Study On The Mechanism Of Tuckahoe Polysaccharide Affecting Breast Cancer Cell Proliferation Through LEP/SREBP1/FASN Pathwa

Purpose:1.Gene targets related to glucose and lipid metabolism in the pathogenesis of breast cancer were screened by GEO database.CCK8 method was used to obtain the optimal administration concentration and time of Poria cocos polysaccharide on breast cancer cell MCF-7,and verify the changes of glycolipid metabolism genes after Poria cocos polysaccharide administration.2.To observe the effect of Poria cocos polysaccharide on the expression of genes and proteins in the lipid metabolism pathway of LEP/SREBP1/FASN in MCF-7 cells,to observe the effect of Poria cocos polysaccharide on the expression of proteins and phosphorylated proteins in PI3K/AKT signal pathway,and to explore the mechanism of Poria cocos polysaccharide inhibiting the proliferation of breast cancer cells.Material and method:1.Retrieve the data set of patients with breast cancer in GEO database.The data set contains the cancerous tissue of breast cancer and matched paracancerous tissue or normal tissue.The differentially expressed genes(DEG)between the cancer group and the normal group was analyzed by using the GEO2 R analysis system,and the differential gene was intersected with VENNY2.1.0.Apply the selected target gene intersection to DAVID 6.8 database for gene Ontology(GO)function analysis,select genes related to glucose metabolism and lipid metabolism in Biological Process(BP),and use STRING online data analysis website to build a Protein-Protein Interaction Network.2.Using the CCK8 method to detect different dosing concentrations(10,50,100,200,400μg/ml),cell proliferation at different administration times(24,48,72 hours).To screen the best concentration and time of Poria cocos polysaccharide on breast cancer cell MCF-7.3.MCF-7 cells were divided into a control group and a treatment group and the treatment group was added with the optimal drug concentration obtained through CCK8 method screening,and the optimal action time was processed.Detection of m RNA expression levels of genes related to glucose metabolism and lipid metabolism in the control group and treatment group using RT q PCR method.4.RT-q PCR method was used to detect the expression level of lipid metabolism pathway genes LEP,SREBP1,FASN in the control group and the treatment group.5.Western Blot was used to detect the protein expression levels of lipid metabolism pathway proteins LEP,SREBP1,FASN and PI3 K,AKT,p-PI3 K,p-AKT,and to verify the specific molecular mechanism of Poria cocos polysaccharide on inhibiting the proliferation of breast cancer cells.Results:1.The data sets GSE139038 and GSE15852 are obtained by filtering the retrieval content.A total of 526 differential genes were obtained from GSE139038 and 57 differential genes were obtained from GSE15852.The two were intersected to obtain 27 differential genes,of which25 genes were up-regulated and 2 genes were down-regulated.The functional analysis of GO showed that the biological functions mainly involved in gluconeogenesis,glucose metabolism,lipid metabolism,lipid storage and fatty acid metabolism.A total of 7 genes related to glucose metabolism and lipid metabolism(PCK1,RBP4,CD36,LEP,LPL,CAV1,ACACB)were selected.Seven genes were imported into STRING online data,and PPI network interaction diagram was drawn.2.CCK8 results showed that compared with other time,the 48-hour treatment group had the most obvious effect on inhibiting cell proliferation.The inhibitory effect on cell proliferation was better with the increase of administration concentration,the inhibition effect of cell proliferation is better,400 μg /ml has the best inhibition effect.3.The results of RT-q PCR showed that the m RNA expression level of seven glycolipid metabolism genes after intervention with Poria cocos polysaccharide was significantly lower than that of the control group,with a statistically significant difference(P<0.01).4.The results of RT-q PCR showed that after the intervention of Poria cocos polysaccharide administration,the relative m RNA expression of lipid metabolism related genes LEP,SREBP1 and FASN in the treatment group was significantly lower than that in the control group,with a statistically significant difference(P<0.05).5.Western blot results showed that the protein expression levels of lipid metabolism-related proteins LEP,SREBP1,FASN and cancer pathway PI3 K,AKT,p-PI3 K,p-AKT were significantly decreased in the treatment group after the intervention of Poria cocos polysaccharide administration(P<0.05).Poria cocos polysaccharide can regulate the LEP/SREBP1/FASN pathway and mediate the PI3K/AKT pathway to affect the proliferation of MCF-7 cells in the level of protein expression.Conclusion:1.Through GEO database mining,seven genes(PCK1,RBP4,CD36,LEP,LPL,CAV1,ACACB)related to glucose metabolism and lipid metabolism in breast cancer were obtained.The CCK8 method was used to screen that the optimal drug concentration of Poria cocos polysaccharide for treating MCF-7 cells was 400 μ g/ml,the best treatment time is 48 hours.After the treatment of Poria cocos polysaccharide,7 glycolipid metabolism genes were significantly changed.2.Poria cocos polysaccharide can inhibit the proliferation of breast cancer cells,and the specific mechanism may be related to regulating the lipid metabolism LEP/SREBP1/FASN pathway and mediating the protein expression of PI3K/AKT pathway.