第二临床医学院知识库

目的：观察ELAV1基因对去势抵抗性前列腺癌PC-3细胞的作用及相关机制研究。 方法：应用qRT-PCR、Western blot、免疫组化、免疫荧光等方法检测去势抵抗性前列腺癌PC-3细胞、正常前列腺上皮RWPE-1细胞中ELAV1基因和蛋白表达水平，同时收集12例去势抵抗性前列腺癌和癌旁标本采用免疫组化法检测ELAV1蛋白在细胞核和细胞质中的表达情况。将Ad5.-ELAV1 shRNA-GFP转染前列腺癌PC-3细胞来对细胞中的ELAV1进行沉默，通过CCK-8检测PC-3细胞增殖能力的变化、流式细胞仪检测细胞周期的变化和Western blot检测细胞中COX-2、Cyclin D1、PI3K、Akt、pAkt的蛋白表达变化。建立前列腺癌PC-3细胞皮下移植瘤模型27只，采用随机数字表法将其分为3组，每组9只，分别给予瘤内注射Ad5.- ELAV1 shRNA-GFP、Ad5.-GFP、生理盐水，每3天测量1次移植瘤体积观察移植瘤生长情况，并于2周后收取移植瘤进行称重并进行数据统计。 结果：qRT-PCR、Western blot结果显示PC-3细胞中ELAV1 基因和蛋白表达水平均高于RWPE-1细胞，免疫荧光、细胞免疫组化显示PC-3细胞中的ELAV1蛋白主要集中表达于细胞质，而在RWPE-1细胞中则主要表达于细胞核。12例前列腺癌和癌旁组织标本免疫组化结果也同样显示ELAV1蛋白在前列腺癌中主要表达细胞质，在癌旁组织中主要表达于细胞核。CCK-8、细胞周期等实验结果显示PC-3细胞中ELAV1基因被沉默后其增殖能力下降、细胞周期被阻滞在G1期。Western-blot实验证实与肿瘤细胞生长有关的COX-2、与肿瘤细胞周期阻滞有关的Cyclin D1表达明显降低；此外，肿瘤信号通路相关分子PI3K、Akt及磷酸化Akt的表达均下降。裸鼠移植瘤收取后显示Ad5.- ELAV1 shRNA-GFP移植瘤的体积和重量与对照病毒Ad5.-GFP组相比明显减小（P<0.05）。 结论：ELAV1蛋白在前列腺癌中高表达且主要表达于细胞质；ELAV1蛋白通过PI3K/Akt经典肿瘤信号通路对前列腺癌细胞的生长起着促进作用。

Objective: To observe the effect and mechanism of ELAV1 on the castration-resistant prostate cancer cell PC-3. Methods: The expressions of ELAV1 gene and protein in castration-resistant prostate cancer PC-3 cells and normal prostate epithelial RWPE-1 cells were detected by qRT-PCR, Western blot, immunohistochemistry, immunofluorescence and so on. Immunohistochemistry was used to detect the expression of ELAV1 protein of nucleus and cytoplasm in 12 cases of castrated resistant prostate cancer and paracancerous specimens. Ad5.-ELAV1 shRNA-GFP was transfected into prostate cancer PC-3 cells. Cell proliferation was detected by CCK-8 and cell cycle was measured by flow cytometry. Western Blot was used to detect the protein expression of COX-2, Cyclin D1, PI3K, Akt and pAkt after infection of Ad5.-ELAV1 shRNA-GFP. A total of 27 prostate cancer PC-3 xenografts models by subcutaneous injection of prostate cancer cell line PC-3 were established and were divided into 3 groups by random number table, 9 in each group. The Ad5.- ELAV1 shRNA-GFP, Ad5.-GFP and normal saline were injected into the tumor, respectively. The tumor volume was measured once 3 days to observe the growth. After 2 weeks, the volume and weight of the xenograft tumor were measured and the data were statistically analyzed. Results: The results of qRT-PCR and Western blot showed that the expression levels of ELAV1 gene and protein in PC-3 cells were higher than those of RWPE-1 cells. Immunofluorescence and immunohistochemistry showed that the ELAV1 protein in PC-3 cells was mainly expressed in the cytoplasm, while the main expression was in the nucleus in RWPE-1 cells. Immunohistochemical results of 12 cases of prostate cancer and paracancerous specimens also showed that ELAV1 protein mainly expressed in cytoplasm of prostate cancer and mainly expressed in the nucleus of adjacent tissues. The results of CCK-8 and cycle test showed that the proliferation ability of PC-3 cells decreased after Ad5.- ELAV1 shRNA-GFP infection, and the cycle was arrested in the G1 phase. Western Blot confirmed that the expreesions of COX-2 related to tumor cell proliferation, Cyclin D1 related to cell cycle, and PI3K, Akt and phosphorylated Akt of tumor classical signaling pathway were significantly reduced after silencing of ELAV1 gene. The volume and weight in Ad5.- ELAV1 shRNA-GFP group was significantly lower than those in the control group(P<0.05). Conclusion: ELAV1 protein is highly expressed in prostate cancer and mainly expressed in cytoplasm. The ELAV1 gene promotes the growth of prostate cancer cells by classical PI3K/Akt signaling pathway.