Dentin sialophosphoprotein (DSPP) gene-silencing inhibits key tumorigenic activities in human oral cancer cell line, OSC2.
Dentin sialophosphoprotein (DSPP) gene-silencing inhibits key tumorigenic activities in human oral cancer cell line, OSC2.
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牙本质唾液酸磷蛋白 (DSPP) 基因沉默可抑制人口腔癌细胞系 OSC2 的关键致瘤活性。
DOI：
10.1371/journal.pone.0013974
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发表时间：
2010-11-12
期刊：
影响因子：
3.7
通讯作者：
Ogbureke KU
中科院分区：
文献类型：
--
作者：
Joshi R;Tawfik A;Edeh N;McCloud V;Looney S;Lewis J;Hsu S;Ogbureke KU
我们最近发现牙本质涎磷蛋白(DSPP)是小整合素结合配体N-连接糖蛋白家族中的一员，它在口腔鳞状细胞癌(OSCC)中高度上调，而OSCC的上调与肿瘤的侵袭性有关。为探讨DSPP沉默对口腔癌细胞系OSC2致瘤性的影响，采用短发夹状RNA(ShRNA)干扰沉默OSC2细胞中的DSPP。利用设计用于沉默DSPP基因表达的hDSP-shRNA慢病毒颗粒，针对DSPP转录本的多个区域进行shRNA干扰。用编码不能降解任何已知细胞mRNA的加扰序列的对照shRNA质粒作为阴性对照。通过Western印迹和RT-PCR分析、细胞活力、集落形成、改良Boyden小室(迁移和侵袭)和流式细胞术(细胞周期和细胞凋亡)分析口腔癌变的表型特征。DSPP沉默的OSC2细胞表现出细胞形态改变，存活率下降，克隆形成能力下降，迁移和侵袭减少，G0/G1期细胞周期停滞，肿瘤细胞对顺铂诱导的凋亡敏感性增加。此外，MMP2、MMP3、MMP9、血管内皮生长因子、Ki 67、P53和EGFR表达下调。经最小二乘回归分析表明：基质金属蛋白酶-2{(y = 0.850x，p<；0.001)(y = 1.156x，p<；0.001)}，基质金属蛋白酶-3{(y = 0.994x，p<；0.001)(y = 1.324x，p = 0.004)和MMP9{(y = 1.248x，p = 0.005，y = 0.809，p = 0.013)}。DSPP在OSC2细胞中的沉默降低了口腔肿瘤发生的显著特征，并为DSPP在口腔癌生物学中潜在的关键作用提供了第一个功能性证据。在DSPP沉默的OSC2细胞中，MMP2、MMP3、MMP9、P53和VEGF的表达下调，为阐明DSPP在口腔癌生物学中的作用机制提供了重要的功能/分子框架。
We determined recently that dentin sialophosphoprotein (DSPP), a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins, is highly upregulated in human oral squamous cell carcinomas (OSCCs) where upregulation is associated with tumor aggressiveness. To investigate the effects of DSPP-silencing on the tumorigenic profiles of the oral cancer cell line, OSC2, short-hairpin RNA (shRNA) interference was employed to silence DSPP in OSC2 cells. Multiple regions of DSPP transcript were targeted for shRNA interference using hDSP-shRNA lentiviral particles designed to silence DSPP gene expression. Control shRNA plasmid encoding a scrambled sequence incapable of degrading any known cellular mRNA was used for negative control. Following puromycin selection of stable lines of DSSP-silenced OSC2 cells, phenotypic hallmarks of oral carcinogenesis were assayed by western blot and RT-PCR analyses, MTT (cell-viability), colony-formation, modified Boyden-Chamber (migration and invasion), and flow cytometry (cell-cycle and apoptosis) analyses. DSPP-silenced OSC2 cells showed altered cell morphology, reduced viability, decreased colony-formation ability, decreased migration and invasion, G0/G1 cell-cycle arrest, and increased tumor cell sensitivity to cisplatin-induced apoptosis. Furthermore, MMP-2, MMP-3, MMP-9, VEGF, Ki-67, p53, and EGFR were down-regulated. There was a direct correlation between the degree of DSPP-silencing and MMP suppression, as indicated by least squares regression: MMP-2 {(y = 0.850x, p<0.001) (y = 1.156x, p<0.001)}, MMP-3 {(y = 0.994x, p<0.001) (y = 1.324x, p = 0.004)}, and MMP-9 {(y = 1.248x, p = 0.005, y = 0.809, p = 0.013)}. DSPP-silencing in OSC2 cell decreased salient hallmarks of oral tumorigenesis and provides the first functional evidence of a potential key role for DSPP in oral cancer biology. The down-regulation of MMP-2, MMP-3, MMP-9, p53 and VEGF in DSPP-silenced OSC2 cells provides a significant functional/molecular framework for deciphering the mechanisms of DSPP activities in oral cancer biology.
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