Zhang Shurong, Cui Zhe
Department of Ophthalmology, The Third Affiliated Hospital of Qiqihar Medical University, Qiqihar, Heilongjiang 161000, P.R. China.
Exp Ther Med. 2021 Mar;21(3):255. doi: 10.3892/etm.2021.9686. Epub 2021 Jan 25.
Retinoblastoma (RB) is one of the most common forms of childhood intraocular cancer. While the occurrence of RB is traditionally associated with dysregulation of the gene, efforts have been made to assess the role of several other pathways that may result in RB. The Notch signaling pathway has been identified as one of the sentinel pathways in retinal development and has been indicated to serve as a tumor suppressor. However, epigenetic modifications of the Notch signaling pathway, and their consequences on tumor establishment and progression, have received little attention. The present study attempted to elucidate the microRNA (miR)-mediated dysregulation of the Notch signaling pathway and its implications on tumor initiation. Upon recruitment of patients with RB (age, 4-25 months), the levels of miR-34b-5p were determined in tumor and adjacent healthy tissues. Simultaneously, the serum levels of miR-34b-5p were measured in tumor and healthy samples using reverse transcriptase-quantitative PCR (RT-qPCR). Binding of miR-34b-5p to Notch1 and Notch2 were confirmed bioinformatically. studies were performed in Y79 and Weri-Rb-1 RB cell lines. The cell lines were transfected with miR-34b-5p constructs and miR-34b-5p overexpression was confirmed using RT-qPCR. The impact of miR-34b-5p overexpression on cell growth and cancer stemness markers (Sox-2, Nanog, and CD133) was examined. The expression levels of Notch1 and Notch2 were evaluated in the presence of miR-34b-5p. The rescue of cell growth and cancer stemness phenotypes was evaluated by co-transfection of miR-34b-5p with Notch1 or Notch2. The results of the present study indicated that the expression levels of miR-34b-5p were reduced in patient tissues and serum samples compared with those in healthy tissues and samples. Notch1 and Notch2 expression level was negatively correlated with the expression level of miR-34b-5p. Overexpression of miR-34b-5p resulted in reduced cell proliferation, migration, invasion and cancer stemness compared with the control group. Further experiments confirmed the inhibitory effects of miR-34b-5p on RB cell proliferation. Upon co-transfection of miR-34b-5p with Notch1 or Notch2, these phenotypes were rescued with reversal of cell growth and tumor sphere formation. Collectively, the results indicated that miR-34b-5p functions as a tumor suppressor in RB via regulating the Notch signaling pathway. Therefore, miR-34b-5p may be explored for its utility as a therapeutic target in RB.
视网膜母细胞瘤（RB）是儿童眼内癌最常见的形式之一。虽然传统上认为RB的发生与该基因的失调有关，但人们已努力评估其他几种可能导致RB的信号通路的作用。Notch信号通路已被确定为视网膜发育中的关键信号通路之一，并被认为具有肿瘤抑制作用。然而，Notch信号通路的表观遗传修饰及其对肿瘤发生和进展的影响却很少受到关注。本研究试图阐明微小RNA（miR）介导的Notch信号通路失调及其对肿瘤起始的影响。招募4至25个月大的RB患者，测定肿瘤组织和相邻健康组织中miR-34b-5p的水平。同时，使用逆转录定量聚合酶链反应（RT-qPCR）测定肿瘤样本和健康样本中miR-34b-5p的血清水平。通过生物信息学方法证实了miR-34b-5p与Notch1和Notch2的结合。在Y79和Weri-Rb-1 RB细胞系中进行了实验。用miR-34b-5p构建体转染细胞系，并使用RT-qPCR确认miR-34b-5p过表达。检测miR-34b-5p过表达对细胞生长和癌症干细胞标志物（Sox-2、Nanog和CD133）的影响。在存在miR-34b-5p的情况下评估Notch1和Notch2的表达水平。通过将miR-34b-5p与Notch1或Notch2共转染来评估细胞生长和癌症干细胞表型的恢复情况。本研究结果表明，与健康组织和样本相比，患者组织和血清样本中miR-34b-5p的表达水平降低。Notch1和Notch2的表达水平与miR-34b-5p的表达水平呈负相关。与对照组相比，miR-34b-5p过表达导致细胞增殖、迁移、侵袭和癌症干细胞特性降低。进一步的实验证实了miR-34b-5p对RB细胞增殖的抑制作用。当miR-34b-5p与Notch1或Notch2共转染时，这些表型得以恢复，细胞生长和肿瘤球形成逆转。总体而言，结果表明miR-34b-5p通过调节Notch信号通路在RB中发挥肿瘤抑制作用。因此，可探索miR-34b-5p作为RB治疗靶点的效用。
