CAJ | 학술논문

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：qRT-PCR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。
배경여목적：미소RNA(microRNA，miRNA，miR)재종류적발생、발전중구유중요적작용。miR-222재다충종류조직중표체상조，이기재신암(renal cell carcinoma，RCC)중적표체급기작용궤제상불청초。본연구의통과검측RCC조직급상응암방조직중miR-222적표체정황，탐토miR-222재RCC중적작용。병재체외조건하，통과검측miR-222적하유작용파점，탐토miR-222적항RCC작용궤제。방법：채용실시정량취합매련반응(quantitative real-time polymerase chain reaction，qRT-PCR)검측RCC조직화암방조직중miR-222적표체；채용CCK-8법검측세포적증식활력；채용단백[질]인적법(Western blot)검측DDIT4단백급LC3-Ⅱ적표체；채용형광소매실험험증miR-222적작용파점；전염EGFP-LC3후재격광공취초현미경하관찰세포자서상태。결과：qRT-PCR검측결과현시，miR-222재RCC조직중표체명현상조。재인신투명세포암786-O세포주중고저miR-222표체후현저억제세포적증식활력，이과표체miR-222가증강786-O세포적증식활력(P<0.01)。재786-O세포중고저miR-222후，기파기인DDIT4단백적표체현저상조，과표체miR-222후，DDIT4표체명현하조。형광소매실험결과표명，DDIT4위miR-222적직접작용파점。RCC조직적DDIT4표체하조。재786-O세포중억제miR-222표체후，LC3-Ⅱ적표체수평현저상조，자서체수목현저증가。결론：miR-222재RCC중고표체，병가능통과파향억제DDIT4적표체삼여조공RCC세포자서。
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 speciifc 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 up-regulated in 786-O cells transfected with miR-222 antagomiR, whereas overexpression of miR-222 induced the down-regulation 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 signiifcant 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 down-regulating the expression of DDIT4.