Ji Zhou-Jing, Wang Jian-Li, Chen Li
Department of Dermatology and Venereology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, P.R. China.
Department of Pathological Anatomy, Nantong University, Nantong, Jiangsu, P.R. China.
Oncol Rep. 2015 Aug;34(2):811-22. doi: 10.3892/or.2015.4062. Epub 2015 Jun 15.
The simultaneous silencing of multiple upregulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. In the present study, skin squamous cell carcinoma (SSCC) tissue microarray was constructed and the expression of NET-1 and survivin was identified. The high expression of NET-1 and survivin gene in SSCC was confirmed as an important event for the formation and development of the cancer. A total of 100 primary SSCC patients were included in the present study. Expression of NET-1 and survivin in cancer cells was evaluated immunohistochemically in tissue microarrays. The interaction between NET-1 and survivin in SSCC by co-immunoprecipitation was subsequently verified by producing the siRNA sequence targeting the single gene (siRNA-NET-1 and siRNA-survivin) as well as NET-1 and survivin gene (one-chain-double-target siRNA). The levels of NET-1 and survivin mRNA and protein expression in A431 cells were detected by RT-qPCR and western blotting, and the expression of related genes including vascular endothelial growth factor (VEGF), cortactin, Bcl-2, caspase-3 and -8 was identified using RT-qPCR. The protein localization and expression of NET-1 and survivin in A431 cells were documented by immunohistochemistry and immuno-fluorescence staining. The proliferation and apoptosis of A431 cells were detected by CCK-8 assay and flow cytometry (FCM). The tissue microarray showed that NET-1 and survivin were highly expressed in SSCC, while the correlation analysis showed NET-1 expression was positively associated with survivin. In addition, we reported that using the one-chain-double-target siRNA conjugate composed of NET-1 and survivin siRNA sequences in the same backbone inhibited proliferation and promoted apoptosis of SSCC. The one-chain-double-target siRNA showed further downregulation on NET-1 and survivin mRNA and protein levels compared with NET-1 siRNA or survivin siRNA. It also exhibited greater suppression on proliferation and triggering of apoptosis in A431 cells than NET-1 siRNA or survivin siRNA. This result may be explained by the significant downregulation of VEGF, cortactin and Bcl-2, and upregulation of caspase-3 and -8. NET-1 and survivin were overexpressed in SSCC and an interaction between NET-1 and survivin was identified. The one-chain-double-target siRNA appears to be superior in inhibiting cell proliferation and promoting apoptosis compared with the single target siRNA. NET-1 and survivin may have correlative signaling pathways with VEGF, cortactin, Bcl-2, caspase-3 and -8. Simultaneous silencing of NET-1 and survivin using one-chain-double-target siRNA thus provides an advantageous alternative in the development of therapeutics for SSCC.
同时沉默多个上调基因是治疗包括癌症在内的许多不治之症的一种有吸引力且可行的策略。在本研究中，构建了皮肤鳞状细胞癌（SSCC）组织芯片，并鉴定了NET-1和生存素的表达。NET-1和生存素基因在SSCC中的高表达被确认为癌症形成和发展的重要事件。本研究共纳入100例原发性SSCC患者。通过免疫组织化学在组织芯片中评估癌细胞中NET-1和生存素的表达。随后，通过产生靶向单个基因的siRNA序列（siRNA-NET-1和siRNA-生存素）以及NET-1和生存素基因（单链双靶点siRNA），通过免疫共沉淀验证了SSCC中NET-1和生存素之间的相互作用。通过RT-qPCR和蛋白质印迹法检测A431细胞中NET-1和生存素mRNA及蛋白质表达水平，并使用RT-qPCR鉴定包括血管内皮生长因子（VEGF）、皮层肌动蛋白、Bcl-2、半胱天冬酶-3和-8在内的相关基因的表达。通过免疫组织化学和免疫荧光染色记录A431细胞中NET-1和生存素的蛋白质定位和表达。通过CCK-8法和流式细胞术（FCM）检测A431细胞的增殖和凋亡。组织芯片显示NET-1和生存素在SSCC中高表达，而相关性分析显示NET-1表达与生存素呈正相关。此外，我们报道使用由NET-1和生存素siRNA序列组成的单链双靶点siRNA偶联物可抑制SSCC的增殖并促进其凋亡。与NET-1 siRNA或生存素siRNA相比，单链双靶点siRNA在NET-1和生存素mRNA及蛋白质水平上表现出进一步下调。它在A431细胞中对增殖的抑制和凋亡的触发也比NET-1 siRNA或生存素siRNA更强。这一结果可能是由于VEGF、皮层肌动蛋白和Bcl-2的显著下调以及半胱天冬酶-3和-8的上调所致。NET-1和生存素在SSCC中过表达，并且鉴定出NET-1和生存素之间存在相互作用。与单靶点siRNA相比，单链双靶点siRNA在抑制细胞增殖和促进凋亡方面似乎更具优势。NET-1和生存素可能与VEGF、皮层肌动蛋白、Bcl-2、半胱天冬酶-3和-8具有相关的信号通路。因此，使用单链双靶点siRNA同时沉默NET-1和生存素为SSCC治疗药物的开发提供了一种有利的选择。
