Identification of TM9SF2 as a Candidate of the Cell Surface Marker Common to Breast Carcinoma Cells

OBJECTIVE We aimed identification of cell surface molecules, which might serve as diagnostic biomarkers or useful targets for therapies, in breast cancer. METHODS We developed unique DNA microarray coupled with spherical self-organizing map （sSOM） analysis to characterize cells and tissues by the cell surface markers. In the microarray 1,797 probes for human genes coding membrane bound proteins were spotted. With this microarray the gene expression profiles of eight breast carcinoma cell lines were compared to identify the genes that were commonly expressed in breast carcinomas but not in normal cells. RESULTS The gene expression profiles of sSOM from the eight breast carcinoma cell lines were successfully distinguished from that of normal breast tissue derived cells suggesting the presence of genes of interest, sSOMon the data extensively filtered revealed several candidate genes, of which expression was significant in carcinoma cells but low in normal cells. Finally, TM9SF2 was nominated through validations of PCR procedures together with CD24 and ErbB3, which are known breast carcinoma markers. TMgSF2 expression was further confirmed by immunological staining. Interestingly, TMgSF2 was found to be expressed in all the cell lines evaluated while CD24 and ErbB3 were not in all of the carcinoma cells, supporting their relationship in sSOM. Although physiological significance of TMgSF2 is unknown yet, siRNA treatment significantly inhibited the growth of MDA- MB-231 cells. CONCLUSION We propose TM9SF2 as a novel and useful diagnostic marker as well as a potential molecular target specific to breast carcinoma cells covering wide range of breast cancer.

TM9SF2促进三阴性乳腺癌MDA-MB-231细胞的增殖与转移

探究9次跨膜超家族蛋白2(transmembrane 9 superfamily protein member 2,TM9SF2)对于三阴性乳腺癌MDA-MB-231细胞增殖和转移的影响及其分子机制。采用Western blot实验检测三阴性乳腺癌细胞株MDA-MB-231和非致瘤的乳腺上皮细胞株MCF-10A中TM9SF2蛋白表达的情况;对高表达TM9SF2的三阴性细胞株MDA-MB-231进行基因沉默;采用MTS法检测细胞增殖活性,采用Transwell实验和划痕实验检测细胞的转移能力;采用Western blot实验检测细胞内增殖相关蛋白(PI3K、AKT、SRC和ERK)和转移相关蛋白(Snail、Slug和N-cadherin)的表达情况。Western blot实验证明,MDA-MB-231中TM9SF2蛋白的表达量高于MCF-10A细胞。与对照组相比,siRNA-TM9SF2转染组TM9SF2蛋白表达下调,细胞增殖活性降低,细胞转移能力减弱,PI3K、Snail、Slug和N-cadherin表达水平均降低,AKT蛋白磷酸化激活降低。研究结果表明,TM9SF2基因能促进三阴型乳腺癌MDA-MB-231细胞的增殖和转移。

鸡TM9SF2蛋白胞外结构域的原核表达及其家兔抗血清的制备

为了探究鸡源九次跨膜超家族成员2(TM9SF2)的生物学功能,通过软件分析TM9SF2蛋白的氨基酸序列保守性,选择其胞外区,经密码子优化后进行基因合成,命名为chTM9ECD;将该基因亚克隆至原核表达载体pET-28a(+)中,成功构建重组表达质粒pET-chTM9ECD,并在大肠杆菌BL21(DE3)中诱导表达;用纯化的重组TM9SF2蛋白与佐剂混合后免疫新西兰大白兔制备多抗血清。SDS-PAGE和Western-blot均证实重组TM9SF2蛋白以包涵体形式表达,分子质量约为33.6 ku。经ELISA检测,抗体效价达1∶128 000,Western-blot证实家兔抗重组TM9SF2血清可特异性识别293细胞中表达的TM9SF2。结论,本试验应用大肠杆菌成功表达了重组TM9SF2胞外蛋白,具有免疫原性,并获得家兔源阳性血清,为TM9SF2生物学功能研究奠定基础。