Wang Shiyang, Wang Tianlong, Wang Li, Zhong Liansheng, Li Kai
Department of Geriatric Surgery, The First Hospital, China Medical University, Shenyang 110001, People's Republic of China.
Department of Surgical Oncology, The First Hospital, China Medical University, Shenyang 110001, People's Republic of China.
Onco Targets Ther. 2020 Oct 28;13:10917-10929. doi: 10.2147/OTT.S271855. eCollection 2020.
RING finger protein 126 (RNF126), as a novel E3 ubiquitin ligase, plays an oncogenic role in several solid cancers. But its potential role in colorectal cancer (CRC) that harbored 50% mutant p53, to our knowledge, is rarely reported.
We investigated the clinical significance and relationship of RNF126 and p53 in CRC tissues and cells. Meanwhile, WB, qRT-PCR, co-IP, MTT, and transwell were used to investigate the function and molecular mechanism of RNF126 in regulating malignant biology in vitro.
RNF126 was overexpressed in human CRC specimens, which was tightly associated with tumor size (=0.021), T stage (=0.030), lymph node metastasis (=0.006), TNM stage (=0.001), and the poor survival (=0.003) of CRC patients. RNF126 had no association with p53 mutation in CRC specimens, and in p53 mutant Colo-205 and SW620 cells. However, in p53 wildtype HCT116 and HCT-8 cells, RNF126 silencing upregulated p53 and p21 but inhibited Rb phosphorylation at Serine 780 (pRb), which was inhibited by p53siRNA. Conversely, RNF126 overexpression downregulated p53 and p21 but promoted pRb expression, which was reversed by a classic proteasome inhibitor, MG132. However, the mRNA levels of above target genes were unchanged, implying a ubiquitination dependent post-translational modification involving in above regulation. Meanwhile, RNF126 was co-immunoprecipitated with p53 and p21 to form a triple complex. RNF126 silencing and overexpression inhibited and promoted p53 ubiquitination and degradation in vitro, respectively. In addition, p53siRNA reversed RNF126 silencing-inhibited cell proliferation, drug resistance, and cell mobility in HCT116 cells. Conversely, MG132 inhibited RNF126 overexpression-promoted above cell biology in HCT-8 cells.
Overexpression of RNF126 was remarkably associated with multiple advanced clinical characters of CRC patients independent of mutant p53. RNF126 promotes cell proliferation, mobility, and drug resistance in CRC via enhancing p53 ubiquitination and degradation.
环状指蛋白126（RNF126）作为一种新型E3泛素连接酶，在多种实体癌中发挥致癌作用。但据我们所知，其在50%携带p53突变的结直肠癌（CRC）中的潜在作用鲜有报道。
我们研究了RNF126和p53在CRC组织和细胞中的临床意义及关系。同时，运用蛋白质免疫印迹法（WB）、定量逆转录聚合酶链反应（qRT-PCR）、免疫共沉淀（co-IP）、噻唑蓝比色法（MTT）和Transwell实验来研究RNF126在体外调节恶性生物学行为的功能及分子机制。
RNF126在人类CRC标本中过表达，这与肿瘤大小（=0.021）、T分期（=0.030）、淋巴结转移（=0.006）、TNM分期（=0.001）以及CRC患者的不良生存（=0.003）密切相关。在CRC标本以及p53突变的Colo-205和SW620细胞中，RNF126与p53突变无关联。然而，在p53野生型的HCT116和HCT-8细胞中，RNF126沉默上调了p53和p21，但抑制了丝氨酸780位点的视网膜母细胞瘤蛋白（Rb）磷酸化（pRb），而p53小干扰RNA（siRNA）可抑制这种作用。相反，RNF126过表达下调了p53和p21，但促进了pRb表达，经典蛋白酶体抑制剂MG132可逆转这种作用。然而，上述靶基因的mRNA水平未发生变化，这意味着存在一种依赖泛素化的翻译后修饰参与上述调节过程。同时，RNF126与p53和p21进行免疫共沉淀形成三聚体复合物。RNF126沉默和过表达分别在体外抑制和促进了p53的泛素化及降解。此外，p53siRNA逆转了RNF126沉默对HCT116细胞增殖、耐药性及细胞迁移能力的抑制作用。相反，MG132抑制了RNF126过表达对HCT-8细胞上述生物学行为的促进作用。
RNF126的过表达与CRC患者的多个晚期临床特征显著相关，且与p53突变无关。RNF126通过增强p53的泛素化及降解促进CRC细胞的增殖、迁移和耐药性。
