Department of Ophthalmology, Jiangxi Children's Hospital, Nanchang, Jiangxi 330006, P.R. China.
Department of Ultrasound, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi 330006, P.R. China.
Int J Mol Med. 2018 Mar;41(3):1651-1658. doi: 10.3892/ijmm.2018.3379. Epub 2018 Jan 10.
The aim of the present study was to investigate the effects of high-mobility group protein B1 (HMGB1) silencing on the susceptibility of retinoblastoma (RB) cells to chemotherapeutic drugs and the underlying molecular mechanisms. Western blot analysis revealed that vincristine (VCR), etoposide (ETO) and carboplatin (CBP) significantly increased the expression of HMGB1 in Weri‑Rb-1 and Y79 cells compared with the untreated control (P<0.01). siRNA HMGB1 and siRNA negative control (NC) were transfected to Y79 cells by Lipofectamine™ 2000 and, following VCR treatment, the expression of HMGB1 and nuclear factor-κB (NF-κB) was analyzed. siRNA HMGB1 transfection silenced HMGB1 expression. The cytotoxicity of VCR to cells with and without siRNA HMGB1 was investigated by methyl thiazolyl tetrazolium (MTT) assay. siRNA HMGB1 markedly reduced the IC50 value of VCR to RB cells through downregulating the expression of NF-κB, similar to pyrrolidinedithiocarbamate (PDTC). Moreover, following siRNA HMGB1, siRNA NC and ammonium PDTC treatment, the apoptosis of RB cells with VCR incubation was evaluated by Hoechst staining, and the expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), Beclin 1 and p62 were determined with western blot analysis. The LC3 puncta were determined with immunofluorescence assay. The results demonstrated that VCR treatment significantly downregulated the expression of cleaved caspase-3, cleaved PARP and p62, and upregulated the expression of Beclin 1 in RB cells (P<0.01). Similar to the NF-κB inhibitor PDTC, siRNA HMGB1 significantly promoted apoptosis and suppressed autophagy of VCR‑treated RB cells through reversing the effects of VCR on these signaling molecules (P<0.01). Therefore, HMGB1 silencing promoted the susceptibility of RB cells to chemotherapeutic drugs through downregulating NF-κB.
本研究旨在探讨高迁移率族蛋白 B1（HMGB1）沉默对视网膜母细胞瘤（RB）细胞对化疗药物敏感性的影响及其潜在的分子机制。Western blot 分析显示，与未处理的对照组相比，长春新碱（VCR）、依托泊苷（ETO）和卡铂（CBP）显著增加了 Weri-Rb-1 和 Y79 细胞中 HMGB1 的表达（P<0.01）。通过 Lipofectamine™2000 将 siRNA HMGB1 和 siRNA 阴性对照（NC）转染至 Y79 细胞，并在 VCR 处理后分析 HMGB1 和核因子-κB（NF-κB）的表达。siRNA HMGB1 转染可沉默 HMGB1 的表达。通过甲基噻唑基四唑（MTT）测定法研究了 VCR 对有无 siRNA HMGB1 的细胞的细胞毒性。siRNA HMGB1 通过下调 NF-κB 的表达，显著降低了 VCR 对 RB 细胞的 IC50 值，与吡咯烷二硫代氨基甲酸盐（PDTC）相似。此外，在用 siRNA HMGB1、siRNA NC 和铵 PDTC 处理后，通过 Hoechst 染色评估了 VCR 孵育后 RB 细胞的凋亡，并用 Western blot 分析测定了裂解的 caspase-3、裂解的多聚（ADP-核糖）聚合酶（PARP）、Beclin 1 和 p62 的表达。用免疫荧光测定法测定 LC3 斑点。结果表明，VCR 处理显著下调了 RB 细胞中裂解的 caspase-3、裂解的 PARP 和 p62 的表达，并上调了这些信号分子的表达（P<0.01）。与 NF-κB 抑制剂 PDTC 相似，siRNA HMGB1 通过逆转 VCR 对这些信号分子的作用，显著促进了 VCR 处理的 RB 细胞的凋亡并抑制了自噬（P<0.01）。因此，HMGB1 沉默通过下调 NF-κB 促进了 RB 细胞对化疗药物的敏感性。
