Role of microRNA9 in spinal dorsal horn neurons in over-expression of calcium homeostasis modulator 1 in rats with diabetic neuropathic pain

Abstract

Objective To evaluate the role of microRNA9 (miR-9) in spinal dorsal horn neurons in over-expression of calcium homeostasis modulator 1 (CALHM1) in rats with diabetic neuropathic pain (DNP). Methods Ninety-three healthy male Sprague-Dawley rats, aged 2 months, weighing 180-200 g, were used in the study.Diabetes mellitus was induced by intraperitoneal 1% streptozocin (STZ) 60 mg/kg.The experiment was performed in two parts.Experiment Ⅰ The rats were divided into control group (group C, n=10) and DNP group (n=83) using a random number table.The mechanical paw withdrawal threshold (MWT) was measured before STZ injection and at 1, 2, 3, 4, 5 and 6 weeks after STZ injection.The expression of miR-9 in the spinal dorsal horn was determined using the in situ hybridization at 6 weeks after STZ injection.Experiment Ⅱ The rats with DNP were selected at 6 weeks after STZ injection, and the spinal dorsal horn neurons were isolated and cultured.The neurons were seeded in culture plates at the density of 5×106 cells/ml (2 ml/well) and divided into 2 groups (n=18 each) using a random number table: control group (group C) and miR-9 antisense oligonucleotide group (group ASO). The neurons were cultured in normal culture atmosphere in group C. In group ASO, the single nucleotide sequence of miR-9 antisense oligonucleotide sequence 5′-UUCUCCGAACGUGUCACGUTT-3′ was added with the final concentration of 100 pmol/L.The expression of miR-9 and CALHM1 mRNA was detected using quantitative real-time polymerase chain reaction at 48 h of incubation.The expression of CALHM1 was detected by Western blot at 72 h of incubation. Results Experiment Ⅰ Compared with group C, the MWT was significantly decreased at 2-6 weeks after STZ injection, and the expression of miR-9 in the spinal dorsal horn was up-regulated in group DNP (P<0.05). Experiment Ⅱ Compared with group C, the expression of miR-9 and CALHM1 protein and mRNA in spinal dorsal horn neurons was significantly down-regulated in group ASO (P<0.05). Conclusion miR-9 in spinal dorsal horn neurons probably mediates the over-expression of CALHM1 in rats with DNP. Key words: microRNAs; Calcium channels; Spinal cord; Neurons; Diabetes mellitus; Neuralgia