Study On RFRP-3 On Differentially Expressed Proteins In Hypothalamus Of OEP Rats And The Inhibition Of MCF-7 Cell Line Of Human Breast Cancer

Gonadotropin inhibitory hormone(GnIH)is a new hypothalamic neuropeptide that can inhibit gonadotropin-releasing hormone(GnRH)found in quail brain by Tsutsui team.Hypothalamus is a high-level center regulating reproduction and energy metabolism,which is mainly involved in the dorsomedial hypothalamic nucleus(DMH),ventromedial hypothalamic nucleus(VMH),paraventricular hypothalamic nucleus(PVH)and arcuate nucleus(ARC).At the same time,RFRP neuropeptide with C-terminal sequence Arg-Phe-NH2(RFamide related peptide)was found in vertebrates,which mainly includes RFRP-1 and RFRP-3.In mammals,GnIH/RFRP-3 mainly exists in the dorsomedial hypothalamic nucleus(DMH)or its adjacent area.In addition,bonini et al.Found two kinds of receptors GPR147 and GPR74 of GnIH,among which GPR147 is the main receptor of RFRP-3,and this receptor widely exists in hypothalamus,ovary,uterus and testis of mammals.It has been confirmed that GnIH can bind to GPR147 through hypothalamus pituitary gonad axis,inhibit GnRH secretion,inhibit the release of luteinizing hormone(LH)and follicle stimulating hormone(FSH),inhibit ovarian development,reduce estrogen synthesis and secretion,and regulate neuroendocrine system.Proteomics is a new way for life science to study after entering the protein era,and it is also the direction for future science to reveal the mystery of life.Bioinformatics is a combination of life science and natural science.It uses computer as a tool and complex big data to reveal the mystery of organisms.Breast cancer is the most common malignant tumor in women and ranks as one of the three most common cancers in the world alongside lung cancer and colon cancer.Studies have found that the increase in estrogen levels is closely related to the occurrence and development of breast cancer,and the reason may be related to the hypothalamus releasing GnRH and stimulating the secretion of FSH and LH.FSH can stimulate the synthesis of estrogen in the ovarian follicles,and then act on the hypothalamus to induce the production of LH.A sharp increase in LH triggers ovulation and development of the corpus luteum.After menopause,the level of estrogen produced by the ovaries is negligible.Early menarche and delayed menopause are associated with a high risk of breast cancer,which highlights the importance of gonadal hormone production in normal breast development and breast cancer.The previous research of this group showed that after ovarian extraction supplemented with estrogen(Ovariectomized Estrogen Primed,OEP)rats lateral ventricle microinjection of RFRP-3,it can affect the release of GnRH,LH,FSH and other hormones,but for hormone-dependent tumors less.Gonadotropin has a regulatory effect on the release of estrogen,progesterone and other sex hormones in the ovary.Therefore,this project assumes that the lateral ventricle micro injection of RFRP-3 can affect the secretion of hypothalamic proteins,and this regulation may affect the secretion of GnRH,and further Regulate the release of pituitary gonadotropin,thereby affecting the physiological or pathological changes of downstream target organs.In this study,proteomics and bioinformatics methods were used to investigate the changes of protein expression in hypothalamus of OEP rats after microinjection of RFRP-3 into lateral ventricle.It is known that glycolysis can stimulate the release of GnRH,so it is speculated that the glycolysis pathway may be one of the mechanisms of GnRH inhibition.GnRH can regulate the release of estrogen,progesterone and other gonadal hormones,and the abnormality of estrogen is related to the occurrence and development of breast cancer.Therefore,this experiment hypothesized that RFRP-3 may regulate the occurrence and development of hormone dependent breast cancer through this pathway.To test this hypothesis,we treated MCF-7 cells with RFRP-3.The results showed that RFRP-3 of 10000 ng/ml could inhibit glycolysis and induce apoptosis of MCF-7 cells by blocking PI3K/Akt/mTOR/HIF-1α pathway.Part Ⅰ Proteomic study on the regulation of hypothalamic proteins by microinjection of RFRP-3 into lateral ventricleObjective:To explore the effect of microinjection of RFRP-3 into lateral ventricle on hypothalamic protein regulation by proteomics and bioinformatics.Methods:1.Methods:Thirty adult female SD rats were selected to establish ovariectomized estrogen primed(OEP)rat model.They were randomly divided into experimental group injected with GnIH and control group injected with normal saline The rats were injected with RFRP-3 and normal saline(16μl/kg,2μg/μl),0.5μlevery 30 seconds,and the injection was completed in 4 minutes.Six hours after administration,the hypothalamus was taken out to prepare protein samples.2.The hypothalamic protein samples were enzymolyzed and analyzed by LC-MS/MS using the Dionex ultimate 3000 nano LC system.Maxquant-1.5.2.0 algorithm was used to quantitatively analyze the unlabeled proteomics of peptides,and the original data of mass spectrometry was obtained.3.Go gene ontology analysis,KEGG signal pathway enrichment analysis and protein-protein interaction analysis were used to screen differentially expressed proteins and signal pathways.4.Identification of key protein expression in tumor by CPTAC database.Results:1.A total of 253 differentially expressed proteins were identified,129 of which were up-regulated and 124 down regulated.2.Go results showed that the biological process(BP)of differential proteins was mainly enriched in single organism and small molecule metabolism,the cell component(CC)differential proteins were mainly enriched in cytoplasm and nucleus,and the molecular function(MF)was mainly reflected in the function of binding with small molecules.3.KEGG results showed that in addition to 10 important signaling pathways involved in metabolic pathway,endocrine and other factors regulating calcium reabsorption,differential proteins were also enriched in glycolysis pathway,which was closely related to the occurrence and development of tumor(P<0.05).4.Protein protein interaction(PPI)analysis by omicsbean software showed that galm,adpgk,PGM1 and PFKL were up-regulated,ALDH2 were down regulated(P<0.05,FC≥2).5.Cptac database analysis showed that GLAM(P=0.050),ADPGK(P=0.0060),PGM1(P=0.25),PFKL(P=0.0062)and ALDH2(P=0.0092)were differentially expressed in breast cancer tissues and adjacent tissues.Part Ⅱ RFRP-3 inhibits glycolysis pathway through PI3K/AKT/mTOR/HIF-1α pathway to induce apoptosis of human breast cancer cell line MCF-7Objective:To investigate the effect of RFRP-3 on apoptosis and its mechanism of human breast cancer cell line MCF-7.Methods:1.The culture of human breast cancer cell line MCF-7 is the same as the second part.2.Flow cytometry was used to detect the apoptotic rate of MCF-7 cells treated with RFRP-3.3.Western blot was used to detect the expression of Bcl-2,Bax,cytochrome c,Caspase-3,p53,PI3K,Akt/p-Akt,mTOR,HIF-1α and GPR147 related proteins in human breast cancer cell line MCF-7 at the optimal concentration of RFRP-3.4.Quantitative real-time PCR(qRT-PCR)was used to detect the mRNA expression of Bcl-2,Bax,cytochrome c,Caspase-3,p53,PI3K and Akt in human breast cancer cell line MCF-7 at the optimal concentration of RFRP-3.Results:1.The apoptosis rate of MCF-7 cells was detected by flow cytometry.The results of flow cytometry showed that the apoptotic rate of control group and RFRP-3 group were(7.76 ± 1.57)%,(16.14 ± 3.001)%,respectively.The apoptosis rate of the control group was significantly higher than that of the experimental group(P<0.05).2.RFRP-3 upregulated the expression of ADPGK,Bax,Caspase-3,cytochrome c and p53 in MCF-7 cells.Compared with the control group,the expression levels of Bax(P<0.01),caspase-3(P<0.01),cytochrome c(P<0.01),p53(P<0.05)and ADPGK(P<0.01)in the experimental group were significantly increased.3.RFRP-3 down regulated the expression of Bcl-2,PI3K,Akt/p-Akt,mTOR and HIF-1α in MCF-7 cells.The expression levels of Bcl-2(P<0.05),PI3K(P<0.05),Akt/p-Akt(P<0.01),mTOR(P<0.05)and HIF-1α(P<0.05)in MCF-7 cells were significantly lower than those in the control group and the experimental group(P<0.05).4.RFRP-3 upregulated the expression of Bax,caspase-3 and p53 mRNA in MCF-7 cells.Compared with the control group,the expression levels of Bax mRNA,Caspase-3 mRNA and TP53 mRNA in 10μg/ml experimental group were significantly up-regulated(P<0.05).5.RFRP-3 down regulates Bcl-2,PI3K and Akt mRNA in MCF-7 cellsCompared with the control group,the expression levels of Bcl-2 mRNA,mRNA,PI3K mRNA and Akt mRNA in the 10μg/ml experimental group were significantly down regulated(P<0.01,P<0.05,P<0.01,P<0.01,P<0.05,P<0.05).Conclusion:1.Proteomics and bioinformatics analysis showed that microinjection of RFRP-3 into the lateral ventricle caused protein changes in the hypothalamus,and the differential proteins were involved in the glycolysis/gluconeogenesis pathway,which may be related to the occurrence and development of hormone dependent tumors.2.RFRP-3 inhibits the proliferation and induces apoptosis of breast cancer MCF-7 cells through GPR147 receptor,which may be related to PI3K/Akt/mTOR/HIF-1α signaling pathway.