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microRNAs in pain

A functional analysis of microRNAs involved in chronic pain

Dr. Kiran Kumar Bali
Dr. Kiran Kumar Bali is a postdoc in Prof. Rohini Kuner’s lab at the University Clinic Heidelberg in Germany. He has been using microRNA inhibitors in vivo to study microRNAs involved in chronic pain.

What is the main focus of your research?

The laboratory of Prof. Rohini Kuner investigates several key aspects of pain pathophysiology, ranging from molecular mechanisms to structural plasticity of nerves. My specific research areas include identification of novel molecular mediators involved in chronic pain pathophysiology.


How did you come to be interested in microRNAs?

During my graduate studies, my search for novel mediators of pain pathology encouraged me to pursue the then novice and challenging entities, miRNAs. miRNAs allured me especially because of their capability to function as ‘master regulators’ by regulating a set of pain-related genes.


What is the aim of your current project?

This project started with two major goals. The first aim was to identify the expression repertoire and dysregulation of microRNAs in peripheral sensory neurons in cancer-pain states. The second and more challenging aim was to investigate molecular mechanisms of candidate miRNAs in the mediation of cancer pain.


Which specific questions did you want to address?

i) To identify tumor-mediated pain-related miRNAs via a genome-wide miRNA expression screen and to prioritize candidate miRNAs for functional analysis in vivo ii) To functionally validate candidate miRNAs in vivo in animal models of bone metastases and iii) To investigate mRNA targets via which miRNA-mediated nociceptive modulation is observed


Which experiments had you performed leading up to this project?

We performed multidisciplinary experiments including high-throughput screening, molecular, in silico and behavioral experiments in clinically-relevant animal models.


What, if any, was your previous experience with microRNA inhibition?

Our previous experience with miRNA inhibition was limited at the start of this project. For a matter of fact, there were absolutely no attempts made in the literature to inhibit a specific miRNA in peripheral sensory neurons.


How did you perform the experiments and analyze the results?

We started with a genome-wide microRNA expression analysis in dorsal root ganglia (DRG) of mice bearing bone metastases in the calcaneus bone of the hind paw using pre-spotted arrays and identified a subset of 57 miRNAs to be significantly and differentially regulated. After confirming the differential regulation of highly regulated miRNAs via qPCR we proceeded to functional validation of highly regulated miRNAs in vivo by using either miRNA-inhibitors from Exiqon or modified miRNA-mimics. To interfere with pathophysiological expression levels of microRNAs in DRGs in vivo , we established effective protocols for intrathecal delivery of microRNA inhibitors or mimics and demonstrated the efficacy of selective manipulations in miRNA expression in vivo . Our behavioral analyses in the bone-metastases model indicated miR-1a-3p or miR-34c-5p and miR-483-3p as pronociceptive and miR-370-3p as antinociceptive in the mediation of tumor-mediated hyperalgesia. Finally, we also investigated mRNA targets and molecular mechanisms involved in miRNA-mediated cancer pain modulation and established miR-1a-3p-Clcn3 as a novel and functional promising miRNA-mRNA regulation pair in modulation of cancer pain.


What were some specific challenges in your experiments?

Specific challenge was to deliver miRNA-inhibitors (or miRNA-mimics) to peripheral sensory neurons in vivo and to target a specific miRNA without off-target effects. It was a challenge because of inherent complexity of the system under investigation and difficulty to deliver the inhibitors or mimics into the peripheral neurons in vivo .


How did you overcome them?

We established efficient protocols to deliver miRNA-inhibitors (or mimics) into peripheral sensory neurons in vivo following intrathecal delivery. Specific LNA-based inhibitors designed by Exiqon not only helped us for a stable delivery but also facilitated specific targeting of candidate miRNA without effecting the expression of closely related miRNAs. Further, short-design of in vivo miRNA inhibitors facilitated the uptake by neuronal cells following intrathecal delivery in vivo .


How do you feel about your results so far?

These results entail the first study to address the importance of microRNAs in the development and maintenance of tumor-mediated chronic pain and also provided a proof-of-principle that expression of a single miRNA could be altered in peripheral sensory neurons in vivo and that alteration could have beneficial effects on pain perception. However, our results also opened up an interesting bunch of questions which have to be addressed in future studies.


What do you find to be the main advantage of the LNA microRNA inhibitors from Exiqon?

As mentioned above, LNA miRNA inhibitors from Exiqon especially are useful for in vivo applications. Their short design facilitate cellular uptake while their LNA-based design provide long-term inhibition of targeted miRNA.


Why would you recommend Exiqon’s microRNA inhibitors to colleagues?

I recommend Exiqon’s miRNA inhibitors mainly because of their specificity and less off-target effects. Customer friendly handling of orders and providing useful discussions with experienced scientists is another reason for my recommendation.


What would be your advice to colleagues about getting started with microRNA inhibition/ in vivo inhibition?

The crucial point is to select a suitable candidate miRNA for in vivo inhibition in order to save time and money. Hypothesis driven candidate selection should be followed if not performing a comprehensive screening. It is always advisable to run a pilot experiment to test the efficiency of inhibitor, especially when running in vivo experiments.


In your opinion what is the most important factor for a successful microRNA inhibition experiment?

Most important factors will be an efficient design of inhibitor to have minimal off-target effect and cytotoxicity while providing stable inhibition of targeted miRNA.


What are the next steps in the current project and how do you plan to perform them?

The next steps in the current project involve investigation of miRNA regulation in other chronic pain modalities and tissue types. Investigation of miRNA-networks in the context of chronic pain is also immediate question to be addressed.


What are the future perspectives for this research?

Understanding miRNA-mediated mechanisms and network regulation in the context of chronic pain is an immediate need of this field of study. Further, improving their potential to be used as therapeutic targets by developing more potent miRNA-expression modulators and non-invasive means of their delivery into peripheral and central sensory neurons has to be addressed. Investigation of non-coding RNAs is a challenging field of study but also holds enormous potential.


When and where will be read more about your studies?

Read more in Bali et al. Genome-wide identification and functional analyses of microRNA signatures associated with cancer pain. EMBO Mol Med. 2013.
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