Aloin decelerates the progression of hepatocellular carcinoma through circ_0011385/miR-149-5p/WT1 axis

CircRNA/miRNA/mRNA axis has been reported to play crucial regulatory roles in multiple cancers, including hepatocellular carcinoma (HCC). In addition, recent investigations revealed that aloin exerted anti-tumor functions in HCC. However, the underlying mechanism of aloin on anti-tumor functions in HCC remained elusive. Therefore, this study aimed to investigate whether circRNA/miRNA/mRNA axis medicated the anti-tumor effect of aloin in HCC. Cell viability, invasion, apoptosis and autophagy were accessed by cell counting kit-8 (CCK-8), transwell invasion assay, flow cytometry, Western blot and immunofluorescence analysis, respectively. Expression levels of circ_0011385, miR-149-5p and WT1 mRNA were determined using qRT-PCR assay. Binding sites between miR-149-5p and circ_0011385 or WT1 were predicted in starBase database. The binding relationship among circ_0011385, miR-149-5p and WT1 were verified by dual-luciferase reporter assay and RNA immunoprecipitation. Besides, the rescue experiments were performed by co-transfection with cric_0011385 overexpression plasmid, si-cric_0011385, miR-149-5p mimic and inhibitor, WT1 pDNA and si-WT1 in HCC cells. Furthermore, tumor growth was also investigated in the xenograft mouse model. Aloin inhibited HCC proliferation and invasion as well as promoted apoptosis and autophagy both <i>in vitro</i> and <i>in vivo</i>. Besides, aloin suppressed circ_0011385 expression. Overexpressed circ_0011385 partially reversed the anti-tumor effect of aloin on HCC. In addition, it was revealed that the circ_0011385, miR-149-5p and WT1 genes were abnormally expressed in HCC. Furthermore, the binding interactions between circ_0011385, miR-149-5p and WT1 were predicted and confirmed. Moreover, the effect of circ_0011385 on the anti-tumor role of aloin in HCC was rescued by miR-149-5p mimics. MiR-149-5p regulated HCC progression via modulating WT1. Aloin suppressed cell proliferation, invasion and tumor growth and promoted apoptosis and autophagy in HCC through regulating circ_0011385/miR-149-5p/WT1 axis. Aloin may be a potential candidate drug for HCC treatment.<b>Abbrevations:</b> HCC: Hepatocellular carcinoma; ceRNA: competing endogenous RNA; miRNA: microRNA; MREs: miRNA response elements; WT1: Wilms’ tumor 1; MMP-2: Matrix metalloproteinase; EMT: epithelial-mesenchymal transition; GADPH: glyceraldehyde 3-phosphate dehydrogenase; WT: wild type; MUT: mutant type; DMEM: dulbecco’s modified eagle medium.

环状RNA（circular RNA, circRNA）/微小RNA（microRNA, miRNA）/信使RNA（messenger RNA, mRNA）调控轴已被证实在包括肝细胞癌（hepatocellular carcinoma, HCC）在内的多种癌症中发挥关键调控作用。此外，近期研究表明芦荟大黄素（aloin）在肝细胞癌中可发挥抗肿瘤功能，但芦荟大黄素在肝细胞癌中产生抗肿瘤作用的具体分子机制仍尚不明确。因此，本研究旨在探究circRNA/miRNA/mRNA调控轴是否介导了芦荟大黄素在肝细胞癌中的抗肿瘤效应。
本研究分别采用细胞计数试剂盒-8（cell counting kit-8, CCK-8）、Transwell侵袭实验、流式细胞术、蛋白质印迹法及免疫荧光分析，检测细胞活力、侵袭能力、细胞凋亡及自噬水平；通过实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测circ_0011385、miR-149-5p及WT1 mRNA的表达水平。通过starBase数据库预测miR-149-5p与circ_0011385或WT1的结合位点，并采用双荧光素酶报告基因实验及RNA免疫沉淀实验验证三者之间的结合关系。此外，本研究通过在肝细胞癌细胞中共转染circ_0011385过表达质粒、si-circ_0011385、miR-149-5p模拟物及抑制剂、WT1过表达质粒（WT1 pDNA）及si-WT1，完成挽救实验；同时通过异种移植瘤小鼠模型探究肿瘤生长情况。
实验结果显示，芦荟大黄素在体内外均能抑制肝细胞癌的增殖与侵袭，并促进细胞凋亡与自噬；同时可下调circ_0011385的表达。过表达circ_0011385可部分逆转芦荟大黄素对肝细胞癌的抗肿瘤效应。此外，研究发现circ_0011385、miR-149-5p及WT1基因在肝细胞癌中存在异常表达，且三者之间的结合相互作用已被预测并验证。miR-149-5p模拟物可挽救circ_0011385对芦荟大黄素抗肝细胞癌活性的抑制作用；miR-149-5p可通过调控WT1的表达影响肝细胞癌的进展。综上，芦荟大黄素可通过调控circ_0011385/miR-149-5p/WT1调控轴，抑制肝细胞癌细胞的增殖、侵袭及肿瘤生长，并促进细胞凋亡与自噬，有望成为肝细胞癌治疗的潜在候选药物。
缩略语：
HCC：肝细胞癌（hepatocellular carcinoma）
ceRNA：竞争性内源RNA（competing endogenous RNA）
miRNA：微小RNA（microRNA）
MREs：miRNA应答元件（miRNA response elements）
WT1：肾母细胞瘤1（Wilms’ tumor 1）
MMP-2：基质金属蛋白酶-2（Matrix metalloproteinase）
EMT：上皮间质转化（epithelial-mesenchymal transition）
GADPH：甘油醛-3-磷酸脱氢酶（glyceraldehyde 3-phosphate dehydrogenase）
WT：野生型（wild type）
MUT：突变型（mutant type）
DMEM：达尔伯克改良伊格尔培养基（dulbecco’s modified eagle medium）