抑制硬化蛋白基因表达有利于诱导成骨细胞的分化:体外乳腺癌骨转移模型

SOST knockdown promotes differentiation of osteoblasts MG63 and mesenchymal stem cells C3H10 in an in vitro model of bone metastasis of breast cancer

目的探讨在体外乳腺癌骨转移模型中抑制硬化蛋白基因的表达后对成骨细胞分化成熟的影响。方法以干扰硬化蛋白基因表达的si RNA腺病毒感染乳腺癌细胞株MDA-MB-231,RT-PCR检测确认硬化蛋白基因表达的改变。分别以正常培养的MDA-MB-231上清液,感染空病毒的MDA-MB-231上清液,感染SiSOST病毒的MDA-MB-231上清液与MG63或C3H10共培养,并以成骨诱导培养基培养MG63及C3H10为阳性对照,普通培养基培养MG63及C3H10为阴性对照。定量PCR检测成骨细胞分化相关指标的改变,染色检测碱性磷酸酶(ALP)表达的改变。结果感染Ad-si SOST病毒的MDA-MB-231细胞中硬化蛋白表达明显降低。定量PCR结果显示:与单独培养的MG63相比,加成骨培养基后其骨钙素、骨桥蛋白、护骨因子和整合素结合涎蛋白的表达均上升(P<0.01)。在此基础上,与231细胞上清液共培养后其表达降低(P<0.01),差异具有统计学意义;MG63+231-RFP组结果一致,感染SiSOST病毒后,分化指标重新上升(P<0.01),差异具有统计学意义。C3H10细胞实验结果趋势一致。MG63细胞与阴性对照组相比,分化诱导培养基可促进ALP的分泌(P<0.01),但与231细胞共培养后,ALP分泌减少(P<0.01),感染Si-SOST后,可部分扭转ALP分泌的减少(P<0.01),ALP染色得到同样的结果。C3H10细胞结果与MG63细胞趋势一致。结论在体外乳腺癌骨转移模型中,成骨细胞分化受阻;干扰硬化蛋白基因的表达有利于逆转成骨细胞的分化受阻。

Objective To investigate whether SOST is involved in breast cancer MDA-MB-231 cells-induced suppression of differentiation of osteoblast MG63 cells and mesenchymal stem C3H10 cells. Methods SOST-specific small interfering RNA （siRNA） was transfected into breast cancer MDA-MB-231 cells, and the interfering efficiency was verified by RT-PCR. The supernatants were collected from MDA-MB-231 cells in routine culture, cells transfected with SOST siRNA via adenovirus, and cells transfected with empty adenoviral vectors and added in MG63 or C3H10 cell cultures. The changes in the expressions of OPG, OCN, OPN and IBSP in MG63 and C3H10 cells were detected using quantitative real-time PCR, and ALP activity was detected with ALP reading and ALP staining with the cells cultured in routine culture medium and cells in osteogenic induction medium as the negative and positive controls. Results The adenovirus Ad-siSOST effectively knocked down the expression of SOST in MDA-MB-231 cells. MG63 cells and C3H10 cells cultured in osteogenic medium showed significantly upregulated expressions of the osteoblast markers OPG, OPN, OCN and IBSP （P〈0.01）, while co-culture with the supernatant of MDA-MB-231 cells obviously reduced the expressions of the osteoblast markers （P〈0.01）; the expression of the markers increased again in MG63 and C3H10 cells after treatment with the supernatant of MDA-MB-231 cells transfected with ad-siSOST （P〈0.01）. ALP activity in MG63 and C3H10 cells exhibited a similar pattern of variations in response to the treatments （P〈0.01）. Conclusion In the in vitro model of bone metastasis of breast cancer, the differentiation of MG63 or C3H10 cells is suppressed, which can be partly reversed by knocking down the expression of SOST in the bone metastasis microenvironment.