Differential Gene Expression Profile Analysis Of M6A Modified MRNA In Spinal Cord Of Rats With Bone Cancer Pain

Background & Aims: Most tumors have distant bone metastases in advanced stages.Patients suffered pain from bone destruction caused by tumors.m6 A modification is the most abundant modification in eukaryotic RNA.There are numerous evidence suggests that m6 A modification plays a critical role in cancer through various mechanisms.However.Whether it also plays a role in bone cancer pain remains unknown.Therefore,this study aims to investigate whether m6 A modification of m RNAs has an impact on bone cancer pain by establishing BCP animal models.To provide bioinformatics clues for studying the pathological mechanism of bone cancer pain.Methods: We have established BCP rat models and divided the rats into three groups: Normal group,sham-operated group(Sham group),and bone cancer pain group(BCP group).The model was induced by injection of Walker 256 cells into the rat tibia canal.By measuring MWT,TWL,X-ray examination,pathological analysis,body weight and general condition to evaluate the model’s establishment.Spinal intumescentia lumbalis were harvested at 21-day post operation and the differential m6 A modified RNA expression were investigated by gene chips.Identify the expression pattern of m6 A modification between different samples by hierarchical clustering.GO and Pathway enrichment analysis were performed for differential m RNAs.Me RIP and RT-q PCR analysis were performed used to verify the results.Results: BCP group: rats showed hyperalgesia;the X-ray showed tibia destruction;HE staining showed large number of tumor cells infiltration in the bone tissue;Slow weight gain and reduced activity.There was no significant changes in Sham group and Normal group.A total of 454 lnc RNAs,699 m RNAs and 53 other types of RNAs(sno RNA,pre-mi RNA,sn RNA)were modified and regulated by methylation.The results of cluster analysis showed that the methylation degree of m RNA was higher and with more differentially expressed genes.GO and Pathway analysis found that m6 A modification is involved in bone cancer pain by mediating immune activation,inflammatory cell and chemokine migration,and ion channel activation.Me RIP and RT-q PCR were used to verify 11 genes that related to pain,inflammation and cancer signaling pathways or is highly expressed.The m6 A modified expression levels of ALOX5、CACNA1B、EVC、Foxp3、NOS1、P2RX2、and PBK in BCP group were down-regulated,the m6 A modified expression levels of CD74、CXCL10、P2RX4 and CXCL11 in BCP group was up-regulated by m6 A,which were consistent with the microarray detection results.CD74,Foxp3 and CACNA1 B may be involved in bone cancer pain.Conclusions: 1.In this bone cancer pain model,m6 A modification is involved in bone cancer pain by participating in neural immune,inflammatory response,ion channel activation and their related pathways.2.CD74,Foxp3,ALOX5 and CACNA1 B are new molecules found in this study that may be involved in bone cancer pain through m6 A modification.