Experession Significance Of PB3A Induced Differential Expressed MicroRNA And It's Target Genes In SW480 Colon Cancer Cell Line

Propolis flavonoid pinobanksin-3-acetate(Pinobanksin-3-acetate,PB3A)is a kind of light yellow flavonoid monomer which can be isolated and purified from propolis,honey and some plant buds.Studies on PB3 A to date have shown PB3 A exerts anti-cancer effect by inhibiting cell proliferation and inducing apoptosis,It can inhibit proliferation and induce apoptosis in HCT116 cells,SW480 cells,gastric cancer SGC-7901 cells and human hepatocellular carcinoma HepG-2 cells.However,the role and molecular mechanism of miRNA in the inhibition of proliferation and induction of apoptosis of propolis flavonoid PB3 A in tumor cells still remains unclear so far.In this study,we investigated the effect of propolis flavonoid PB3 A on the miRNA expression profile of colon cancer,and sought for differential expression miRNAs closely related to the carcinogenesis and progression of colon cancer,so as to provide new methods and ideas for the target screening of anticancer drugs.In this study cell proliferation assaywas used to detect the effects of different concentrations of PB3A(25,50,75 and 100 ug / mL)on cell proliferation at different times,results showed that with the increase of treatment time and PB3 A concentration,the proliferation inhibition effect was also significantly increase.Then,According to the results of miRNA microarry analysis 12 different expressed miRNA were slected,then the SW480 cells were treated with PB3 A at a concentration of 100?g/mL and the RNA was extracted.,the authenticity and reliability of the miRNA expression microarray were verified by qRT-PCR.The results of qRT-PCR showed that the expression of miR-22-5p,miR-211-5p,mi R-198,mi R-769-3P,miRNA-4321,miRNA-204-5P,miRNA-4326,miRNA-219a-2-3P,miR-4697-3p,miR-142-3p in the 12 slected miRNAs was consistent with the microRNA microarray result,and the difference was statistically significant(P<0.05).The expression of mi R-642a-5P was opposite to that of the microarray results.There was no significant difference in the expression of miR-761 between the two groups(P> 0.05),so unable to verify themiRNA microarry results.Next,by using Lipofectamine?3000 transifection reagent miR-4326 mimics and miRNA mimic Negative Control were transfected to the SW480 cell lines for 48 hours,and then the transfection efficiency of miR-4326 mimics and miRNA Negative Control were detected by Fluorescence microscopy and qRT-PCR.miR-4326 mimics transfection experiment result showed that miR-4326 successfully transfected into colon cancer SW480 cells,compared with the control group,the expression of miR-4326 significantly increased in the miR-4326 mimics transfection group,the difference was statistically significant(P<0.05).Furthermore according to miRNA microarry analysis and qRT-PCR result we selected14 upregulated miRNAs,performed Bioinformatics prediction of those miRNA`s target genes.And gene ontology network analysis,Gene Class,PCass,KEGG pathway analysis were used to better understand the roles of experession altered miRNAs.Target gene prediction results revealed that miR-18a-5p has 762 target genes,miR-802 has 896 target genes,miR-296-5p has 906 target genes,miR-3064-5p has209 target genes,miR-22-5p has 575 miR-2115 p has 1730 target genes,miR-769-3P has 728 target genes,miRNA-4321 has 7 target genes,miRNA-204-5P has 1693 target genes,miRNA-4326 has 299 Target genes,miRNA-219a-2-3P has 626 target genes,miR-4697-3p has 115 target genes,miR-142-3p has 534 target genes,miR-198 has 859 target genes.GO enrichment analysis result showed,these 14 miRNA are involved in a variety of cell component formation,molecular function,and biological processes by regulating their target genes.Among them the largest number of target genes are annotated in biological processes,so these miRNA may play a role in cancer suppression or carcinogenesis by regulating their target genes which involved in a variety of biological processes.The gene function of these miRNAs is mainly enriched in transcription factors,copy number variants,histons,protein kinases,onconcogenes.These miRNAs Protein function is related to Ras pathway,PDGF signaling pathway,integrin signaling pathway,EGF receptor signaling pathway,angiogenesis,FGF signaling pathway,Wnt signaling pathway,p53 pathway,apoptotic signaling pathway and so on.KEGG pathway analysis revealed that the 14 miRNAs target genes involved in tumor pathway,Wnt signaling,endocytosis,axon-directed,chronic myeloid leukemia,MAPK signaling pathway,prostate cancer,insulin signaling pathway,glioma,melanogenesis,Actin cytoskeleton control,colorectal cancer and other signaling pathways.Overall the results of this study reveals that propolis flavonoid PB3 A can affect the expression of miRNA in colon cancer SW480 cells.The results of qRT-PCR suggest that miR-22-5p?miR-211-5p?miR-198?miR-769-3P?miRNA-4321?miRNA-204-5P?miRNA-4326?miRNA-219a-2-3P?miR-4697-3p?mi R-142-3p,which are differentially expressed in SW380 cells after treated with PB3 A,may play an important role in the anti-cancer effect of PB3 A.PB3A induced differentially expressed miRNAs are involved in tumor pathway,Wnt signaling pathway,MAPK signaling pathway,axon-guided pathway,endocytosis and other important signaling pathways through regulating the target genes involved in the formation of multiple cell components,molecular functions and biological processes.Thus inhibiting colorectal cancer cell proliferation,invasion,migration and induce apoptosis.