Expression Of MiRNA-365in Morphine Tolerance Rat’s Spine And DRG And Analysis Of Its Target Gene

Objective:In this study we will investigate the distribution and localization of miR-365in spinal cord and DRG, and find out its potential target genes.Method:1. Establish morphine tolerance model:A total of12Sprague-Dawly (SD) rats weighting250-300g were randomly divided into two groups after intrathecal catheterization following the modified Yaksh method:the morphine tolerant group (MT, n=6) and the normal saline group (NS, n=6). The MT group rats were given10μg morphine intrathecally twice daily for6consecutive days to establish a chronic morphine tolerant model; in contrast, the NS group rats were administrated10μL normal saline (NS) as the same way. Tail-flick test was performed to measure the tail thermal pain threshold of rats on day1,3,5,7, and the results were converted to%MPE.2. In situ hybridization and double labeling immunochemistry staining:DRG and spinal sacral enlargements were harvested and cut frozen sections for in situ hybridization and immunochemistry staining. Double labeling immunochemistry was performed to determine the localization of miR-365, the expression level of miR-365was also recorded.3. Prediction of target gene of miR-365:we used three microRNA targets prediction softwares or databases (target-scan, miRanda and miRDB) to predict our interested target genes of miR-365. Luciferase report systems were used to identify the target genes of miR-365.Result:1.%MPE of NS group had no significant difference from day1to day7(P>0.05).%MPE of MT group was significantly higher on day1compared with NS group and it gradually decreased in the following days, on day7, it had no significant difference compared with NS group.2. In situ hybridization results showed that miR-365expressed thoroughly in grey matter of spinal cord, Double labeling showed miR-365mostly expressed in neurons. In DRG, it expressed in the large, middle and small diameter neurons. The expression level of miR-365in Spine and DRG were down regulated in MT group compared with NS group.3. After searching all three databases, we found that β-arrestin2, the key gene in morphine tolerance, might be the target gene of miR-365. By Luciferase report systems, we identified miR-365was the target gene of miR-365.Conclusion:In our study, we found out miR-365expressed in neurons of spinal dorsal horn and DRG, the expression level of miR-365 was down regulated in morphine tolerance rats, and we identified that β-arrestin2was the target gene of miR-365.