【摘要】 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。更多还原

【Abstract】 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.更多还原