Weng Yanjie, Shi Ying, Xia Xi, Zhou Wenjuan, Wang Hongyan, Wang Changyu
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
Department of Gynecology and Obstetrics, Affiliated Shenzhen Nanshan Hospital, Guangdong Medical College, Shenzhen, Guangdong 518052, P.R. China.
Oncol Lett. 2017 Mar;13(3):1553-1562. doi: 10.3892/ol.2017.5672. Epub 2017 Feb 2.
RNA interference (RNAi) is a powerful technology for suppressing gene function. In most studies, small interfering RNAs (siRNAs) consist of one short hairpin RNA (shRNA) and, therefore, are often unable to achieve loss-of-function of their target genes. In the current study, an RNAi vector containing three shRNAs under the control of three RNA polymerase III U6 promoters was constructed. RNAi vectors containing one or two shRNAs were generated for comparisons. A pilot study targeting exogenously expressed DsRed in the HEK293 cell line revealed promising effects and a high selectivity for the multi-shRNA RNAi vector. Akt2 is constitutively expressed in cultured SKOV3 human ovarian cancer cells, and the multi-shRNA RNAi vector showed a strong efficiency for downregulating the expression of Akt2 in these cells, with no apparent interferon response. In addition, the Akt2-3shRNA vector, containing three shRNAs targeting Akt2, showed the best effect of all the shRNA vectors in reversing paclitaxel-induced resistance in SKOV3 cells. This study developed a widely applicable resource for enhancing the efficiency of gene silencing and a novel technique for performing complex loss-of-function screens in mammalian cells.
RNA干扰（RNAi）是一种抑制基因功能的强大技术。在大多数研究中，小干扰RNA（siRNA）由一个短发夹RNA（shRNA）组成，因此往往无法实现其靶基因的功能缺失。在本研究中，构建了一种RNAi载体，其在三个RNA聚合酶III U6启动子的控制下包含三个shRNA。构建了含有一个或两个shRNA的RNAi载体用于比较。一项针对HEK293细胞系中外源表达的DsRed的初步研究揭示了多shRNA RNAi载体具有良好的效果和高选择性。Akt2在培养的SKOV3人卵巢癌细胞中组成性表达，多shRNA RNAi载体在这些细胞中显示出强烈下调Akt2表达的效率，且无明显的干扰素反应。此外，含有三个靶向Akt2的shRNA的Akt2 - 3shRNA载体在逆转SKOV3细胞中紫杉醇诱导的耐药性方面显示出所有shRNA载体中最佳的效果。本研究开发了一种广泛适用的资源以提高基因沉默效率，并开发了一种在哺乳动物细胞中进行复杂功能缺失筛选的新技术。
