Analysis of Circulating microRNAs Targeting DNA Methyltransferases As a Predictor for Treatment Outcome

Autumn Rogers*, Marieta Gencheva, Kayla Steinberger, E. Hannah Hoblitzell, Malcolm Mattes6, Duaa Dakhlallah, Timothy Eubank, Ph.D.
Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV, 26505.

Presentation Category: Health Sciences (Poster presentation)

Student’s Major: Immunology and Medical Microbiology

Epigenetic pathways can alter gene expression without modifications in the genetic code. These alterations occur naturally during aging but are also observed in cancers and other diseases. DNA methylation is mediated by DNA methyltransferases (DNMTs) which add methyl groups to 5’ carbons of cytosines at cytosine-guanine dinucleotide (CpG) within the gene promoter to inhibit transcription. DNMT-3A and -3B are de novo methyltransferases that mediate new methylation patterns, while DNMT-1 resides at the DNA replication fork and preserves methylation. microRNAs (miRNAs) are non-coding RNAs that silence mRNA targets. Dysregulation of miRNAs lead to altered levels of target mRNAs available for translation into proteins. We recently reported differences in circulating DNMT mRNAs isolated from patient plasma who were critically ill and non-septic, or varying severities of sepsis. We hypothesize that circulating levels of specific DNMT mRNAs can predict patient resiliency by transcriptionally regulating genes essential in recovery or regulate failure in treatment of cancer. We collected 10 mL whole blood from cancer patients with no previous radiation or chemotherapy. We isolated the leukocyte cellular compartment and total RNAs from the serum. cDNA was synthesized from this RNA pool, including mRNAs and miRNAs. We then performed qPCR analysis for miRNAs targeting DMNTs. We aim to correlate specific miRNA expression with patient outcomes, and confirm functionality using the RNA fraction for presence/absence of their target DNMT mRNAs in the patient-matched samples. Finally, we will use the cellular DNA fraction to confirm DNMT activity using bisulfite treatment analysis for global methylation and RNA-Seq for transcriptional gene regulation.

Funding: First2 Network, National Cancer Institute, WVCTSI (GM104942; West Virginia State Startup Funds) (T.D.E), NIH, and Ruby Distinguished Fellowship to (K.J.S).

Program/mechanism supporting research/creative efforts: WVU's Research Apprenticeship Program (RAP) & accompanying HONR 297-level course