The effect of short-trem opioid administration on epigenetic mechanisms

Morphine is an opioid alkaloid with a strong analgesic effect, used in the treatment of severe pain. Abuse of morphine changes neuron functions with the adaptation occuring in brain neurons. Some theories have been proposed to explain morphine addiction, but have not yet been conclusively proven. Indirect epigenetic modifications and influencing different transcription factors cause changes in gene expression. There are also studies revealing the epigenetic effects of morphine and other opioids. It is known that some miRNA levels change and even affect the next generations epigenetically. The aim of this thesis is to investigate the effect of short-term opioid administration on the expression of miRNA biogenesis pathway and DNMT (DNA methyl transferase) genes, which are effective on methylation profile using an in vitro and in vivo model. In the in vivo morphine addiction and withdrawal rat model, hypotalamus tissues of control (C), 7 days of 10 mg/kg morphine (M) and 10 mg/kg morphine addiction+ 1 mg/kg naloxone (M+N) groups were used. In the in vitro model, the SH-SY5Y human neuroblastoma cell line, preferred as the gold standard in neuroscience studies, was used. The control, 10 µM highest non-toxic morphine dose, 10 µM naloxone and 10 µM morphine+10 µM naloxone groups were formed. Total RNA isolation and cDNA synthesis were performed from tissue and cell samples. The expression levels of miRNA biogenesis pathway and DNMT genes were determined by qPCR and the differences between groups were compared by statistical analysis. Based on the XTT analysis, highest non-toxic morphine dose was determined as 10 µM in SH-SY5Y cells, which was used in in vitro morphine addiction model. Expression levels of DNMT1, DNMT2 and DNMT3B were found to be elevated in morphine group; however, naloxone administration downregulated this incraese. Except TRBP2, increased expression levels of miRNA biogenesis genes in morphine group were downregulated in morphine+naloxone groups. Gene expression profiles were generally found to have similar profile in both in in vivo and in vitro models. Steady-state levels DNMTs were upregulated in M groups and downregulated in M+N group of hypothalamus tissues. However, this difference was significant for DNMT3A. Increased levels of DGRC8, XPO5 and AGO1 in morphine group were decreased by naloxone application. Morphine and naloxone administration affected gene expression levels, which indicated an epigenetic regulation mechanisms. This thesis project allows comparison of the effectiveness of in vivo and in vitro subchronic opioid addiction models. Findings suggested that in vitro model is found to be effective. Therefore, it is expected to contribute to a better understanding of different pharmacological applications and the physiopathology of opioid addiction. As an alternative to the need for experimental animal models, it is expected to have a positive effect on similar studies at the translational level due to the ethical and technical conveniences.