 mRNA (Mayr et al. 2008) (See Figure 9A). These data when taken in conjunction with the results provided by Johnson et al. (2005) suggest that mRNA can also display a repression sensitivity based on the number of common homologies present in the 3UTR in addition to the dosage of miRNA available. This variance may explain why cancer related mRNA and its putative miRNA show different magnitudes of repression in response to varying degrees of miRNA dysregation in cancer.

Figure 10 (A) Luminescence repression assays showing the 3UTR of BCL2 enabling miRNA-15/16 regulation. sense and antisense (A or S) miRNA vectors were used. (B) two different mutant 3UTR of Bcl2 mRNA were used. (3M1) had none of the 9bp miRNA/mRNA interaction, (3M2) had the first 5bp deleted in the complementary region. All assays were repeated in triplicate with sense and antisense oligos (A or S)

In the Cimmino et al. (2005) study outlining miRNA-15a/16s potential interaction with Bcl-2 in CLL, the researchers were able to show a similar repression in luminescence when Bcl-2 mRNA tagged with a luciferase reporter was exposed to transfected miRNA- 15a/16. Cimmino et al. (2005) went a step further and used the luminescence repression along with point specific mutation of miRNA-15a/16 to show that a full sequence of 9 complementary nucleotides was need for the successful miRNA-15a/16 interaction with Bcl-2 mRNA (See Figure 10). This corroborates preliminary evidence that drew parallels between miRNA presence and oncogene expression, by yielding data that matches all the

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criteria typical miRNA post-transcriptional repression, i.e. increased miRNA presence and decrease mRNA translation and successful miRNA::mRNA interaction. Finally, LRAs support current trends claiming that let-7 and miRNA-15a/16 exhibit tumor suppressive behaviors by down regulating oncogenes Ras, HMGA2, and Bcl2 respectively. miRNAs exhibiting oncogene like behavior are also shown to function by binding to complementary sequences in the 3UTR of mRNA (Zhu et al. 2008; Galardi et al. in 2007). Sequence analysis of miRNA-21 and its putative Pdcd4 mRNA target revealed that the two molecules share an 13-nucleotide stretch that may be responsible for the outcomes of the correlation studies produced by Zhu et al. (2008) and Asangani et al. (2009). LRAs were performed on human embryonic kidney cells (HEK293) because of their characteristically low expression of miRNA-21. This is an important testing prerequisite because Zhu et al. (2008) wanted to investigate the effects of increased levels of miRNA-21 on luminescence repression. If the basal levels miRNA-21 were already high, luminescence would start out too low to be able to study the effects of increasing miRNA-21 ectopically. While Zhu et al. (2008) did not give any indication of how much miRNA-21 was used to transfected the HEK293 cell lines, the study did claim that the

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luciferase Pdcd4 mRNA construct resulted in a 50% reduction in luminescence when compared to control cell lines. LRAs were also compared with the repression of mutant luciferase Pdcd4 mRNA that lacked the shared

Figure 11 Percent luminescence repression indicating the relative amount of direct interaction between tagged PDCD4 mRNA (luc) and miRNA-21 transfected ectopically in HEK293 cells. (UTR = wt ) (D = deleted UTR)

13-nucleotide homology (See Figure 11). This resulted in a luminescence repression that

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matched the untransfected control with a marginal difference of 5%. Both LRAs, wild type 3UTR and mutant 3 UTR, resulted in statistical significance values (P 0.01) that rejected the null hypothesis suggesting that tested variables and the resulting data were related. Since Zhu et al. (2008) used cell lines that characteristically express miRNA-21 at low levels the changes in luminescence repression in response to both miRNA-21 vector dosage and deletions in shared homologies on the 3UTR of Pcd4 mRNA produced observable results that allowed for positive interaction between the two molecules to be asserted. Bioinformatics analysis of Pdcd4 mRNA between various species revealed that of the 13-nucleotides found similar on the presumptive human Pdcd4 miRNA-binding site, a stretch of 8-nucleotides are 100% conserved across all 7 tested species (Asangani et al. 2008). This suggests a high evolutionary dependence on this 13nt sequence for functionality of the molecule and survival of the individual. The statistical values of the luminescence repression studies strongly support Zhu et al. (2008) and Asangani et al. (2009) findings of miRNA-21 repressing Pdcd4 mRNA translation. This is based of the common trend of miRNA-21 upregulation in breast (Zhu et al. 2008) and colon cancer (Asangani et al. 2008) being correlated to an aberrant under expression of Pdcd4. When taken into account with their common homologies and the results of the LRAs, this supports miRNA-21 is capable of negatively regulating a tumor suppressor, indicative of oncogene like behavior. Questions of whether p27Kip1 mRNA shared any homologies with miRNA-221/222 also became a topic of investigation after data revealed a parallel in patterns of expression in prostate cancer (Galardi et al. in 2007). Since Galardi et al. (2007) and Zhu et al. (2008) had very similar objectives investigating miRNA-based down regulation of tumor

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suppressors the fact that their protocols were so alike was unsurprising. Before any LRAs were performed sequence analysis revealed that miRNA-221/222, because of their similarity in sequence, had 3 common sites on 3UTR of p27Kip1 mRNA that were deemed as potential miRNA binding sites. While the total number of complementary nucleotides varied from 7 - 15, all 3 plausible mRNA binding sites had a stretch of 7 nucleotides in common with both miRNAs. Much like Zhu et al. (2008) with the low miRNA expressing HEK293 cell lines, low miRNA- 221/222 expressing LNCaP cells were chosen so that transfection with luciferase tagged p27Kip1 mRNA and ectopic miRNA-221/222 resulted in repression that was principally due to the
Figure 12 Relative reporter activity indicating the amount of direct interaction between tagged p27 mRNA and miRNA-221/222

exogenous delivery of miRNA under investigation. When the LNCaP cell lines

expressing tagged wild type p27Kip1 mRNA were compared to cell lines expressing tagged mutant p27Kip1 mRNA (antisense), only the cell lines containing wild type mRNA displayed a notable level of luminescence repression in response to ectopic miRNA-221 and/or miRNA-222 (Galardi et al. 2007). Between the wild type p27Kip1 mRNA control receiving no exogenous miRNA and the wild type p27Kip1 mRNA that was exposed miRNA, there was roughly a 50% shared luminescence repression for all three wild type mRNA samples transfected with miRNA-221 and/or miRNA-222 (See Figure 12). As suspected, luminescence repression was dependent on the presence of intact homologies in the 3UTR

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of p27Kip1 mRNA and on the availability of miRNAs-221/222 (Galardi et al. 2007). Nearly identical to the outcome of Zhu et al. (2008) research on miRNA-21, Galardi et al. (2007) research similarly resulted in a collection of evidence from correlation studies, sequence analysis, and LRAs that all argued for miRNAs role as a tumor suppressor. Sequence analysis of let-7 and miRNA-15a/16, presumed to be functioning as tumor suppressors, was used to highlight similar homologies in their suspected mRNA target. This method was also used in investigating miRNA-21 and miRNA-221/222 suspected actions that result in behavior similar to oncogenes. In both cases, miRNA as an oncogene or as a tumor suppressor, the discovered homologies were used in LRAs to ensure that the successful interaction required for post-transcriptional regulation was possible. Collectively, these studies indicates that oncogenes: Bcl2, Ras, and HMGA2, along with tumor suppressors: p27Kip1 and Pdcd4 sensitivity to miRNAs is due to the homologies found in the 3UTRs of these cancer related mRNAs were to blame. This assessment is both significant and reliable for number of reasons. First, it eliminated any invalid correlations based solely on relating the miRNA and mRNA expression levels. Secondly, it showed a successful method for obtaining evidentiary support of miRNA potential as a cancer gene regulator and subsequently supporting its role as an oncogene/tumor suppressor. This was accomplished through experimental procedures that resulted data consistent with both the aberrant expression patterns found common to certain cancers and the mode in which miRNA was traditionally understood to function. The last significant aspect of this assessment was that it set the grounds for future research that would ultimately contribute to understanding how altered miRNA levels can result in certain cancerous behaviors and the therapeutic potential of restoring that change.

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Purposeful dysregulation of miRNA: Accumulating evidence for abnormal expression of miRNA playing a major role in cancer has sparked the next series of experiments identifying how deliberate mutations to miRNA expression can either restore normal cell behavior or lead to oncogenesis. Researchers altering the expression of miRNA in cell lines further verified the cause and effect nature behind how exactly divergent patterns of miRNA levels in cancer can affect cell behavior. Slack et al. 2005 was able to conclusively link the traits of excessive cell proliferation with inversely correlated concentrations of let-7 and Ras by complementary RNA interference (RNAi) of Caenorhabditis elegan lethal-60 (let-60 is an ortholog of human let-7). While the selective inhibition of let-60 resulted in the predicted overexpression of Ras activity, the mutations had characteristically different phenotypes in tumorous defects between C. elagans and human tissues. Even with this being true, the experiment still served as an important model for how altered miRNAs (or more aptly let-60) correlated to tumor formation (Slack et al. 2005). Purposeful dysregulation of miRNA expression was also used to expose how let-7s supplemental involvement with HMGA2 would
Figure 13 Growth of transfected H1299 cells measured optical density at 0, 3rd and 5th day of incubation in 2% serum. (triangle) transfected with mutant 3UTR HMGA2 mRNA (dash) wild type.

result in changes to cellular behavior (Lee et al. 2007). After conclusive evidence was unveiled

for both let-7s correlation to HMGA2 expression and common homologies between the key molecules Lee et. at (2007) sought to determine whether eliminating the confirmed miRNA::mRNA binding site would affect the proliferative capacity lung cancer cell lines.

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The important detail is even though presence of let-7 was left unchanged the alterations to the complementary sites in the 3UTR of HGMA2 mRNA by transfection with a mutated form of HGMA2 mRNA resulted in a 2 fold increase in proliferative capacity of cancerous cell lines (See Figure 13). These examples provide solid evidence for not only miRNAs mode of action but the consequences to cellular division as well. The common final inquiry in every cancer related investigation is how can the

alterations to the processes leading to tumorous transformation be restored. In an attempt to assess the therapeutic potential of miRNA Cimmino et al. (2006) wanted to see if reestablishing normal expression of miRNA 15a/16 would be sufficient to restore Bcl-2 repression (See Figure 14B) and subsequently uninhibit the apoptotic pathway

Figure 14 (A) Reactivation of intrinsic apoptotic pathway (Western blot) through transfection of MEG-01 cultures with miRNA-15/16 vectors. DNA fragments, consequence of apoptotic pathway activation, analyzed with southern blot (B) Restored down regulation of Bcl2 with sense miRNA- 15/16 transfection vectors while no change with mutant (antisense) or control.

responsible for decreasing cancerous behavior (See Figure 14A). Of the cell lines tested in vitro only the cultures transfected with miRNA-15a/16-wt vectors resulted in the

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reactivation of the intrinsic apoptotic pathway. This was evident by the cleaved DNA fragments discovered in the Southern blots and the presence of apaf-1-caspase9-PARP (proteins involved in apoptotic pathway) in the Western blots. This study emphasizes miRNA-15a/16 importance as tumor suppressor by inducing apoptosis albeit indirectly through the down regulation of Bcl-2 gene expression. Additionally, recovery of the apoptotic pathway shows the potential of miRNA as a therapeutic target for future cancer.

Figure 15 (A) transfection analysis of HepG2 cell lines with miRNA-26 vector and the subsequent decrease of Cyclins D2 and E2 (Western blot) (B) Representative images of livers from miR-26a- treated and control animals. Treated with AAV (C) Cell-cycle profiles of retrovirally infected HepG2 cells following treatment with nocodazole (Noc) which was used to synchronize cell lines. Numbers over each histogram indicate the percentage of cells remaining in G1.

In a similar study (Kota et al. 2009) investing the role of miRNA as a tumor

suppressor, researchers were able to use an in vivo model organism to assess the efficacy of

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reestablishing normal levels of miRNA on abnormal oncogene up regulation. miRNA-26as newly discovered role in inhibiting expression of oncoproteins (cyclin D2 and E2) responsible for altering G1 cell cycle progression in Homo sapien and Mus musculus liver cancer provided researchers a vital opportunity to explore the potential of a miRNA based treatment. Using adeno-associated vector (AAV) gene therapy as a delivery mechanism, treated M. musculus showed a significant reduction in tumor size and normal levels of cyclin D2 and E2 indicated by Western blot and qt-PCR (Kota et al. 2009)(See Figures 15A 15B ). The study was also able to show positive changes to the percentage of cells in the G1 cycle for liver cancer cell lines transfected retrovirally (See Figure 15C). While the number of research available showing miRNA expression recovery for in vivo systems is rare especially in cases where miRNAs targeting tumor suppressors are upregulated because of the unique set of problems it presents researchers attempting to restore levels of miRNA expression. The outcomes of this study is still serve as a milestone because of its ability to show at proof of concept in terms of miRNA being used as a potential therapeutic target. Conclusion: It has become increasingly clear that dysregulation of the traditionally understood oncogenes/tumor suppressors (p27, p53, Ras, HMGA2 Bcl-2 etc.) can be set into motion through a variety of mechanisms. Literature has revealed that miRNAs general function, wide variety of mRNA targets, and vulnerability to mutation (deletion, amplification, translocation etc.) offer insight on how cancerous transformation occurs. While the strengths of todays literature gives the reader the impression that cancer researchers are finally on the right track, much work still lies ahead. Its also significant to mention that while a concise and shared method of deducing miRNA involvement in various cancers exists, there is a general lack in understanding on how to apply such knowledge

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therapeutically. Major changes to how cancer is categorized have been made because of previously discussed literature. Slowly turning away from grouping cancer by morphology, tumor location, or affected cell type cancer is view in context of the genomic basis for alteration. The evolution of how cancer is view will potentially lead to more successful downstream therapeutic targets. Future research will continue understanding the various miRNAs and their complicate involvement in regulation various traditionally understood cancer critical mRNA. Further development also needs to be made on the delivery mechanism for adjusting change to miRNA expression from various forms of mutation.

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