MiR-330-mediated Downregulation Of GABA_B2 Receptor In Spinal Dorsal Horn Contributed To Pain In Chronic Pancreatitis

Objective:One of the most important symptoms is constant or recurrent intense abdominal pain that is present in up to 90% of patients with chronic pancreatitis (CP) and pancreatic cancer. Pain in chronic pancreatitis often seriously affects the quality of the patients’ life.The analgesic measures based on the exisiting mechanisms of pain in chronic pancreatitis are often not satisfactory to the patients. At the same time, We observed that inflammatory lesions are present in the majority of patients with pancreatic cancer. So it has profound clinical significance to make clear the mechanisms that inflammation lesions of pancreas cause pain.As posttranscriptional modulators,miRNAs bind through partial sequence complementarity to the 3’-UTR of target mRNAs and cause a block of translation as well as some degree of mRNA degradation. With respect to pain mechanisms, selective miRNAs have been implicated in multiple cellular processes, including neuronal plasticity and neurogenesis, nociceptor excitability, pain threshold, and chronic pain conditions.In our previous study,We used the technology of microarry to detect the expression of miRNAs in the dorsal horn of naked mouse with pancreatic cancer and naive naked mouse. We dicovered the expression of miR-330 is significantly up-regulated in naked mouse with pancreatic cancer pain,while there is no expression difference between pancreatic cancer model without pain hypersensitivity and naive naked mouse.This suggests miR-330 may be closely associatd with the modulation of pain. We further took advantage of the gene database and found the gene GABBR2 for one of the GABA receptor subunits GABAb2 may be the target gene of miR-330. GABAB2-receptor is one of the main subunits of GABAe-receptor,and it is essential to the function of GABAs-receptor.In the central nervous system, GABAB-receptor is one of the receptors that the main inhibitory neurotransmitter GABA acts through. GABAB-receptor is widely distributed in the central nervous system and peripheral nervous system. In the spinal cord, GABA receptors are mainly expressed in the superficial dorsal horn.When activated,they can regulate the synaptic plasticity and excitability, and thus play an important role in the generation and modulation of pain.Therefore, our objective is to detect if miR-330 may downreglulate the expression of GABAB2-R and inhibit the function of GABAB-R which may be involved in the mechanisms of pain in chronic pancreatitis.Methods:(1) we transfered miR-330 to the spinal cord neurons through intrathecal injection of miR-330 lentivirus vector,and we test the pain threshold on the abdomen of rats with transfection of miR-330.(2) Wistar rats, which had received a tail vein injection DBTC, or no manipulation, were tested at 3,7,14, and 21 days after injection. The observer performing the behavioral testing was blind to the animal’s condition.(3) We detected the expression of the GABAB2 receptors of T8-T12 segement spinal dorsal horn in the protein levels with immunofluorescence histochemical and western-blot techniques.(4) We detected the expression of the GABAB receptors of T8-T12 segement DRG neurons in the mRNA levels with RT-PCR.(5) We explored the relationship between miR-330 and GABBR2 by the luciferase reportor array.Results:(1) we found that the pain threshold of rats with transfection miR-330 was significantly down-regulated. Wistar rats, which had received intrathecal injection of lentivirus carrying miR-330, or lentivirus without miR-330, were tested at 5,7,14, and 21 days after injection. The observer performing the behavioral testing was blind to the animal’s condition.At 14 days after intrathecal injection,we found that compared with injection of lentivirus without miR-330 group,abdomen pain threshold of rats received intrathecal injection of lentivirus carrying miR-330 was significantly decreased. The pain threshold is reduced from 50 g to 10 g.(2) Using a tail vein injection DBTC we successfully established model of chronic pancreatitis.The rats injected with DBTC demonstrated increased sensitization to mechanically applied stimuli on the abdominal area. Hematoxylin and eosin staining for the pancreas, we found that the rats at the 7 days after injection DBTC showed signs of chronic pancreatitis including scattered pancreas acinar cell swelling and necrosis, extensive infiltration of inflammatory cells and a large number of fibrous connective tissue. We tested abdominal pain threshold of rats which received injection DBTC at 3d,7d,14d,21d,and normal rats. Rats with dibutyltin dichloride (DBTC) injection begin to demonstrate a greater sensitivity to von Frey stimuli compared with naive rats at 3 days after DBTC injection, and the abdominal pain gradually worsened with the development of inflammation.(3) Using immunofluorescence histochemical technique, we found that GABAb2-R is mainly distributed in the superficial dorsal horn which is closely associated with nociception.(4) We found that compared with the controls and the acute pancreatitis group, the expression of GABAb2-R in the spinal dorsal horn was definitely downregluated in the chronic pancreatitis group. We tested the expression of GAB Ab2-R in the spinal dorsal horn of rats which received injection DBTC at 3d,7d,14d,21d,and normal rats.The results showed after a tail vein injection DBTC there was no significant difference of the expression of GABAb2-R between the normal rats and the aute pancreatitis rats,while in chronic pancreatitis group the expression of GABAb2-R was definetely downregulated to the normal ratsl/3-1/2.At the same time, the trend of the downregulation of GABAb2-R is consistent with the pain threshold value of chronic pancreatitis.(5) With immunofluorescence histochemical technique we found the GABAb-R is widely distributed in the dorsal root ganglion. Compared with the controls and the acute pancreatitis group, the expression of GABAb-R in the dorsal root ganglion was definitely down-regluated in the chronic pancreatitis group.(6) We defined that 3’UTR of GABAB2-R is a target site for miR-330 by the luciferase reporter array.The results showed miR-330 can bind the 3’UTR of GABBR2 and inhibit the expression of GABAb2-R-Conclusion:It is indicated in these results that miR-330 downreglulate the expression of GABAb2-R and inhibit the function of GABAb-R which may be involved in the mechanisms of pain in chronic pancreatitis.