| Background:Neuropathic pain(NP)can be divided into peripheral neuropathic pain(p NP)and central neuropathic pain based on the anatomical location of the injury or disease.At present,the mechanisms of peripheral neuropathic pain mainly include:ion channel changes,peripheral nerve sensitization,and decreased function of the descending inhibitory system.However,the exact pathophysiological mechanism is still unclear.Previous studies have found that long non-coding RNA is an important regulator of neuropathic pain and is related to the progression of neuropathic pain.However,there are few reports on the research of small nucleolar RNA host gene(SNHG)in neuropathic pain.SNHG is non-coding genes containing complete fragments of small nucleolar RNA,exons,and introns,with a length of more than 200 nucleotides,mainly located in the introns of protein-coding genes,and belong to the long non-coding RNA family.Current studies on SNHG4 are related to tumors,but there are no reports on the research on neuropathic pain.Micro RNA(microRNA or miRNA)is a class of non-coding single-stranded RNA molecules encoded by endogenous genes with a length of about 22 nucleotides.It is a huge family and is widely involved in various aspects of the body as a huge gene network.The regulation of physiological and pathological processes.Current studies have confirmed that the occurrence and progression of neuropathic pain are regulated by miRNA networks.For example,miR-142-3p can reduce neuropathic pain by regulating the high mobility group box 1;miR-93 can reduce neuropathic pain by targeting signal transducer and activator of transcription 3.Mi R-423-5p,a homologous gene of the above-mentioned miRNA,is out of control in many diseases,but the role of miR-423-5p in neuropathic pain and its relationship with SNHG4 have not been studied yet.In this study,we intend to observe the expression changes of SNHG4 and miR-423-5p in the spinal cord tissue through the spinal nerve ligation(SNL)rat model,and evaluate the changes in mechanical and thermal hyperalgesia neuropathic pain behavior in rats by knocking down SNHG4.The effect of SNHG4 on miR-423-5p,and the mechanism of SNHG4 in the rat model of spinal nerve ligation was analyzed.In addition,we consulted the bioinformatics analysis network(Http://starbase.sysu.edu.cn/).It is speculated that miR-423-5p is the target of SNHG4,and we plan to perform in vitro cytological RNA immunoprecipitation(RIP)and RNA pull-down experiment to explore the correlation between miR-423-5p and SNHG4.The experiment was divided into the following three parts.Part One: Changes of SNHG4 in Rat Model of Neuropathic Pain Induced by Spinal Nerve LigationObjective: To observe the changes of small nucleolar RNA host gene 4(SNHG4)and miR-423-5p in a rat model of neuropathic pain induced by spinal nerve ligation(SNL).Methods: Thirty clean male Sprague-Dawley(SD)rats were randomly divided into control group(Control,n=15)and experimental group(SNL,n=15).Rats in SNL group were separated and ligated L5 spinal nerves with 6-0 silk thread,while rats in control group were separated but not ligated in the same way.Paw withdrawal threshold(PWT)and paw withdrawal latency(PWL)were performed in each group of rats on the 0th,3th,6th,9th,and 15 th days after operation,and then the spinal cord tissue specimens were immediately collected and stored at-80°C for later experiments.The expression levels of SNHG4 and miR-423-5p at each time point were determined.Results:(1)Compared with the control group,PWT and PWL were significantly reduced on the 3rd,6th,9th,and 15 th day after surgery(P <0.05),indicating that the SNL-induced neuropathic pain model was successfully constructed.(2)The expression of SNHG4 in spinal cord tissue of SNL rats was significantly higher than that of the control group on the 3rd,6th,9th,and 15 th days after surgery(P <0.05).(3)The level of miR-423-5p in spinal cord tissue of SNL rats was significantly lower than that of the control group on the 6th,9th,and 15 th days after surgery(P <0.05).Conclusion: In the SNL rat model,SNHG4 expression was significantly up-regulated and miR-423-5p was significantly reduced.Part Two: Effects of Knockdown of SNHG4 on Inflammatory Factors and miR-423-5p in Spinal Nerve Ligation RatsObjective: In the first part of the study,it was observed that SNHG4 was over expressed in the spinal cord tissue of SNL rats while miR-423-5p was significantly reduced.This part aims to observe the effect of knockdown of SNHG4 on miR-423-5p in SNL rats,and further explore its influence on related inflammatory factors.Methods: Thirty clean male Sprague-Dawley(SD)rats were randomly divided into control group(Control,n=10),lentivirus blank control group(SNL+LV-NC,n=10),and SNHG4 knockdown group(SNL+LV-sh SNHG4,n=10).Rats in SNL+LV-NC and SNL+LV-sh SNHG4 groups were separated and ligated L5 spinal nerves with 6-0 silk sutures.In Control rats,nerves were separated but not ligated in the same way.On the 3rd day after operation,rats in SNL+LV-NC and SNL+LV-sh SNHG groups received intrathecal injection of lentivirus(MOI=100).In each group of rats,PWT and PWL were measured on the 0th,3th,6th,9th,and 15 th days after operation.Spinal cord tissue samples were collected on the 6th day after operation.The protein levels of inflammatory factors TNF-α,IL-12,IL-6,and IL-10 were detected by enzyme-linked immunosorbent assay(ELISA).The m RNA expression of SNHG4,miR-423-5p and TNF-α,IL-12,IL-6,and IL-10 were detected by real-time quantitative PCR(q RT-PCR).Result:(1)Compared with the control group,the PWT and PWL of rats in the SNL+LV-NC group were significantly reduced on the 3rd,6th,9th,and 15 th days after the operation.Compared with the SNL+LV-NC group,knockdown of SNHG4 significantly reduced the reduction of PWT and PWL in SNL rats(P < 0.05).(2)On the 6th day after surgery,compared with the control group,the level of miR-423-5p in the SNL+LV-NC group was significantly reduced.Compared with the SNL+LV-NC group,knockdown of SNHG4 significantly increased the level of miR-423-5p(P <0.05).(3)On the 6th day after the operation,compared with the control group,the levels of pro-inflammatory factors such as TNF-α,IL-12,and IL-6 in the spinal cord tissue samples of the SNL rats increased,while the anti-inflammatory factor IL-10 decreased.Knockdown of SNHG4 inhibited theses changes,reducing inflammation(P < 0.05).Conclusion: Knockdown of SNHG4 could reduce the progression of neuropathic pain in SNL rats.The mechanism may be related to inhibiting pro-inflammatory factors and activating the expression of anti-inflammatory factors,while increasing the level of miR-423-5p.Part Three: SNHG4 affects neuropathic pain by sponging miR-423-5pObjective: To reveal the mechanism of interaction between SNHG4 and miR-423-5p.Methods: Thirty clean male Sprague-Dawley(SD)rats were randomly divided into lentivirus blank control group(SNL+LV-NC,n=10),miR-423-5p inhibition group(SNL+LV-anti-miR-423-5p,n=10),SNHG4 knockdown group(SNL+LV-anti-miR-423-5p+LV-sh SNHG4,n=10).Three groups of rats were separated and ligated L5 spinal nerves with 6-0 silk thread.On the third day after surgery,rats received intrathecal injection of lentivirus(MOI=100),including miR-423-5p gene-suppressing lentiviral vector,short hairpin SNHG4 Gene lentiviral vector and its corresponding negative control lentiviral vector.In each group of rats,PWT and PWL were measured on the 0th,3th,6th,9th,and 15 days after operation.Spinal cord tissue samples were collected on the 6th day after operation,and the protein levels of inflammatory factor TNF-α,IL-12,IL-6,and IL-10 were detected by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescent PCR(q RT-PCR)was performed to detect the m RNA expression of TNF-α,IL-12,IL-6,and IL-10.The cytology experiment object is the PC12 cell line,and the RNA immunoprecipitation test(RIP)was detected by the Magna RNA immunoprecipitation assay kit.Biotin-labeled miR-423-5p-Bio,miR-423-5p-Bio-MUT vectors were constructed and RNA pull-down experiments were performed.Results:(1)Compared with the virus blank control group,the PWT and PWL of rats in the miR-423-5p inhibition group were significantly reduced on the 6th,9th,and 15 th days after surgery(P < 0.05),while knockdown of SNHG4 significantly alleviated the reduction of PWT and PWL induced by miR-423-5p inhibited(P <0.05).(2)On the 6th day after operation,the levels of TNF-α,IL-6,and IL-12 in the spinal cord tissue of rats in the miR-423-5p inhibition group were significantly increased,and IL-10 was significantly reduced.Knockdown of SNHG4 rescued miR-423-5p inhibition influence on inflammatory factors,reducing inflammation.(3)The enrichment rate of SNHG4 and miR-423-5p in Ago2 pellets of PC12 cell line was significantly higher than that in IgG pellets(P < 0.05).The RNA pull down test also confirmed that SNHG4 can directly target miR-423-5p.The biotin-labeled miR-423-5p(miR-423-5p-Bio)group detected high level of SNHG4 than the control(NC-Bio)and miR-423-5p gene mutation(miR-423-5p-Bio-MUT)group(P <0.05).Conclusion: Mi R-423-5p was one of the targets of SNHG4.SNHG4 acted as an endogenous competitive RNA to bind to miR-423-5p,thereby achieving negative regulation of miR-423-5p.Knockdown of SNHG4 can relieve neuropathic pain in rats with spinal nerve ligation. |
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