【摘要】 目的 研究赖氨酸脱甲基酶8(KDM8)、丙酮酸激酶M2(PKM2)在食管鳞状细胞癌(ESCC)中的表达以及对ESCC细胞增殖及迁移的作用。方法 生物信息学分析KDM8在食管癌中的表达及其与PKM2的相互作用关系；收集2008-01-01-2018-12-31新疆医科大学第一附属医院收治的120例ESCC组织及癌旁组织(距离肿瘤组织>3 cm)标本，免疫组织化学法检测KDM8与PKM2表达水平，并分析二者与患者临床参数的关系。采用慢病毒感染技术、细胞计数试剂盒8(CCK-8)、平板克隆形成实验、划痕愈合实验及流式细胞术检测敲低KDM8对ESCC细胞增殖、迁移及凋亡的影响，实时荧光定量聚合酶链式反应(RT-qPCR)及蛋白质印迹实验检测敲低KDM8后PKM2的表达水平，同时检测基质金属蛋白酶-9(MMP-9)蛋白表达变化。结果 生物信息学结果显示，KDM8和PKM2在食管癌组织中呈高表达并且存在相互作用关系。免疫组织化学结果显示，KDM8和PKM2在ESCC组织中高表达率分别为87.5%(105/120)和85.0%(102/120),高于癌旁组织的15.8%(19/120)和16.7%(20/120),χ~2值分别为123.404和112.098,均P<0.001。KDM8高表达与ESCC患者肿瘤大小(χ~2=8.778,P=0.012)和分化程度(χ~2=7.959,P=0.047)有关联；PKM2高表达与ESCC患者肿瘤大小(χ~2=11.755,P=0.003)和浸润深度(χ~2=6.257,P=0.044)有关联。Spearman相关分析提示KDM8与PKM2蛋白表达呈正相关，r=0.688,P<0.001。平板克隆形成实验和CCK-8实验结果均显示敲低KDM8后ESCC细胞增殖能力降低，F值分别为26.37和79.48,P值分别为<0.001和0.001;细胞凋亡率增加，F=37.10,P<0.001;细胞迁移能力降低，F=9.99,P=0.004。沉默KDM8后PKM2 mRNA表达水平降低(F=332.50,P<0.001)、PKM2蛋白表达量降低(F=117.10,P<0.001),MMP-9蛋白表达量降低(F=7.43,P=0.024)。结论 KDM8可能通过促进PKM2蛋白表达，进而促进ESCC恶性进展。更多还原

【Abstract】 Objective To investigate the expression of KDM8 and PKM2 in ESCC and their roles in the proliferation and migration of ESCC cells.Methods Bioinformatics analysis was performed to examine the expression of KDM8 in esophageal cancer and its interaction with PKM2.Collected 120 specimens of ESCC tissue and adjacent non-cancerous tissue(more than 3 cm away from the tumor tissue) from patients admitted to the First Affiliated Hospital of Xinjiang Medical University from January 1,2008 to December 31,2018.Immunohistochemical method was used to detect the expression levels of KDM8 and PKM2,and to analyze the relationship between these two markers and the clinical parameters of patients.At the cellular level,lentiviral infection,CCK-8 assay,colony formation assay,wound healing assay,and flow cytometry were employed to assess the effects of KDM8 knockdown on ESCC cell proliferation,migration,and apoptosis.RT-qPCR and western blot were used to measure PKM2 expression levels after KDM8 silencing,and the protein expression changes of invasion related factor MM P-9 were also detected.Results Bioinformatics results showed that KDM8 and PKM2 were highly expressed in esophageal cancer tissues and had an interactive relationship;Immunohistochemical results showed that the high expression rates of KDM8 and PKM2 in ESCC tissues were 87.5%(105/120) and 85.0%(102/120),higher than 15.8%(19/120) and 16.7%(20/120) in adjacent normal tissues,the differences was statistically significant(χ~2=123,404,P<0.001;χ~2=112.098,P<0.001).The high expression of KDM8 was mainly associated with tumor size(χ~2=8.778,P=0.012) and differentiation degree(χ~2=7.959,P=0.047),while the high expression of PKM2 was correlated with tumor size(χ~2=11.755,P=0.003) and invasion depth(χ~2=6.257,P=0.044).Spearman correlation analysis suggested a positive correlation between KDM8 and PKM2(r=0,688,P<0.001).Both the colony formation assay and CCK-8 assay results indicated a decrease in the proliferation ability of ESCC cells after KDM8 knockdown,with F values of 26.37 and 79.48,respectively,and P values of <0.001 and 0.001,respectively.The apoptosis rate of tumor cells was increased(F=37.10,P<0.001),and the cell migration ab ility was decreased(F=9.99,P=0.004),After silencing KDM8,the expression level of PKM2 mRNA decreased(F=332.50,P<0.001),as did the protein expression(F=117.10,P<0.001);the protein expression of MMP-9 also decreased(F=7.43,P=0.024).Conclusion KDM8 may promote the malignant progression of esophageal squamous cell carcinoma by upregulating PKM2 protein expression.更多还原