ABSTRACT

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death, and radiotherapy is currently one of the main treatments. Long non-coding RNAs (lncRNAs) are associated with the radiosensitivity and tumorigenesis of HCC. However, the role and molecular mechanism of potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) in HCC are still unclear. The relative expression of KCNQ1OT1, microRNA-146a-5p (miR-146a-5p) and alkaline ceramidase 3 (ACER3) was quantified by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Clonogenic assay was used to assess the radiosensitivity of cells. Cell apoptosis and metastasis were evaluated by flow cytometry and transwell assays, respectively. The protein levels of apoptosis markers, metastasis markers and ACER3 were detected by western blot (WB) analysis. The relationship between miR-146a-5p and KCNQ1OT1 or ACER3 was determined by dual-luciferase reporter assay. Additionally, animal experiments were carried out to explore the effect of KCNQ1OT1 silencing on HCC tumor growth in vivo. KCNQ1OT1 was highly expressed in HCC, and its knockdown hindered the proliferation and metastasis, while increased the radiosensitivity and apoptosis of HCC cells. MiR-146a-5p could interact with KCNQ1OT1, and its inhibition reversed the effects of silenced-KCNQ1OT1 on the radiosensitivity and tumorigenesis of HCC cells. Besides, ACER3 was a target of miR-146a-5p, and its overexpression inversed the effects of miR-146a-5p mimic on the radiosensitivity and tumorigenesis of HCC cells. The expression of ACER3 was regulated by KCNQ1OT1 and miR-146a-5p. Furthermore, KCNQ1OT1 also could reduce the growth of HCC by regulating the miR-146a-5p/ACER3 axis in vivo. Our study suggested that KCNQ1OT1 improved ACER3 expression to regulate the radiosensitivity and tumorigenesis of HCC through sponging miR-146a-5p, indicating that KCNQ1OT1 might be a new therapeutic target for HCC.

摘要

肝细胞癌（HCC）是癌症相关死亡的第三大原因，放疗是目前主要的治疗方法之一。长链非编码 RNA (lncRNA) 与 HCC 的放射敏感性和肿瘤发生有关。然而，钾电压门控通道亚家族 Q 成员 1 重叠转录物 1 (KCNQ1OT1) 在 HCC 中的作用和分子机制仍不清楚。KCNQ1OT1、microRNA-146a-5p (miR-146a-5p) 和碱性神经酰胺酶 3 (ACER3) 的相对表达通过定量实时聚合酶链反应 (qRT-PCR) 进行量化。通过 3-(4, 5-二甲基噻唑-2-基)-2, 5-二苯基溴化四唑 (MTT) 测定法测量细胞增殖。克隆形成试验用于评估细胞的放射敏感性。细胞凋亡和转移分别通过流式细胞术和 transwell 测定进行评估。通过蛋白质印迹（WB）分析检测细胞凋亡标志物、转移标志物和ACER3的蛋白质水平。miR-146a-5p 与 KCNQ1OT1 或 ACER3 之间的关系由双荧光素酶报告基因测定确定。此外，还进行了动物实验以探讨 KCNQ1OT1 沉默对 HCC 肿瘤生长的影响体内。KCNQ1OT1在HCC中高表达，其敲低阻碍了HCC细胞的增殖和转移，同时增加了HCC细胞的放射敏感性和凋亡。MiR-146a-5p 可以与 KCNQ1OT1 相互作用，其抑制作用逆转了沉默的 KCNQ1OT1 对 HCC 细胞放射敏感性和肿瘤发生的影响。此外，ACER3 是 miR-146a-5p 的靶标，其过表达逆转了 miR-146a-5p 模拟物对 HCC 细胞放射敏感性和肿瘤发生的影响。ACER3 的表达受 KCNQ1OT1 和 miR-146a-5p 的调控。此外，KCNQ1OT1还可以通过在体内调节miR-146a-5p/ACER3轴来减少HCC的生长. 我们的研究表明，KCNQ1OT1 通过海绵 miR-146a-5p 提高 ACER3 表达以调节 HCC 的放射敏感性和肿瘤发生，表明 KCNQ1OT1 可能是 HCC 的新治疗靶点。