Prohibitin2 knockdown decreases glioma malignant phenotypes and radio-resistance by inhibiting mitophagy

Prohibitin2 (PHB2), located in inner mitochondrial membrane (IMM), is an important receptor to induce mitophagy. PHB2 was identified as a cancer-promoting factor in most cancers. However, the function of PHB2 in glioma cells remains unclear. This study delved into the impact of PHB2 knockdown on the phenotype, radiosensitivity and mitophagy of glioma cells. PHB2 expression and its clinical relevance in glioma were investigated by western blot, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and TCGA databases. The malignant phenotypes of glioma cells were analyzed in vitro using cell proliferation, cell cycle, wound healing and transwell assay. The radiosensitivity of glioma cells was detected by colony forming assay. The potential mechanism by which PHB2 regulated mitophagy was investigated by coimmunoprecipitation assay. The expression of PHB2 was significantly upregulated in glioma cells and closely correlated with the malignant degree of glioma. The knockdown of PHB2 inhibited the proliferation, migration and invasion activities of glioma cells. Furthermore, PHB2 knockdown enhanced the radiosensitivity of normoxic and hypoxic glioma cells and suppressed the ionizing radiation-induced mitophagy in glioma cells. Cyanide 3-chlorophenylhydrazone (CCCP), a mitophagy agonist, could reverse the phenotypes and radiosensitivity changes elicited by PHB2 knockdown. Additionally, PHB2 regulated the expression of PGAM5 and PINK1 by directly binding to PARL. Our findings revealed that PHB2 knockdown decreased glioma malignant phenotypes and radio-resistance by inhibiting mitophagy <i>via</i> PARL–PGAM5–PINK1–Parkin pathway. PHB2 is a promising candidate target for the development of new therapeutic strategy to enhance the efficacy of radiotherapy for glioma.

定位于线粒体内膜（inner mitochondrial membrane, IMM）的抑制素2（Prohibitin2, PHB2）是诱导线粒体自噬的重要受体。PHB2在多数癌症中被鉴定为促癌因子。然而，PHB2在胶质瘤细胞中的功能仍未明确。本研究探讨了PHB2敲低对胶质瘤细胞表型、放射敏感性及线粒体自噬的影响。研究通过蛋白质印迹（western blot）、定量反转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction, qRT-PCR）及TCGA数据库，探究了PHB2在胶质瘤中的表达情况及其临床相关性。本研究通过细胞增殖实验、细胞周期实验、划痕愈合实验及Transwell实验，体外分析了胶质瘤细胞的恶性表型。采用集落形成实验检测胶质瘤细胞的放射敏感性。通过免疫共沉淀实验（coimmunoprecipitation assay）探究了PHB2调控线粒体自噬的潜在分子机制。结果显示，PHB2在胶质瘤细胞中表达显著上调，且与胶质瘤的恶性程度密切相关。敲低PHB2可抑制胶质瘤细胞的增殖、迁移及侵袭能力。此外，敲低PHB2可增强常氧及缺氧胶质瘤细胞的放射敏感性，并抑制电离辐射诱导的胶质瘤细胞线粒体自噬。线粒体自噬激动剂氰化3-氯苯基腙（Cyanide 3-chlorophenylhydrazone, CCCP）可逆转PHB2敲低所引发的表型及放射敏感性改变。此外，PHB2可通过直接结合PARL，调控PGAM5与PINK1的表达。本研究结果表明，敲低PHB2可通过抑制PARL–PGAM5–PINK1–Parkin通路介导的线粒体自噬，降低胶质瘤的恶性表型与放射抵抗性。PHB2有望成为开发新型治疗策略以增强胶质瘤放射治疗疗效的潜在候选靶点。