Oncogenic Forkhead box D3 antisense RNA 1 promotes cell survival and confers temozolomide resistance in glioblastoma cells through the miR-128-3p/WEE1 G2 checkpoint kinase axis

Although temozolomide (TMZ) is recommended for glioblastoma (GBM) treatment, patients treated with TMZ usually develop TMZ resistance. Thus, there is an urgent need to elucidate the mechanism through which GBM cells acquire TMZ resistance. FOXD3-AS1, a recently discovered lncRNA, shows high expression in diverse cancer types. Nonetheless, its role in GBM remains unclear. This study found that FOXD3-AS1 was overexpressed in GBM cells and associated with dismal prognostic outcome in GBM patients. Functional studies revealed that depletion of FOXD3-AS1 inhibited cell growth and induced apoptosis of GBM cells. Results also showed that FOXD3-AS1 participates in the tolerance of GBM cells to TMZ. Specifically, TMZ-resistant cells exhibited higher FOXD3-AS1 expression compared to parental cells. Overexpression of FOXD3-AS1 increased TMZ tolerance in TMZ sensitive cells, whereas depletion of FOXD3-AS1 sensitized TMZ-resistant cells to TMZ treatment. Mechanistically, WEE1 was positively expressed with FOXD3-AS1. Given that both FOXD3-AS1 and WEE1 contain a binding site for miR-128-3p, FOXD3-AS1 could act as a competing endogenous RNA (ceRNA) to promote WEE1 expression by sponging miR-128-3p. Furthermore, we demonstrated that WEE1 was upregulated in TMZ-resistant GBM cells. Overexpression of WEE1 increased TMZ tolerance in TMZ sensitive cells, whereas deletion of FOXD3-AS1 promoted TMZ-resistant cells to be more sensitive to TMZ. Importantly, depletion of WEE1 could reverse TMZ resistant phenotype in FOXD3-AS1-overexpressed GBM cells. Collectively, our findings reveal a critical role of FOXD3-AS1 in the survival of GBM cells and TMZ resistance, which suggests that FOXD3-AS1 is a potential biomarker for the diagnosis and treatment of GBM.

尽管替莫唑胺（temozolomide, TMZ）是胶质母细胞瘤（glioblastoma, GBM）的推荐治疗方案，但接受TMZ治疗的患者通常会产生TMZ耐药性。因此，迫切需要阐明胶质母细胞瘤细胞获得TMZ耐药性的机制。FOXD3-AS1是近期发现的长链非编码RNA（long non-coding RNA, lncRNA），在多种癌症类型中呈高表达，然而其在胶质母细胞瘤中的作用仍不明确。本研究发现，FOXD3-AS1在胶质母细胞瘤细胞中过表达，且与胶质母细胞瘤患者的不良预后显著相关。功能实验显示，敲低FOXD3-AS1可抑制胶质母细胞瘤细胞的增殖并诱导其凋亡。研究结果还表明，FOXD3-AS1参与了胶质母细胞瘤细胞对TMZ的耐受过程：具体而言，与亲本细胞相比，TMZ耐药细胞的FOXD3-AS1表达水平更高；过表达FOXD3-AS1可增强TMZ敏感细胞对TMZ的耐受性，而敲低FOXD3-AS1则可使TMZ耐药细胞对TMZ治疗重新敏感。从机制层面分析，WEE1的表达与FOXD3-AS1呈正相关。鉴于FOXD3-AS1与WEE1均含有miR-128-3p的结合位点，FOXD3-AS1可作为内源竞争RNA（competing endogenous RNA, ceRNA），通过海绵吸附miR-128-3p促进WEE1的表达。此外，本研究证实WEE1在TMZ耐药的胶质母细胞瘤细胞中呈上调表达；过表达WEE1可增强TMZ敏感细胞对TMZ的耐受性，而敲低FOXD3-AS1则可使TMZ耐药细胞对TMZ治疗更为敏感。值得注意的是，敲低WEE1可逆转过表达FOXD3-AS1的胶质母细胞瘤细胞的TMZ耐药表型。综上，本研究结果揭示了FOXD3-AS1在胶质母细胞瘤细胞存活及TMZ耐药性形成中的关键作用，提示FOXD3-AS1可作为胶质母细胞瘤诊断与治疗的潜在生物标志物。