| Purpose:Based on the thought of integrated traditional Chinese and western medicine,“Phlegm” and“Dyslipidemia” associated.To explore the differentially expressed genes of abnormal lipid metabolism in hepatocellular carcinoma(HCC)by data mining,and to analyze the molecular mechanism of pachymaran-mediated SQLE affecting the Pyroptosis of NLRP3/GSDMD.The aim of this study is to analyze the mechanism of“Turbid phlegm” in HCC and the intervention mechanism of Pachyman from the perspective of modern biology,so as to provide the research strategy and experimental basis for studying the pathogenesis of HCC.Material and methods:1.The first part First of all,to investigate the effects of“Phlegm”and“Abnormal lipid metabolism”on liver cancer,and then combined with bioinformatics technology,downloaded the gene chip GSE84402 data set matrix from GEO public database,using GEO2R analysis to analyze the data and screen the differentially expressed genes between HCC and its adjacent tissues.David Online Database and GSEA database were used for analysis,and R language was used to analyze GO and KEGG enrichment analysis.Venny diagram was used to screen the co-expression genes of liver cancer lipid metabolism genes in David and GSEA database,and Cytoscape software was used to draw the protein interaction network.Using the GEPIA database,we further validated the core differentially expressed genes in the above-mentioned cholesterol metabolic pathways and analyzed their prognosis and survival.SQLE mRNA was detected in the samples from patients with liver cancer.CCK-8 was used to screen the concentration of pachymaran,and the inhibitory effect of pachymaran on cell growth was observed under microscope.The ability of cell migration was measured by scratch test;RT-qPCR was used to detect the change of SQLE mRNA expression level after pachymaran intervention in HepG2 cells.2.The second part Compared with the control group,the mRNA expression of NLRP3,GSDMD and Caspase-1,the protein expression of NLRP3,GSDMD-N,Caspase-1,IL-1β and IL-18 were significantly increased in pachyman group(P<0.05),and the expression of Caspase-1 was significantly increased in pachyman group(P<0.05);Compared with MCC950 group,MCC950+Pachymaran group had a significant inhibitory effect on HepG2 cells,and the mRNA expressions of NLRP3,GSDMD,Caspase-1 were significantly increased(P<0.05),and the protein expressions of NLRP3,GSDMD-N,Caspase-1,IL-1β,IL-18 were significantly increased(P<0.05);The expression of Caspase-1 was increased by immunofluorescence.3.The third part HepG2 cells were divided into 5 groups: control group,OE-NC Group,OE-SQLE Group,OE-NC + Pachyman Group,OE-SQLE + Pachyman Group.The expression levels of SQLE,NLRP3 and Caspase-1 mRNA were detected by RT-qPCR.The expression levels of NLRP3,Caspase-1,GSDMD and GSDMD-N were detected by Western Blot.LDH expression was measured by colorimetry,IL-1β and IL-18 levels were measured by ELISA,and cell necrosis was measured by Flow cytometry staining.Results:1.The first part results Through GEO,GSEA and David databases,22 differentially expressed genes of lipid metabolism in hepatocellular carcinoma were screened,and 6 genes related to cholesterol metabolism pathway were selected,the differentially expressed genes SQLE were identified by using GEPIA database.GEPIA and KM-plotter showed that SQLE mRNA expression were closely related to the overall survival of HCC patients.The expression of SQLE mRNA in HCC tissues was detected.Compared with para-cancer group,SQLE was significantly increased and APOA5 was significantly decreased in HCC tissues(P<0.01)..In vitro: CCK-8test showed that Pachymaran at 800μg/mL inhibited HepG2 cells significantly after 48 hours.The results of RT-qPCR showed that compared with the control group,the expression of SQLE mRNA was significantly decreased(P<0.05).2.The second part results Compared with those of the control group,the viability of pachymaran group was significantly reduced,and 800 μg/mL Pachymaran had the best inhibitory effect on cells after 48h intervention,and the levels of NLRP3,GSDMD,Caspase-1,IL-1β mRNA were increased(P<0.05),and expressions of NLRP3,GSDMD,Caspase-1,IL-1β,IL-18 protein were significantly increased(P<0.05),and the expression of Caspase-1 was promoted.Compared with the MCC950 group,the MCC950+pachymaran group had significant inhibitory effect on HepG2 cells,and the levels of NLRP3,GSDMD,Caspase-1,IL-1β mRNA were increased(P<0.05),and expressions of NLRP3,GSDMD,Caspase-1,IL-1β,IL-18 protein were significantly increased(P<0.05),and the expression of Caspase-1 was promoted.3.The third part results After overexpression of SQLE,there was no difference in mRNA and protein between OE-NC Group and control group;Compared with OE-NC group,the expression levels of SQLE mRNA and protein,NLRP3,GSDMD,Caspase-1 mRNA and protein,and the expression levels of GSDMD-N protein in OE-SQLE group were significantly higher,and the expression levels of NLRP3,GSDMD,Caspase-1 mRNA and protein were significantly lower than those in OE-NC group,The level of cell necrosis,the release of LDH,IL-1β and IL-18 decreased significantly(P<0.05);The expression levels of SQLE mRNA and protein,NLRP3,GSDMD,Caspase-1 mRNA and protein,and GSDMD-N protein were significantly increased in OE-NC+pachymaran group(P<0.01),The level of cell necrosis,the release of LDH,IL-1βand IL-18 increased significantly(P<0.05);Compared with OE-SQLE group,the expression levels of SQLE mRNA and protein,NLRP3,GSDMD,Caspase-1 mRNA and protein,and GSDMD-N protein in OE-SQLE+pachymaran group were significantly decreased(P<0.01),the level of cell necrosis and the release of LDH,IL-1β and IL-18 increased significantly(P<0.05);Compared with the OE-NC+Pachyman group,the expression levels of SQLE mRNA and protein,NLRP3,GSDMD,Caspase-1 mRNA and protein,and GSDMD-N protein in the OE-SQLE+Pachyman group were significantly increased(P<0.01),the level of cell necrosis and the release of LDH,IL-1β and IL-18 were significantly decreased(P<0.05).Conclusions:1.SQLE was abnormally highly expressed in HCC2.Pachyman polysaccharide can inhibit the occurrence and development of liver cancer by activating NLRP3/GSDMD Pyroptosis pathway.3.Pachyman polysaccharide can affect the growth of hepatoma cells by mediating SQLE to activate NLRP3/GSDMD Pyroptosis pathway. |
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