CaMKK2调控肝细胞癌化疗耐药性的作用和机制  

Role and mechanism of CaMKK2 in regulating chemoresistance of hepatocellular carcinoma

作　　者：惠博[1] 张健[1] 李韧[1] 李江伟 杨正安[1] HUI Bo;ZHANG Jian;LI Ren;LI Jiangwei;YANG Zheng′an(Fourth Cadre Ward,Second Affiliated Hospital of Xi′an Jiaotong University,Xi′an 710004,China)

机构地区：[1]西安交通大学第二附属医院干四病区,西安710004

出　　处：《山西医科大学学报》2024年第6期671-679,共9页Journal of Shanxi Medical University

基　　金：陕西省自然科学基础研究计划项目(2016JM0084)。

摘　　要：目的 探讨钙/钙调蛋白依赖性蛋白激酶激酶2(calcium/calmodulin-dependent protein kinase kinase 2,CaMKK2)调控肝细胞癌(hepatocellular carcinoma, HCC)化疗耐药性的作用及其机制。方法 (1)为了检测CaMKK2在HCC耐药细胞株中的表达变化,将实验分为亲本组和耐药组。采用浓度梯度递增法建立奥沙利铂(oxaliplatin, OXA)耐药细胞株MHCC97H/OXA和Hep3B/OXA。采用Western blot检测CaMKK2的磷酸化和总蛋白表达水平。(2)为了检测CaMKK2对肝细胞癌化疗药性的调控作用,将实验分为对照组和CaMKK2敲除组。采用CRISPR/Cas9技术敲除MHCC97H/OXA和Hep3B/OXA细胞株中的CaMKK2基因表达,采用Western blot验证CaMKK2敲除效率。采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)实验检测CaMKK2敲除对MHCC97H/OXA和Hep3B/OXA细胞株细胞存活率的影响。采用流式细胞术检测CaMKK2敲除对MHCC97H/OXA和Hep3B/OXA细胞株凋亡的影响。采用Western blot检测CaMKK2敲除对微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、p62、腺苷酸活化蛋白激酶(adenosine 5′-monophosphate-activated protein kinase, AMPK)和UNC-51样激酶1(UNC51-like kinase 1,ULK1)蛋白表达水平的影响。(3)为了验证CaMKK2对肝细胞癌化疗药性的调控作用,将实验分为CaMKK2敲除+空载体组和CaMKK2敲除+CaMKK2载体组。采用Western blot检测CaMKK2的蛋白表达水平。采用CCK-8实验检测重新表达CaMKK2对CaMKK2敲除的细胞耐药性的影响。采用Western blot检测重新表达CaMKK2对CaMKK2敲除的细胞中AMPK、ULK1和LC3蛋白表达水平的影响。结果 (1)与亲本组相比,耐药组HCC细胞株中CaMKK2的总蛋白表达水平无显著变化(P>0.05),而CaMKK2的磷酸化水平显著升高(P<0.01)。(2)与对照组比较,CaMKK2敲除组细胞中CaMKK2表达水平显著减少(P<0.01)。与对照组比较,CaMKK2敲除组HCC耐药细胞对OXA的敏感性显著提高(P<0.05),OXA细胞凋亡率显著升高(P<0.01)。与对照组比较,CaMKK2敲除�Objective To explore the role of calcium/calmodulin-dependent protein kinase kinase 2(CaMKK2)in regulating the chemoresistance of hepatocellular carcinoma(HCC)and its underlying mechanism.Methods①To examine the expression of CaMKK2 in chemoresistant HCC cells,the experiments were divided into parental group and chemoresistant group.The Oxaliplatin(OXA)-resistant cell lines MHCC97H/OXA and Hep3B/OXA were established by concentration gradient increasing method.The total and phosphorylated levels of CaMKK2 were detected by Western blot.②To investigate the role of CaMKK2 in regulating the chemoresistance of HCC cells,the experiments were divided into control group and CaMKK2 knockout group.CaMKK2 in MHCC97H/OXA and Hep3B/OXA cell lines was knocked out by CRISPR/Cas9 technology.The knockout efficiency of CaMKK2 was verified by Western blot.The effect of CaMKK2 knockout on the survival rate of MHCC97H/OXA and Hep3B/OXA cell lines was detected by cell counting kit-8(CCK-8)assay.The effect of CaMKK2 knockout on the apoptosis of MHCC97H/OXA and Hep3B/OXA cell lines was measured by flow cytometry.The effect of CaMKK2 knockout on the expressions of microtubule-associated protein1 light chain 3(LC3),p62,adenosine 5′-monophosphate-activated protein kinase(AMPK),and UNC51-like kinase 1(ULK1)was examined by Western blot.③To confirm the role of CaMKK2 in regulating the chemoresistance of HCC cells,the experiments were divided into CaMKK2 knockout+empty vector group and CaMKK2 knockout+CaMKK2 vector group.The expression of CaMKK2 was confirmed by Western blot.The effect of CaMKK2 re-expression on the chemosensitivity of CaMKK2 knockout cells was assessed by CCK-8 assay.The effect of CaMKK2 re-expression on the expressions of AMPK,ULK1,and LC3 was evaluated by Western blot.Results①Compared with parental group,the total level of CaMKK2 showed no significant change in chemoresistant group(P>0.05),while the phosphorylated level of CaMKK2 was significantly increased(P<0.01).②Compared with control group,CAMKK2 expression le

关 键 词：肝细胞癌 化疗耐药性 细胞自噬 CaMKK2 奥沙利铂 基因敲除 

分 类 号：R735.7[医药卫生—肿瘤]