| 3) Dual luciferase report gene assay method has determined the site of miR-330 regulating GABBR2, in which miRNA-330 can bind the 3'UTR site of target gene GABBR2, inhibiting the expression of target gene (p<0.05).4) The expression of GABAB2 was found decreased in neuronal membrane with glutamate treatment, which revealed that excitatory neurotransmitters could regulate membrane GABAB2 protein.To sum up, in nude mice model with the pancreatic cancer pain, the expression of miR-330 of spinal cord increased significantly, and GABAB2 receptors decreased, parralleled with pancreatic pain sensitivity. miR-330 is likely to be an attractive potential therapeutic target in cancer pain.MicroRNAs (miRNAs) are little non-coding, endogenous 19-23 nucleotide (nt) RNAs that regulate target-gene expression by either translation inhibition or mRNA degradation. More and more researches have showed that miRNAs are likely to play an important role in the induction and maintenance of neurological diseases. Owing to their uncommon targeting properties, a single miRNA species can bind and regulate multiple targets, some have been suggested that miRNAs are major switches of the genome, and this regulation is essential for normal physical activity, when affected, can lead to drastic pathological conditions. However, the number of miRNAs is huge. So far, the confirmed number in miRNA database (miRbase) has reached more than 1000. Therefore, these mechanisms triggers pathological states are as well a large number of contents waiting to be discovered. MicroRNAs in pain are reported to be related to the regulation of neural activity, and has been becoming a hot concern in recent years.The patients with pancreatic cancer suffer from pain frequently and seriously in clinic, so the quality of life is decreased obviously, but there are still no effective analgesic methods because of the poor understanding of the mechanisms involved in the pain induction and maintenance. In this study, for the first time by gene chip analysis, we found that miR-330, which is a kind of non-coding RNA, is up-regulated in a naked mouse model with pancreatic cancer pain, and there is no expression difference between pancreatic cancer model without pain hypersensitivity and naive naked mouse, that strongly suggests that miR-330 play an important role in the process of pancreatic cancer pain. Further, we discovered through gene database screening analysis, the gene GABBR2 for one of the central main metabotropic GABA receptor subunits GABAB2 may be the target gene of miR-330. So based on our previous study of which was focused on peripheral nerves in pancreatic cancer pain, this paper will use the cultured spinal cord dorsal horn neurons, combining with gene manipulation, molecular biology and electrophysiology techniques, and pay attention to the spinal cord dorsal horn neurons that could regulate pain signaling from dorsal root ganglion significantly. Further, we will try to make sure that up-regulated miR-330 can decrease the expression of GABAB2 subunits, and then find the regulation site on GABBR2 by miR-330, also the pre- and post- synaptic mechanism of decrease GABAB receptor function during pancreatic cancer pain. This study will improve our understanding of the pancreatic cancer pain and provide new ideas for basic researches and clinical treatments. The experimental results are as follows:1. The high expression of miR-330 in the spinal dorsal horn may lead to pancreatic cancer pain.1) In the spinal cord dorsal horn of a nude mice model with pancreatic cancer pain, compared with that in no obvious hyperalgesia mice model of pancreatic cancer and normal mice, the expression of miR-330 increased significantly after day7 and day21 of surgery, there was statistical significance (p=0.0214, p<0.05). And the expression of miR-330 did not show significant changes in a nude mice group with no pancreatic cancer pain hypersensitivity compared with that in the normal mice group (p>0.05).2) In the cultured spinal cord neurons with the high expression of miR-330 via lentivirus transfection, the excitability of neurons dramatically increased by electrophysiological experiments. The amplitude and frequency of sEPSCs increased significantly, comparring with that in the empty vector group (p<0.01), which could be inhibited by 10?M baclofen administration (p<0.01). miR-330 could regulate neurons activity in the spinal cord through presynaptic and postsynaptic mechanisms.2. The mechanism of miR-330 inhibition of GABAB2 receptor function.1) In a nude mice model, the expression of GABAB2 receptor in the spinal cord dorsal horn after 7 days,14 days,21 days,28 days of surgery was decreased, in 14 days GABAB2 receptor reached peak, while in 21 days this situation has become restored. It coincided with the time of hyperalgesia behavior.2) The spinal cord neurons were transfected by lentivirus after 7 days of culture, which was randomly divided into three groups, normal control group, empty virus transfection group (vehicle group) and miR-330 lentiviruses transfection group. In vehicle group GABAB2 receptor expression slightly decreased, the transfection miR-330 transfection group has a obviously statistical significance compared with normal control group, (p<0.0001). Unlike GABAB2 expression, in the vehicle group and transfection miR-330 group, the expression of GABAB1 is not affected, compared with that in normal group (p>0.05). |
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