Gao Zhuo, Jiang Junnan, Hou Lijian, Zhang Bin
Department of Gastroenterology and Endoscopy Center, China-Japan Union Hospital of Jilin University, Changchun 130033, Jilin Province, China.
Department of Gastrointestinal Colorectal Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, Jilin Province, China.
J Transl Int Med. 2022 Mar 26;10(1):65-75. doi: 10.2478/jtim-2021-0043. eCollection 2022 Mar.
RING finger protein 187 (RNF187) belongs to RING domain-containing E3 ligases family, which was recently reported to be involved in oncogenesis and development of several cancers. This research aims to clarify the role of RNF187 in colorectal cancer (CRC) development.
The expression of RNF187 and miR-144-4p were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The levels of RNF187 protein were assessed by western blot analysis. Cell Counting Kit-8 (CCK8) assay, clonogenic assay, cell scratch test and transwell assay were used to determine the proliferation, migration and invasion of CRC cells in vitro. The binding of miR-144-5p and RNF197 mRNA was validated by luciferase reporter assays. Tumor-bearing nude mice were used to determine CRC cells growth in vivo.
RNF187 expression significantly increased in CRC specimens and cell lines compared to normal colon tissues and normal colonic mucosa cell line, respectively. Upregulation of RNF187 expression was inversely correlated to poor prognosis in CRC patients. In addition, knockdown of RNF187 expression inhibited the proliferation, migration, and invasion but promoted the apoptosis of CRC lines Caco-2 and SW480 cells. Further studies validated that RNF187 was the direct target of miR-144-5p. The expression of miR-144-5p was downregulated in CRC tissues, which was negatively correlated to the expression of RNF187. Restoration of miR-144-5p significantly inhibited the progression of CRC cells and its anti-tumor effects could be abrogated by overexpression of RNF187.
Our findings demonstrate the deregulation of miR-144-5p/ RNF187 axis in CRC, as well as its role in regulation of the tumor progression, thus providing a novel therapeutic strategy for CRC treatment.
环指蛋白187（RNF187）属于含环指结构域的E3泛素连接酶家族，最近有报道称其参与多种癌症的发生和发展。本研究旨在阐明RNF187在结直肠癌（CRC）发生发展中的作用。
采用定量实时聚合酶链反应（qRT-PCR）检测RNF187和miR-144-4p的表达。通过蛋白质免疫印迹分析评估RNF187蛋白水平。使用细胞计数试剂盒-8（CCK8）检测、克隆形成试验、细胞划痕试验和Transwell试验来测定CRC细胞在体外的增殖、迁移和侵袭能力。通过荧光素酶报告基因试验验证miR-144-5p与RNF197 mRNA的结合。利用荷瘤裸鼠来测定CRC细胞在体内的生长情况。
与正常结肠组织和正常结肠黏膜细胞系相比，RNF187在CRC标本和细胞系中的表达显著增加。RNF187表达上调与CRC患者的不良预后呈负相关。此外，敲低RNF187表达可抑制CRC细胞系Caco-2和SW480细胞的增殖、迁移和侵袭，但促进其凋亡。进一步研究证实RNF187是miR-144-5p的直接靶点。miR-144-5p在CRC组织中的表达下调，与RNF187的表达呈负相关。恢复miR-144-5p的表达可显著抑制CRC细胞的进展，而RNF187的过表达可消除其抗肿瘤作用。
我们的研究结果表明，miR-144-5p/RNF187轴在CRC中失调，以及其在调节肿瘤进展中的作用，从而为CRC治疗提供了一种新的治疗策略。
