| Objective Neuropathic pain(neuropathic pain,NP),also known as neuralgia,has become a serious problem that seriously affects human health.Research shows that neuropathic pain is due to the peripheral or central nervous system damage or dysfunction caused by chronic pain caused by its clinical features are usually for hyperalgesia or allodynia,spontaneous pain,the pathogenesis of neuropathic pain is not clear,there is no effective treatment.A large number of studies have confirmed that the pathogenesis of neuropathic pain is complex,which may be closely related to the peripheral nervous receptor sensitivity,central nervous system abnormal neuronal reorganization and the ectopic excitation of the afferent nerve.Due to the lack of effective treatment in clinical practice,it is difficult to effectively cure neuropathic pain,the scope of the current global number of patients more and more,but due to the intense pain caused great physical and mental pain to the patient,can not be ignored to treat health problems more.In the clinical treatment for neuropathic pain is taking antidepressants,anticonvulsants and local anesthetics,but the efficacy of these drugs is still not ideal,and serious side effects,the pathogenesis of neuropathic pain and therefore clearly accelerate the development of high efficiency and low toxicity of drugs,has become a hotspot in the field of clinical research.The imbalance of genome expression is the key factor of neuropathic pain.It is clinically instructive to explain the pathogenesis of disease from gene level.Mi RNA is the body of a kind of expression of endogenous factor function of regulatory genes involved in the regulation of cell proliferation and apoptosis,and tissue differentiation and maintenance of function of the role of mi RNA in eukaryotic cells play a role in regulation of gene expression level,is mainly achieved by combining with the target gene m RNA 3 'non encoding region.On the one hand,mi RNA binds to proteins in the cytoplasm,the formation of RNA interference silencing ribonucleoprotein complex,this complex can occur with complementary target gene m RNA and protein degradation of m RNA,thereby inhibiting the transcription of target genes;in addition,mi RNA can also be used with m RNA incomplete complementary transcription inhibition protein.In fact,neuropathic pain can be regarded as the stress reaction of nervous system to injury.The pathological changes are closely related to the expression of various proteins,including signal transduction molecules,neurotransmitters,inflammatory factors and ion channel proteins.Therefore,mi RNA plays a different regulatory role in the pathological process of neuropathic pain by regulating the protein expression related to different signal pathways.Mi RNA research on its antitumor effects and mechanism of more extensive,and there has been some research into clinical application stage,but the role of mi RNA in the nervous system and its mechanisms are relatively scarce,in recent years,studies have revealed the occurrence and development of the differential expression of mi RNA in neuropathic pain,neuropathic pain by synthetic inhibitors related mi RNA or agonist drugs for neuropathic pain,is expected to provide a new effective target,but at the present stage,mi RNA and neuropathic pain complex regulating relationship still needs further study.Method 1.The sciatic nerve branch selective injury(SNI)method was used to construct the mouse model of neuropathic pain.The Dorsal rootganglia(DRG)was isolated from mice.2.The animal behavior of SNI mouse model includes behavioral assessment of pain induced by mechanical stimulation,behavioral analysis of pain induced by heat stimulation,and pain detection by cold stimulation.3 After DRG cells were isolated and cultured for 48 hours,the extraction and preamplification of total mi RNA were carried out,and the experimental operation was carried out according to the instructions of the kit.4.The mi RNA of differential expression in each sample was screened using Taq Man low density microarray chip,and the experimental procedure was carried out according to the reagent instructions.5.The real-time fluorescence quantitative PCR was further identified for the screening of mi RNA.6.We use Targetscan,mi RBase and mi RGen Targets three databases,combined with the existing literature,predict and analyze the downstream target genes of mi R-449 a,and then select the target genes which are most closely related to neuropathic pain as the research objectives.7.Using luciferase reporter gene to detect the interaction of transcription factor and target gene promoter DNA 8.DRG microinjection was used to detect the effect of exogenous mi RNA-449 a on neuropathic pain.9.The level of m RNA and protein expression of the target gene was detected by realtime fluorescent quantitative PCR and western.Results 1.The mice in the model group recovered well after SNI operation,and the physiological function returned to normal.The mechanical withdrawal threshold determination results showed that preoperative MWT based values of the two groups of mice had no significant difference in the third day after SNI,observed in the model group the MWT value decreased to 1.13±0.28 g,compared with the sham operation group(p<0.01);there were significant differences in postoperative fifth,seventh days in the model group the MWT value decreased slightly,and reaches the lowest value in seventh days MWT.However,there was no significant difference in the MWT value between the two groups on the nonoperative contralateral side.It was confirmed that the pain threshold of the mechanical side of the model group decreased significantly,and it decreased with the prolongation of the operation time.The threshold of thermal stimulation and thermal stimulation of the left hind limbs of the model group was basically the same as that of the mechanical stimulation.2.The results of mi RNA screening of Taq Man low density microarray chip,as shown in Table 1.1,showed significant difference between mi RNA-449 a and mi RNA-185 expression in DRG of SNI mouse model(P<0.05).Real time quantitative PCR was used to detect the expression of mi R-449 a and mi R-185 in DRG mice of SNI mice and control mice,so as to exclude false positive results.PCR quantitative detection showed that there were significant differences in mi R-449 a and mi RNA-185 expression in DRG of SNI mice on the third day after operation,and mi RNA-449 a expression level was the highest.The expression level of mi R-449 a and mi RNA-185 decreased with the prolongation of postoperative time,and the differential level of mi RNA-449 a was more significant.3.We predict the target base of mi R-449 a by online database Target Scan,mi RBase and mi RGen Targets,because KCNMA1 is preliminarily combined with Blast database validation.We initially assume that KCNMA1's 3 'UTR region has a structural sequence that interacts with the 5' end of mi R-449 a.The relative activity of luciferase was mi R-449a-NC-3'UTR-WT(0.91 + 0.07),mi R-449a-H+3'UTR-WT(0.52 + 0.06),mi R-449a-NC+3'UTR-MUT(0.96 + 0.09)and mi R-449a-H+3'UTRMUT(0.94 + 0.08).4.by dual luciferase reporter gene assay showed that KCNMA1 of wild type 3'UTR gene and mi R-449 a reporter plasmids were transfected into DRG cells,visible luciferase activity was significantly decreased(P<0.01,figure 4.1),while the KCNMA1 mutant 3'UTR luciferase reporter gene plasmid and mi R-449 a were transfected into DRG cells after the luciferase activity had no obvious effect.This result shows that the 3'UTR region of the KCNMA1 gene can be associated with mi R-449 a,and its expression is directly regulated by mi R-449 a.5.Of mi RNA-449 a m RNA and protein expression of KCNMA1 in DRG cells were detected by fluorescence quantitative PCR results showed that KCNMA1 mi RNA-449 a overexpression in DRG cells and the level of m RNA was significantly decreased to 0.58 + 0.04(p<0.05,compared with the negative control group);and no significant differences compared to blank control group and negative control group.Western blot was used to detect the expression of KCNMA1 protein in DRG cells,with the results of m RNA detection,in the over expression of mi RNA-449 a,KCNMA1 protein level in DRG cells was significantly decreased to 0.59 + 0.04(p<0.05,compared with negative control group,figure 2.3);and no significant differences compared to blank control group and negative control group.It was confirmed that mi RNA-449 a could inhibit the expression of KCNMA1 in DRG cells.6.Mice were operated on SNI or Sham after third days of DRG microinjection,and the behavioral changes of mice at different time points were detected in the previous experiments.The results showed that the preoperative mice in each group were no significant differences in MWT values;and in each control group after injection of DRG mice MWT value had no significant difference;and after SNI were observed in mi R-449 a mimics+Sham group had no significant change(compared with group Naive,p>0.05),mi R-449 a mimics+SNI group in postoperative 3,5 The 7 day,the MWT value and the basic values for this group(zeroth days)was significantly decreased(P <0.05);Vehicle+SNI group and NC+SNI group MWT of mice after SNI in the detection of all time points were significantly reduced(P <0.05,compared with the baseline value).The results of TWL and CWL in each group were the same as that of MWL.7.DRG mice were microinjected third days after SNI or Sham surgery,the surgical side were isolated.The expression of DRG was detected by real-time fluorescent quantitative PCR KCNMA1 m RNA level and protein level changes,test results showed that the KCNMA1 Vehicle+SNI group,NC+SNI group and mi R-449 a mimics+SNI group in DRG m RNA and protein levels increased significantly,compared with the in Naive group,there was significant difference(p<0.05);no significant change of mi R-449 a group mimics+Sham KCNMA1 and protein levels of m RNA(p>0.05).Conclusion 1.The expression of mi R-449 a in SNI neuropathic pain mice was significantly reduced.2.In DRG cells,mi RNA-449 a could target the expression of inhibition of KCNMA1.3.Exogenous mi RNA-449 a can improve the pain level of SNI neuropathic pain mouse model,and its effect may be closely related to the inhibition of the expression of KCNMA1. |
PDF Full Text Request