Supplementary Material for: Downregulation of lncRNA FOXD2-AS1 Confers Radiosensitivity to Gastric Cancer Cells via miR-1913/SETD1A Axis

Long noncoding RNA FOXD2 adjacent opposite strand RNA1 (FOXD2-AS1) plays an oncogenic role in various cancers, including gastric cancer (GC). However, the function of FOXD2-AS1 in regulating radiosensitivity of GC cells and its underlying molecular mechanisms have not been elucidated. This study aimed to figure out the potential mechanisms of FOXD2-AS1 in regulating GC cell radiosensitivity. RT-qPCR revealed upregulation of FOXD2-AS1 in GC cells exposed to radiation. Subcellular fractionation assay was used to localize FOXD2-AS1 in GC cells. Colony formation, MTT, EdU, and flow cytometry assays were performed to investigate the role of FOXD2-AS1 in regulating cell proliferation, cell cycle progression, and cell apoptosis. Western blotting was used to assess protein levels of apoptosis-associated markers and SET domain containing 1A (SETD1A). Homologous recombination reporter assay was conducted to explore the effect of FOXD2-AS1 on DNA damage repair. The downstream molecules of FOXD2-AS1 were identified with RNA pulldown, luciferase reporter, and RNA immunoprecipitation assays. The results showed that FOXD2-AS1 knockdown suppressed cell proliferation and cell cycle progression and promoted cell apoptosis and radiosensitivity of GC. FOXD2-AS1 could bind with miR-1913 in GC cells. In addition, miR-1913 targeted SETD1A, which was highly expressed in GC cells. Overexpression of SETD1A reversed FOXD2-AS1 silencing-induced effects on proliferation, apoptosis, and radiosensitivity of GC cells. In conclusion, knocking down FOXD2-AS1 enhances the radiosensitivity of GC cells by sponging miR-1913 to upregulate SETD1A expression.

长链非编码RNA FOXD2相邻反向链RNA1（Long noncoding RNA FOXD2 adjacent opposite strand RNA1，FOXD2-AS1）在包括胃癌（GC）在内的多种癌症中发挥致癌作用。然而，FOXD2-AS1对胃癌细胞放射敏感性的调控作用及其潜在分子机制尚未阐明。本研究旨在明确FOXD2-AS1调控胃癌细胞放射敏感性的潜在机制。
实时定量PCR（RT-qPCR）结果显示，经辐射处理的胃癌细胞中FOXD2-AS1表达上调。采用亚细胞分级实验定位胃癌细胞中FOXD2-AS1的亚细胞分布。通过克隆形成实验、MTT实验、EdU实验及流式细胞术，探究FOXD2-AS1对细胞增殖、细胞周期进程及细胞凋亡的调控作用。采用蛋白质印迹法（Western blotting）检测凋亡相关标志物及SET结构域包含蛋白1A（SET domain containing 1A，SETD1A）的蛋白表达水平。采用同源重组报告基因实验探究FOXD2-AS1对DNA损伤修复的影响。通过RNA下拉实验（RNA pulldown）、荧光素酶报告基因实验及RNA免疫沉淀实验（RNA immunoprecipitation，RIP）鉴定FOXD2-AS1的下游靶分子。
结果显示，敲低FOXD2-AS1可抑制胃癌细胞的增殖与细胞周期进程，同时促进细胞凋亡并增强其放射敏感性。FOXD2-AS1可在胃癌细胞中与miR-1913结合。此外，miR-1913可靶向调控在胃癌细胞中高表达的SETD1A。过表达SETD1A可逆转FOXD2-AS1沉默对胃癌细胞增殖、凋亡及放射敏感性的影响。
综上，敲低FOXD2-AS1可通过海绵吸附miR-1913上调SETD1A的表达，从而增强胃癌细胞的放射敏感性。