骨膜蛋白：前列腺癌治疗干预的一个有前景的靶点。
Periostin: a promising target of therapeutical intervention for prostate cancer.
机构信息
Department of Urology, Huashan Hospital, Fudan University, Shanghai, 200040 China.
出版信息
J Transl Med. 2011 Jun 30;9:99. doi: 10.1186/1479-5876-9-99.
BACKGROUND
In our recent study, Periostin was up-regulated in prostate cancer(PCa) compared with benign prostate hyperplasia (BPH) by proteomics analysis of prostate biopsies. We investigated the effect of sliencing Periostin by RNA interference (RNAi) on the proliferation and migration of PCa LNCap cell line.
METHODS
All the prostate biopsies from PCa, BPH and BPH with local prostatic intraepithelial neoplasm(PIN) were analyzed by iTRAQ(Isobaric tags for relative and absolute quantification) technology. Western blotting and immunohistochemical staining were used to verify Periostin expression in the tissues of PCa. Periostin expression in different PCa cell lines was determined by immunofluorescence staining, western blotting and reverse transcription PCR(RT-PCR). The LNCap cells with Periostin expression were used for transfecting shRNA-Periostin lentiviral particles. The efficancy of transfecting shRNA lentiviral particles was evaluated by immunofluorescence, western blotting and Real-time PCR. The effect of silencing Periostin expression by RNAi on proliferation of LNCap cells was determined by MTT assay and tumor xenografts. The tissue slices from theses xenografts were analyzed by hematoxylin and eosin(HE) staining. The expression of Periostin in the xenografts was deteminned by Immunohistochemical staining and western blotting. The migration of LNCap cells after silencing Periostin gene expression were analyzed in vitro.
RESULTS
Periostin as the protein of interest was shown 9.12 fold up-regulation in PCa compared with BPH. The overexpression of Periostin in the stroma of PCa was confirmed by western blotting and immunohistochemical staining. Periostin was only expressed in PCa LNCap cell line. Our results indicated that the transfection ratio was more than 90%. As was expected, both the protein level and mRNA level of Periostin in the stably expressing shRNA-Periostin LNCap cells were significantly reduced. The stably expressing shRNA-Periostin LNCap cells growed slowly in vitro and in vivo. The tissues of xenografts as PCa were verificated by HE staining. Additionally, the weak positive Periostin expressed tumor cells could be seen in the tissues of 6 xenografts from the group of down-regulated Periostin LNCap cells which had a significant decrease of the amount of Periostin compared to the other two group. Furthermore, our results demonstrated that sliencing Periostin could inhibit migration of LNCap cells in vitro.
CONCLUSIONS
Our data indicates that Periostin as an up-regulated protein in PCa may be a promising target of therapeutical intervention for PCa in future.
背景
在我们最近的研究中，通过前列腺活检的蛋白质组学分析，发现骨膜蛋白在前列腺癌（PCa）中上调，而在良性前列腺增生（BPH）中则下调。我们研究了通过 RNA 干扰（RNAi）沉默骨膜蛋白对 PCa LNCap 细胞系增殖和迁移的影响。
方法
通过 iTRAQ（相对和绝对定量的同位素标记）技术分析所有来自 PCa、BPH 和 BPH 伴局部前列腺上皮内瘤变（PIN）的前列腺活检标本。Western 印迹和免疫组织化学染色用于验证 PCa 组织中骨膜蛋白的表达。免疫荧光染色、Western 印迹和逆转录 PCR（RT-PCR）用于确定不同 PCa 细胞系中骨膜蛋白的表达。用表达骨膜蛋白的 LNCap 细胞转染 shRNA-骨膜蛋白慢病毒颗粒。通过免疫荧光、Western 印迹和实时 PCR 评估转染 shRNA 慢病毒颗粒的效率。通过 MTT 测定和肿瘤异种移植评估 RNAi 沉默骨膜蛋白表达对 LNCap 细胞增殖的影响。对这些异种移植的组织切片进行苏木精和伊红（HE）染色。通过免疫组织化学染色和 Western 印迹确定异种移植中骨膜蛋白的表达。体外分析沉默 Periostin 基因表达后 LNCap 细胞的迁移。
结果
骨膜蛋白作为研究的蛋白，在 PCa 中与 BPH 相比上调了 9.12 倍。Western 印迹和免疫组织化学染色证实了 PCa 基质中骨膜蛋白的过表达。骨膜蛋白仅在 PCa LNCap 细胞系中表达。我们的结果表明转染率超过 90%。正如预期的那样，稳定表达 shRNA-骨膜蛋白的 LNCap 细胞中的骨膜蛋白蛋白水平和 mRNA 水平均显著降低。体外和体内稳定表达 shRNA-骨膜蛋白的 LNCap 细胞生长缓慢。HE 染色证实异种移植组织为前列腺癌。此外，在下调 Periostin 的 LNCap 细胞的异种移植组织中，可以看到肿瘤细胞呈弱阳性表达的 Periostin，与其他两组相比，其 Periostin 数量明显减少。此外，我们的结果表明沉默 Periostin 可抑制 LNCap 细胞的体外迁移。
结论
我们的数据表明，骨膜蛋白作为 PCa 中上调的蛋白，可能是未来 PCa 治疗干预的一个有前途的靶点。
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