Mechanism Of Melatonin Inhibiting Apoptosis In Bovine Ovarian Granulosa Cells Via TRF-Tyr-GTA-020

Follicle is the functional unit of ovary.During the follicle development,the metabolic rate rised with granulosa cells proliferation,the level of reactive oxygen species（ROS）,a by-product of aerobic metabolism,increased as well.the accumulation of ROS induces ovarian granulosa cells apoptosis.Studies have shown that the apoptosis of granulosa cells is the initiating factor resulting in follicle atresia.Melatonin,which exists in follicular fluid,can inhibit the apoptosis of bovine ovarian granulosa cells and maintain the survival of granulosa cells through various ways under stress conditions.However,its molecular mechanism remains unclear.t RNA derived fragments（tRFs）are small non-coding RNAs of 14-30nts in length which are specifically cleaved by mature t RNA and t RNA precursors when organisms are suffered from oxidative stress,heat/cold shock,ultraviolet irradiation and other stressors.tRFs have been reported in viruses,archaea,bacteria,protozoa,plants,chickens,mice and humans,and are widely involved in diverse physiological and pathological processes such as ribosome biogenesis,viral reverse transcription,cancer and apoptosis.Since endogenous nucleotide cleavage of t RNAs is a widely conserved reaction in eukaryotes.Therefore,tRFs may play an important role in regulating the apoptosis of bovine ovarian granulosa cells.The aim of this study was to clarify the molecular mechanism by which melatonin inhibits apoptosis of bovine ovarian granulosa cells through tRF.In this study,we first established the model of melatonin inhibiting H₂O₂ induced apoptosis of bovine ovarian granulosa cells,and obtained the differential expression profiles in this model by tRF&ti RNA sequencing in order to search for tRF that may be involved in melatonin regulating apoptosis of bovine ovarian granulosa cells.The effect of tRF on the inhibition of H₂O₂ induced apoptosis of bovine ovarian granulosa cells by melatonin was determined by flow cytometry,fluorescence quantitative PCR and western blot.tRF target genes were screened by Targetscan and mi Randa software,and the signal pathways involved in regulation were screened by GO and KEGG analysis and dual luciferase reporter assay,flow cytometry,fluorescence quantitative PCR and western blot were carried out to verify that.Therefore,we clarify the molecular mechanism of melatonin inhibiting H₂O₂ induced apoptosis of bovine ovarian granulosa cells via tRF.The results are as follows:1.Screen the tRF differentially expressed in melatonin inhibiting H₂O₂ induced apoptosis of bovine ovarian granulosa cells.CCK8 experiment was used to screen the optimal treatment conditions of melatonin and H₂O₂.The results showed that10nmol/L melatonin could inhibit the apoptosis of bovine ovarian granulosa cells induced by 400μmol/L H₂O₂.H₂O₂ treatment significantly increased the apoptosis rate（P<0.05）,the m RNA and protein expressions of pro-apoptotic genes Caspase3and Bax were significantly increased（P<0.05）,and the m RNA and protein expressions of anti-apoptotic gene Bcl2 were significantly decreased（P<0.05）.Melatonin pretreatment could reverse these apoptotic changes.tRF&ti RNA sequencing was used to screen for differentially expressed tRFs in this model.The results showed that there were four differentially expressed tRFs in the inhibition of H₂O₂ induced apoptosis of bovine ovarian granulosa cells by melatonin,among which tRF-Cys-GCA-079 and tRF-Gly-TCC-017 were up-regulated,and tRF-Gly-TCC-027and tRF-Tyr-GTA-020 were down-regulated.The m RNA expressions of tRF-Gly-TCC-017,tRF-Gly-TCC-027,tRF-Tyr-GTA-020 in melatonin inhibiting H₂O₂ induced apoptosis of bovine ovarian granulosa cells were consistent with sequencing results.GO and KEGG analysis showed that GO analysis significantly enriched transcription coregulator activity（GO:0003712）and kinase activity（GO:0016301）.KEGG was significantly enriched in AKT3/RAF1（bta04210）signaling pathway.2.Demonstrate the role of tRF-Tyr-GTA-020 in melatonin inhibiting H₂O₂induced apoptosis of bovine ovarian granulosa cells.The results showed that overexpression of tRF-Tyr-GTA-020 significantly reversed melatonin inhibiting the H₂O₂ induced apoptosis rate of bovine ovarian granulosa cells（P<0.05）,increased the m RNA and protein expression of pro-apoptotic genes Caspase3 and Bax（P<0.05）,and decreased the m RNA and protein expression of anti-apoptotic gene Bcl2（P<0.05）.Inhibition of tRF-Tyr-GTA-020 expression significantly enhanced the inhibitory effect of melatonin on the H₂O₂ induced apoptosis rate of bovine ovarian granulosa cells,significantly decreased the m RNA and protein expression of pro-apoptotic genes Caspase3 and Bax（P<0.05）,and increased the m RNA and protein expression of anti-apoptotic gene Bcl2（P<0.05）.3.Screen the target genes and downstream signaling pathways of tRF-Tyr-GTA-020 and identify the molecular mechanism of melatonin inhibiting apoptosis in bovine ovarian granulosa cells via tRF-Tyr-GTA-020.The results showed that overexpression of tRF-Tyr-GTA-020 significantly decreased the m RNA and protein expression of RAF1（P<0.05）.Inhibition of tRF-Tyr-GTA-020 significantly increased the m RNA and protein expression of RAF1（P<0.05）.The results of dual luciferase reporter gene assay indicated that tRF-Tyr-GTA-020 binds to RAF1 3’UTR.Interference with RAF1 significantly increased apoptosis rate of melatonin inhibiting apoptosis in bovine ovarian granulosa cells（P<0.05）,increased the m RNA and protein expression of pro-apoptotic genes Caspase3 and Bax（P<0.05）,and decreased the m RNA and protein expression of anti-apoptotic gene Bcl2（P<0.05）.At the same time,RAF1 interference could significantly reduce the phosphorylation levels of AKT and ERK signaling pathway（P<0.05）,and activation of AKT and ERK signaling pathway could significantly reverse the changes in apoptosis level caused by RAF1interference.In conclusion,this study demonstrated that melatonin inhibits H₂O₂ induced apoptosis of bovine ovarian granulosa cells by inhibiting tRF-Tyr-GTA-020,targeting up-regulated RAF1 gene expression and activated AKT and ERK signaling pathways.