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. 2015 Aug 7;8:2067-74.
doi: 10.2147/OTT.S77709. eCollection 2015.

PP4R1 Accelerates Cell Growth and Proliferation in HepG2 Hepatocellular Carcinoma

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Free PMC article

PP4R1 Accelerates Cell Growth and Proliferation in HepG2 Hepatocellular Carcinoma

Gang Wu et al. Onco Targets Ther. .
Free PMC article

Abstract

Hepatocellular carcinoma (HCC), as the fifth most common cancer worldwide, has become the third leading cause of cancer-related deaths. It is reported that protein phosphatase 4 (PP4) is an essential protein for nucleation, growth, and stabilization of microtubules in centrosomes/spindle bodies during cell division. Besides, previous studies have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a constitutive interaction partner of PP4 catalytic subunit PP4C. The PP4C-PP4R1 PP4 complex plays a role in dephosphorylation, regulation of histone acetylation, and NF-κB activation. However, little is known about the pathological functions of PP4R1 in human cancers. Thus, in order to investigate how PP4R1 functions in human HCC, two common hepatocarcinogenesis HCC cell lines HepG2 and SMMC-7721 were employed, transduced with recombinant lentivirus expressing PP4R1 short hairpin RNA. Compared with the controls, the cells treated with Lv-shPP4R1 showed a significant decrease in cell proliferation and colony formation. The results of flow cytometry showed that the knockdown of PP4R1 caused HepG2 cells arrest at G2/M phase in the cell cycle. Furthermore, the transduction of Lv-shPP4R1 into HepG2 cells led to the inactivation of two major mitogen-activated protein kinase signaling cascades: p38 and c-Jun N-terminal kinase (JNK), indicating that PP4R1 could promote cell proliferation, which might be regulated by p38 and c-Jun N-terminal kinase pathways. In a word, this study highlights the crucial role of PP4R1 in promoting HCC cell growth, which might elucidate the pathological mechanism of HCC.

Keywords: PP4R1; apoptosis; hepatocellular carcinoma; proliferation; shRNA.

Figures

Figure 1
Figure 1
Silencing of PP4R1 expression in Hepg2 cells and SMMC-7721 cells by Lv-shPP4R1. Notes: (A) Fluorescence microscopy examination of lentivirus infection efficiency in HepG2 cells and SMMC-7721 cells (scale bar: 100 μm). (B) RT-qPCR analysis of the PP4R1 mRNA level in HepG2 cells after lentivirus infection. (C) Western blot analysis of the PP4R1 protein level in HepG2 cells and SMMC-7721 cells. After lentivirus infection, data represent as means ± SD from three independent experiments. *P<0.001. Abbreviations: Con, control; Lv-shCon, control shRNA; Lv-shPP4R1, lentivirus expressing PP4R1 shRNA; PP4R1, protein phosphatase 4 regulatory subunit 1; RT-qPCR, real-time quantitative polymerase chain reaction; shRNA, short hairpin ribonucleic acid; SD, standard deviation.
Figure 2
Figure 2
Lv-shPP4R1 inhibited the viability and proliferation of HepG2 cells. Notes: (A) Growth curves of HepG2 cells and SMMc-7721 cells in three groups (Con, Lv-shCon, and Lv-shPP4R1) measured by MTT assay. (B) Statistical analysis revealed that the number of colonies in the Lv-shPP4R1 group was significantly smaller than that in the control groups. (C) Images recorded under micro and macro views, representing the size and the number of colonies in each group (scale bar: 250 μm). Data represented as means ± SD from three independent experiments. *P<0.001. Abbreviations: Con, control; GFP, green fluorescent protein; Lv-shCon, control shRNA; Lv-shPP4R1, lentivirus expressing PP4R1 shRNA; OD, optical density; PP4R1, protein phosphatase 4 regulatory subunit 1; SD, standard deviation; shRNA, short hairpin ribonucleic acid.
Figure 3
Figure 3
Lv-shPP4R1 blocked the cell cycle progression of HepG2 cells. Notes: (A) FACS analysis of cell cycle distribution of HepG2 cells in three groups (Con, Lv-shCon, Lv-shPP4R1). (B) Knockdown of PP4R1 in HepG2 cells led to an increase of cells in the G2/M phase and, concomitantly, a decrease of cells in the G0/G1 phase. Data represent means ± SD from three independent experiments. *P<0.05, **P<0.01. Abbreviations: Con, control; FACS, fluorescence activated cell sorting; Lv-shCon, control shRNA; Lv-shPP4R1, lentivirus expressing PP4R1 shRNA; PP4R1, protein phosphatase 4 regulatory subunit 1; SD, standard deviation; shRNA, short hairpin ribonucleic acid.
Figure 4
Figure 4
Lv-shPP4R1 caused inactivation of p38 and JNK. Notes: (A) Cell extracts were prepared and analyzed using the PathScan® Intracellular Signaling Array Kit (Chemiluminescent Readout). Images were acquired by briefly exposing the slide to standard chemiluminescent film. (B) Western blot analysis of p38 and JNK protein level in HepG2 cells and SMMC-7721 cells after lentivirus infection. Abbreviations: JNK, c-Jun N-terminal kinase; Lv-shCon, control shRNA; Lv-shPP4R1, lentivirus expressing PP4R1 shRNA; PP4R1, protein phosphatase 4 regulatory subunit 1; shRNA, short hairpin ribonucleic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 5
Figure 5
Lv-shPP4R1 had no effect on the activity of NF-κB. Notes: Western blot analysis of p65 protein level in HepG2 cells and SMMC-7721 cells after lentivirus infection. Abbreviations: Lv-shCon, control shRNA; Lv-shPP4R1, lentivirus expressing PP4R1 shRNA; PP4R1, protein phosphatase 4 regulatory subunit 1; shRNA, short hairpin ribonucleic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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