| It has been reported that about 15 million patients suffering from cancer pain all over the world.About 60%-90%of advanced cancer will develop skeletal metastasis,such as breast,prostate,lung cancer and multiple myeloma（MM）The bone would be the prior metastatic site for these cancer.Bone cancer pain has become one of the most serious problems affecting the quality of life of patients with advanced cancer.The characteristics of bone cancer pain is related to bone metastases parts continued chronic pain and intermittent breakthrough pain intensified.Now,the occurrence and development mechanism of bone cancer pain has not been clarified,so there are still lacks of effective measures to treat the bone cancer pain.Can we find some clue if we focus on the bone cancer pain related gene?In this study,RNAseq（RNA Sequencing）technique was used to analyze the dorsal root ganglion（DRG）of bone cancer pain rats,and the key genes involved in bone cancer pain were screened.The differential expression of the key gene in dorsal root ganglion of bone cancer rats was confirmed by real-time quantitative PCR,immunohistochemistry,Elisa and western-blot.The effect of the key gene on the pain behavior of rats with bone cancer pain was studied by the method of intrathecal administration.Through the above research,it will help to further elucidate the mechanism of bone cancer pain developmen and provide a theoretical basis for finding new targets for the treatment of bone cancer pain.Part I:Transcriptome study of dorsal root ganglion in rats with bone cancer pain based on RNAseq（RNA Sequencing）Objective:In this study,RNAseq technique was used to analyze the transcriptomics of dorsal root ganglion（DRG）in Sham operation and bone cancer pain group.The differentially expressed genes were screened out.Then the difference genes were further analyzed by GO（Gene Ontology）、KEGG（Kyoto Encyclopedia of Genes and Genomes）to find out the key target gene that involved in bone cancer pain sensitization.We hope these findings could provid some experimental evidence for further explaining the molecular mechanism of bone cancer pain.Twenty healthy female SD rats were randomly divided into Sham group and Bone cancer pain（BCP）group.BCP group was injected with walker256 breast cancer cell 2×104/μl（10μl）to establish the rat tibial cancer pain model.The Sham group was injected with equal volume of saline into the right tibial bone marrow as control.The paw mechanical withdrawal threshold（PMWT）and limb use score during normal ambulation were measured at 1 d（baseline level）and 4,7,11,14,17,19,21d after operation.Using this method of pain detection and surgical tibial radiographic examination to determine whether the success of modeling.The rats were randomly selected from the control group and the BCP group,and the RNA of the ipsilateral L3-L5 dorsal root ganglion（DRG）was detected by RNAseq.The expression level of the whole transcriptome of DRG tissues was detected by RNAseq technique.The differentially Expressed Genes（DEGs）were screened out by the statistical model of Poisson distribution,and then the biologically GO analysis and KEGG pathway enrichment analysis to further screen out the genes that play a key role in pain sensitization of bone cancer.Results:The DRG tissue of Sham group and BCP group was analyzed by RNAseq technique.The results showed that 725 gene expression was changed in DRG neurons of BCP rats compared with Sham group,among which 536 genes were up-regulated and 189 genes were down-regulated（P<0.01）.Using GO to annotate the cell localization,molecular function and biological function of differentially expressed genes,it was found that the differential expression genes in DRG neurons of BCP group were mainly located in cell membrane,synapse and Golgi apparatus compared with Sham group.These differentially expressed genes are closely related to protein binding and enzyme catalytic activity or regulatory activity,and are involved in many biological processes such as inflammatory response,neurotransmitter synthesis and transport,synaptic plasticity,signal transduction etc.Further use of KEGG to analyze differentially expressed and enriched signaling pathways.The results showed that the main enrichment pathways of BCP group and Sham group were Hippo,mTOR,TGF-β,steroid biosynthesis,digestion and absorption of vitamins,histidine metabolism and other signal pathways,among which Hippo,MTOR,TGF-βand other signal pathways are activated,and steroid biosynthesis,vitamin digestion and absorption,histidine metabolism and other signaling pathways are suppressed.The expression of BMP2,BMP4,BMP15 and BMP2 receptor Bmpr1b in the TGF-βfamily was significantly different from in the BCP group and the Sham group(P=BMP2:4.65×10-4;BMP4:7.9×10-4;BMP15:9.87×10-5;Bmpr1b:1.54×10-4),and in the GO tree and KEGG tree these are in a critical position.Conclusion:A variety of gene levels in the dorsal root ganglion of bone cancer pain rats were significantly changed,such as the expression of BMP and its receptor gene.Part II:BMP2 is involved in the formation of peripheral pain sensitization in rats with bone cancer painObjective:To investigate the role of BMP2 in peripheral sensory sensitization of rats with bone cancer pain.Methods:The expression of BMP2 in ipsilateral L3-5DRG tissues of Sham group and BCP group was detected by immunohistochemistry and real-time quantitative PCR,Elisa and western-blot.Seven days after surgery,the rats in Sham group and BCP group were randomly divided into two groups respectively by either intrathecal injection of RNA fragments（BMP2-siRNA）that interfere with DRG tissue BMP2expression or negative control RNA fragments（NC-siRNA）,namely bone cancer pain+BMP2-siRNA group,bone cancer pain+NC-siRNA group,Sham operation+BMP2-siRNA group,Sham operation+NC-siRNA group.The pain behavior of PMWT and limb use score during normal ambulation were tested every other day post operatively.Results:The BMP2 mRNA and BMP2 level was significantly higher in DRG tissues of BCP group than that of Sham group（P<0.05）.Intrathecal injection of BMP2-siRNA significantly increased the mechanical pain threshold on day 4,6,8,10,12 and the limb use score on day 6,8,10 of bone cancer rats with a reduction of DRG tissue BMP2 expression.There was no significant difference on day 4 and 12 days after intrathecal interference.However intrathecal injection of BMP2-siRNA to Sham operation groups has no significant effect on pain behavior.Conclusion:The expression of BMP2 in DRG tissue of rats with bone cancer was significantly increased.The expression of BMP2 in DRG could have involed in the sensory sensitization of bone cancer pain. |
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