Role and mechanism of CaMKK2 in regulating chemoresistance of hepatocellular carcinoma
Objective To explore the role of calcium/calmodulin-dependent protein kinase kinase 2(CaMKK2)in regulating the che-moresistance of hepatocellular carcinoma(HCC)and its underlying mechanism.Methods ①To 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 phos-phorylated 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'-mono-phosphate-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.Results ①Compared 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 signifi-cantly increased(P＜0.01).②Compared with control group,CAMKK2 expression level was significantly decreased in CaMKK2 knockout group(P＜0.01).Compared with control group,the sensitivity of OXA-resistant cell lines to OXA was significantly increased in CaMKK2 knockout group(P＜0.05),and OXA-induced cell apoptosis was significantly enhanced(P＜0.01).Compared with control group,LC3 Ⅱ/LC3 Ⅰ was significantly decreased,p62 level was increased,and p-AMPK/AMPK and p-ULK1/ULK1 were significantly reduced in CaMKK2 knockout group(all P＜0.01).③Compared with CaMKK2 knockout+empty vector group,the chemo-sensitivity to OXA was significantly decreased,and p-AMPK/AMPK,p-ULK1/ULK1,and LC3 Ⅱ/LC3 Ⅰ were significantly upregulated in CaMKK2 knockout+CaMKK2 vector group(all P＜0.01).Conclusion Knockout of CaMKK2 can effectively reverse the chemoresistance of HCC possibly through the AMPK/ULK1-mediated autophagy pathway.
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