Fang Quangang, Liu Haiyun, Zhou Aiqun, Zhou Huaping, Zhang Zhiyong
Department of Laboratory, Jiangxi Provincial People's Hospital Affiliated to Nanchang University, Nanchang 330006, Jiangxi, People's Republic of China.
Cancer Manag Res. 2020 Aug 4;12:6849-6860. doi: 10.2147/CMAR.S253510. eCollection 2020.
Many studies have confirmed that circular RNAs (circRNAs) play a key role in the biological progression of cancers. However, the function of a novel circRNA, circ_0046599, in hepatocellular carcinoma (HCC) progression has not been explored.
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression of circ_0046599, microRNA (miR)-1258 and Ribophorin II (RPN2). Subcellular fractionation location assay was used to localize circ_0046599 in HCC cells. The circular characteristic of circ_0046599 was verified using Ribonuclease R (RNase R) digestion assay. Besides, cell counting kit 8 (CCK8) assay, colony formation assay, wound healing assay and transwell assay were used to detect cell proliferation, migration and invasion, respectively. The lactate production and glucose level were determined by Lactate and Glucose Assay Kits. Furthermore, the protein levels of glycolysis, metastasis and proliferation-related marker proteins, as well as RPN2 were tested by Western blot (WB) analysis. Moreover, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to confirm the interactions among circ_0046599, miR-1258 and RPN2. In addition, mice xenograft models were applied to observe the effect of circ_0046599 silencing on HCC tumor growth in vivo.
Circ_0046599 was highly expressed in HCC tissues and cells, and its knockdown could suppress HCC cell proliferation, migration, invasion and glycolysis process. MiR-1258 could be targeted by circ_0046599, and its inhibitor could invert the suppressing effect of circ_0046599 knockdown on HCC progression. Further, RPN2 was a target of miR-1258. Overexpressed RPN2 could reverse the inhibiting effect of miR-1258 overexpression on HCC progression. Also, knockdown of circ_0046599 could restrain HCC tumor growth in vivo.
Our results provided new evidence that circ_0046599 could promote the progression of HCC by increasing RPN2 expression via sponging miR-1258.
许多研究已证实环状RNA（circRNA）在癌症的生物学进展中起关键作用。然而，一种新型circRNA，即circ_0046599，在肝细胞癌（HCC）进展中的功能尚未得到探索。
采用定量实时聚合酶链反应（qRT-PCR）检测circ_0046599、微小RNA（miR）-1258和核糖体结合蛋白II（RPN2）的表达。亚细胞分级定位试验用于在肝癌细胞中定位circ_0046599。使用核糖核酸酶R（RNase R）消化试验验证circ_0046599的环状特征。此外，分别使用细胞计数试剂盒8（CCK8）试验、集落形成试验、伤口愈合试验和Transwell试验检测细胞增殖、迁移和侵袭。通过乳酸和葡萄糖检测试剂盒测定乳酸产量和葡萄糖水平。此外，通过蛋白质印迹（WB）分析检测糖酵解、转移和增殖相关标记蛋白以及RPN2的蛋白质水平。此外，采用双荧光素酶报告基因试验和RNA免疫沉淀（RIP）试验来证实circ_0046599、miR-1258和RPN2之间的相互作用。此外，应用小鼠异种移植模型观察circ_0046599沉默对体内HCC肿瘤生长的影响。
Circ_0046599在HCC组织和细胞中高表达，其敲低可抑制HCC细胞增殖、迁移、侵袭和糖酵解过程。MiR-1258可被circ_004659靶向，其抑制剂可逆转circ_0046599敲低对HCC进展的抑制作用。此外，RPN2是miR-1258的靶标。过表达RPN2可逆转miR-1258过表达对HCC进展的抑制作用。此外，circ_0046599的敲低可抑制体内HCC肿瘤生长。
我们的结果提供了新的证据，表明circ_0046599可通过海绵化miR-1258增加RPN2表达来促进HCC的进展。
