MicroRNAs in cancers and aging, and back-to-the-nematode

MicroRNAs (miRNAs) are generating increased excitement among cancer, neurobiology and longevity researchers.  I wrote an introduction to MicroRNAs is in my earlier blog post MicroRNAs, diseases and yet-another view of aging, and readers might want to review that information before proceeding further here.  The purpose of this post is to zero in on some recent research findings in the areas of cancer and aging. 

Again, miRNAs are short strands of RNA encoded by genes which do not themselves encode proteins, proteins being the expression products of “regular” genes.  Instead, MiRNAs can profoundly affect the expression of genes that do encode proteins by post-translational operation on the messenger RNA produced by such genes.  “MicroRNAs downregulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation(ref).”  That is, miRNAs can halt or slow the creation of proteins of “regular”genes by damaging or interfering with their messenger RNA (mRNA) before it is converted into proteins or by interfering with the final protein-creation step.  See the earlier post for more detailed discussion and links on what miRNAs do, how many of them there are, their roles in stem cells, and how they enter into several disease processes.  I also mentioned the possible roles of miRNAs in aging in that post and promised I would elaborate on that topic in a subsequent post, which is this one.

One aspect of miRNAs emphasized in this post is that many of them are ancient and evolutionary conserved in species as different as nematode worms (Caenorhabditis elegans), flies, fish, and humans. This means that aging research done on species that only live a few days might well be applicable to humans. Nematodes are “–  simple enough to be studied in great detail. Strains are cheap to breed and can be frozen. When subsequently thawed they remain viable, allowing long-term storage. — In addition, C. eleagans is transparent, facilitating the study of cellular differentiation and other developmental processes in the intact organism. The developmental fate of every single somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped out(ref).^[8][9]“ 

MiRNAs and cancers

Quoting from a January 2010 review study The role of let-7 in cell differentiation and cancer: “MicroRNAs (miRNAs or miRs) are small noncoding RNAs capable of regulating gene expression at the translational level. Current evidence suggests that a significant portion of the human genome is regulated by microRNAs, and many reports have demonstrated that microRNA expression is deregulated in human cancer. The let-7 family of microRNAs, first discovered in Caenorhabditis elegans, is functionally conserved from worms to humans. The human let-7 family contains 13 members located on nine different chromosomes, and many human cancers have deregulated let-7 expression. A growing body of evidence suggests that restoration of let-7 expression may be a useful therapeutic option in cancers, where its expression has been lost. In this review, we discuss the role of let-7 in normal development and differentiation, and provide an overview of the relationship between deregulated let-7 expression and tumorigenesis. The regulation of let-7 expression, cancer-relevant let-7 targets, and the relationship between let-7 and drug sensitivity are highlighted.” A 2008 publication indicates that the let-7 miRNA works by targeting the miRNA processing enzyme Dicer within its coding sequence.

It appears that miRNAs could play important roles in prognostication of cancer outcomes.  The February 2010 research publication A MicroRNA Expression Signature for Cervical Cancer Prognosis states “Invasive cervical cancer is a leading cause of cancer death in women worldwide, resulting in about 300,000 deaths each year. The clinical outcomes of cervical cancer vary significantly and are difficult to predict. Thus, a method to reliably predict disease outcome would be important for individualized therapy by identifying patients with high risk of treatment failures before therapy. In this study, we have identified a microRNA (miRNA)-based signature for the prediction of cervical cancer survival. miRNAs are a newly identified family of small noncoding RNAs that are extensively involved in human cancers. Using an established PCR-based miRNA assay to analyze 102 cervical cancer samples, we identified miR-200a and miR-9 as two miRNAs that could predict patient survival. A logistic regression model was developed based on these two miRNAs and the prognostic value of the model was subsequently validated with independent cervical cancers. — Our results suggest that both miR-200a and miR-9 could play important regulatory roles in cervical cancer control. In particular, miR-200a is likely to affect the metastatic potential of cervical cancer cells by coordinate suppression of multiple genes controlling cell motility.”

A stream of publications explores the role of the miRMA miR-34a in neuroblastoma. “miR-34a acts as a suppressor of neuroblastoma tumorigenesis by targeting the mRNA encoding E2F3 and reducing E2F3 protein levels(ref).” “A functional screen identifies miR-34a as a candidate neuroblastoma tumor suppressor gene(ref).”  Also see MicroRNA involvement in the pathogenesis of neuroblastoma: potential for microRNA mediated therapeutics, MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells, and The MYCN oncogene is a direct target of miR-34a.The 2008 publication Diagnostic and prognostic microRNAs in stage II colon cancer states “Functional studies showed that miR-145 potently suppressed growth of three different colon carcinoma cell lines. In conclusion, our results suggest that perturbed expression of numerous miRNAs in colon cancer may have a functional effect on tumor cell behavior, and, furthermore, that some miRNAs with prognostic potential could be of clinical importance.”  And the report of a November 2009 study indicates “Human colon cancer profiles show differential microRNA expression depending on mismatch repair status and are characteristic of undifferentiated proliferative states.”A December 2009 study Gene networks and microRNAs implicated in aggressive prostate cancer reports “Prostate cancer, a complex disease, can be relatively harmless or extremely aggressive. To identify candidate genes involved in causal pathways of aggressive prostate cancer, we implemented a systems biology approach by combining differential expression analysis and coexpression network analysis to evaluate transcriptional profiles using lymphoblastoid cell lines from 62 prostate cancer patients with aggressive phenotype (Gleason grade 8) and 63 prostate cancer patients with nonaggressive phenotype (Gleason grade 5). From 13,935 mRNA genes and 273 microRNAs (miRNA) tested, we identified significant differences in 1,100 mRNAs and 7 miRNAs with a false discovery rate (FDR) of <0.01. We also identified a coexpression module demonstrating significant association with the aggressive phenotype of prostate cancer (P = 3.67 x 10–11). The module of interest was characterized by overrepresentation of cell cycle–related genes (FDR = 3.50 x 10–50). From this module, we further defined 20 hub genes that were highly connected to other genes. Interestingly, 5 of the 7 differentially expressed miRNAs have been implicated in cell cycle regulation and 2 (miR-145 and miR-331-3p) are predicted to target 3 of the 20 hub genes. Ectopic expression of these two miRNAs reduced expression of target hub genes and subsequently resulted in cell growth inhibition and apoptosis. These results suggest that cell cycle is likely to be a molecular pathway causing aggressive phenotype of prostate cancer.”The concept of sets of hub genes that are highly interconnected with other genes is an important one characterized in this article. Hub genes were discovered in the nematode and are evolutionary conserved across many species including humans.  “–their normal function is to act as genetic buffers, minimizing the effects of mutations in other genes. — “RNAi (RNA interference) in the nematode is a particularly simple and elegant method to study the effects of genetic variation,” explained Dr Andy Fraser, Principal Investigator at the Wellcome Trust Sanger Institute, “and the worm is ideally suited to high-throughput analysis. If we want to understand how genes work in concert, we have to look at the activities of combinations of genes in living organisms.” — “We found specific roles for many new genes in individual known pathways that are key in human disease but, more important, identified a class of hub genes, which affect many different pathways. We predict that other classes of hub genes will be identified using this method.”

The 2009 study Profiling of 95 microRNAs in pancreatic cancer cell lines and surgical specimens by real-time PCR analysis reports “The expression of miRNAs for pancreatic cancer tissue samples or cancer cell lines was normalized to U6 RNA and compared with those in relatively normal pancreatic tissues or normal human pancreatic ductal epithelial (HPDE) cells. Human pancreatic tissue with chronic pancreatitis also was included for analysis. RESULTS: In the initial analysis, the expression of most 95 miRNAs was substantially changed in pancreatic cancer tissues (n=5) and cell lines (n=3) compared with relatively normal pancreatic tissues and HPDE cells. However, each pancreatic cancer tissue or cell type had a substantially different profiling pattern with other cases or cell types as well as chronic pancreatitis tissue, indicating the individual diversity of pancreatic cancer. Further analysis was performed on 10 pancreatic cancer cell lines and 17 pairs of pancreatic cancer/normal tissues. Eight miRNAs were significantly upregulated in most pancreatic cancer tissues and cell lines, including miR-196a, miR-190, miR-186, miR-221, miR-222, miR-200b, miR-15b, and miR-95. The incidence of upregulation of these eight genes between normal control subjects and tumor cells or tissues ranged from 70-100%. The magnitude of increase of these miRNAs in pancreatic cancer samples ranged from 3- to 2018-fold of normal control subjects.”

The above-quoted studies are just a sampling of the extensive work going on investigating the roles of miRNAs in various types of cancers.  The outcomes of this work are likely to be 1.  a lot of new fundamental insights into cancer processes, 2.  a new generation of miRNA-based diagnostic and prognostic tests related to specific cancers and 3. At some point, new miRNA-based cancer therapies.  I point out that this discovery work is greatly empowered by the availability of new-generation highly efficient analysis technology like that embodied in the Quantimir Small RNA Quantitation System.

MiRNAs and aging

It is fascinating that miRNAs known as lin-4 and lin-14 appear to govern how long tiny Caenorhabditis elegans worms (nematodes) live.  The 2007 publication A developmental timing microRNA and its target regulate lifespan in Caenorhabditis elegans relates “The microRNA lin-4 and its target, the putative transcription factor lin-14, control the timing of larval development in Caenorhabditis elegans. Here, we report that lin-4 and lin-14 also regulate life span in the adult.  Reducing the activity of lin-4 shortened life span and accelerated tissue aging, whereas overexpressing lin-4 or reducing the activity of lin-14 extended life span. Lifespan extension conferred by a reduction in lin-14 was dependent on the DAF-16 and HSF-1 transcription factors, suggesting that the lin-4-lin-14 pair affects life span through the insulin/insulin-like growth factor-1 pathway. This work reveals a role for microRNAs and developmental timing genes in life-span regulation.”  Actually, lin-4 and lin-14 were the first two miRNAs to be intensely studied(ref).  A great history on the discovery of miRNAs going back to 1979 can be found here.

The question of course, is whether there are evolutionary-conserved homologs of lin-4 and lin-14 that control human longevity, a bit of a push since these two genes are involved in larval development of nematodes.  But it is not necessarily an impossible push.   The first item under cancers above points out how “The let-7 family of microRNAs, first discovered in Caenorhabditis elegans, is functionally conserved from worms to humans.”  A great many other miRNAs are similarly evolutionary conserved  and are found in diverse species including worms, flies, zebrafish, mice and humans(ref).  It appears that when evolution finds a biological pathway that works for certain essential life functions in primitive species, when possible it carries this pathways forward with minimum required tinkering as it evolves higher species.  This seems to be a general principle in genomics where many genes are the same in us and in yeast cells. 

An interesting 2007 blog comment quotes research indicating that there seems to be no aging-related effect on gene expression for 256 miRNAs in lung tissues.  “ — we have used a highly-sensitive, semi-quantitative RT-PCR based approach to measure the expression profile of 256 of the 493 currently identified miRNAs in the lungs from 6 month (adult) and 18 month (aged) old female BALB/c mice. We show that, despite the characteristic changes in anatomy and gene expression associated with lung aging, there were no significant changes in the expression of 256 miRNAs. Conclusion: Overall, these results show that miRNA transcription is unchanged during lung aging and suggests that stable expression of miRNAs might instead buffer age related changes in the expression of protein-encoding genes.” 

Some researchers think we can take a lesson from the situation for Caenorhabditis elegans and look for miRNAs that are directly related to aging pathways.  We know that the insulin/insulin-like growth factor pathway, the one affected in the nematode by the lin-4-lin-14 pair, is also very important for longevity in humans.  It is the pathway affected by calorie restriction and by taking resveratrol. 

The 2009 publication Epigenetic Control of MicroRNA Expression and Aging suggests that with aging miRNA expression tends to increase creating increased quieting of target genes, slowly shutting down our protein-making and gene expression.  “Our recent results with several model systems show that upon aging, there is a trend of up-regulation of microRNA expression, with concomitant inverse down-regulation of target genes. This review addresses molecular mechanisms that may provide the underlying control for this up-regulating trend, focusing on activation by various microRNAs’ own promoters, through binding with pivotal transcription factors, stress response, methylation of clustered DNA domains, etc.  Thus, epigenomic control of aging may be due in part to heightened promoter activation of unwanted microRNA expressions, which in turn down-regulate their target gene products. Overriding and dampening the activation of these noncoding RNAs may prove to be a new frontier for future research, to delay aging and extend healthy life-span.”  This suggests another mechanism behind the 13^th theory of aging covered in my treatise, Programmed Epigenomic Changes.  Why, exactly, there is increasing expression of miRNA genes with age is not clear to me, however. 

The 2007 publication MicroRNA, the putative molecular control for mid-life decline  suggests “– possible derailment of these negative regulators for gene expression (miRNAs)in mid-life may be the putative force inducing molecular frailty in individual cell signaling, and in time leading to tissue-wide dysfunction.”  

In my earler post on miRNAs I mentioned a somewhat different take on miRNAs and aging.  “Quoting again from the Gen article, “Eugenia Wang, Ph.D., professor at the University of Louisville, has proposed that miRNAs have a critical role in “a universal or system-specific programmatic shift of signaling control” that occurs at mid-life and brings about a decline in cellular health status associated with aging, which may precipitate increased risk of late-life diseases. In her presentation, she will review the hypothesis that the changes in expression of most if not all aging-related genes are controlled by underlying hubs and the belief that miRNAs, acting as molecular master switches, are candidate hubs.”   This seems more or less consistent with the ideas in the previous paragraph.   

Another line of research applicable to both cancers and longevity has looked at at our old friend, the P53 tumor-suppressor gene and its relationship to miRNAs.  The 2009 publication The p53 Pathway Encounters the MicroRNA World has this to say: “Recently, microRNAs (miRNAs) have been reported to be directly transactivated by p53. Equally, p53 and components of its pathway have been shown to be targeted by miRNA thereby affecting p53 activities. In this review, we focus our discussion on the biological and pathological roles of miRNAs in the p53 pathway.”  P53 is of course a key apoptosis gene and when it is incorrectly turned off cancers can result.  When it comes to molecular biology of cells, it seems to me that often everything has to do with everything else. 

In summary, while many researchers think miRNAs are implicated in aging, there are several quite different hypotheses about what they might be doing.  As far as I can tell we don’t yet have a “smoking gun” for the  aging control in humans like lin-14-Lin4 miRNAs are for nematodes. 

The 2009 publication MicroRNAs in C. elegans Aging: Molecular Insurance for Robustness? suggests that there may be a lot more for us to learn about aging by studying the impacts of miRNAs on lifespans in those little critters. “It has been proposed that miRNAs ensure biological robustness, and aging has been described as a progressive loss of system and cellular robustness, but relatively little work to date has addressed roles of miRNAs in longevity and healthspan (the period of youthful vigor and disease resistance that precedes debilitating decline in basic functions). The C. elegans model is highly suitable for testing hypotheses regarding miRNA impact on aging biology: the lifespan of the animal is approximately three weeks, there exist a wealth of genetic mutations that alter lifespan through characterized pathways, biomarkers that report strong healthspan have been defined, and many miRNA genes have been identified, expression-profiled, and knocked out. 50/114 C. elegans miRNAs change in abundance during adult life, suggesting significant potential to modulate healthspan and lifespan. Indeed, miRNA lin-4 has been elegantly shown to influence lifespan and healthspan via its lin-14 mRNA target and the insulin signaling pathway. 27 of the C. elegans age-regulated miRNAs have sequence similarity with both fly and human miRNAs. We review current understanding of a field poised to reveal major insights into potentially conserved miRNA-regulated networks that modulate aging.”  

Our understanding of how miRNAs work in humans is just getting off the ground.  As the last publication suggests back to the nematode may be a good strategy for now.  Let us fully exploit the fact that evolution carries biological machinery forward from species to species, including possibly machinery that controls aging.  If we can thoroughly investigate various combinations of miRNAs already suspected or known to keep C. elegans young,  and find homologs in humans of the same miRNAs, they may well keep us young too. We already know that the normal lifespans of nematodes of 20 days can be extended to 125 days by genetic manipulation(ref).  If that life extension factor of more than six could be translated to humans, it would mean normal lifespans of around 480 years.