| Gonadotropin inhibitory hormone(Gn IH),a new hypothalamic neuropeptide,has been discovered in 2000.The Gn IH orthologs have been further identified in mammalian,called RFamide-related peptide 1 and3(RFRP-1 and-3).Among them,RFRP-3 plays a major role in various biological processes.Uterus is an important reproductive organ in females.Researches have demonstrated that the Intracerebroventricular injection(ICV)of RFRP-3 would delay the uterine development.Therefore,it is of great significance to investigate the molecular mechanisms underlying the effects of RFRP-3 on uterus.Endometrial carcinoma(EC)is one of most common malignant tumors in women that is serious threats women’s life and health.The incidence rate of EC is increasing year by year and showing a younger trend.At present,the main treatment measures are surgery,radiotherapy,chemotherapy and hormone therapy.But for advanced cancer,recurrent cancer and to meet the reproductive needs of young sick women,the treatment effect is not good.Therefore,finding more effective treatment methods,delayed recurrence,reduced mortality and improving the quality of patients’ life,is the focus of scholars at home and abroad.Our previous experiment showed that intracerebroventricular injection of RFRP-3 for 6 hours had a significant effect on postoperative estrogen replacement after ovariectomy,The results showed that the effect of Gn RH on Gn RH neurons was significant in rats.RFRP-3 could indirectly affect Gn RH neurons by inhibiting POMC and kisspeptin neurons and stimulating NPY neurons,so as to inhibit the release of Gn RH,and then inhibit the release of pituitary gonadotropin,which regulated the hypothalamic pituitary reproductive axis.The uterus is the downstream target organ of hypothalamic pituitary reproductive axis.It is not clear whether RFRP-3 can regulate it through hypothalamic pituitary reproductive axis.In this study,liquid chromatography tandem mass spectrometry(LC-MS/MS)was used to identify the proteins in the uterine fluid of OEP rats microinjected with RFRP-3 and normal saline.In order to explore the potential molecular mechanism and influence of RFRP-3 on uterus,Genetic Ontology(GO)functional enrichment analysis,Kyoto Encyclopedia of genes and genomes(KEGG)pathway analysis,protein-protein interaction(PPI)network and other bioinformatics analysis were carried out.The purpose of this study was to elucidate the molecular mechanism of RFRP-3 on endometrial cancer cell line HEC-1A and explore the effect of RFRP-3 on endometrial cancer cells.The results provide a new idea for further exploring the effect of RFRP-3 on uterus.Part 1 Effects of intracerebroventricular injection of RFRP-3 uterine fluid in OEP rats based on proteomic study Objective:To investigate the effect of intracerebroventricular injection of gonadotropin inhibitory hormone on protein histochemical changes in uterine fluid of OEP rats and its underlying molecular mechanism.Methods:1.Collection and grouping of uterine cavity fluid: 30 adult female SD rats were selected to establish ovariectomized estrogen primed(OEP)rat model.Thirty OEP rats were randomly divided into normal saline control group and RFRP-3 experimental group(n = 15).Uterine fluid was taken 6hours after injection,and the uterine fluid of experimental group(n=15)and Control group(n=15)were mixed and divided into three parts.2.Liquid chromatography tandem mass spectrometry(LC-MS/MS)was used to compare the protein components of experimental group(n=15,16μl/kg)and Control group(n=15,16 μl/kg)after intracerebroventricular injection of RFRP-3 for 6 hours.The qualitative analysis was performed by Maxquant software,and the differential proteins were screened by uniport database.3.Go enrichment analysis was used to analyze the biological process,cell composition and molecular function of the differential proteins.4.KEGG pathway analysis was used to analyze the signal pathways involved in different proteins.5.Screening of central node proteins by protein-protein interaction network.Results:1.Effect of intracerebroventricular injection of RFRP-3 on protein changes in uterine cavity fluidThe results showed that compared with Control group,the protein composition and expression level in the uterine cavity fluid of OEP rats were significantly affected by intracerebroventricular injection of RFRP-3 for 6hours(P<0.05).According to the standard of P<0.05,FC=2,417 differential proteins were screened,including 279 up-regulated and 138 down regulated proteins.2.Effect of intracerebroventricular injection of RFRP-3 on the biological function of DEPsTo analyze the biological process(BP),cellular component(CC)and molecular function(MF)of differentially expressed proteins(DEPs),functional enrichment analyses were performed using GO.Analysis results showed that changes in BP of differentially expressed proteins were significantly enriched in response to organic substance,response to oxygen-containing compound,regulation of biological quality and response to external stimulus.Changes in MF were mainly enriched in protein binding,protein complex binding and macromolecular complex binding.Changes in CC of differentially expressed proteins were mainly enriched in the extracellular region and membrane-bounded vesicle.3.Analysis of signal pathways involved in DEPs after intracerebroventricular injection of RFRP-3KEGG pathway analysis revealed that the differentially expressed proteins were mainly enriched in Carbon metabolism,Gap junction,Long-term depression,Regulation of actin cytoskeleton,Biosynthesis of amino acids,Complement and coagulation cascades,Glycolysis/Gluconeogenesis,Proteoglycans in cancer,Platelet activation,Thyroid hormone synthesis,and involved in multiple signal transduction pathways.4.Protein interaction analysis of DEPs after intracerebroventricular injection of RFRP-3The data of protein-protein interaction are from string database.PPI network is built online by using string database and visualized by omicsbean software.By analyzing and comparing the degree of interaction between proteins,five proteins were selected as central node proteins,namely Gna13,Gnaq,Gnai3,Kras,Mmp9.Mmp9 was up-regulated,while Gna13,Gnaq,Gnai3 and Kras were down regulated.Conclusion:RFRP-3 may changes the protein expression profile of uterine fluid in ovariectomized rats with estrogen supplement by up regulating Mmp9 and down regulating the expression of Gna13,Gnaq,Gnai3,Kras.Part 2 Effect and mechanism of RFRP-3 on human endometrial carcinoma cell line HEC-1A Objective:To explore the effect and the molecular mechanism of RFRP-3 on proliferation and apoptosis of human endometrial carcinoma cell line HEC-1A.Methods:1.The expression of central node protein in endometrial carcinoma was searched by using UALCAN database.2.CCK8 assay was used to detect the proliferation ability of endometrial cancer cells(HEC-1A,Ishikawa)treated with different concentrations of RFRP-3 for 24 and 48 hours.3.The apoptosis of endometrial cancer cells HEC-1A after 24 hours of adding RFRP-3 were used by Annexin V-FITC/PI double staining flow cytometry.4.The expression levels of apoptosis related proteins Bcl-2 and Bax in HEC-1A were used by Western blot.5.Western blot was used to detect the expression of KRAS(central node protein)in HEC-1A Control group and RFRP-3 group.6.RT-qPCR was used to detect the transcription level of PI3K/AKT/mTOR pathway gene in HEC-1A Control group and RFRP-3group.7.The expression of PI3K/AKT/mTOR pathway protein in HEC-1A were used by Western blot.8.RT-qPCR was used to detect the transcription level of ERK pathway gene in HEC-1A Control group and RFRP-3 group.9.Western blot was used to detect the expression of ERK pathway protein in HEC-1A Control group and RFRP-3 group.10.RT-qPCR and Western blot were used to detect the autophagy of endometrial cancer cell HEC-1A in Control group and RFRP-3 group.11.Western blotting was used to detect the expression of GPR147 in HEC-1A cells.Results:1.The expressions of Gna13,Gnaq,Kras and Mmp9 were significantly changed in endometrial carcinomaAfter searching the five central node proteins in the UALCAN database,the results showed that Gna13,Gnaq,Kras and Mmp9 were different between endometrial carcinoma and normal tissues.Compared with normal tissues,the expressions of Kras and Mmp9 were up-regulated in endometrial carcinoma,while the expressions of Gna13 and Gnaq were down regulated in endometrial carcinoma.2.RFRP-3 inhibits the proliferation of endometrial carcinoma cell line HEC-1AIn Ishikawa endometrial cancer cell line and HEC-1A endometrial cancer cell line,six concentrations of RFRP-3(0.1,1,10,100,1000,10000ng/ml)were selected for CCK8 test at 24 h and 48 h.The results showed that10000 ng/ml RFRP-3 could inhibit the growth of endometrial cancer cells HEC-1A,and the best effect was 24 h.There was no effect on Ishikawa endometrial cancer cell line at the concentrations of 0.1,1,10,100,1000,10000 ng/ml,24 h and 48 h.3.Apoptosis of HEC-1A induced by RFRP-3Detected and analyzed the apoptosis rate of endometrial cancer HEC-1A cells 24 hours after addition RFRP-3 by Annexin V-FITC/PI double staining method.The sum of late apoptosis and early apoptosis(UR+ LR)is the rate of apoptosis in this experiment.Compared with the Control group,the apoptosis rate of 10000 ng/ml RFRP-3 group was significantly higher(P<0.05).4.Expression of apoptosis related proteins in HEC-1A changed by RFRP-3Western blot results showed that the expression of Bcl-2 protein in10000 ng/ml RFRP-3 group was lower than Control,and the expression of Bax protein in 10000 ng/ml RFRP-3 group was higher than Control(P<0.05).5.RFRP-3 reduces the expression of Kras protein in HEC-1AWestern blot results showed that the expression of Kras protein in10000 ng/ml RFRP-3 group was lower than Control(P<0.05).6.RFRP-3 can down regulate the m RNA expression level of PI3K/AKT/mTOR pathway in HEC-1A and the gene transcription levelRT-qPCR results showed that: compared with the Control group,the relative expression levels of PI3 K m RNA(P<0.001),AKT m RNA(P<0.01)and mTOR m RNA(P<0.01)in 10000 ng/ml RFRP-3 group were significantly lower,respectively.7.RFRP-3 down regulates PI3K/AKT/mTOR pathway protein expression in HEC-1AWestern blot results showed that: the expression level of PI3 K protein in10000 ng/ml RFRP-3 group was significantly lower than Control(P<0.01);the expression level of p-AKT protein in RFRP-3 group was significantly lower than Control(P<0.05);the expression level of AKT protein in RFRP-3group had no change with Control;the expression level of mTOR protein in RFRP-3 group was significantly lower than Control(P<0.05).8.RFRP-3 down regulates the transcription of ERK pathway gene in HEC-1ART-qPCR results showed that the relative expression level of ERK m RNA in the 10000 ng/ml RFRP-3 group was significantly lower than Control group(P<0.001).9.RFRP-3 down regulates ERK pathway protein expression in HEC-1AWestern blot results showed that the expression level of p-ERK protein in 10000 ng/ml RFRP-3 group was significantly lower than Control group(P<0.05);compared with Control,the expression level of ERK1/2protein had no change.10.Autophagy of HEC-1A induced by RFRP-3RT-qPCR results showed that the relative expression level of LC3-Ⅱm RNA in 10000 ng/ml RFRP-3 group was significantly higher than Control(P<0.01).Western blot results showed that compared with Control,the ratio of LC3-Ⅱ/LC3-Ⅰ in the 10000 ng/ml RFRP-3 group was increased(P<0.05);the relative expression level of p62 protein was decreased(P<0.05).11.RFRP-3 increases the expression of GPR147 in HEC-1AWestern blot results showed that compared with the Control group,the protein expression of GPR147 in the 10000 ng/ml RFRP-3 group was increased(P<0.05).Conclusion:The inhibitive effect of RFRP-3 on HEC-1A cells may be through activating receptor gpr147 and binding to it,affecting the central node protein Kras,activating its downstream PI3K/AKT/mTOR pathway and ERK pathway,reducing the expression of Bcl-2,promoting the expression of Bax and increasing autophagy. |
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