目的:GPX8,在内质网腔中发现,是谷胱甘肽过氧化物酶(GPXs)家族的成员。其在肝细胞癌(HCC)中的作用尚不清楚。
方法:免疫组织化学染色用于检测HCC组织微阵列中GPX8的蛋白水平。短发夹RNA慢病毒用于敲除GPX8,并使用转录组测序和磷酸化激酶阵列研究主要信号通路。球体形成测定,克隆形成试验和细胞迁移试验用于评估HCC细胞的干性和迁移能力。通过免疫沉淀和蛋白质质谱实现GPX8相互作用蛋白的鉴定。
结果:肝癌患者GPX8蛋白水平下调。GPX8蛋白低表达与HCC患者早期复发及预后不良有关。GPX8敲低可增强肝癌细胞的干性和迁移能力。始终如一,基于转录组分析,多种信号通路,包括PI3K-AKT和调节干细胞多能性的信号通路,在GPX8敲除后被激活。GPX8的下调可以增加肿瘤干性标志物KLF4、OCT4和CD133的表达。GPX8在体内下调还可促进肝癌细胞皮下成瘤和迁移能力。MK-2206是AKT的小分子抑制剂,可以在体内和体外逆转促肿瘤作用。我们发现GPX8和71kDa热休克同源蛋白(Hsc70)具有直接相互作用。AKT的磷酸化促进了Hsc70转位进核和PI3Kp110亚基的表达,从而增加GPX8的下调。
结论:这项研究的发现证明了GPX8通过灭活Hsc70/AKT途径在HCC中的抗癌活性。结果提示了HCC的可能治疗靶点。
OBJECTIVE: GPX8, which is found in the endoplasmic reticulum lumen, is a member of the Glutathione Peroxidases (GPXs) family. Its role in hepatocellular carcinoma (HCC) is unknown.
METHODS: Immunohistochemical staining was used to detect the protein levels of GPX8 in HCC tissue microarrays. A short hairpin RNA lentivirus was used to knock down GPX8, and the main signaling pathways were investigated using transcriptome sequencing and a phosphorylated kinase array. The sphere formation assays, cloning-formation assays and cell migration assays were used to evaluate the stemness and migration ability of HCC cells. Identifying the GPX8-interacting proteins was accomplished through immunoprecipitation and protein mass spectrometry.
RESULTS: The GPX8 protein levels were downregulated in HCC patients. Low expression of GPX8 protein was related to early recurrence and poor prognosis in HCC patients. GPX8 knockdown could enhance the stemness and migration ability of HCC cells. Consistently, Based on transcriptome analysis, multiple signaling pathways that include the PI3K-AKT and signaling pathways that regulate the pluripotency of stem cells, were activated after GPX8 knockdown. The downregulation of GPX8 could increase the expression of the tumor stemness markers KLF4, OCT4, and CD133. The in vivo downregulation of GPX8 could also promote the subcutaneous tumor-forming and migration ability of HCC cells. MK-2206, which is a small-molecule inhibitor of AKT, could reverse the tumor-promoting effects both in vivo and in vitro. We discovered that GPX8 and the 71-kDa heat shock cognate protein (
Hsc70) have a direct interaction. The phosphorylation of AKT encouraged the translocation of
Hsc70 into the nucleus and the expression of the PI3K p110 subunit, thereby increasing the downregulation of GPX8.
CONCLUSIONS: The findings from this study demonstrate the anticancer activity of GPX8 in HCC by inactivating the
Hsc70/AKT pathway. The results suggest a possible therapeutic target for HCC.