抑制Sema 4D表达介导的成骨前体细胞AKT磷酸化水平上调在乳腺癌骨转移中的作用

Role of Inhibition of Sema 4D-mediated AKT Phosphorylation of Osteogenic Precursor Cells in Breast Cancer Bone Metastasis

目的探讨抑制Sema 4D介导的成骨前体细胞AKT磷酸化水平上调在乳腺癌骨转移中的作用。方法通过siRNA技术构建稳定的Sema 4D低表达人乳腺癌MCF-7细胞株,采用实时荧光定量PCR检测细胞Sema 4D mRNA表达,MTT法检测各组细胞增殖能力,Transwell法检测各组细胞侵袭能力;采用Transwell小室建立成骨细胞和乳腺癌细胞的共培养体系,茜素红染色实验检测骨矿化能力,Western blot检测AKT、p-AKT蛋白表达。结果 MCF-7细胞Sema 4D mRNA相对表达量显著高于Hs578Bst细胞(P<0.05)。Sema 4D siRNA转染后,Sema 4D siRNA组Sema 4D mRNA相对表达量显著低于Control组和NC组(均P<0.05),Control组和NC组Sema 4D mRNA相对表达量比较,差异无统计学意义(P>0.05)。Sema 4D siRNA组细胞增殖率显著低于NC组和Control组(均P<0.05),Sema 4D siRNA组细胞侵袭率亦显著低于NC组和Control组(均P<0.05)。MCF-7+OM组和Sema 4D siRNA+OM组骨结节面积显著低于Control+OM组(均P<0.05),Sema 4D siRNA+OM组骨结节面积显著高于MCF-7+OM组(均P<0.05)。Control+OM组、MCF-7+OM组、Sema 4D siRNA+OM组AKT蛋白表达水平比较差异无统计学意义(P>0.05);MCF-7+OM组和Sema 4D siRNA+OM组p-AKT蛋白表达水平显著低于Control+OM组(均P<0.05),Sema4D siRNA+OM组p-AKT蛋白表达水平显著高于MCF-7+OM组(P<0.05)。结论人乳腺癌细胞系MCF-7 Sema4D表达显著上调,并与细胞增殖、侵袭等生物学行为有关;下调Sema 4D表达,能够提高AKT磷酸化水平,减弱对成骨细胞分化的抑制作用。

Objective To explore the role of inhibition of sema 4D-mediated AKT phosphorylation of osteogenic precursor cells in breast cancer bone metastasis.Methods The stable low expression of Sema 4D in human breast cancer cell line MCF-7was constructed by siRNA technique.Sema 4D mRNA expression was detected by real-time fluorescence quantitative PCR;cell proliferation activity was detected by MTT assay;cell invasion ability was detected by Transwell method.The co-culture system of osteoblasts and breast cancer cells was established by Transwell coculture system.Bone mineralization ability was detected by alizarin red staining.AKT and p-AKT protein expression were determined by Western blotting.Results The relative expression of Sema 4D mRNA was significantly higher in MCF-7 cells than in Hs578 Bst cells（P〈0.05）.After Sema 4D siRNA transfection,Sema 4D mRNA was significantly lower in Sema 4D siRNA group than in control group and NC group（P〈0.05）,and there was no significant difference in Sema 4D mRNA expression between control group and NC group（P〈0.05）.The cell proliferation rate was significantly lower in Sema 4D siRNA group than in control group and NC group（P〈0.05）,and invasion rate in Sema 4D siRNA group was also significantly lower than that in control group and NC group（P〈0.05）.The bone nodule area in MCF-7＋OM group and Sema 4D siRNA＋OM group was significantly lower than that of the control ＋ OM group（P〈0.05）,and bone nodule area of Sema 4D siRNA＋OM group was significantly higher than that of the MCF-7＋ OM group（P〈0.05）.There was no significant difference in AKT protein expression between control ＋ OM group,MCF-7＋OM group and Sema 4D siRNA＋OM group（P〉0.05）.The p-AKT protein expression in MCF-7 ＋ OM group and Sema 4D siRNA＋OM group was significantly lower than that in control＋OM group,and p-AKT protein expression in Sema 4D siRNA＋OM group was significantly higher than that in MCF-7 ＋ OM group（P〈0.05）.Conclusion Sema 4D MCF-7 expression is significantly up-regulated in human breast cancer cell line MCF-7,and it is related to proliferation and invasion biological behavior.Down-regulation of the expression of Sema 4D in MCF-7 cells can improve the AKT phosphorylation levels,and weaken the inhibitory effect on osteoblast differentiation.