Kawahara Naoki, Mizutani Ayano, Matsubara Sho, Takeda Yoshinori, Kobayashi Hiroshi
Department of Obstetrics and Gynecology, Nara Medical University, Kashihara, Nara 634-8522, Japan.
Exp Ther Med. 2020 Nov;20(5):122. doi: 10.3892/etm.2020.9250. Epub 2020 Sep 21.
Deubiquitinase USP28 is a target gene of the transcription factor HNF1 homeobox β (HNF-1β), which promotes the survival of ovarian clear cell carcinoma (OCCC) cell lines. However, the pharmacological inhibition of HNF-1β can cause several adverse effects as it is abundantly expressed in numerous organ systems, including the kidney, liver, pancreas and digestive tract. Therefore, small interfering RNA (siRNA) screening was performed in the current study to identify other potential downstream targets of the HNF-1β-mediated pathway. The results revealed that glycogen synthase kinase-3β (GSK-3β) may be a potential downstream target affecting cell viability. To further clarify the effects of GSK-3β, two human OCCC cell lines, TOV-21G (HNF-1β overexpressing line) and ES2 (HNF-1β negative) were transfected with siRNA targeting GSK-3β or control vectors. Loss-of-function studies using RNAi-mediated gene silencing indicated that HNF-1β facilitated GSK-3β expression, resulting in the loss of phosphorylated nuclear factor-κB (p-NFκB) and the reduction of TOV-21G cell proliferation. The cell proliferation assay also revealed that GSK-3β inhibitors rescued the effects of HNF-1β silencing on cell viability in a dose-dependent manner. Furthermore, the GSK-3β inhibitor, AR-A014418, effectively inhibited tumor cell proliferation in a xenograft mouse model. In conclusion and to the best of our knowledge, the current study was the first to determine that GSK-3β is a target gene of HNF-1β. In addition, the results of the present study revealed the novel HNF-1β-GSK-3β-p-NFκB pathway, occurring in response to DNA damage. Targeting this pathway may therefore represent a putative, novel, anticancer strategy in patients with OCCC.
去泛素化酶USP28是转录因子肝细胞核因子1同源框β（HNF-1β）的靶基因，HNF-1β可促进卵巢透明细胞癌（OCCC）细胞系的存活。然而，对HNF-1β的药理抑制会导致多种不良反应，因为它在包括肾脏、肝脏、胰腺和消化道在内的众多器官系统中大量表达。因此，本研究进行了小干扰RNA（siRNA）筛选，以确定HNF-1β介导途径的其他潜在下游靶点。结果显示，糖原合酶激酶-3β（GSK-3β）可能是影响细胞活力的潜在下游靶点。为了进一步阐明GSK-3β的作用，用靶向GSK-3β的siRNA或对照载体转染了两个人OCCC细胞系，TOV-21G（HNF-1β过表达细胞系）和ES2（HNF-1β阴性细胞系）。使用RNAi介导的基因沉默进行的功能丧失研究表明，HNF-1β促进GSK-3β表达，导致磷酸化核因子κB（p-NFκB）缺失以及TOV-21G细胞增殖减少。细胞增殖试验还显示，GSK-3β抑制剂以剂量依赖性方式挽救了HNF-1β沉默对细胞活力的影响。此外，GSK-3β抑制剂AR-A014418在异种移植小鼠模型中有效抑制了肿瘤细胞增殖。总之，据我们所知，本研究首次确定GSK-3β是HNF-1β的靶基因。此外，本研究结果揭示了响应DNA损伤而发生的新的HNF-1β-GSK-3β-p-NFκB途径。因此，靶向该途径可能代表一种针对OCCC患者的新型抗癌策略。
