Huang Jia-Yi, Guo Dan
1Department of Pathophysiology, 2Research Center of Molecular Medicine and Cancer, Chongqing Medical University, Chongqing 400016, China.E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2017 Aug 20;37(8):1035-1039. doi: 10.3969/j.issn.1673-4254.2017.08.06.
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.
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.
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).
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.
研究硬化蛋白（SOST）是否参与乳腺癌MDA - MB - 231细胞诱导的成骨细胞MG63细胞和间充质干细胞C3H10细胞分化抑制。
将SOST特异性小干扰RNA（siRNA）转染至乳腺癌MDA - MB - 231细胞中，通过逆转录聚合酶链反应（RT - PCR）验证干扰效率。收集常规培养的MDA - MB - 231细胞、经腺病毒转染SOST siRNA的细胞以及转染空腺病毒载体的细胞的上清液，并添加到MG63或C3H10细胞培养物中。使用定量实时PCR检测MG63和C3H10细胞中骨保护素（OPG）、骨钙素（OCN）、骨桥蛋白（OPN）和骨涎蛋白（IBSP）表达的变化，以常规培养基培养的细胞和在成骨诱导培养基中培养的细胞作为阴性和阳性对照，通过碱性磷酸酶（ALP）读数和ALP染色检测ALP活性。
腺病毒Ad - siSOST有效敲低了MDA - MB - 231细胞中SOST的表达。在成骨培养基中培养的MG63细胞和C3H10细胞显示成骨细胞标志物OPG、OPN、OCN和IBSP的表达显著上调（P<0.01），而与MDA - MB - 231细胞的上清液共培养明显降低了成骨细胞标志物的表达（P<0.01）；用Ad - siSOST转染的MDA - MB - 231细胞的上清液处理后，MG63和C3H10细胞中标志物的表达再次增加（P<0.01）。MG63和C3H10细胞中的ALP活性在响应处理时表现出类似的变化模式（P<0.01）。
在乳腺癌骨转移的体外模型中，MG63或C3H10细胞的分化受到抑制，通过敲低骨转移微环境中SOST的表达可部分逆转这种抑制。
