Zhang Hongyan, Li Wanbin
Department of Gynecology, Affiliated Hospital of Jining Medical University, Jining, Shandong 272029, P.R. China.
Jining Medical University, Jining, Shandong 272113, P.R. China.
Oncol Rep. 2016 Dec;36(6):3605-3610. doi: 10.3892/or.2016.5148. Epub 2016 Oct 4.
The objective of the present study was to identify the association between mir-143-3p and RalA-binding protein 1 (RALBP1), and their roles in regulating the development of ovarian cancer. Overexpression of RALBP1 induced apoptosis of the ovarian cancer cells, and developed ovarian cancer. In silico analysis and luciferase assay were used to identify whether RALBP1 was the target of mir-143-3p. Subsequently, real‑time PCR and western blotting were used to determine the expression level of mir-143-3p, RALBP1 mRNA and protein in different groups, furthermore, MTT assay and flow cytometry were used to detect the viability and apoptosis of cells in different treatment groups. We identified RALBP1 as a target gene of miR-143-3p using computational analysis, and the luciferase activity of cells transfected with wild-type RALBP1 and RALBP1 siRNA were much lower than the scramble control, however, the luciferase activity of cells transfected with mutant RALBP1 was similar with scramble control. The real-time PCR and western blot results suggested that the miR‑143-3p level was markedly lower in participants with ovarian cancer compared with normal control, while the expression of RALBP1 mRNA and protein were evidently overexpressed in participants with ovarian cancer compared with normal control. Furthermore, the RALBP1 mRNA and protein level in cells transfected with miR-143-3p mimics and RALBP1 siRNA were downregulated, while notably upregulated subsequent to transfection with miR-143-3p inhibitor, when compared with scramble control. Additionally, the viability of cells were inhibited following transfection with miR-143-3p mimics and RALBP1 siRNA, while notably promoted subsequent to transfection with miR-143-3p inhibitor. Apoptosis of cells were promoted following transfection with miR-143-3p mimics and RALBP1 siRNA, while notably inhibited subsequent to transfection with miR-143-3p inhibitor. These findings provide support that downregulation of the miR-143-3p is associated with a decreased risk of ovarian cancer.
本研究的目的是确定mir-143-3p与RalA结合蛋白1(RALBP1)之间的关联,以及它们在调控卵巢癌发展中的作用。RALBP1的过表达诱导卵巢癌细胞凋亡,并引发卵巢癌。通过计算机分析和荧光素酶测定来确定RALBP1是否为mir-143-3p的靶标。随后,使用实时PCR和蛋白质印迹法测定不同组中mir-143-3p、RALBP1 mRNA和蛋白质的表达水平,此外,使用MTT法和流式细胞术检测不同处理组细胞的活力和凋亡情况。我们通过计算机分析确定RALBP1为miR-143-3p的靶基因,转染野生型RALBP1和RALBP1 siRNA的细胞的荧光素酶活性远低于乱序对照,然而,转染突变型RALBP1的细胞的荧光素酶活性与乱序对照相似。实时PCR和蛋白质印迹结果表明,与正常对照相比,卵巢癌患者中miR-143-3p水平显著降低,而RALBP1 mRNA和蛋白质的表达在卵巢癌患者中明显高于正常对照。此外,与乱序对照相比,转染miR-143-3p模拟物和RALBP1 siRNA的细胞中RALBP1 mRNA和蛋白质水平下调,而在用miR-143-3p抑制剂转染后显著上调。此外,用miR-143-3p模拟物和RALBP1 siRNA转染后细胞活力受到抑制,而在用miR-143-3p抑制剂转染后显著促进。用miR-143-3p模拟物和RALBP1 siRNA转染后细胞凋亡增加,而在用miR-143-3p抑制剂转染后显著抑制。这些发现支持miR-143-3p的下调与卵巢癌风险降低相关。