Qi Yuying, Hu Tinghui, Li Kai, Ye Renqing, Ye Zuodong
Department of Laboratory, The Affiliated Ningde Municipal Hospital of Fujian Medical University, Ningde, China.
Department of Oncological Surgery, The Affiliated Ningde Municipal Hospital of Fujian Medical University, Ningde, China.
J Breast Cancer. 2015 Sep;18(3):218-24. doi: 10.4048/jbc.2015.18.3.218. Epub 2015 Sep 24.
Protein phosphatase 4 regulatory subunit 1 (PP4R1), as an interaction partner of the catalytic serine/threonine-protein phosphatase 4 catalytic subunit has been shown to involve in cellular processes and nuclear factor κB signaling. However, the functions of PP4R1 in human breast cancers remain unclear. This study is designed to explore the effect of PP4R1 knockdown on the biological characteristics of breast cancer cells.
A lentivirus-mediated short hairpin RNA (shRNA) was designed to knockdown the expression of PP4R1 in ZR-75-30 breast cancer cells. The efficiency of lentivirus-mediated shRNA infection was determined using fluorescence microscopy to observe lentivirus-mediated green fluorescent protein expression and confirmed to be over 80%. PP4R1 expression in infected ZR-75-30 cells was detected by quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation and colony formation ability were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay, respectively. Flow cytometry was used to measure cell cycle progression and cell apoptosis. In addition, apoptosis makers, including poly-ADP-ribose polymerase (PARP) and caspase-3, were investigated in PP4R1-silenced ZR-75-30 cells by western blot assay.
We successfully constructed lentivirus-mediated shRNA to target PP4R1 in ZR-75-30 cells. MTT assay and colony formation assay showed the loss of PP4R1 suppressed the proliferation of ZR-75-30 cells. Flow cytometry analysis indicated cell cycle arrest and increased cell apoptosis in PP4R1 knockdown cells. Further, the apoptosis response in cells depleted of PP4R1 was illustrated by downregulation of PARP and upregulation of caspase-3.
Our results suggest that PP4R1 could promote breast cancer cell proliferation and might play a vital role in breast cancer occurrence.
蛋白磷酸酶4调节亚基1（PP4R1）作为催化性丝氨酸/苏氨酸蛋白磷酸酶4催化亚基的相互作用伴侣，已被证明参与细胞过程和核因子κB信号传导。然而，PP4R1在人类乳腺癌中的功能仍不清楚。本研究旨在探讨敲低PP4R1对乳腺癌细胞生物学特性的影响。
设计慢病毒介导的短发夹RNA（shRNA）以敲低ZR-75-30乳腺癌细胞中PP4R1的表达。使用荧光显微镜观察慢病毒介导的绿色荧光蛋白表达来确定慢病毒介导的shRNA感染效率，确认其超过80%。通过定量实时聚合酶链反应和蛋白质印迹分析检测感染的ZR-75-30细胞中PP4R1的表达。分别通过3-（4,5-二甲基-2-噻唑基）-2,5-二苯基-2-H-四氮唑溴盐（MTT）法和集落形成试验测量细胞增殖和集落形成能力。使用流式细胞术测量细胞周期进程和细胞凋亡。此外，通过蛋白质印迹分析在PP4R1沉默的ZR-75-30细胞中研究凋亡标志物，包括聚ADP-核糖聚合酶（PARP）和半胱天冬酶-3。
我们成功构建了慢病毒介导的shRNA以靶向ZR-75-30细胞中的PP4R1。MTT法和集落形成试验表明，PP4R1的缺失抑制了ZR-75-30细胞的增殖。流式细胞术分析表明PP4R1敲低细胞中细胞周期停滞且细胞凋亡增加。此外，PP4R1缺失细胞中的凋亡反应通过PARP的下调和半胱天冬酶-3的上调得以体现。
我们的结果表明，PP4R1可促进乳腺癌细胞增殖，可能在乳腺癌发生中起重要作用。
