Hosseini Ebrahim, Hosseini Seyed Younes, Hashempour Tayebeh, Fattahi Mohammad-Reza, Sadeghizadeh Majid
Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 1411713116, Iran.
Bacteriology and Virology Department, School of Medicine, Shiraz University of Medical Sciences, Shiraz 71348‑14336, Iran.
Mol Med Rep. 2017 Jan;15(1):495-501. doi: 10.3892/mmr.2016.6009. Epub 2016 Dec 8.
The melanoma differentiation-associated gene‑7 (MDA-7) gene, also termed interleukin‑24 (IL‑24), is a tumor suppressor gene that induces apoptosis in a broad scope of malignant neoplastic cells. The apoptosis induction capacity of the MDA‑7/IL‑24 gene is partially associated with adhering to cognate receptors. The current study aimed to enhance the antitumor effect of IL‑24. The intrinsic signal sequence of IL-24 replaced with a fused artificial signal (secrecon)-RGD4C sequence and its impact was evaluated in HepG2 cells. The modified SP.RGD.IL‑24 and native IL‑24 cDNA sequences were cloned into the pcDNA3.1 expression vector. Subsequently, the expression level, secretion efficacy and targeting propensity of the modified SP.RGD.IL‑24 product compared with normal IL‑24 by were determined by enzyme‑linked immunosorbent assay. The constructs were then transfected into HepG2 and LX‑2 cells as tumor and normal hepatic cell lines, respectively. The expression level of the pro‑apoptotic DNA damage inducible transcript 3 (Gadd153) and BCL2 associated X apoptosis regulator (Bax) genes in the different groups were compared by reverse transcription-quantitative polymerase chain reaction. Additionally, the rate of apoptosis induction of modified and intact IL‑24 sequences was compared by flow cytometry analysis of cells following their propidium iodide/annexin V staining. SP.RGD-IL-24 protein was expressed and secreted in a similar manner to native IL‑24, however, the modified SP.RGD.IL-24 adhered to tumor cells more efficiently than IL‑24 (P<0.05). SP.RGD.IL‑24 significantly induced upregulation of Gadd153 and Bax in HepG2 cells compared with native IL‑24 (P<0.05). However, neither had a significant impact on the expression level of pro-apoptotic genes in LX‑2 cells. Flow cytometry analysis also indicated that modified SP.RGD.IL-24 induced apoptosis more than native IL‑24 in HepG2 cells (P<0.05). In conclusion, the novel generated RGD‑coupled IL-24 construct exhibited sufficient anticancer activity compared with the native IL‑24. The results of the current study provide novel insights for the future of cytokine targeting and indicates its potential capacity as a valuable candidate for gene therapy methods.
黑色素瘤分化相关基因-7（MDA-7）基因，也称为白细胞介素-24（IL-24），是一种肿瘤抑制基因，可在多种恶性肿瘤细胞中诱导凋亡。MDA-7/IL-24基因的凋亡诱导能力部分与粘附同源受体有关。当前研究旨在增强IL-24的抗肿瘤作用。将IL-24的内在信号序列替换为融合的人工信号（secrecon）-RGD4C序列，并在HepG2细胞中评估其影响。将修饰的SP.RGD.IL-24和天然IL-24 cDNA序列克隆到pcDNA3.1表达载体中。随后，通过酶联免疫吸附测定法测定修饰的SP.RGD.IL-24产物与正常IL-24相比的表达水平、分泌效率和靶向倾向。然后将构建体分别转染到作为肿瘤细胞系的HepG2细胞和作为正常肝细胞系的LX-2细胞中。通过逆转录-定量聚合酶链反应比较不同组中促凋亡DNA损伤诱导转录本3（Gadd153）和BCL2相关X凋亡调节因子（Bax）基因的表达水平。此外，通过碘化丙啶/膜联蛋白V染色后的细胞流式细胞术分析比较修饰的和完整的IL-24序列的凋亡诱导率。SP.RGD-IL-24蛋白的表达和分泌方式与天然IL-24相似，然而，修饰的SP.RGD.IL-24比IL-24更有效地粘附于肿瘤细胞（P<0.05）。与天然IL-24相比，SP.RGD.IL-24在HepG2细胞中显著诱导Gadd153和Bax的上调（P<0.05）。然而，两者对LX-2细胞中促凋亡基因的表达水平均无显著影响。流式细胞术分析还表明，修饰的SP.RGD.IL-24在HepG2细胞中比天然IL-24诱导更多的凋亡（P<0.05）。总之，与天然IL-24相比，新生成的RGD偶联IL-24构建体表现出足够的抗癌活性。当前研究结果为细胞因子靶向治疗的未来提供了新的见解，并表明其作为基因治疗方法有价值候选物的潜在能力。
