Research On The Gene Expression Profiles And Autophagy Of Peiminine In HCT-116 Cells

Background and Objective:Colon cancer is a common malignant tumor of the digestive system,the incidence of colon cancer increases year by year in our country.At present the treatment of colon cancer are generally the surgery,chemotherapy,targeted therapy.However,the survival and prognosis of patients are not very ideal.Traditional Chinese Medicine has advantage in the prevention of recurrence of colon cancer and improve survival quality of patients,but the clinical effects are different,mechanism of action remains to be further defined.Before our study found that peiminine inhibited the proliferation of colorectal carcinoma HCT-116 cells obviously in vitro experiment,this study is to explore the influence of peiminine on the gene expression profile in HCT-116 cells and observe the autophagy induced by the peiminine,The aim is to clear molecular targets of peiminine on HCT-116 cells for the development of the drug and provide theoretical basis for clinical application.Methods:1.The whole genome oligonucleotide microarray(Affymetrix PrimeView ? Human Gene Expression Array)is applied to explore the influence of peiminine on the gene expression profile in HCT-116 cells.The experiment is divided into the peiminine group and the control group,and total RNA is extracted after dealed with human colon cancer HCT-116 cells respectively,then the gene chip hybridization steps are followed by CapitalBio,and data is processed and analysed by ourselves.2.GFP-LC3 plasmids are successful transfected to human colon cancer HCT-116 cells,the postive group deal with different concentrations(0 uM,50 uM,100 uM,200 uM)of peiminine,the control group treat with the same volume of DMSO,autophagy is confirmed by confocal microscope,HCT-116 cells are dealed with different dosage and length of peiminine,Western blotting is applied to detect autophagy related proteins expression.RESULT:1.A total of 37 differentially expressed genes are observed,among which,5 are up-regulated,and the other 32 are down-regulated.With the comparison between the experimental group and the control group,differences in genes mainly are concentrated on the biological processes and molecular functions,and have no effect on cell components after GO enrichment analysis;From biological processes differences in gene mainly are focused on the regulation of heat generation,regulation of locomotion involved in locomotory behavior,uridine metabolic process,regulation of adrenergic receptor signaling pathway,fat pad development,and regulation of G-protein coupled receptor protein signaling pathway,From molecular function differences in genes mainly are concentrated on adrenergic receptor binding activity and uridine phosphorylase activity.Through KEGG analysis differences in genes mainly are concentrated on drug metabolism enzymes,pyrimidine metabolism,synthesis and degradation of ketone bodies,vitamin B6 metabolism,ribosomes.2.With the comparison between the peiminine group and the control group,multiple green LC3 dots gathered brightly could be seen by confocal microscope in the former,the dots are scattered in the latter.And with the higher concentration of peiminine,the more cells with autophagy increase.Western blotting shows the increase expression of LC3B-II/ LC3B-I and SQSTM1 after 200 uM,400 uM peiminine at different dosage and 200 uM peiminine deal with colorectal carcinoma HCT-116 cells at different length(0 hour,4 hours,8 hours,12 hours,24 hours).Conclusions:From the results of gene microarray suggest the different genes are 37,the change of these genes maybe anti-tumor targets of peiminine,and mainly reflected in the impact on fluorouracil metabolic pathways,cell proliferation,energy metabolism and regulation of YY1 transcription factor.YY1 raised,combinationation with accumulation of LC3 dots by confocal microscope and the expression of autophagy related protein LC3B-II and SQSTM1,suggest peiminine can be concerned with autophagy of HCT-116 cells.It is advantageous for the research of pharmacological activities of peiminine,and provide theoretical basis for the clinical application of antitumor effect of bulbus fritillaria thunbergii.