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, 21 (7), 1329-1341

Elevated PRC1 in Gastric Carcinoma Exerts Oncogenic Function and Is Targeted by Piperlongumine in a p53-dependent Manner

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Elevated PRC1 in Gastric Carcinoma Exerts Oncogenic Function and Is Targeted by Piperlongumine in a p53-dependent Manner

Bin Zhang et al. J Cell Mol Med.

Abstract

Gastric carcinoma is one of the most common malignancies worldwide and the second most frequent cause of cancer-related death in China. Protein regulator of cytokinesis 1 (PRC1) is involved in cytokinesis and plays key roles in microtubule organization in eukaryotes. This study was aimed to analyse the expression and to investigate the functional role of PRC1 in gastric tumorigenesis. The expression of PRC1 was evaluated by qRT-PCR, Western blot and immunohistochemistry. The biological function of PRC1 was determined by CCK-8 proliferation assays, monolayer colony formation, xenografted nude mice and cell invasion assays by shRNA-mediated knockdown in AGS and HGC27 cells. The regulation of PRC1 expression by piperlongumine was also investigated using dual-luciferase reporter assay and ChIP-qPCR analysis. PRC1 was up-regulated in primary gastric cancers. Overexpression of PRC1 in gastric cancers was associated with poor disease-specific survival and overall survival. PRC1 knockdown in AGS and HGC27 cell lines suppressed proliferation, reduced monolayer colony formation, inhibited cell invasion and migration ability and induced cell-cycle arrest and apoptosis. Inhibition of PRC1 also suppressed tumour growth in vivo. We finally confirmed that PRC1 is a novel downstream target of piperlongumine in gastric cancer. Our findings supported the oncogenic role of PRC1 in gastric carcinogenesis. PRC1 might serve as a prognostic biomarker and potential therapeutic target for gastric carcinoma.

Keywords: PRC1; gastric cancer; oncogene; p53; piperlongumine.

Figures

Figure 1
Figure 1
PRC1 overexpression in gastric cancers and its association with patients’ survival. (A)Recapitulated is the gene expression of PRC1 mRNA in gastric cancer tissues (T) and adjacent non‐tumoural tissues (N) based on quantitative real‐time analysis of paired specimens (N = 17). P values were generated using the paired Student's t‐test. (B) PRC1 gene expressions in gastric cancer tissues (T) and adjacent non‐cancerous tissues (AN) as determined by gene expression array. Data were from NCBI, GEO database GSE63089 (N = 45), GSE27342 (N = 80) and GSE65801 (N = 32), respectively. P values were generated using the unpaired Student's t‐test. (C) Kaplan–Meier plot showing overall survival and time to first progression of gastric cancer patients stratified by high or low PRC1 mRNA expression. These data are from 593 gastric tumour samples using publicly available data sets (http://kmplot.com/analysis/index.php?p=service&cancer=gastric). (D) Western blot analysis of PRC1 in a subset of paired gastric cancer tissues. β‐Actin was used as the endogenous loading control. N, non‐tumour; T, tumour. (E) Representative immunohistochemistry (IHC) staining for PRC1 expression in gastric cancer tissues. Scale bar, 200 μm for left panel and 50 μm for right panel, respectively. (F) Kaplan–Meier plot showing disease‐specific survival curve according to PRC1 staining status in gastric adenocarcinoma.
Figure 2
Figure 2
shRNA‐mediated PRC1 silencing suppresses tumour growth both in vitro and in vivo. (A) and (B) AGS and HGC27 cells were treated with non‐target control shRNA (shCont) or shRNA against PRC1 (sh1 and sh2). Quantitative real‐time PCR and Western blot analysis were performed to detect PRC1 expression. β‐Actin was used as loading controls.*P < 0.01, Student's t‐test. (C)The cell growth rates were determined with CCK‐8 proliferation assay. *P < 0.05, Student's t‐test. (D) Colony formation assay of gastric cells transduced with lentiviruses expressing the indicated shRNA. (E) Representative images of the tumours formed in nude mice induced by HGC27 cells transduced with indicated shRNA. (F) Mean xenograft tumour volumes were plotted against days after injection. (G) Weights of the excised xenograft tumours were summarized. **P < 0.01, Student's t‐test. Three independent experiments were conducted in A, C and D, each in triplicate. Values are means ± standard deviation (S.D.) for triplicate determinations from three different cultures. Data obtained from a representative experiment are shown.
Figure 3
Figure 3
Inhibition of PRC1 leads to cell‐cycle arrest and apoptosis. (A) Cell‐cycle analysis of cells transduced indicated shRNA using PI and flow cytometric analysis. *P < 0.01, when compared with shCont. (B) Western blot analysis of the protein expression of cell‐cycle regulators. β‐Actin was used as the loading control. (C) Flow cytometry analysis of the apoptotic cells using Annexin V/PI double‐staining assay. (D) Positive Annexin V cells were displayed as histogram. *P < 0.05, when compared with shCont. (E) Western blotting was used to analyse the expression of full‐length and cleaved PARP, cleaved caspase‐3 and cleaved caspase‐9. β‐Actin was used as the loading control. (A‐D), all experiments were carried out in triplicate and representative results from three independent experiments are shown. Bar means mean ± S.D. for triplicate determinations from three different cultures.
Figure 4
Figure 4
Knockdown of PRC1 impedes gastric cancer cell migration and invasion. Significant reduction in cell migration (A) and cell invasion (B) was found in cells expressing sh1 and sh2, compared with cells expressing shCont. Representative images of migrated AGS and HGC27 cells in each group are shown in the left. Cells migrated through the pores of transwell plates were counted and reported in the right. Data are presented as mean ± S.D. of three independent experiments. *P < 0.05, when compared with shCont.
Figure 5
Figure 5
Knockdown of PRC1 selectively impairs cytokinesis in AGS but not HGC27 cells. (A) Cells expressing indicated shRNA were analysed using phalloidin (FITC, green) and DAPI (blue). Scale bar, 50 μm. (B) The cell membrane was stained using the fluorescent dye PKH67 after continuous subculture for 2 generations. Scale bar, 100 μm. (C) Quantitative real‐time analysis of mRNA levels of genes related to mitosis. β‐Actin mRNA expression was used as an internal control. Experiment was conducted in triplicate. Values represent the mean ± S.D. of 3 independent experiments. *P < 0.01, when compared with shCont.
Figure 6
Figure 6
Piperlongumine inhibits PRC1 expression via a p53‐dependent mechanism. (A) Quantitative real‐time analysis of PRC1 mRNA level in AGS and HGC27 cells transfected with p53‐expressing plasmid or empty vector. β‐Actin mRNA expression was used as an internal control. Experiment was conducted in triplicate. Values represent the mean ± S.D. of three independent experiments. *P < 0.01, when compared with cells transfected with empty vector. (B) Western blotting analysis of p53 and PRC1 proteins in AGS and HGC27 cells transfected with p53‐expressing plasmid or empty vector. β‐Actin was used as the loading control. (C) Quantitative real‐time analysis of PRC1 mRNA level in AGS and HGC27 cells treated with or without increased concentrations of piperlongumine for 24 hrs. β‐Actin mRNA expression was used as an internal control. Experiment was conducted in triplicate. Values represent the mean ± S.D. of three independent experiments. *P < 0.01, when compared with cells treated with vehicle control. (D) Western blotting analysis of p53 and PRC1 in AGS and HGC27 cells treated with various concentrations of piperlongumine for 24 hrs. β‐Actin was used as the loading control. (E) AGS cells were transfected with either negative control‐siRNA or p53‐specific siRNA for 48 hrs and then followed by treatment of 10 μM of piperlongumine for 24 hrs, and whole‐cell lysates were separated by SDS‐PAGE and then reacted with indicated antibodies. (F) Normalized luciferase activity of various PRC1 promoter reporters in AGS cells treated with piperlongumine at 10 μM or with vehicle control for 24 hrs. pRL‐TK reporter plasmid was cotransfected as a control for transfection efficiency. The relative activity of pGL3‐SV40, a luciferase reporter gene construct driven by the SV40 promoter in control cells was designated as 1.0. Results are the mean ± S.D. of triplicate measurements. *P < 0.01.

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