MicroRNAs as biomarkers for treatment response in rheumatoid arthritis
Methotrexate is a first line treatment of rheumatoid arthritis, but is only effective in a proportion of patients. This project aims to identify variations in microRNA (miRNA) expression profiles in different immune cells before and after methotrexate treatment in order to reveal biomarkers for good treatment reponse. The EPIRA (epigenetics of rheumatoid arthritis) project investigates rheumatoid arthritis patient samples from two different cohorts (NOR-VEAC and ARTIC-REWIND). For including new patients into the cohorts, we are cooperating with the Diakonhjemmet and Martina Hanssen Hospital. The NOR-VEAC cohort comprises newly diagnosed rheumatoid arthritis (RA) patients. A blood sample is taken from the patient before starting the methotrexate treatment and three month later. The ARCTIC-REWIND cohort includes patients undergoing methotrexate treatment who have been in remission for more than a year. The patients was randomized such that one half of the patients receive a 50% reduction in methotrexate dose while the other 50% got unchanged doses. The first blood sample from the patients is taken before randomization. Some patients with reduced methotrexate dose will experience a flare of the symptoms. One new blood sample is collected at this time and another when the patient is back in remission after increasing the methotrexate dose again. Patients without a flare are invited to give a second blood sample after eight months. As of today our study includes approximately 30 patients, with samples taken at two or three time points, as well as nine controls. To increase the statistical power in the data analysis, we will continue including new patients throughout the project. From the blood samples of the two cohorts, we separate pathologically relevant immune cells (B cells and five different subtypes of T cells). The wet-lab process continues by extracting total RNA, followed by the creation of small RNA libraries for next generation sequencing. The sequenced small RNA libraries are bioinformatically analysed for investigating changes in the miRNA expression signatures of the controls and RA patients at different time points of methotrexate treatment. 2016 has been used to collect further RA patients. A laboratory protocol was established to simultaneously extract total RNA and DNA. The extracted DNA was used for DNA methylation profiling studies, EPIRAs second research project financed by Helse Sør-Øst. From the extracted total RNA, small RNA libraries were generated which will be used for miRNA-sequencing. This part of the project is performed in close collaboration with the core facility at the Norwegian Sequencing Center. Approximately half of the samples (48 individuals x 4 cell types) have completed the sequencing step and are at the moment being analysed using our in house developed bioinformatics pipeline. The remaining samples will be completed during spring 2017. Furthermore, we will integrate DNA methylation, isomiR and gene expression datasets into the pipeline. The first preliminary results of this work were sent in as an abstract to the NGS 2017- International society for computational biology conference. The doctoral candidate has also attended bioinformatics courses to obtain the knowledge needed to work on large amounts of data and analysing next-generation sequencing data. In strong cooperation with another research group at the Department of Medical Genetics, we are combining the sequencing data from miRNA, isomiR, mRNA and DNA methylation studies of the RA patients. By doing so, we will obtain a comprehensive understanding of molecular changes that are occurring during treatment in RA which helps us in investigating the efficiency of the drug treatment.
We will investigate if variations in microRNA expression profiles are suitable for determining the efficiency in drug treatment response in rheumatoid arthritis, which is a chronic inflammatory disorder of the joints. Rheumatoid arthritis (RA) is a chronic inflammatory disorder of the joints. RA occurs in almost 1% of the Norwegian population. Multiple treatments exist but the challenge is to determine the optimal treatment as efficacy varies amongst patients and treatments may have considerable side effects. The cause of RA is unclear but it is hypothesised that genetic disposition and environmental factors play an important role. Furthermore, those factors are suspected to influence the different outcomes of the RA treatment. Methotrexate is often chosen as a first line treatment in RA but is only effective in a proportion of patients. The primary goal of the EPIRA research group is to understand at a molecular level why some RA patients respond to a methotrexate treatment while others do not. These findings will provide valuable knowledge for the development of individualized medicine in RA. The aim of the part in the EPIRA project described here is to study variations in microRNA expression profiles in different immune cells of patients before and after treatment with methotrexate. Together with the results of the DNA methylation profiling we expect to get a better understanding of methotrexate drug treatment in RA patients. The EPIRA project investigates patient samples from two different cohorts. We are cooperating with the Diakonhjemmet and Martina Hanssen Hospital for including new patients to our study. The NOR-VEAC cohort comprises newly diagnosed RA patients. Blood samples have been taken from these patients before they started a methotrexate treatment and three month later. The ARCTIC-REWIND cohort includes patients undergoing methotrexate treatment who have been in remission for more than a year. The patients are randomized such that one half of the patients receive a 50% reduction in methotrexate dose. Blood samples are taken before randomization. Some patients with reduced methotrexate dose will experience a flare of the symptoms. Blood samples will be collected at this time and once they are back in remission after increasing the dose again. Patients without a flare are invited to give a blood sample after eight months. As of today our study includes approximately 30 patients, with samples taken at two or three time points, as well as ten controls. To increase the statistical power in the analysis of the data, we will continue including new patients throughout the project. From the blood samples of the two cohorts, we separate relevant immune cells (B cells and five subtypes of T cells). The first half year of this project has been used to collect patients, isolate cells, test laboratory protocols and start establish an bioinformatics pipeline. An initial method has been developed for isolating total RNA/microRNA and DNA from these immune cells. For studying microRNA profiles, state of the art next generation sequencing technologies will be used. This part of the project will be performed in close collaboration with the Illumina Sequencing node of the Norwegian Sequencing Center. A protocol for the microRNA analysis is under development. The doctoral candidate has attended several bioinformatics courses, and we started developing an analysis pipeline that can be used to analyse microRNA profiles. Furthermore, we will integrate DNA methylation, isomiR and gene expression datasets into the pipeline.