Xue Han1, Wen Cheng, Hui Jing, Jiu-Wei Zhang, Li-Li Tang. 1. Department of Ultrasound, Third Affiliated Hospital of Harbin Medical University, 150 Haping Rd, Nangang District, 150081 Harbin, China.
Abstract
OBJECTIVES: Short interfering RNA (siRNA) has been used to knock down the expression of targeted genes in a process known as RNA interference. However, the key to RNA interference is the efficient intracellular delivery of the siRNA. In this study, we sought to enhance the efficiency of transduction and find a novel therapy for hepatic carcinoma. METHODS: Three types of neuroepithelial transforming protein 1 (NET-1) siRNAs (labeled fluorescent) were designed and transduced into HepG2 cells. Then the most effective one in silencing NET-1 was determined. The HepG2 cells were divided into 5 groups: untreated control; delivery of siRNA; delivery of siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA; group L); delivery of siRNA using ultrasound exposure and microbubbles (group US); and delivery of siRNA using Lipofectamine, ultrasound exposure, and microbubbles (group LUS). The efficiency of siRNA transfer was determined by detection of luciferase activity on microscopy; NET-1 expression was assayed by reverse transcription-polymerase chain reaction and western blotting; and proliferation investigations of the HepG2 cells were performed. RESULTS- The transfection efficiency of microbubbles combined with ultrasound exposure was nearly equal to Lipofectamine-mediated transfection (P = .609). More importantly, the combination of Lipofectamine, microbubbles, and ultrasound exposure effectively reduced NET-1 expression compared with the other groups (P < .01). Furthermore, the proliferation of cells in groups L, US, and LUS was visibly inhibited between 24 and 72 hours. CONCLUSIONS: The use of a microbubble contrast agent combined with ultrasound exposure could be a potent physical method for increasing gene delivery efficiency. This technique is a promising nonviral approach that can be used in liver cancer.
OBJECTIVES: Short interfering RNA (siRNA) has been used to knock down the expression of targeted genes in a process known as RNA interference. However, the key to RNA interference is the efficient intracellular delivery of the siRNA. In this study, we sought to enhance the efficiency of transduction and find a novel therapy for hepatic carcinoma. METHODS: Three types of neuroepithelial transforming protein 1 (NET-1) siRNAs (labeled fluorescent) were designed and transduced into HepG2 cells. Then the most effective one in silencing NET-1 was determined. The HepG2 cells were divided into 5 groups: untreated control; delivery of siRNA; delivery of siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA; group L); delivery of siRNA using ultrasound exposure and microbubbles (group US); and delivery of siRNA using Lipofectamine, ultrasound exposure, and microbubbles (group LUS). The efficiency of siRNA transfer was determined by detection of luciferase activity on microscopy; NET-1 expression was assayed by reverse transcription-polymerase chain reaction and western blotting; and proliferation investigations of the HepG2 cells were performed. RESULTS- The transfection efficiency of microbubbles combined with ultrasound exposure was nearly equal to Lipofectamine-mediated transfection (P = .609). More importantly, the combination of Lipofectamine, microbubbles, and ultrasound exposure effectively reduced NET-1 expression compared with the other groups (P < .01). Furthermore, the proliferation of cells in groups L, US, and LUS was visibly inhibited between 24 and 72 hours. CONCLUSIONS: The use of a microbubble contrast agent combined with ultrasound exposure could be a potent physical method for increasing gene delivery efficiency. This technique is a promising nonviral approach that can be used in liver cancer.