Xiao Wen, Lou Ning, Ruan Hailong, Bao Lin, Xiong Zhiyong, Yuan Changfei, Tong Junwei, Xu Guanghua, Zhou Yali, Qu Yan, Hu Wenjun, Gao Yaoying, Ru Zeyuan, Liu Lei, Xiao Haibing, Chen Ke, Yang Hongmei, Zhang Xiaoping
Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Pathogenic Biology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan, China.
Cell Physiol Biochem. 2017;43(6):2420-2433. doi: 10.1159/000484395. Epub 2017 Oct 27.
BACKGROUND/AIMS: We previously performed microRNA (miRNA) microarray to identify effective indicators of clear cell renal cell carcinoma (ccRCC) tissue samples and preoperative/postoperative plasma in which we identified miR-144-3p as an oncomiRNA. However, the molecular mechanism of miR-144-3p remains unclear. This study aims to explore the roles of miR-144-3p in the invasion, migration and Sunitinib-resistance in ccRCC and to elucidate the underlying mechanisms.
Gain and loss of function approaches were used to investigate the cell proliferation, cycle distribution, clonogenicity, migration, invasion, chemosensitivity of miR-144-3p in vitro. The xenograft model was used to assess the effects of miR-144-3p overexpression on tumorigenesis. Bioinformatics analysis and dual-luciferase reporter assay were used to indentify AT-rich interactive domain 1A (ARID1A) as a direct target gene of miR-144-3p. Quantitative RT-PCR, Western blotting, and immunohistochemical (IHC) staining were used to explore ARID1A expression level of the mRNA and protein.
We found that miR-144-3p overexpression enhanced cell proliferation, clonogenicity, migration, invasion, and chemoresistance in ccRCC cells. Notably, the oncotumor activities of miR-144-3p were mediated by repressing the expression of ARID1A. The downregulation of ARIDIA could promote the function of miR-144-3p in cell proliferation, metastasis and chemoresistance. Consistently, ARID1A mRNA and protein levels were decreased in ccRCC and in nude mice, and they negatively correlated with miR-144-3p.
Higher miR-144-3p may enhance malignancy and resistance to Sunitinib in ccRCC by targeting ARID1A, the observations may uncover novel strategies of ccRCC treatment.
背景/目的：我们之前进行了微小RNA（miRNA）微阵列分析，以鉴定肾透明细胞癌（ccRCC）组织样本以及术前/术后血浆中的有效指标，在此过程中我们将miR-144-3p鉴定为一种致癌miRNA。然而，miR-144-3p的分子机制仍不清楚。本研究旨在探讨miR-144-3p在ccRCC的侵袭、迁移和对舒尼替尼耐药中的作用，并阐明其潜在机制。
采用功能获得和功能缺失方法在体外研究miR-144-3p对细胞增殖、周期分布、克隆形成能力、迁移、侵袭和化学敏感性的影响。利用异种移植模型评估miR-144-3p过表达对肿瘤发生的影响。通过生物信息学分析和双荧光素酶报告基因检测，确定富含AT的相互作用结构域1A（ARID1A）为miR-144-3p的直接靶基因。采用定量逆转录聚合酶链反应（RT-PCR）、蛋白质印迹法和免疫组织化学（IHC）染色来检测ARID1A的mRNA和蛋白质表达水平。
我们发现miR-144-3p过表达增强了ccRCC细胞的增殖、克隆形成能力、迁移、侵袭和化学抗性。值得注意的是，miR-144-3p的致癌活性是通过抑制ARID1A的表达介导的。ARID1A的下调可促进miR-144-3p在细胞增殖、转移和化学抗性方面的功能。同样，ccRCC组织和裸鼠中ARID1A的mRNA和蛋白质水平均降低，且与miR-144-3p呈负相关。
较高水平的miR-144-3p可能通过靶向ARID1A增强ccRCC的恶性程度和对舒尼替尼的耐药性，这些观察结果可能揭示ccRCC治疗的新策略。
