茯苓多糖通过SQLE/NLRP3/GSDMD信号通路调控肝癌细胞焦亡

Pachymaran regulates pyroptosis of liver cancer cells via SQLE/NLRP3/GSDMD signaling pathway

目的:应用生物信息学分析技术初步探索肝细胞癌(HCC)脂代谢异常差异表达基因,并结合实验验证,探讨茯苓多糖通过角鲨烯环氧酶(SQLE)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/消皮素D(GSDMD)信号通路影响细胞焦亡的分子机制。方法:(1)利用GEO、GSEA、DAVID、STRING和GEPIA数据库筛选HCC脂代谢异常差异表达基因。(2)收集HCC患者肿瘤组织(n=9)验证SQLE基因的差异表达。(3)培养人HCC细胞系HepG2,采用CCK-8法筛选茯苓多糖浓度。(4)将HepG2细胞分为对照组和茯苓多糖(800 mg/L)组,划痕实验观察细胞迁移能力,RT-qPCR检测细胞中SQLE的mRNA表达水平。(5)进一步采用构建过表达SQLE(OE-SQLE)的HepG2细胞,再用茯苓多糖处理,分为5组:对照组、过表达阴性对照(OE-NC)组、OE-SQLE组、OE-NC+茯苓多糖组和OESQLE+茯苓多糖组。RT-qPCR和Western blot检测SQLE和焦亡相关因子的mRNA及蛋白表达水平;比色法检测乳酸脱氢酶(LDH)活性;ELISA法检测白细胞介素1β(IL-1β)和IL-18释放水平;流式细胞术检测细胞坏死水平。结果:生物信息学分析筛选出差异表达基因SQLE。HCC患者肿瘤标本验证SQLE的mRNA表达水平显著升高(P<0.01)。800 mg/L茯苓多糖干预48 h对HepG2细胞活力具有显著抑制作用,且可显著降低SQLE的mRNA表达水平(P<0.01)。SQLE过表达后,焦亡相关因子的mRNA和蛋白表达水平、细胞坏死水平,以及LDH、IL-1β和IL-18释放水平均显著降低(P<0.05);茯苓多糖干预后,上述指标均显著升高(P<0.05)。结论:SQLE在HCC中异常高表达;茯苓多糖可通过SQLE激活NLRP3/GSDMD焦亡通路,从而影响HCC细胞生长。

AIM:Using bioinformatics analysis and experiment validation to explore the differential expression genes related to abnormal lipid metabolism in hepatocellular carcinoma(HCC)and the molecular mechanism of pachymaran affecting pyroptosis through squalene epoxidase(SQLE)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)/gasdermin D(GSDMD)signaling pathway.METHODS:(1)The GEO,GSEA,DAVID,STRING and GEPIA databases were employed to screen abnormal lipid metabolism-related differentially expressed genes in HCC.(2)The tumor tissues from HCC patients(n=9)were collected to verify the differential expression of SQLE.(3)The inhibitory effect of pachymaran on the viability of human HCC cell line HepG2 was measured by CCK-8 assay.(4)The HepG2 cells were divided into control group and pachymaran(800 mg/L)group.The cell migration was analyzed by wound-healing assay,and RT-qPCR was used to measure SQLE mRNA expression.(5)The HepG2 cells with overexpression of SQLE(OE-SQLE)were divided into 5 groups as follows:control group,overexpression negative control(OE-NC)group,OE-SQLE group,OE-NC+pachymaran group,and OE-SQLE+pachymaran group.The mRNA and protein expression levels of SQLE and pyroptosis-related factors were determined by RT-qPCR and Western blot.Colorimetric method and ELISA were used to measure lactate dehydrogenase(LDH),interleukin-1β(IL-1β)and IL-18 levels.The necrosis of HepG2 cells was analyzed by flow cytometry.RESULTS:The SQLE gene was screened through bioinformatics analysis,and its mRNA expression was significantly increased in tumor tissues from HCC patients(P<0.01).In cell experiments,treatment with 800 mg/L pachymaran for 48 h had a significant inhibitory effect on HepG2 cell viability,and the expression of SQLE mRNA was reduced(P<0.01).After overexpression of SQLE,the mRNA and protein levels of pyroptosis-related factors,necrotic rate,and LDH,IL-1βand IL-18 levels were significantly decreased(P<0.05).After treatment with pachymaran,the above indicators were significantly increased(P<0.05).CONCLUSION:The SQLE is abnormally highly expressed in HCC,and pachymaran can affect the growth of HCC cells by activating the NLRP3/GSDMD pyroptosis pathway through SQLE.