miR-222通过下调DDIT4抑制肾癌细胞自噬

doi:10.3969/j.issn.1007-3969.2015.03.001

中国癌症杂志 ›› 2015, Vol. 25 ›› Issue (3): 161-166.doi: 10.3969/j.issn.1007-3969.2015.03.001

miR-222通过下调DDIT4抑制肾癌细胞自噬

倪晓辰，赵志红，马永良，任宗涛，刘彬，张爱莉

河北医科大学第四医院泌尿外科，河北石家庄 050011

出版日期:2015-03-30 发布日期:2015-05-18
通信作者: 张爱莉　E-mail:z13930409899@163.com
基金资助:
河北省医学科学研究重点课题计划(20110142)。

miR-222 can inhibit the autophagy of renal cell carcinoma cells through down-regulating the expression of DDIT4

NI Xiaochen, ZHAO Zhihong, MA Yongliang, REN Zongtao, LIU Bin, ZHANG Aili

Department of Urology, the Fourth Hospital of Hebei Medical University, Shijiazhuang Hebei 050011, China

Published:2015-03-30 Online:2015-05-18
Contact: ZHANG Aili E-mail: z13930409899@163.com

摘要/Abstract

摘要： 背景与目的：微小RNA(microRNA，miRNA，miR)在肿瘤的发生、发展中具有重要的作用。miR-222在多种肿瘤组织中表达上调，而其在肾癌(renal cell carcinoma，RCC)中的表达及其作用机制尚不清楚。本研究拟通过检测RCC组织及相应癌旁组织中miR-222的表达情况，探讨miR-222在RCC中的作用。并在体外条件下，通过检测miR-222的下游作用靶点，探讨miR-222的抗RCC作用机制。方法：采用实时定量聚合酶链反应(quantitative real-time polymerase chain reaction，qRT-PCR)检测RCC组织和癌旁组织中miR-222的表达；采用CCK-8法检测细胞的增殖活力；采用蛋白[质]印迹法(Western blot)检测DDIT4蛋白及LC3-Ⅱ的表达；采用荧光素酶实验验证miR-222的作用靶点；转染EGFP-LC3后在激光共聚焦显微镜下观察细胞自噬状态。结果：qRTPCR检测结果显示，miR-222在RCC组织中表达明显上调。在人肾透明细胞癌786-O细胞株中敲低miR-222表达后显著抑制细胞的增殖活力，而过表达miR-222可增强786-O细胞的增殖活力(P<0.01)。在786-O细胞中敲低miR-222后，其靶基因DDIT4蛋白的表达显著上调，过表达miR-222后，DDIT4表达明显下调。荧光素酶实验结果表明，DDIT4为miR-222的直接作用靶点。RCC组织的DDIT4表达下调。在786-O细胞中抑制miR-222表达后，LC3-Ⅱ的表达水平显著上调，自噬体数目显著增加。结论：miR-222在RCC中高表达，并可能通过靶向抑制DDIT4的表达参与调控RCC细胞自噬。

关键词: 　miR-222, 肾癌, DDIT4, 自噬

Abstract: Background and purpose: MicroRNA (miRNA, miR) plays an important regulatory role in cancer. miR-222 is reported to be up-regulated in various tumors, but its role in renal cell carcinoma (RCC) remains unclear. In this study, we detected the expression of miR-222 in both RCC and adjacent tissue samples. The aim of this study was to investigate the role of miR-222 in RCC. Methods: The expression levels of miR-222 in RCC tissue samples were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). DDIT4 and LC3-Ⅱ protein expressions were determined by Western blot. Dual luciferase assay was performed to verify the target of miR-222. EGFP-LC3 microscopy assay was performed to assess autophagy. Results: Results from qRT-PCR showed that the expression of miR-222 was up-regulated in RCC tissues. Knockdown of miR-222 with specific antagomiR decreased the cell viability of 786-O cells, whereas overexpression of miR-222 increased the cell viability (P<0.01). The levels of DDIT4 were upregulated in 786-O cells transfected with miR-222 antagomiR, whereas overexpression of miR-222 induced the downregulation of DDIT4 expression. Data from dual luciferase assay indicated that miR-222 directly targeted the expression of DDIT4. Consistently, the expression of DDIT4 in RCC tissues was down-regulated compared with adjacent tissues. Knockdown of miR-222 in 786-O cells induced a significant increase of autophagosome formation and LC3 lipidation. These results supported that miR-222 could inhibit autophagy in RCC cells, which may affect the clinical characteristcs of RCC. Conclusion: miR-222 is up-regulated in RCC and can inhibit the autophagy of RCC cells through downregulating the expression of DDIT4.

Key words: miR-222, Renal cell carcinoma, DDIT4, Autophagy

倪晓辰,赵志红,马永良,等. miR-222通过下调DDIT4抑制肾癌细胞自噬[J]. 中国癌症杂志, 2015, 25(3): 161-166.

NI Xiaochen, ZHAO Zhihong, MA Yongliang, et al. miR-222 can inhibit the autophagy of renal cell carcinoma cells through down-regulating the expression of DDIT4[J]. China Oncology, 2015, 25(3): 161-166.

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miR-222通过下调DDIT4抑制肾癌细胞自噬

miR-222 can inhibit the autophagy of renal cell carcinoma cells through down-regulating the expression of DDIT4

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