To Investigate The Intervention Effect And Mechanism Of Curcumin On Glycolysis In Lung Cancer A549 Cells Based On NF-κB-Snail/HK3 Pathwa

Purpose:1.Screening differentially expressed genes related to glycolysis in lung cancer and searching for potential targets of curcumin in the treatment of lung cancer.2.To investigate whether curcumin improves the promotion of lipopolysaccharide on glycolysis in lung cancer cells through the NF-κB-Snail/HK3 pathway,and to preliminarily elucidate the mechanism of action of curcumin in regulating HK3.Material and method:Paper 1: Preliminary study on the mechanism of curcumin intervention in lung cancer based on glycolysis-related genes1.To obtain data sets of differentially expressed genes in lung cancer and genes related to diseases with abnormal glycolysis using the GEO and GSEA databases,respectively,and to jointly analyze and screen target genes related to glycolysis and lung cancer.2.Detection of the expression trend of target genes in lung cancer A549 cells versus lung normal epithelial cells BEAS-2B by RT-qPCR.3.The inhibition of cell growth of lung cancer A549 cells treated with curcumin at different concentrations(5μmol/L,10μmol/L,20μmol/L,40μmol/L,80μmol/L)for 12 h,24h and 48 h was determined by CCK-8 method to screen the optimum curcumin concentration and action time.4.The effect of curcumin intervention on the expression of target genes in lung cancer A549 cells was detected by RT-qPCR.Paper 2: Curcumin ameliorates lipopolysaccharide-stimulated glycolysis in lung cancer cells via NF-κB-Snail/HK3 pathway1.LPS inhibition of lung cancer A549 cells growth at different concentrations(0.1μg/mL,1μg/mL,10μg/mL)was determined using CCK-8 method to screen the optimal LPS action concentration.2.The experiment was divided into five groups: control group(Control group);lipopolysaccharide group(LPS group);inhibitor group(JSH-23 group);lipopolysaccharide +inhibitor group(LPS+JSH-23 group);lipopolysaccharide + curcumin group(LPS+CUR group).The expression levels of NF-κB,Snail,HK3 mRNA,and protein in each group of cells were detected by RT-qPCR and Western Blot techniques,respectively.The contents of glucose and lactate acid in the supernatant were measured by colorimetric method.Results:1.The lung cancer-related dataset GSE118370 was obtained from the GEO database,screening 1069 differentially expressed genes in lung cancer,including 680 up-regulated genes and 389 down-regulated genes.The glycolysis-related dataset REACTOME＿GLYCOLYSIS was obtained from the GSEA database,containing 72 genes related to abnormal glycolytic diseases.The final two glycolysis-related genes with up-regulated expression in lung cancer were obtained by joint analysis,namely ALDOB and HK3.2.The results of RT-qPCR showed that the expression of ALDOB and HK3 mRNA in lung cancer A549 cells was significantly higher than that in human normal lung epithelial BEAS-2B cells(P<0.01).3.CCK-8 results showed that the inhibitory effect of cell proliferation became more and more obvious with the increase of curcumin administration concentration compared with the control group.The inhibition rate of the 24 h treatment group was significantly higher than that of the 12 h treatment group and 48 h treatment group(P<0.01).The 24 h treatment group,with an IC50 of 26.44 μmol/L,was calculated to be closer to the 20 μmol/L group.Therefore,subsequent experiments used a curcumin concentration of 20 μmol/L to treat lung cancer A549 cells for 24 h.4.RT-qPCR results showed that the expression levels of ALDOB and HK3 mRNA in the CUR group were significantly down-regulated in lung cancer A549 cells compared with the control group(P<0.01).5.The CCK-8 results showed that the cell proliferation effect became more and more obvious with the increase of LPS stimulation concentration compared with the control group(P<0.01).However,the difference between the 1μg/mL group and the 10μg/mL group was not statistically significant,and 1μg/mL was chosen as the optimal effect concentration of LPS to avoid toxic effects from high drug concentrations.6.Compared with the Control group,NF-κB,Snail,HK3 mRNA and protein expression levels were significantly upregulated in the cells of the LPS group(P<0.01),and both glucose levels in the cells and lactate levels in the supernatant were significantly increased(P<0.01).Compared with the LPS group,NF-κB,Snail,HK3 mRNA and protein expression levels were significantly downregulated in the LPS+JSH-23 group(P<0.05),and both glucose levels in cells and lactate levels in the supernatant were significantly decreased(P<0.05).It is suggested that LPS may accelerate glycolysis in lung cancer cells through the NF-κB-Snail/HK3 pathway.7.Compared with the LPS group,the mRNA and protein expression levels of NF-κB,Snail,and HK3 were significantly downregulated in the LPS+CUR group(P<0.05),and intracellular glucose levels and supernatant lactate levels were significantly reduced(P<0.05),suggesting that curcumin reverses the effects of LPS stimulation on NF-κB-Snail/HK3 pathway and glycolysis promotion.Conclusions:1.Curcumin can affect the glycolysis of lung cancer cells by inhibiting the expression of ALDOB and HK3.2.Lipopolysaccharide promotes glycolysis in lung cancer cells through the NF-κB-Snail/HK3 pathway.3.Curcumin improves lipopolysaccharide stimulation-induced glycolysis in lung cancer cells via the NF-κB-Snail/HK3 pathway.