Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan 430060, People's Republic of China.
Graefes Arch Clin Exp Ophthalmol. 2011 Sep;249(9):1399-407. doi: 10.1007/s00417-010-1607-3. Epub 2011 Jan 14.
To determine the presence of integrin-linked kinase (ILK) in tissue samples of retinoblastoma patients, and to explore the function of ILK in human Y79 retinoblastoma cells.
The expression of ILK was studied in samples of retinoblastoma patients by immunohistochemistry. In vitro, specific small interfering RNA (siRNA) targeting ILK was transfected into Y79 retinoblastoma cells using liposome. Silencing of ILK expression was measured by reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR and Western blotting assays. Then the regulation of cell proliferation and apoptosis was assessed by Cell Counting Kit-8(CCK-8), Annexin V-FITC/ propidium iodide (PI) immunofluorescence, and flow cytometry assays. Furthermore, the involvement of c-Jun N-terminal kinase signal pathway was tested by JNK signal transduction inhibitor assay.
Positive staining for ILK was detected in 15 of the 17 retinoblastoma tissue samples. Specific siRNA targeting ILK significantly silenced ILK expression in Y79 retinoblastoma cells, as confirmed by RT-PCR, real-time PCR and Western blotting assays (P < 0.01). This was accompanied by decreased cell proliferation (P < 0.05) and enhanced apoptosis (P < 0.01). The phosphorylation status of JNK and c-Jun was constitutively activated by ILK siRNA (P < 0.01), and JNK inhibitor simultaneously reversed the effects on cell proliferation and apoptosis induced by ILK siRNA.
Our results demonstrated that ILK promoted proliferation and suppressed apoptosis via repressing phosphorylations of the JNK signal pathway in human retinoblastoma cells. This might provide a potential therapeutic target in the treatment of this deadly disease.
检测整合素连接激酶（ILK）在视网膜母细胞瘤患者组织样本中的表达，探讨 ILK 在人 Y79 视网膜母细胞瘤细胞中的作用。
采用免疫组织化学法检测视网膜母细胞瘤患者组织样本中 ILK 的表达。体外采用脂质体转染法将针对 ILK 的特异性小干扰 RNA（siRNA）转染至 Y79 视网膜母细胞瘤细胞中。采用逆转录聚合酶链反应（RT-PCR）、实时 PCR 和 Western blot 检测 ILK 表达的沉默。采用细胞计数试剂盒-8（CCK-8）、Annexin V-FITC/碘化丙啶（PI）免疫荧光和流式细胞术检测细胞增殖和凋亡的调控。进一步采用 c-Jun N 末端激酶信号通路抑制剂检测 JNK 信号转导通路的参与情况。
在 17 例视网膜母细胞瘤组织样本中，有 15 例检测到 ILK 阳性染色。针对 ILK 的特异性 siRNA 显著沉默了 Y79 视网膜母细胞瘤细胞中的 ILK 表达，这通过 RT-PCR、实时 PCR 和 Western blot 检测得到证实（P<0.01）。这伴随着细胞增殖减少（P<0.05）和凋亡增强（P<0.01）。ILK siRNA 持续激活 JNK 和 c-Jun 的磷酸化状态（P<0.01），JNK 抑制剂同时逆转了 ILK siRNA 诱导的细胞增殖和凋亡的作用。
我们的结果表明，ILK 通过抑制 JNK 信号通路的磷酸化促进人视网膜母细胞瘤细胞的增殖并抑制凋亡。这可能为治疗这种致命疾病提供一个潜在的治疗靶点。
Zotero 插件