Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
J Cell Biochem. 2019 Jun;120(6):9203-9212. doi: 10.1002/jcb.28196. Epub 2018 Dec 2.
BACKGROUND: Considering the complex nature of gastrointestinal cancer, different methods including surgery, radiotherapy, and chemotherapy are considered for the treatment. Novel strategies including silencing of oncogenes using safe delivery systems could be considered as a novel approach in colorectal cancer treatment. The aim of this study was to investigate the silencing effect of high mobility group A2 (HMGA2) small interfering RNA (siRNA)-loaded nanoliposomes on gastrointestinal cancers. METHODS: The siRNA-lipoplexes were prepared using dioleoyl trimethylammonium propane (DOTAP)/cholesterol (Chol)/1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) through the freeze-drying of a monophase solution method. The size, polydispersity index (PDI), and zeta-potential of nanoliposomes were determined using Zetasizer analyzer. The morphology of the nanoliposomes was determined by transmission electron microscopy (TEM). The agarose gel-retardation assay was carried out to confirm the loading of siRNAs into liposome. The silencing of the HMGA2 in cancer cells was evaluated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of liposomes on cell cytotoxicity was studied by MTT assay. The inhibitory effect of siRNA-loaded liposomes was evaluated by a wound-healing assay. The apoptosis induction was investigated via the annexin V/propidium iodide assay. RESULTS: The size, PDI, and zeta-potential of the prepared liposomes were found to be 350 nm, 0.67, and 86.3 mV, respectively. They were spherical in shape and could efficiently associate with siRNA. The results of gene silencing showed that the optimum condition of HMGA2 silencing was 80 pmol HMGA2 and 24 hours after treatment in each cancer cell lines. MTT assays indicated that silencing of HMGA2 in optimal condition could reduce the viability of the cancer cells more than 60% in the three cell lines. The result of the apoptosis assay showed more than 50% of the cell deaths related to the apoptosis in all three cell lines. The gene expression evaluation confirmed that apoptosis was induced via the intrinsic pathway inducing both caspase-3 and -9 expressions. Also, the reduction in Bcl2 expression confirmed the activation apoptosis pathway in the treated cancer cells. The wound-healing assay showed the suppression of cancer cell migration after treatment with the prepared nanoliposomes. CONCLUSION: The results of this study showed the HMGA2 siRNA-loaded nanoliposomes could be effective in the treatment of gastrointestinal cancers.
背景：考虑到胃肠道癌症的复杂性，包括手术、放疗和化疗在内的多种方法都被认为是治疗方法。使用安全的递送系统沉默致癌基因等新策略可以被视为结直肠癌治疗的一种新方法。本研究旨在研究高迁移率族蛋白 A2 (HMGA2) 小干扰 RNA (siRNA) 负载的纳米脂质体对胃肠道癌症的沉默效果。
方法：通过单相溶液法的冷冻干燥法，使用二油酰基三甲基铵丙烷 (DOTAP)/胆固醇 (Chol)/1,2-二油酰基-sn-甘油-3-磷酸乙醇胺 (DOPE) 制备 siRNA-脂质体。使用 Zetasizer 分析仪测定纳米脂质体的粒径、多分散指数 (PDI) 和 zeta 电位。通过透射电子显微镜 (TEM) 确定纳米脂质体的形态。琼脂糖凝胶阻滞实验证实 siRNA 载入脂质体。通过定量逆转录聚合酶链反应 (qRT-PCR) 评估癌细胞中 HMGA2 的沉默。通过 MTT 测定研究脂质体对细胞毒性的影响。通过划痕愈合试验评估载有 siRNA 的脂质体的抑制作用。通过 Annexin V/碘化丙啶测定法研究凋亡诱导。
结果：所制备的脂质体的粒径、PDI 和 zeta 电位分别为 350nm、0.67 和 86.3mV。它们呈球形，能够与 siRNA 有效结合。基因沉默的结果表明，HMGA2 沉默的最佳条件是在三种癌细胞系中，每种癌细胞系的 80pmol HMGA2 和 24 小时后处理。MTT 测定表明，在最佳条件下沉默 HMGA2 可使三种细胞系中癌细胞的活力降低 60%以上。凋亡试验的结果表明，三种细胞系中与凋亡相关的细胞死亡超过 50%。基因表达评估证实，凋亡是通过诱导 caspase-3 和 -9 表达的内在途径诱导的。此外，Bcl2 表达的减少证实了处理后的癌细胞中凋亡途径的激活。划痕愈合试验表明，用制备的纳米脂质体处理后，抑制了癌细胞的迁移。
结论：本研究结果表明，HMGA2 siRNA 负载的纳米脂质体可有效治疗胃肠道癌症。
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