Department of Laboratory, Ningbo No.2 Hospital, Ningbo, 315010, P.R. China.
Department of Hepatopancreatobiliary Surger, Ningbo No.2 Hospital, Ningbo, 315010, P.R. China.
J Cell Biochem. 2018 Jan;119(1):659-668. doi: 10.1002/jcb.26229. Epub 2017 Jul 31.
This study explored the effects involved in silencing CLIC4 on apoptosis and proliferation of mouse liver cancer Hca-F and Hca-P cells. A CLIC4-target small interfering RNA (siRNA) was designed to compound into two individual complementary oligonucleotide chains. A process of annealing and connection to a pSilencer vector was followed by transfection with Hca-F and Hca-P cells. Quantitative real-time polymerase chain reaction and Western blotting techniques were used to determine CLIC4 mRNA and protein expressions. CCK8 assay and flow cytometry were employed for analysis of the survival and apoptosis rate as well as the cell cycle in an octreotide-induced apoptosis model. Expressions of caspase 3, caspase 9, and cleaved PARP were measured using Western blotting. The CLIC4 mRNA and protein expressions in Hca-F and Hca-P cells transfected by pSilencer-CLIC4 siRNA plasmid in the blank group displayed remarkably decreased levels of expression, when compared with both the control and negative control (NC) groups. Decreased survival rates and cleaved PARP expression, increased cell apoptosis rate,expressions of caspase 3 and caspase 9 in Hca-F and Hca-P cells were detected in groups that had been cultured in a medium containing octreotide. The pSilencer-CLIC4 siRNA-2 group when compared with the control and NC groups exhibited decreased survival rates, cleaved PARP expression, increased cell apoptosis rates, and increased expressions of caspase 3 and caspase 9 of Hca-F and Hca-P cells. The results demonstrated that siRNA-induced down-regulation of CLIC4 could proliferation, while in turn promoting apoptosis of mouse liver cancer Hca-F and Hca-P cells. J. Cell. Biochem. 119: 659-668, 2018. © 2017 Wiley Periodicals, Inc.
本研究旨在探讨沉默 CLIC4 对小鼠肝癌 Hca-F 和 Hca-P 细胞凋亡和增殖的影响。设计了针对 CLIC4 的小干扰 RNA(siRNA)，并将其复合成两条互补的寡核苷酸链。经过退火和连接到 pSilencer 载体后，转染 Hca-F 和 Hca-P 细胞。采用实时定量聚合酶链反应和 Western blot 技术检测 CLIC4mRNA 和蛋白表达。采用 CCK8 法和流式细胞术分析奥曲肽诱导的凋亡模型中细胞的存活率、凋亡率和细胞周期。采用 Western blot 法检测 caspase 3、caspase 9 和裂解型 PARP 的表达。pSilencer-CLIC4siRNA 质粒转染 Hca-F 和 Hca-P 细胞后，空白组 CLIC4mRNA 和蛋白表达水平明显低于对照组和阴性对照组（NC 组）。与对照组和 NC 组相比，奥曲肽培养的 Hca-F 和 Hca-P 细胞的存活率降低，裂解型 PARP 表达增加，细胞凋亡率增加，caspase 3 和 caspase 9 表达增加。与对照组和 NC 组相比，pSilencer-CLIC4siRNA-2 组 Hca-F 和 Hca-P 细胞的存活率降低，裂解型 PARP 表达增加，细胞凋亡率增加，caspase 3 和 caspase 9 表达增加。结果表明，siRNA 诱导的 CLIC4 下调可促进小鼠肝癌 Hca-F 和 Hca-P 细胞的增殖，进而促进其凋亡。J. Cell. Biochem. 119: 659-668, 2018. © 2017 Wiley Periodicals, Inc.
