转染LKB1基因乳腺癌细胞与胚胎发育信号通路及cAMP/PKA通路的关系

Interactions between the Sonic Hedgehog signaling pathway and the cAMP/PKA signaling pathway in LKB1 transfected breast cancer cells

目的探讨抑癌基因LKB1与胚胎发育(Sonic Hedgehog,SHH)信号通路、cAMP/PKA信号通路之间的相互关系。方法抑癌基因LKB1转染人乳腺癌细胞MDA-MB-231,分MDA-MB-231组(231组)和转染LKB1基因的MDA-MB-231(LKB1组)两组,每组分别采用0、5×10-6、1×10-5、2×10-5 mol/L等4种浓度的SHH信号通路抑制剂环靶明(cyclopamine)作用于细胞,各小组细胞分别用RT-PCR法和Western blot法检测SHH信号通路相关基因SHH、Smo的mRNA表达水平和蛋白表达水平,用cAMP、PKA试剂盒检测cAMP、PKA数值水平。结果 LKB1组的细胞通过不同剂量环靶明抑制后,SHH信号通路相关基因SHH、Smo的mRNA和蛋白表达水平相应较231组明显抑制,且与环靶明剂量呈正相关,抑制效果在环靶明浓度为10×10-6 mol/L时最明显,继续增加环靶明浓度到20×10-6 mol/L,抑制效果保持不变。231组和LKB1组中细胞的PKA和cAMP数值均随环靶明浓度增加而增高,至10×10-6 mol/L时达到最高,继续增加环靶明浓度到20×10-6 mol/L,两组中的PKA和cAMP数值保持不变。在相同环靶明浓度下,LKB1组中PKA和cAMP数值高于231组中相应数值。结论抑癌基因LKB1协同环靶明抑制乳腺癌MDA-MB-231细胞的SHH信号通路的同时亦协同增加cAMP/PKA信号通路的表达,且在环靶明浓度为10×10-6 mol/L时效果最明显;推测存在LKB1-SHH-cAMP/PKA通路。

Objective To investigate the interactions between the LKB1 tumor suppressor,the Sonic Hedgehog（SHH） signaling pathway and the cAMP/PKA signaling pathway.Methods The LKB1 gene was reintroduced into MDA-MB-231 breast cancer cells which were lack of LKB1.And then two groups were classified：MDA-MB-231 group（231 group） and LKB1 transfected MDA-MB-231 group（LKB1 group）.The cells in each group were treated with the SHH signaling inhibitor cyclopamine at different concentration levels（0,5×10-6,1×10-5 and 2×10-5mol/L）,respectively.The levels of SHH,Smo genes mRNA and protein expressions related to the SHH signaling pathway were detected with the methods of RT-PCR and Western blot analysis.Meanwhile,the activities of cAMP and PKA were determined with corresponding kits.Results After treated with different concentration of SHH signaling inhibitor cyclopamine,SHH,Smo genes mRNA and protein expressions related to the SHH signaling pathway in LKB1 group were significantly inhibited in comparison with those in 231 group.The inhibition effect was positively correlated to the concentration of cyclopamine,which reached the highest when the concentration of cyclopamine was 1×10-5mol/L.Above this concentration,the inhibition effect remained unaffected.Meanwhile,the values of PKA and cAMP in 231 group and LKB1 group both increased as cyclopamine concentration increased,and these values simultaneously achieved maxima when the concentration of cyclopamine was 1×10-5mol/L.Further increase of cyclopamine concentration to 2×10-5mol/L did not lead to any raise of PKA or cAMP values.Under the treatment with the same concentration of cyclopamine,higher values of PKA and cAMP were found in LKB1 group compared with 231 group.Conclusions In breast cancer MDA-MB-231 cells,the SHH signaling pathway is inhibited,under the synergetic affection of the LKB1 tumor suppressor and the cyclopamine inhibitor,while the expression of the cAMP/PKA signaling pathway is enhanced.When the concentration of cyclopamine was 1×10-5mol/L,the highest effect was observed.Therefore,it is supposed that there exists a LKB1-SHH-cAMP/PKA pathway.