J Breast Cancer.  2015 Sep;18(3):218-224. 10.4048/jbc.2015.18.3.218.

Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer

Affiliations
  • 1Department of Laboratory, The Affiliated Ningde Municipal Hospital of Fujian Medical University, Ningde, China. yuying_qi@126.com
  • 2Department of Oncological Surgery, The Affiliated Ningde Municipal Hospital of Fujian Medical University, Ningde, China.

Abstract

PURPOSE
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 kappaB 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.
METHODS
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.
RESULTS
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.
CONCLUSION
Our results suggest that PP4R1 could promote breast cancer cell proliferation and might play a vital role in breast cancer occurrence.

Keyword

Apoptosis; Breast neoplasms; Cell proliferation; Protein phosphatase 4 regulatory subunit 1

MeSH Terms

Apoptosis
Blotting, Western
Breast Neoplasms*
Breast*
Caspase 3
Catalytic Domain
Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation
Down-Regulation
Flow Cytometry
Humans
Microscopy, Fluorescence
Population Characteristics
Real-Time Polymerase Chain Reaction
RNA*
RNA, Small Interfering
Up-Regulation
Caspase 3
RNA
RNA, Small Interfering

Figure

  • Figure 1 Establishment of protein phosphatase 4 regulatory subunit 1 (PP4R1)-silenced breast cancer cells. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of PP4R1 was performed in T-47D and ZR-75-30 cells, respectively. (B) Western blot analysis of PP4R1 was performed in T-47D and ZR-75-30 cells, respectively. (C) Fluorescence microscopy examination was performed for the infection efficiency lentivirus vector in ZR-75-30 cells (scale bar, 10 µm). (D) qRT-PCR analysis was performed after the knockdown of PP4R1 by lentivirus-mediated RNA interference (RNAi) in ZR-75-30 cells. (E) Western blot analysis of the knockdown of PP4R1 was performed by lentivirus-mediated RNAi in ZR-75-30 cells. GAPDH=glyceraldehyde-3-phosphate dehydrogenase; Con=control; sh=short hairpin; shCon=the lentivirus-mediated negative control RNAi group; shPP4R1=the lentivirus-mediated PP4R1 RNAi group; GFP=green fluorescent protein. *p<0.01; †p<0.001, compared with shCon.

  • Figure 2 Suppression of proliferation and colony formation of ZR-75-30 cells by protein phosphatase 4 regulatory subunit (PP4R1) silencing. (A) Proliferation of PP4R1-silenced ZR-75-30 cells was assessed by MTT assay, in parallel with Con and shCon group as control. (B) The image of the colony formation assay is represented (scale bar, 50 µm; magnification, ×40). (C) Statistical analyses of colonies number results in ZR-75-30 cells, in parallel with Con and shCon group as control, is represented. Con=control; sh=short hairpin; shCon=the lentivirus-mediated negative control RNAi group; shPP4R1=the lentivirus-mediated PP4R1 RNAi group. *p<0.001, compared with shCon.

  • Figure 3 Cell cycle arrest of ZR-75-30 cells induced by protein phosphatase 4 regulatory subunit 1 (PP4R1) silencing. (A) Flow cytometry analysis of cell cycle progression was performed on the three groups (Con, shCon, and shPP4R1 groups). (B, C) Statistical analysis of cell cycle including the apoptotic cells (sub-G1 cell) is represented. Con=control; sh=short hairpin; shCon=the lentivirus-mediated negative control RNAi group; shPP4R1=the lentivirus-mediated PP4R1 RNAi group. *p<0.05, compared with shCon.

  • Figure 4 Apoptosis in ZR-75-30 cells induced by protein phosphatase 4 regulatory subunit 1 (PP4R1) silencing. (A) Representative flow cytometric dot plots are presented. Cell in the upper right and lower right quadrant were characterized as early apoptotic cells and late apoptotic cells, respectively. (B) Statistical analysis on early and late apoptotic cell percentage in ZR-75-30 cells is represented. Con=control; sh=short hairpin; shCon=the lentivirus-mediated negative control RNAi group; shPP4R1=the lentivirus-mediated PP4R1 RNAi group. *p<0.001.

  • Figure 5 Western blot analysis of several critical apoptosis markers, including caspase-3 and poly-ADP-ribose polymerase (PARP) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. The figure shows the representative images of three independent experiments. Con=control; sh=short hairpin; shCon=the lentivirus-mediated negative control RNAi group; shPP4R1=the lentivirus-mediated PP4R1 RNAi group.


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