Recent research from members of the ExRNA Communication Consortium (ERCC) suggests that extracellular RNAs (exRNAs) circulating in plasma play an active role in insulin resistance (IR). Insulin resistance is an incurable but manageable syndrome where the body stops reacting efficiently to the insulin hormone, which stimulates the uptake of glucose in the blood into cells and inhibits the body from using fat for energy, resulting in high blood sugar levels. The study, by Shav et al., points to certain exRNAs, particularly miR-122 and miR-192, as indicators and active players in IR regardless of the age, sex, or BMI of a person, which suggests that they may serve as more than metabolic markers and that they perhaps have functional, trans-organ roles in mediating IR.

Previous research has demonstrated that exRNAs have different functions in pathways relating to metabolic syndrome, which is a series of conditions that increase the risk of heart disease, stroke, and diabetes. For example, there is measurable miRNA dysregulation in obesity and in the progression of cardiometabolic disease. Other miRNAs are involved in brown/white fat specification, adipose tissue inflammation (Karbiener and Scheideler, 2014), and hepatic steatosis (fatty liver disease, Becker et al., 2015). Although these studies have identified specific exRNAs and miRNA networks that also have roles in IR, they have had either small sample sizes and lack validation in large populations or have been carried out in non-human models. In order to validate these data, the authors carried out a large-scale human translational study in which they analyzed detailed obesity-related phenotypic data from over 2,500 participants (most of whom were non-diabetic) from the Framingham Heart Study (FHS), an unrelated cardiovascular disease study (Feinleib et al., 1975).

To begin, the investigators analyzed blood samples from 2,317 non-diabetic study participants and quantified the plasma extracellular circulating exRNAs. They looked at RNAs [including piwi-interacting RNA (piRNA) and small nucleolar RNA (snoRNA)] expressed above a threshold level and excluded RNAs that were not found in at least 100 participants. From the resulting panel of 391 exRNAs, the investigators identified 16 microRNAs (miRNA), 1 piRNA, and 1 snoRNA that were associated with insulin after controlling for age, sex, and BMI. Of note, the abundance of miR-122 was shown to increase in a stepwise fashion as levels of insulin increased across the population. Higher levels of both miR-122 and miR-192 in the plasma were also consistently associated with a series of metabolic phenotypes, such as greater BMI and waist circumference, visceral fat quantity and quality, and liver attenuation. On the other hand, neither miRNA was associated with subcutaneous fat. These results were consistent whether the analysis included only the non-diabetic participants or the entire FHS population.

miRNAs function to regulate gene expression, so the authors conducted a pathway analysis to determine the targets of the 16 identified miRNAs. Almost unsurprisingly, all 16 miRNAs target insulin signaling pathways such that there is ample crosstalk and targeting of multiple IR-related genes by multiple miRNAs. This analysis validated findings from previous studies that implicated several miRNA target genes in the pathogenesis of IR, notably protein tyrosine phosphatase, nonreceptor type 1 (PTP1B) (Stull et al., 2012), mitogen-activated protein kinases (MAPKs) (Wang, Goalstone & Draznin, 2004), and 5′ adenosine monophosphate-activated protein kinase (AMPK) (Ruderman et al., 2013).

For the second part of the study, the investigators determined whether the miR-122 and miR-192 associations to age, sex, and BMI held true in a separate study population, a cohort of 90 overweight or obese young adults involved in the POOL study. Analyses of the youths’ plasma samples indicated that miR-122 (but not miR-192) was associated with greater IR after adjusting for age, sex, and BMI, and that this association remained even after the miRNA was analyzed independently of age, sex, BMI, or metabolite profile.

This study provides additional evidence and translational support for the role of exRNAs in IR. The findings indicate not only an association of exRNAs with insulin levels, but that the exRNAs may be playing an active role in the development or sustainment of IR. It is therefore critical to conduct further mechanistic investigations into the role of exRNAs in the metabolic architecture of IR.

Dr. Alissa Weaver, Vanderbilt University professor and Extracellular RNA Communication consortium (ERCC) member, will be inducted as an AAAS Fellow this Saturday, February 18, 2016. Dr. Weaver joins Dr. James Patton, also of Vanderbilt, and Dr. David Wong of UCLA as consortium members who are also current AAAS Fellows. This honor is bestowed upon her for her contributions to the field of cancer biology and studies of extracellular vesicles (EVs) in cell motility and cancer metastasis.

Alissa Weaver

Dr. Weaver’s academic career began at Stanford University where she double majored in Biology and Political Science. Always aspiring to be a physician, she then attended medical school at the University of Virginia, Charlottesville. However, along the way, she realized that she missed the academics of a PhD. “When I was in medical school, I realized that I really missed thinking about scientific discovery and was not being taught to do research,” she explained. “I really wanted to have the formal training of getting a PhD so I applied for the program from medical school.” After completing her MD/PhD at UVA, she traveled to Washington University, Saint Louis for 5 years where she did a Laboratory Medicine residency and a postdoctoral fellowship in the Department of Cell Biology and Physiology with Dr. John Cooper.

Finally in 2003, she accepted a faculty position at Vanderbilt University where she now remains as a full time researcher. Her lab focuses on all aspects of extracellular vesicles. The interest originally stemmed from her investigations of cell invasion, migration and cancer metastasis. The lab’s focus shifted as they learned that many of the secreted molecules that facilitated invasion were transported by EVs.

Part of the invasive nature of cancer cells in metastasis involves structures called invadopodia, actin-based protrusions of the plasma membrane that facilitate degradation of the extracellular matrix. For cells to invade, they secrete matrix-degrading proteinases. Work in Weaver’s lab demonstrated that not only were these proteinases carried by EVs but that hot spots for their secretion actually aligned with invadopodia.

Specifically, Weaver’s lab established that invadopodia are important sites for the docking and secretion of exosomes. Exosomes are extracellular vesicles secreted from many different cell types. They originate from multivesicular bodies (MVB), which are mature endosomes that contain many smaller vesicles. Secretion of exosomes occurs when these MVBs fuse with the cell membrane, releasing the molecules contained inside. Though normal cells may use environmental cues to regulate exosome secretion, cancerous cells constitutively turn it on.

Exosome cargoes mediate invadopodia biogenesis, stability, and activity

Exosome cargoes mediate invadopodia biogenesis, stability, and activity.
Source: Hoshino, et al. Cell Rep 2013

“One of the big questions we are working on is the cell biological aspects of these vesicles,” Weaver explained. “How they are made, how cargo gets sorted there, and what does that mean for their biological function after they are secreted? So that is where our work with the ERCC comes in.”

She hopes that working with the scientists of the consortium, they can understand how RNA and RNA binding proteins are trafficked into vesicles. Last year, in a paper published in Cell Reports, her group demonstrated one possible mechanism for the sorting of microRNAs into EVs. They demonstrated that Argonaute 2 (Ago2), part of the RISC machinery that binds to miRNAs, is transported in microvesicles and exosomes. Organization of Ago2 into exosomes is regulated by KRAS-MEK signaling. Dr. Weaver highlighted the study in a blog here mid-last year.

Despite these initial findings, Dr. Weaver admits it is difficult to determine how important extracellular RNA and miRNAs are in regulating cancer metastasis. “I honestly don’t think we know yet, and I think that the field is just now really trying to figure out what are the cargo components that are driving all of these phenotypes we have been trying to characterize so well.” She elaborated, “I think for both the protein and the RNA, the next big step for the field is trying to pin individual EV functions back to specific cargo molecules.”

Asked to reflect on her AAAS fellowship, Dr. Weaver turned the focus on her colleagues in the consortium. “I continue to be very impressed by the quality of investigators and the research being done by the ERCC. I mean really top people who are driving forward what I think is a tough problem.” She and fellow AAAS fellows Dr. Patton and Dr. Wong are, as Dr. Weaver pointed out, “just a small snapshot of fabulous investigators that are part of the consortium.”

The microRNA miR-155 plays a significant role in physiological and pathological processes in humans by blocking the functions of functionally important messenger RNAs of protein coding genes. We found that miR-155 was present in higher levels in cancers resistant to chemotherapy. By studying the association of miR-155 and tumor suppressor TP53 with cancer survival in 956 patients with lung cancer, chronic lymphocytic leukemia and acute lymphoblastic leukemia, we demonstrated that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that blocking or down-regulating miR-155 successfully resensitizes tumors to chemotherapy in vivo. We found that high levels of miR-155 and low levels of TP53 characterize the tumors from lung cancer patients with shorter survival time. Our findings support the existence of a miR-155/TP53 feedback loop involved in resistance to chemotherapy. To target this feedback loop and effectively alter resistance to therapy, we have developed a therapeutic nanoformulation of anti-miR-155 in a lipid nanoparticle (DOPC) and have shown it to be non-toxic in vivo for further pre-clinical work.

We thank our co-authors for their work and discussions that led to this blog.

miR-155 TP53 and resistance to chemotherapy

The first version of the miRandola database has been published in 2012. It contained 89 papers and miRNA data. Now, we have updated the database with 272 papers and we redesigned the website!

We are starting to add more RNA molecules such as lncRNAs and circRNAs.

The miRandola database 2017 includes:

  • 272 articles
  • 2704 entries
  • 6 extracellular RNA forms
  • 673 microRNAs
  • 12 long non-coding RNAs
  • 8 circular RNAs
  • 21 drugs
  • 9 organisms and animal models
  • 173 diseases and cell lines
  • More features are coming soon!

The exrna.org Research Portal is linked in our web page.

Website: http://mirandola.iit.cnr.it/

The study of RNAs that do not produce proteins, so-called noncoding RNAs, has been an active area of research for many years. Recently, new kinds of non-coding RNAs have been described that have poorly defined activities. Circular RNA (circRNAs) are one of these more enigmatic biomolecules. They are formed when the 5′ head and 3′ tail of a messenger RNA precursor are spliced together. Next-generation sequencing studies have recently shown that circRNAs are abundant and widely expressed in mammals. While other non-coding RNAs have been shown to play critical roles in cancer, the association between circRNAs and cancer is largely unknown. In addition, the degree to which circRNAs are secreted outside the cell has not been well explored.

To study the presence and regulated release of circRNAs during colorectal cancer (CRC) progression, we used three related colon cancer cell lines that differ only in the mutation status of KRAS, an enzyme that acts at the beginning of a wide array of cellular signaling pathways. The parental cell line (DLD-1) contains both wild-type and G13D mutant KRAS alleles, whereas the derivative cell lines contain only a mutant KRAS (DKO-1) or wild-type KRAS (DKs-8) allele (Shirasawa et al. 1993). The G13D mutation locks KRAS into an active state. KRAS mutations occur in approximately 34–45% of CRCs and have been associated with a wide range of tumor-promoting effects (Vogelstein et al. 1988, Wong and Cunningham 2008). We performed deep RNA-Seq analysis of ribosomal RNA-depleted total RNA libraries to characterize circRNA expression in these cell lines and in the exosomes they release. The results from this study were recently published in the journal Scientific Reports (Dou et al. 2016).

Using a unique pipeline developed by our group, we identified hundreds of high-quality candidate circRNAs in each cell line. Remarkably, circRNAs were significantly down-regulated at a global level in the cell lines with mutant KRAS alleles (DLD-1 and DKO-1) compared to wild type (DKs-8), indicating a widespread effect of mutant KRAS on circRNA abundance (see Figure 1). This finding was confirmed in another pair of cell lines. In all of these cell lines, circRNAs were found associated with secreted exosomes, and circRNAs were more abundant there than in cells. Although circRNAs were down-regulated in cell lines with mutant KRAS alleles, it is difficult to conclude that KRAS directly regulates circRNAs. Nevertheless, our analysis did show that down-regulation of circRNAs in KRAS mutant cells was not caused by their increased export to exosomes.
 

Figure 1.

Figure 1. The blue highlight shows that expression of most circRNAs is lower in the KRAS-mutant cell lines than in the KRAS wild-type cell line.
FDR = False Discovery Rate; a higher number indicates a more confident prediction of a difference in expression.

There are complex regulatory mechanisms for expression of both circRNA and the host genes from which they derive. Figure 2 shows that lower expression of circRNA in the mutant KRAS vs. wild-type cell lines was not matched by a similar lower expression of host gene mRNA. We found a similar lack of correlation in circRNA and host gene mRNA expression level in all the exosome populations we studied. These results imply that regulation of circRNAs can occur independent of their host genes, and different regulatory processes might direct secretion of circRNA and host gene mRNA.
 

Figure 2.

Figure 2. The blue highlight shows that while expression of most circRNAs is lower in the KRAS-mutant than in the wild-type cell line, host gene mRNA expression shows no such pattern.

To further delineate how circRNA biogenesis could be affected by mutant KRAS, we also examined the expression levels of the RNA-editing enzyme ADAR and the RNA-binding protein QKI, which have been reported as circRNA regulators (Ivanov et al. 2015, Conn et al. 2015) (see Figure 3). Here we obtained contradictory results. The level of ADAR was decreased in the KRAS mutant cells; reduced ADAR activity could lead to an increase of circRNAs. QKI was also down-regulated in KRAS mutant cells, which could lead to a decrease of circRNAs.
 

Figure 3. Effect of ADAR and QKI on pre-mRNA circularization

Figure 3. Effect of ADAR and QKI on pre-mRNA circularization

More broadly, we studied the expression levels of all RNA-binding proteins within the RBPDB database (Cook et al. 2011). Six were found to be significantly differentially expressed in KRAS mutant cell lines compared with wild-type KRAS cell lines (ELAVL2, RBMS3, BICC1, MSI1, RBM44, and LARP6). These genes may serve as candidate circRNA regulators. However, our previous work shows that the correlation between mRNA and protein expression level is low for RNA-binding proteins (Zhang et al. 2014), and thus RNA levels for these RNA-binding proteins might not reflect their true protein levels. Further investigation will be needed to precisely define how circRNAs are regulated. Nevertheless, our results show that oncogenic mutations can change circRNA composition in cells and exosomes and suggest that circRNAs may serve as promising cancer biomarkers.

References

Conn, S.J., et al. The RNA binding protein Quaking regulates formation of circRNAs. Cell (2015) 160: 1125-1134. PMID 25768908.

Cook, K.B., et al. RBPDB: a database of RNA-binding specificities. Nucleic Acids Res (2011) 39: D301-D308. PMID 21036867.

Dou, Y., et al. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes. Sci Rep (2016) 6: 37982. PMID 27892494.

Ivanov, A., et al. Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals. Cell Rep (2015) 10:170-177. PMID 25558066.

Shirasawa et al. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science (1993) 260:85-88. PMID 8465203.

Vogelstein, B., et al. Genetic alterations during colorectal-tumor development. N Engl J Med (1988) 319:525-532. PMID 2841597.

Wong, R. and Cunningham, D. Using predictive biomarkers to select patients with advanced colorectal cancer for treatment with Epidermal Growth Factor Receptor antibodies. J Clin Oncol (2008) 26:5668-5670. PMID 19001346.

Zhang, B., et al. Proteogenomic characterization of human colon and rectal cancer. Nature (2014) 513:382-387. PMID 25043054.

Alzheimer’s Disease (AD) accounts for a large number of dementia cases resulting in impaired memory, thinking, and behavior. Risk factors for AD include age and family history, but unfortunately there is not yet a definitive way to predict if an individual will develop the disease. There are reference biomarkers that can indicate a higher risk of developing AD, such as APOE genotype. Carriers of the APOE4 allele, present in ~20% of the population, are at increased risk for AD. Cerebrospinal fluid (CSF) is a body fluid found in the brain and spine that cushions and protects the brain from injury. CSF protein biomarkers, such as Aβ42, tau and phospho-tau, are important in screening for brain disease, but these reference markers often lack the sensitivity and specificity necessary for clinical utility.

Extracellular RNA, specifically microRNA (miRNA), has been found in CSF and may serve as a useful resource for improved AD biomarkers. In a recently published study, the Saugstad lab from Oregon Health and Science University examined CSF from a large group of living donors to identify unique miRNA biomarkers enriched in AD patients. In the study, miRNA expression levels from 50 AD and 49 control subjects were assessed using TaqMan Low Density Arrays containing probes for 754 validated miRNAs. Each miRNA was given a “Multitest Score” combining the results of four statistical tests, and miRNAs that passed two or more of the tests were considered for further analyses.

Two statistical tests, log-rank and logistic regression, were used to identify candidates that were twice as likely to be associated with AD status as not. The other tests were two variants of random forest classifier, CART and CHAID, designed to select biomarker candidates able to reliably distinguish AD from non-AD status when grouped with random subsets of other miRNAs. 36 miRNA biomarker candidates were identified by at least two of these analyses. The researchers found that linear combinations of subsets of miRNA, and the addition of ApoE genotyping status, further increased the sensitivity and specificity of AD detection (Figure 1).

 

Figure 1. CSF miRNA biomarkers and APOE genotype predict AD status better together. AUC - Area Under the Curve; higher AUC indicates higher predictive power.

Figure 1. CSF miRNA biomarkers and APOE genotype predict AD status better together. AUC – Area Under the Curve; higher AUC indicates higher predictive power.

Reprinted with permission from IOS Press.


 

This study shows the potential use of miRNAs isolated from CSF as AD biomarkers. The stringent statistical analyses and large sample size together provided strength to these initial studies. These 36 candidate biomarkers are currently being tested in further validation studies in CSF from a new group of 120 donors, which will also include APOE genotyping and Aβ42 and tau protein levels. Ultimately, a combination of miRNA CSF biomarkers with existing reference biomarkers (APOE, Aβ42, tau) may provide a specific and sensitive tool for the diagnosis of AD in the clinic.

Citation:
MicroRNAs in Human Cerebrospinal Fluid as Biomarkers for Alzheimer’s Disease
Lusardi T, Phillips J, Wiedrick, J, Harrington C, Lind B, Lapidus J, Quinn J, Saugstad J. Journal of Alzheimer’s Disease (2017) 55: 1223-1233. doi: 10.3233/JAD-160835

Cancer cells actively reprogram gene expression to promote their ability to produce tumors. One way this reprogramming is carried out is by subverting the main routes of cell-to-cell communication by loading exosomes (vesicles that bud off from cells) with specific miRNAs that either promote or suppress tumors and then releasing them into the tumor microenvironment. In a Cancer Research paper published online recently (Kanlikilicer et al., 2016), we found that miR-6126, a miRNA that was reported to be correlated with better overall survival in high-grade serous ovarian cancer patients, is ubiquitously removed from ovarian cancer cells via exosomes.

miR-6126 is ubiquitously removed from ovarian cancer cells via exosomes

We found that miR-6126 suppresses tumors by directly targeting integrin ß1, a key regulator of cancer cell metastasis. Treatment of orthotopic mouse models of ovarian cancer with miR-6126 reduced tumor growth, proliferating cells, and microvessel density. Our findings provide new insights into the role of exosomes in mediating tumor progression and suggest a new therapeutic approach to disrupt the origin and growth of tumors.

Citation:
Ubiquitous release of exosomal tumor suppressor miR-6126 from ovarian cancer cells
Pinar Kanlikilicer, Mohammed Saber, Recep Bayraktar, Rahul Mitra, Cristina Ivan, Burcu Aslan, Xinna Zhang, Justyna Filant, Andreia M Silva, Cristian Rodriguez-Aguayo, Emine Bayraktar, Martin Pichler, Bulent Ozpolat, George A Calin, Anil K. Sood and Gabriel Lopez-Berestein*
Cancer Res. October 14, 2016 doi: 10.1158/0008-5472.CAN-16-0714
*Corresponding author

The concept of a hype cycle is a well-established business concept, in which novel ideas are said to have an initial wave of hype followed by disillusionment. Only after that, the novel concept takes off and become truly useful entering a so-called plateau of productivity. In biomedical science, the field of microRNAs (miRNAs) certainly had a peak of interest in the end of the last decade. This led by high impact publications (1) and characterization of both novel miRNA-entities as well as their associations to a broad range of diseases. Nonetheless, no clear pharmaceutical successes emerged: miRNA targets are being pursued as therapeutic targets, but none have as of yet successfully made it through clinical trials (2). Likewise the use of miRNA-based treatment strategies targeting regular mRNA is an area of interest (3). In this editorial we focus on a third aspect of miRNAs: the use of miRNAs as prognostic biomarkers in disease, asking the question if miRNAs are now entering this plateau of productivity in which actual benefit will be seen.

We focus on the recent paper by Bye et al.: “Circulating microRNAs predict future fatal myocardial infarction in healthy individuals – The HUNT study” (https://www.ncbi.nlm.nih.gov/pubmed/27192016).

 
The hunt for fatal myocardial infarction biomarkers: predictive circulating microRNAs.
Russo F, Rizzo M, Belling K, Brunak S, Folkersen L.

Ann Transl Med. 2016 Oct;4(Suppl 1):S1.

PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27867969

Journal: http://atm.amegroups.com/article/view/11314

As part of the Extracellular RNA Communication Consortium (ERCC) seminar series, on 30 June 2016, Dr. Janusz Rak from McGill University presented his work on “Extracellular Oncogenes as Biological Effectors and Biomarkers.”

When focusing on a single cancer cell, we consider how particular mutations alter the cell’s properties, causing it to grow, replicate, evade immune detection, and do all the other things cancer cells can do. But it’s clear that cancer is more than a collection of cells operating autonomously. Perhaps the most interesting and frightening part of cancer is that tumors are complex, heterogeneous entities that operate and interact with each other and with their environment in characteristic ways. Researchers have been studying cancer cell signaling pathways for years. An example is paracrine interactions with neighboring cells that induce irregular vascular growth. What is new and exciting about Dr. Rak’s work is the focus on oncogenes as cargo in extracellular vesicles (EVs), which is a nontraditional means of communication between cancer cells and from cancer to non-cancer cells.

Dr. Rak has followed several interesting lines of research. He addresses fundamental questions; for example:

  • With which cell types do cancer-cell-derived EVs (CCEV) interact?
  • Are tumor-associated mutant proteins found on or in CCEVs?
  • Are these mutant proteins then expressed on recipient cells?

Preliminary work in these areas has led to the question: Can cancer cells transform non-cancer cells (i.e. make them cancer-like) via EV-mediated transfer of oncogenic proteins? And is this process complete and permanent? Numerous lines of inquiry, from in vitro to mouse studies, lead to the conclusion that the answer is probably no. Dr. Rak argues that such a finding is not surprising: if horizontal oncogenic transformation were occurring regularly between cell types, we would see more cancer patients with mixed tumor types. Secondly, phylogenetic approaches have been used to trace tumors back to single cell ancestors, another argument against the model of horizontal transformation. The current leading hypothesis is that CCEVs, rather than inducing oncogenic transformation, play a modulatory role, regulating interactions between cancer cells and between cancer and host cells but without changing their identity.

A better understanding of cancer EV biology has promise for improving cancer therapy and diagnosis. CCEVs and their oncogene cargo may represent interesting and novel pharmaceutical targets. They also constitute a unique reservoir of mutant molecules in blood, far away from an inaccessible tumor. Efforts are underway to advance cancer diagnosis through detection of CCEVs, particularly in tumor classes like glioblastoma multiforme (GBM) where no other methods of early detection are available. Research in humans has indicated that GBM patients can be identified by EVs in blood serum. As research such as Dr. Rak’s progresses, and EV biology is further elucidated, we will continue moving towards a future where we can utilize EVs in clinical practice to improve health outcomes.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). Currently, magnetic resonance imaging (MRI) is the most commonly used method to diagnose and monitor MS, but there is a poor correlation between MRI disease measures and clinical disability or disease progression in MS. MRI is also an expensive tool that might carry potential risks due to brain accumulation of contrast material (Kanda et al., 2015). In the last few years, a lot of effort has been invested in the identification of biomarkers for MS; however, to date, few of these findings have proven clinically useful. Thus, there is a strong unmet clinical need for objective body fluid biomarkers to assist in early diagnosis, predicting long-term prognosis, monitoring treatment response, and predicting potential adverse effects in MS.

Circulating miRNAs have been detected in several body fluids (Cortez et al., 2011) where they are highly stable as they are resistant to circulating ribonucleases (Mitchell et al., 2008). Their stability, along with the development of sensitive methods for their detection and quantification (Guerau-de-Arellano M. et al., 2012), makes them ideal candidates for biomarkers. We previously reported changes in circulating plasma miRNAs in MS patients (Gandhi R. et al., 2013). In a new study, our group investigated serum miRNAs as biomarkers in MS as part of an NCATS-funded UH2 initiative. We found that several serum miRNAs were differentially expressed in MS, were associated with disease stage, and correlated with disability.

Study Design (Figure 1): Serum from 296 participants including patients with MS, other neurologic diseases (Alzheimer’s disease and amyotrophic lateral sclerosis), inflammatory diseases (rheumatoid arthritis and asthma), and healthy controls (HC) were tested. miRNA profiles were determined using LNA (locked nucleic acid) based qPCR. MS patients were categorized according to disease stage and disability. In the discovery phase, 652 miRNAs were measured from the serum of 26 MS patients and 20 healthy controls. Those miRNAs from the discovery set that were significantly differentially expressed (p <0.05) in cases vs controls were validated using qPCR in 58 MS patients and 30 healthy controls.

 

Serum miRNA biomarkers in MS - Figure 1

 

Note: Results in the current study were normalized to the four most stably expressed miRNA across all the subjects. We agree with other blogs posted on exRNA.org suggesting that there is an immediate need to identify reference miRNA/exRNA that could be used for data normalization.

 

Figure 2: Differentially expressed circulating miRNAs as biomarkers in Multiple Sclerosis (MS). Up to top five miRNAs with p<0.05 are represented for each group comparison; a) MS, b) relapsing remitting MS (RRMS) and secondary progressive (SPMS) compared to the healthy control (HC), c) RRMS vs. SPMS, and d) the correlation of miRNA with the expanded disease severity scale (EDSS).

 

Results: We found 7 miRNAs (p<0.05 in both discovery phase and validation) that differentiate MS patients from healthy controls; miR-320a up-regulation was the most significantly changing serum miRNA in MS patients. We found 8 miRNAs that differentiated relapsing-remitting MS (RRMS) from HC. Among these, miR-484 up-regulation in RRMS patients showed the strongest association. When comparing secondary progressive MS (SPMS) patients to HC, 34 miRNAs significantly differentiated between the groups in both phases, with miR-320a up-regulation showing the strongest link. We also identified two miRNAs linked to disease progression, with miR-27a-3p being the most significant. Ten miRNAs correlated with degree of disability according to the Kurtzke Expanded Disability Status Scale (EDSS), of which miR-199a-5p had the strongest correlation with disability. Of the 15 unique miRNAs we identified in the different group comparisons, 12 have previously been reported to be associated with MS, but not in serum. Kegg Pathway Analysis showed that significant and differentially expressed miRNAs target important immune functions and are related to the maintenance of neuronal homeostasis. For example, miR-27a-3p, the strongest miRNA to distinguish RRMS from SPMS and progressive MS (PMS) (up-regulated in the relapsing form as compared to the progressive forms) shows a strong link to both the neurotrophin signaling pathway and the T cell receptor signaling pathway. Other studies have shown that miR-27a-3p targets multiple proteins of intracellular signaling networks that regulate the activity of NF-κB and MAPKs 6. As a consequence, miR-27a inhibits differentiation of Th1 and Th17 cells and promotes the accumulation of Tr1 and Treg cells (Min S. et al., 2012). It has also been shown that miRa-27-3p is up-regulated in MS active brain lesions and that the level of miR-27a-3p in CSF is reduced in patients with dementia due to Alzheimer’s disease (AD) (Frigerio C.S. et al., 2013). Of all the miRNAs, miR-486-5p was identified in the largest number of comparisons. It correlates with EDSS and is up-regulated in MS compared to HC, to other neurological diseases, and to other inflammatory diseases. This particular miRNA was found to be associated with TGF-beta signaling pathways and is a known tumor suppressor (Oh H.K. et al., 2011). miR-320a has been previously described to be highly expressed in B cells of MS patients and was suggested to contribute to increased blood-brain barrier permeability due to regulation of MMP-9 (Aung L.L. et al., 2015). Pathway analysis links this miRNA to cell-to-cell adhesion pathways, another indication that it may be linked to blood-brain barrier permeability.

The current study is the most comprehensive evaluation to date of the role of serum miRNAs as biomarkers in MS, with the largest sample size and employing two independent cohort designs. One limitation of our study is that participant subject samples were collected from a single MS center. Further external validation of our results will require investigating samples from patients at other centers. We are currently performing such multicenter studies, which may also increase the power of our results. A second limitation of our study is the relatively small number of participants who contributed to each group comparison. Future work will require larger sample sizes to ensure that we have sufficient power to detect miRNAs with smaller effect sizes. Although miRNAs have been studied in cells and the CNS of MS patients, ours is the first comprehensive investigation of serum miRNAs.

Conclusions: Our findings identify circulating serum miRNAs (Figure 2) as potential biomarkers to diagnose and monitor disease status in MS. These findings are now being tested using patient samples obtained from other international MS centers. We are now investigating the role of miRNA as biomarkers for disease prognosis and treatment response in MS.

Acknowledgements: This study is a highly collaborative project, and I thank my whole team at the Ann Romney Center for Neurologic Diseases & MS Center for their contribution. The grant TR000890 is supported by the NIH Common Fund, through the Office of Strategic Coordination / Office of the NIH Director.