重组人Maspin蛋白通过调控SIRT3介导的糖酵解过程抑制宫颈癌血管生成

Recombinant Maspin Protein Inhibits the Tumor Angiogenesis by Regulating SIRT3-Mediated Glycolysis in Cervical Cancer

目的探讨重组人乳腺丝氨酸蛋白酶抑制物(mammary serine protease inhibitor,Maspin)对宫颈癌细胞血管生成及糖酵解的影响及其分子作用机制。方法噻唑盐(MTS)法研究重组人Maspin蛋白对人宫颈癌细胞HeLa增殖的影响;Western blot法检测重组人Maspin蛋白对HeLa细胞中SIRT3蛋白表达的影响;商业化构建SIRT3 siRNA质粒并转染HeLa细胞,采用小管形成实验研究SIRT3干扰后重组人Maspin蛋白对宫颈癌血管生成的影响;采用试剂盒检测SIRT3干扰后重组人Maspin蛋白对HeLa细胞葡萄糖消耗、丙酮酸、乳酸及ATP生成的影响;Western blot法检测SIRT3干扰后重组人Maspin蛋白对HeLa细胞中葡萄糖转运蛋白1(glucose transporter 1,GLUT1)、单羧酸转运蛋白-1(monocarboxylate transporter-1,MCT-1)、己糖激酶2(hexokinase,HK2)、6-磷酸果糖激酶1(fructokinase 6-phosphate,PFK1)和丙酮酸激酶2(pyruvic acid 2,PKM2)表达的影响。结果重组人Maspin蛋白处理HeLa细胞后,肿瘤细胞的体外增殖较对照组明显受到抑制;重组人Maspin蛋白能够促进SIRT3蛋白的表达;干扰SIRT3能够减弱重组人Maspin蛋白抑制肿瘤细胞血管生成的能力;干扰SIRT3显著减弱重组人Maspin蛋白抑制HeLa细胞糖酵解的能力。重组人Maspin蛋白能够显著下调GLUT1、MCT-1、HK2、PFK1和PKM2的表达,而干扰SIRT3则能够恢复上述糖酵解相关蛋白的表达。结论重组人Maspin蛋白能够通过抑制SIRT3介导的糖酵解过程,进而发挥抑制宫颈癌血管生成的能力。

OBJECTIVE To investigate the effect of recombinant mammary serine protease inhibitor(Maspin)on tumor angiogenesis and tumor metabolism of HeLa cervical cancer and its mechanisms.METHODS MTS was used to test the effect of recombinant Maspin on proliferation of HeLa cells.Western blotting was used to determine the effect of recombinant Maspin on the expression of SIRT3 in HeLa cells.SIRT3 siRNA was constructed by commercial and then transfected into HeLa cells.Tube formation assay was used to study the effect of SIRT3 intervene on tumor angiogenesis.Detection kits were used to examine the effects of recombinant Maspin on the glucose consumption and the generation of pyruvic acid,lactic acid as well as ATP after SIRT3 intervene,respectively.Western blotting was used to study the effect of SIRT3 intervene on expression of glucose transporter 1(GLUT1),monocarboxylate transporter-1(MCT-1),hexokinase(HK2),fructokinase 6-phosphate(PFK1)and pyruvic acid 2(PKM2).RESULTS Compared with the control group,the proliferation of HeLa cervical cancer cells was significant inhibited by recombinant Maspin in vitro.SIRT3 siRNA transfection could significantly decrease the inhibition effect of recombinant Maspin on tumor angiogenesis.SIRT3 siRNA transfection could significantly ameliorate the inhibition effect of recombinant Maspin on glucose consumption and the contents of pyruvic acid,lactic acid as well as ATP,respectively.Recombinant Maspin inhibited the expressions of GLUT1,MCT-1,HK2,PFK1 and PKM2,however,SIRT3 siRNA transfection significantly reversed the expression of above glycolysis-related proteins.CONCLUSION Recombinant Maspin inhibits the tumor angiogenesis by suppressing glycolysis in HeLa cells,and such effect is dependent on SIRT3 expression.