Ma Xin, Fan Yang, Gao Yu, Zhang Yu, Huang Qingbo, Ai Qing, Ni Dong, Chen Weihao, Zhang Peng, Song Erlin, Wang Baojun, Shi Taoping, Zheng Tao, Zhang Xu
State Key Laboratory of Kidney Diseases, Department of Urology, Military Postgraduate Medical College, Chinese People's Liberation Army General Hospital, Beijing, People's Republic of China.
Medical School, Nankai University, Tianjin, People's Republic of China.
Urol Oncol. 2014 Jan;32(1):46.e9-17. doi: 10.1016/j.urolonc.2013.06.011. Epub 2013 Oct 4.
Although emerging evidence has shown that the deregulation of micro-ribonucleic acid (RNA) biogenesis machinery is involved in various human malignancies, this role has not been investigated in clear cell renal cell carcinoma (ccRCC). This study aims to determine whether Dicer, a key enzyme responsible for biogenesis of microRNA, is deregulated in ccRCC. The biological roles of Dicer in vitro are also determined.
The expression of Dicer at messenger RNA and protein levels was detected by real-time quantitative polymerase chain reaction and western blot, respectively, in human kidney tubule epithelial cell line, nonmetastatic 786-O ccRCC cell line, and metastatic ACHN ccRCC cell line, as well as in 42 cases of ccRCC surgical specimens including 14 cases with distant metastasis and their corresponding adjacent normal renal tissues. Dicer expression levels in specimens were also measured by immunohistochemical staining. Knockdown of Dicer expression in 786-O and ACHN ccRCC cell lines was achieved by transfecting short interfering RNA against Dicer. The effects of Dicer on cell proliferation, migration, and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, flow cytometric analyses, and Boyden chamber Transwell assay, respectively.
Compared with human kidney tubule epithelial cell line, Dicer expression levels were significantly down-regulated in 786-O and ACHN ccRCC cell lines, with the metastatic ACHN ccRCC cell line having even lower levels. Meanwhile, Dicer expression levels were significantly down-regulated in ccRCC surgical specimens compared with adjacent normal renal tissues, with the metastatic ones further reduced, and Dicer messenger RNA levels were significantly correlated with overall tumor-node-metastasis stage of ccRCC. In vitro, the knockdown of Dicer significantly promoted cell proliferation, migration, and invasion.
Reduced expression of Dicer may play a role in the tumorigenesis of ccRCC and further decline may be associated with distant metastasis of ccRCC.
尽管新出现的证据表明微小核糖核酸（RNA）生物合成机制的失调与多种人类恶性肿瘤有关，但在透明细胞肾细胞癌（ccRCC）中尚未对这一作用进行研究。本研究旨在确定微小RNA生物合成的关键酶Dicer在ccRCC中是否失调。同时也确定Dicer在体外的生物学作用。
分别通过实时定量聚合酶链反应和蛋白质印迹法检测人肾小管上皮细胞系、非转移性786 - O ccRCC细胞系和转移性ACHN ccRCC细胞系，以及42例ccRCC手术标本（包括14例有远处转移的病例）及其相应的癌旁正常肾组织中Dicer在信使RNA和蛋白质水平的表达。还通过免疫组织化学染色测量标本中Dicer的表达水平。通过转染针对Dicer的小干扰RNA实现786 - O和ACHN ccRCC细胞系中Dicer表达的敲低。分别通过3 -（4,5 - 二甲基噻唑 - 2 - 基）- 5 -（3 - 羧甲氧基苯基）- 2 -（4 - 磺基苯基）- 2H - 四唑内盐（MTS）试验、流式细胞术分析和Boyden小室Transwell试验检测Dicer对细胞增殖、迁移和侵袭的影响。
与人类肾小管上皮细胞系相比，786 - O和ACHN ccRCC细胞系中Dicer表达水平显著下调，转移性ACHN ccRCC细胞系中的水平更低。同时，与癌旁正常肾组织相比，ccRCC手术标本中Dicer表达水平显著下调，有远处转移的标本中进一步降低，且Dicer信使RNA水平与ccRCC的总体肿瘤 - 淋巴结 - 转移分期显著相关。在体外，Dicer的敲低显著促进细胞增殖、迁移和侵袭。
Dicer表达降低可能在ccRCC的肿瘤发生中起作用，其进一步下降可能与ccRCC的远处转移有关。
