Phuah Neoh Hun, Azmi Mohamad Nurul, Awang Khalijah, Nagoor Noor Hasima
Faculty of Science, Institute of Biological Science (Genetics and Molecular Biology).
Faculty of Science, Department of Chemistry, Centre for Natural Product Research and Drug Discovery (CENAR).
Onco Targets Ther. 2017 Mar 20;10:1695-1705. doi: 10.2147/OTT.S117492. eCollection 2017.
Cervical cancer is the fourth most frequent malignancy affecting women worldwide, but drug resistance and toxicities remain a major challenge in chemotherapy. The use of natural compounds is promising because they are less toxic and able to target multiple signaling pathways. The 1'S-1'-acetoxychavicol acetate (ACA), a natural compound isolated from wild ginger , induced cytotoxicity on various cancer cells including cervical cancer. MicroRNAs (miRNAs) are short noncoding RNAs that regulate numerous biological processes, such as apoptosis and chemosensitivity. Past studies reported that miR-629 is upregulated in many cancers, and its expression was altered in ACA-treated cervical cancer cells. However, the role of miR-629 in regulating sensitivity toward ACA or other anticancer agents has not been reported. Hence, this study aims to investigate the role of miR-629 in regulating response toward ACA on cervical cancer cells.
The miR-629 expression following transfection with miR-629 hairpin inhibitor and hairpin inhibitor negative control was measured using quantitative real-time polymerase chain reaction (RT-qPCR). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to investigate sensitivity toward ACA. Apoptosis was detected using Annexin V/propidium iodide and Caspase 3/7 assays. The gene target for miR-629 was identified using miRNA target prediction programs, luciferase reporter assay and Western blots. Gene overexpression studies were performed to evaluate its role in regulating response toward ACA.
Transfection with miR-629 hairpin inhibitor downregulated its expression in both cervical cancer cell lines. Suppression of miR-629 increased sensitivity toward ACA by reducing cell proliferation and inducing apoptosis. Luciferase reporter assay confirmed RSU1 as a direct target of miR-629. Overexpression of miR-629 decreased RSU1 protein expression, while inhibition of miR-629 increased RSU1 protein expression. Overexpression of RSU1 augmented antiproliferative and apoptosis-inducing effects of ACA.
Our findings showed that combination of ACA with miR-629 and RSU1 may provide a potential strategy in treating cervical cancer.
宫颈癌是全球影响女性的第四大常见恶性肿瘤，但耐药性和毒性仍是化疗中的主要挑战。天然化合物的使用前景广阔，因为它们毒性较小且能够靶向多种信号通路。1'S-1'-乙酰氧基胡椒酚乙酸酯（ACA）是一种从野生姜中分离出的天然化合物，可对包括宫颈癌细胞在内的多种癌细胞诱导细胞毒性。微小RNA（miRNA）是短的非编码RNA，可调节众多生物学过程，如细胞凋亡和化学敏感性。过去的研究报道，miR-629在许多癌症中上调，并且其表达在ACA处理的宫颈癌细胞中发生改变。然而，miR-629在调节对ACA或其他抗癌药物的敏感性中的作用尚未见报道。因此，本研究旨在探讨miR-629在调节宫颈癌细胞对ACA反应中的作用。
使用定量实时聚合酶链反应（RT-qPCR）检测用miR-629发夹抑制剂和发夹抑制剂阴性对照转染后miR-629的表达。采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法研究对ACA的敏感性。使用膜联蛋白V/碘化丙啶和半胱天冬酶3/7检测法检测细胞凋亡。使用miRNA靶标预测程序、荧光素酶报告基因检测和蛋白质印迹法鉴定miR-629的基因靶标。进行基因过表达研究以评估其在调节对ACA反应中的作用。
用miR-629发夹抑制剂转染下调了两种宫颈癌细胞系中miR-629的表达。抑制miR-629通过减少细胞增殖和诱导细胞凋亡增加了对ACA的敏感性。荧光素酶报告基因检测证实RSU1是miR-629的直接靶标。miR-629的过表达降低了RSU1蛋白表达，而抑制miR-629则增加了RSU1蛋白表达。RSU1的过表达增强了ACA的抗增殖和诱导凋亡作用。
我们的研究结果表明，ACA与miR-629和RSU1联合使用可能为治疗宫颈癌提供一种潜在策略。
