MicroRNA-449a-5p Targets The Calcitonin Gene-Related Peptide MRNA In The Trigeminal Ganglion Of Rat Model Of Migraine

Background— Migraine is a common primary headache in neurology clinic,and the prevalence of it in china is approximately 10%.It is characterized by episodic unilateral or bilateral throbbing headache,and can be accompanied by nausea,vomiting,phonophobia,photophobia,neurological disturbances and so on.In recent years,a large amount of studies have demonstrated that the calcitonin gene-related peptide(CGRP)which is generated in and released from trigeminal ganglion plays an important role in migraine.MicroRNAs(miRNAs),which was found ten years ago,are endogenously 19-25 nucleotide(nt)non-coding RNAs.It turned into the research hotspot in the field of life science since its discovery.It is suggested that,miRNAs could lead to target gene degradation or translation inhibition through perfect or partly complementary to its 3?-UTR by 6–8 nt “seed” sequences in their 5?-terminals,respectively.miRNAs have been demonstrated to participate in the modulation of a variety of physiological or pathological processes.For example,they were found to be necessary regulators for normal neuronal function and key players in the functional changes in nerves and neurons.And nearly 60% of the genes are deemed to be regulated by miRNAs.There is one research demonstrated the change of miRNA expression in rat trigeminal ganglion following inflammatory pain.But,the miRNAs that involved in the occurrence and development of migraine,as well as the miRNAs target CGRP mRNA(CALCA),have not been investigated.Objective— The study aimed to explore the abnormal expression of miRNAs and the miRNAs directly target CALCA in the trigeminal ganglion of migraine model rat,compared with sham-operated rat,via bioinformatics analysis and various test methods.Methods— Firstly,we used three prediction programs of TargetScan,miRanda and PITA to identify the miRNAs putatively regulate the expression of rat CALCA.Secondly,after carring out a special operation and dural inflammation stimulation,we successfully modeled migraine in rats.Then we used western blot and miRNA profiling to evaluate the change of CGRP level and miRNA expression in trigeminal ganglion of migraine model rats,respectively.And reverse transcription and quantitative real-time polymerase chain reaction(RT-qPCR)was utilized to authenticate the differential expression of the miRNAs.Thirdly,according to all the results of the above experiments,we found out the potential miRNAs target CALCA and determined the location of them in trigeminal ganglion with the experiment of Fluorescence in-situ hybridization(FISH).Lastly,the experiments of luciferase report assays and lentivirus transfection were performed to evaluate if the final selected miRNAs could directly regulate CALCA expression through recognizing the putative binding site in its 3?-UTR.Results— We identified significantly increased CGRP level and a mount of differential expressed miRNAs in trigeminal ganglion of migraine model rats,compared to sham-operated group rats.After that,according to the fold changes and the predicted interactions with CALCA,seven miRNAs were specially chosen for RT-qPCR.The results of PCR showed that,in the seven specially chosen miRNAs,the expression of miR-449a-5p and miR-30a-3p were significantly decreased.And they were proved to locate in certain neurons of rat trigeminal ganglion in FISH.So that,we speculated that miR-449a-5p and miR-30a-3p may be co-localize with CGRP in some neurons.However,in the following experiments of luciferase report assays and lentivirus transfection,only miR-449a-5p was demonstrated to directly regulate CALCA expression through recognizing the putative binding site in its 3?-UTR.Conclusions— Our study was the first research to evaluate the abnormal expressed miRNAs and the miRNAs directly modulated CALCA expression in the trigeminal ganglion of rat model of migraine in the world up to now.And according to all the results,miR-449a-5p was proved to be involved in the regulation of CALCA expression in rat.Moreover,on the basis of the analysis,the conservation of the putative binding site of miR-449a-5p in CALCA 3?-UTR was found in many species,for example the rat,mouse and humans.This showed that miR-449a-5p may also participate in the modulation of CALCA expression in humans.So,it may become a new target for diagnosis and treatment of migraine.But,further investigations are still needed.