Based On Proteomics To Explore The Inhibitory Effect Of LAF On The Cell Cycle Of Human Rectal Cancer SW480 Cell

ObjectiveTo explore the effect of Lappaol F on the cell cycle of human colorectal cancer sw480 cell,proteomics and experimental validation were used to study the related mechanism of cell cycle arrest.MethodsThe effect of LAF on the proliferation activity of human colorectal cancer cells SW480,HCT116 and HCT15 was detected by sulfonyl rhodamine B assay.The effect of LAF on the cell cycle of SW480 was detected by PI single stain and flow cytometry.The dynamic changes of the whole proteome of SW480 cells after LAF treatment were quantified by the TMT markers and liquid chromatographytandem mass spectrometry(LC-MS/MS)technology.Compared with the control group,the differentially expressed proteins were screened according to the standard(up-regulated or down-regulated>1.5 times,P<0.05).And then,we use the G0 and KEGG database to classify the function and enrich the possible targets and pathway of these differentially expressed protein.The relationship between these key proteins was analyzed by PPI network which constructed by STRING database and Cytoscape software.The prediction of cell cycle-related gene/protein expression by proteomics was verified by both RT-qPCR and Western Blotting.RNAinterfering adenovirus was designed and constructed according to the sequence of cyclin-dependent kinase inhibitor 1C(CDKN1C).After that,SW480 cells were infected with adenovirus and CDKN1C stable low expression cell line(SW480-CDKN1C RNAi)was established.SW480-CDKN1C RNAi cell line was used to explore the effect of CDKN1C on the expression of cyclindependent kinases(CDK1,CDK2 and CDK6),cyclins(cyclin A2 and cyclin B1),cell cycle and cell proliferation.By comparing the effects of LAF on SW480 and SW480-CDKN1C RNAi cell lines,the differences in the effects of LAF on CDKN1C,cyclin-dependent kinase,cyclin expression and cell proliferation were investigated to verify whether LAF regulates cell cycle with CDKN1C as the target to inhibit the proliferation of tumor cells.ResultsLAF significantly inhibited the proliferation of SW480,HCT116 and HCT15 cells(48 h,the half inhibitory concentration was 47.07±6.67μmol/L,32.81 ± 7.31 μ mo1/L,51.34 ± 4.40 μmol/L14,respectively).Proteomic results showed that compared with the control group,50μmol/L LAF significantly altered the expression of 127 proteins in SW480 cells.KEGG pathway enrichment analysis showed that LAF regulates tumor cell proliferation mainly through the cell cycle pathway.It is worth noting that cyclin A2,cyclin B1,TTK,BUB1,CHEK1,BUB1B,CDC20,CDC25C,and PLK1 showed significant changes in the expression levels of the cell cycle pathway.The results of cell cycle detection showed that 50 μmol/L LAF could induce S-phase arrest of SW480 cells.Quantitative PCR and Western Blotting results showed that LAF could significantly up—regulate CDKN1C,downregulate CDK1,CDK2,CDK6,cyclin A2 and cyclin B1 expression levels.CDK1,CDK2,CDK6,cyclin A2 and cyclin B1 protein expressde levels were increased after RNA interference inhibition of CDKN1C expression,and survival rates of different CDKN1C RNAi interfered cells were increased by 14.56±2.88%and 18.41 ± 4.08%,respectively.These results indicated that CDKN1C could reduce the proliferation of tumor cells by inhibiting the core molecules(CDK and cyclin)regulated by cell cycle.In addition,the inhibition of CDKN1C expression also resulted in the LAF-induced inhibition of the expression of CDK1,CDK2,CDK6,cyclin A2,and cyclin B1,as well as the reduced inhibition of cell proliferation.ConclusionLAF may be a promoter of CDKN1C.By upregulating the expression of CDKN1C LAF can enhance the negative regulation of CDK and cyclin,induce cell cycle arrest and inhibit the proliferation of colorectal cancer SW480 cells.