| Circadian rhythms of melatonin synthesis and secretion in the pineal gland are common in vertebrates,with melatonin expression levels always rising at night.Arylalkylamine nacetyltransferase(AANAT)and acetylserotonin methyltransferase(ASMT)are important in the synthesis of melatonin.ASMT is the last enzyme that regulates melatonin synthesis,but its expression in pineal gland shows no diurnal difference.Therefore,it is speculated that the transfer efficiency of methyl group may be related to the substrate in melatonin synthesis.S-adenosine methionine(SAM)is an important methyl donor in mammals,which can participate in the synthesis of melatonin as a substrate.It is known that MAT can catalyze the synthesis of methionine and ATP into SAM and has diurnal differences.Whether MAT can participate in the regulation of melatonin synthesis remains to be further studied.Circ RNA is an endogenous non-coding RNA with miRNA reaction element(MRE)on its molecule,which can participate in a variety of biological processes as a molecular sponge of miRNA.However,whether circ RNA in the pineal gland can participate in melatonin synthesis through ce RNA mechanism remains unclear.In this study,based on the complete transcriptome sequencing results of rat pineal RNA at different time points of day and night,the targeting relationships of differentially expressed circrnas,mirnas and mrnas were predicted.mat2 a was identified as an important molecule in melatonin synthesis in rats by RT-q PCR,NE treatment,mat2 a siRNA treatment and ELISA.After that,miR-125a-5p and circ-ERC2 were screened by software prediction,cell transfection and double luciferase reporting assay,and then the structure and location of circ-ERC2 were identified to evaluate the effects of circ-ERC2 knockdown on MAT2 A and melatonin synthesis and secretion in rat pineal primary cells.Finally,the experimental model of superior cervical ganglion resection in rats was used to verify the experimental results in vivo.The results show that:1.mat2 a has diurnal differential expression pattern similar to that of aanat and melatonin,and both are regulated by NE.After transfection with mat2 a siRNA,there was no significant difference in melatonin synthesis-related genes,and the expression level of mat2 a and secretion level of melatonin were significantly down-regulated,which preliminarily indicated that mat2 a plays an important role in the melatonin synthesis process of rat pineal gland.2.miR-125a-5p showed diurnal differences in the pineal gland of rats,and was downregulated after NE treatment,which was directly targeted to mat2 a.Compared with other tissues of rats,the expression level of miR-125a-5p was higher in the pineal gland of rats.Dual luciferase reporting assay and transfection assay showed that miR-125a-5p could down-regulate the expression of mat2 a.3.circ-ERC2 can act as a molecular sponge of miR-125a-5p to participate in the secretion of melatonin in rat pineal gland by regulating the expression of MAT2 A.circ-ERC2 was derived from the ERC2 gene by genomic comparison,and circ-ERC2 was identified in the pineal gland of rats using fluorescence in situ hybridization(FISH),first-generation sequencing,and linear RNase treatment.The results of RT-q PCR showed that circ-ERC2 was significantly up-regulated at night and showed an up-regulation trend after NE treatment.The dual luciferase reporting experiment and transfection experiment also jointly indicated that circ-ERC2 had a targeting relationship with miR-125a-5p.4.In order to verify the results of in vitro experiments,this study successfully constructed a rat model of superior cervical ganglion resection,and the experimental results showed that the rat pineal gland was involved in regulating the synthesis and secretion of melatonin by regulating the circERC2/miR-125a-5p/MAT2 A axis under the action of the superior cervical ganglion.In conclusion,this study found that differentially expressed circ-ERC2 can participate in the synthesis and secretion of melatonin in rat pineal gland through miR-125a-5p/MAT2 A pathway,providing a basis for the research on the role of circ RNA in melatonin synthesis and secretion regulation in pineal gland. |
