KIF20A Regulates The Chemosensitivity Of Gastric Cancer Cells To 5-FU Through The P53 Signaling Pathway

Objective:To investigate the differential expression of kinesin 20A(Kinesin Family Member 20A,KIF20A)in gastric cancer and to analyze the clinicopathological parameters and prognosis of patients with KIF20A and gastric cancer.To investigate the effect of silencing KIF20A gene on the sensitivity of 5-FU chemotherapy in gastric cancer cells.Methods:1.First,mRNA data and clinical information data of gastric cancer patients were downloaded from The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)respectively.The differences in KIF20A expression in cancerous and normal tissues could be analyzed using the Wilcox rank sum test in the "limma" package of R software.Survival analysis was performed using the "Survival" R package and the Kaplan-Meier plotter online website(https://www.kmplot.com).Afterwards,chi-square test and Cox proportional regression risk model were used to verify the correlation between KIF20A and clinicopathological characteristics of gastric cancer patients.Gene Set Enrichment Analysis(GSEA)software and R software were used to perform enrichment analysis of the KEGG(Kyoto Encyclopedia)pathway for the TCGA dataset and GEO dataset,respectively.2.In the in vitro experiments,two gastric cancer cell lines with relatively high KIF20A expression were selected based on the results of protein immunoblotting assays(Western blot)performed on our four existing gastric cancer cell lines.Small interfering RNA(si RNA)was used to silence the expression of KIF20A in gastric cancer cells,and Western blot assay was performed to verify the transfection effect.The experimental cells were cultured in the presence of 5-fluorouracil(fluorouracil,5-FU)at serial concentrations(0ug/ml,5ug/ml,10ug/ml,25ug/ml,50ug/ml)by CCK-8 cytotoxicity assay,and the half-inhibitory concentration(IC50)of the experimental cells to 5-FU was calculated.The experimental cells were divided into 4 groups,which were(ⅰ)control group: si RNA-KIF20A(-)with 5-FU treatment(-);(ⅱ)transfection group: si RNA-KIF20A(+)with 5-FU treatment(-);(ⅲ)control group + 5-FU:si RNA-KIF20A(-)with 5-FU treatment(+);(ⅳ)transfection group + 5-FU: si RNA-KIF20A(+)with 5-FU treatment(+).The proliferation difference between the 4 groups of cells can be verified by CCK-8 cell proliferation assay;the migration difference between the 4 groups of cells can be verified by cell scratch assay to compare the scratch healing rate on the one hand,and the expression of matrix metalloprotein(MMP2 and MMP9)on the other hand can be compared by Western blot assay;then the DNA damage between the 4 groups of cells can be verified by cell immunofluorescence assay;the apoptosis between the4 groups of cells can be verified by flow cytometry;and finally the expression difference of P53-related pathway protein between the 4 groups of cells can be verified by Western blot assay.Results:1.Analysis of TCGA and GEO gastric cancer datasets showed that KIF20A was highly expressed in both cancerous tissues(P<0.001).Their corresponding ROC curves indicated that KIF20A has strong sensitivity and specificity for predicting gastric carcinogenesis.2.TCGA and GEO survival analyses suggested that gastric cancer patients with high KIF20A expression had a poorer prognosis,and in the 5-FU chemotherapy dataset for gastric cancer,the prognosis of patients with high KIF20A expression was similarly lower than that of those with low expression,suggesting that high KIF20A expression may reduce the sensitivity of gastric cancer patients to 5-FU chemotherapy.Clinical correlation analysis showed that high KIF20A expression was associated with the N stage of gastric cancer patients,and Cox proportional regression risk model analysis indicated that KIF20A could be considered an independent prognostic factor for gastric cancer patients.3.Pathway analysis showed that KIF20A was enriched in KEGG analysis in GSE15459 dataset suggesting its enrichment in regulation of cell cycle,P53 signaling pathway and calcium signaling pathway,and GSEA enrichment analysis in TCGA dataset suggesting its enrichment in cell cycle,DNA damage repair and P53 signaling pathway.Therefore,it is considered that KIF20A regulates the malignant behavior of gastric cancer cells may be related to the P53 signaling pathway.4.In the in vitro experiment,the results of Western blot assay showed that the expression of KIF20A was relatively high in two gastric cancer cell lines,MGC803 and HGC-27.5.CCK-8 cell proliferation and toxicity assay showed that the IC50 value of MGC803 gastric cancer cells to 5-FU was about 11.246 ug/ml,and the IC50 value of HGC-27 gastric cancer cells to 5-FU was about 16.444 ug/ml.Silencing KIF20A could inhibit the proliferation ability of these two gastric cancer cells,and silencing KIF20A combined with 5-Fu treatment further decreased their proliferation ability than KIF20A normal combined with 5-Fu treatment further decreased their proliferation ability,and the difference was statistically significant,indicating that silencing KIF20A enhanced the chemotherapeutic sensitivity of these two gastric cancer cells to 5-Fu.6.The results of the scratch assay showed that silencing KIF20A inhibited the migratory ability of these two types of gastric cancer cells,and the migratory ability was further reduced by silencing KIF20A combined with 5-Fu treatment than by KIF20A normal combined with 5-FU treatment.analysis of the results of the Western blot assay showed that the expression of MMP2 and MMP9 was reduced in the KIF20A silenced group compared with the control group,and the expression of protein was more significantly reduced by combined with 5-Fu treatment.The decrease in protein expression was even more obvious after 5-FU treatment.It showed that silencing KIF20A enhanced the sensitivity of these two types of gastric cancer cells to 5-FU and reduced chemoresistance.7.Cellular immunofluorescence showed that DNA damage was more obvious in the 5-FU-treated group,with the most obvious DNA damage after silencing KIF20A and combined 5-FU treatment,indicating that silencing KIF20A enhanced the sensitivity of these two gastric cancer cells to 5-FU and increased DNA damage.8.Flow cytometry showed that silencing KIF20A promoted apoptosis in these two gastric cancer cells and increased apoptosis after combined 5-Fu treatment.western blot assay showed that the highest expression levels of phosphorylated P53 and Caspase-3 were observed after silencing KIF20A and combined 5-Fu treatment,while Bcl-2 expression was significantly decreased.It indicated that silencing KIF20A could enhance the anti-tumor properties of 5-FU and promote apoptosis of these two gastric cancer cells.Conclusion:The expression of KIF20A is significantly increased in gastric cancer patients.KIF20A expression can be an independent risk factor and has a significant impact on their prognosis,and those with high KIF20A expression have a poorer prognosis.Silencing KIF20A could enhance the chemosensitivity of gastric cancer cells to 5-FU by regulating the P53 signaling pathway.The above results suggest that KIF20A is important for the process of gastric cancer development.It provides a new direction for the future treatment and research of gastric cancer.