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. 2017 Nov;51(5):1497-1507.
doi: 10.3892/ijo.2017.4137. Epub 2017 Sep 27.

The receptor for activated protein kinase C promotes cell growth, invasion and migration in cervical cancer

Shan Liao  1 , Songshu Xiao  2 , Hongxiang Chen  3 , Manying Zhang  1 , Zhifang Chen  4 , Yuehua Long  1 , Lu Gao  1 , Junyu He  1 , Yanshan Ge  1 , Wei Yi  1 , Minghua Wu  1 , Guiyuan Li  1 , Yanhong Zhou  1
Affiliations

The receptor for activated protein kinase C promotes cell growth, invasion and migration in cervical cancer

Shan Liao et al. Int J Oncol. 2017 Nov.

Abstract

Cervical cancer is one of the most common malignant tumors in women all over the world. However, the exact etiology of cervical cancer remains unclear. The receptor for activated protein kinase C (RACK1) is reported to be involved in tumorigenesis and tumor progression. Besides, the prognostic value of RACK1 in several kinds of tumors has been identified. However, there are limited studies on the functional role of RACK1 in cervical cancer. In this study, we tested the expression level of RACK1 by immunohistochemistry and western blot technologies and find that it is upregulated in cervical cancer. Colony formation and CCK8 assays indicate that RACK1 promotes cell proliferation in CaSki cervical cancer cells. While the silence of RACK1 decreases the cell proliferation in CCK8 analysis. β-galactosidase staining suggests that RACK1 decreases cell senescence in cervical cancer cells. Invasion and migration assay show that RACK1 promotes the invasion and migration of cervical cancer cells. Also, when RACK1 was silenced, it exerts the opposite result. Furthermore, the mRNA expression levels of MMP‑3, MMP‑9 and MMP‑10 were upregulated in RACK1‑overexpressed CaSki cells by qPCR analysis. RACK1 also induces S phase accumulation in cell cycle analysis and suppresses cell apoptosis in cervical cancer cells. Flow cytometry analysis of mitochondria functions suggests that RACK1 increases the mitochondrial membrane potential (Δψm) levels to prevent mitochondrial apoptosis in cervical cancer cells. To explore the possible mechanism of RACK1, we tested and found that RACK1 upregulates the expression of NF-κB, cyclin D1 and CDK4 and downregulates the expression of p53, p38, p21 and STAT1 in cervical cancer cells. These results suggest that RACK1 promotes cell growth and invasion and inhibits the senescence and apoptosis in cervical cancer cells probably by affecting the p53 pathway.

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Figures

Figure 1
Figure 1
The detection of expression levels of RACK1 in the cervical cancer tissues and the adjacent non-cancerous tissues by western blot analysis and IHC. (A) The expression levels of RACK1 were tested by western blot analysis. T, cervical cancer tissues and N, the adjacent non-cancerous tissues. Data are representative of three independent experiments. (B) Immunohistochemistry analysis of the expression of RACK1 protein was detected in 25 pairs of cervical cancer tissues and the adjacent non-cancerous tissues. Antibody of mouse anti-RACK1 was used; brown grains denote positive signal. Representative images of H&E staining of cervical epithelial tissues and cervical cancer tissues, negative control, RACK1 staining of cervical epithelial tissues and cervical cancer tissues are shown. T, cervical cancer tissues and N, the adjacent non-cancerous tissues. Original magnification (×200).
Figure 2
Figure 2
RACK1 promotes the cell proliferation and migration in cervical cancer cells. (A) Identification the RACK1 expression in RACK1-overexpressed stable CaSki cells and RACK1-RNAi transiently transfected cells by western blot analysis. (B) Colony formation assay. Vector represents cells transfected with the pEGFP-N1 plasmid and RACK1 represents cells transfected with the pEGFP-N1-RACK1 plasmid. (C and D) CCK8 analysis was performed in RACK1-overexpressed CaSki cells and RACK1-silenced CaSki cells. CaSki-vector represents CaSki cells transfected with the pEGFP-N1 plasmid and CaSki-RACK1 represents CaSki cells transfected with the pEGFP-N1-RACK1 plasmid. RNAi-NC represents CaSki cells transfected with RNAi-NC and RNAi represents CaSki cells transfected with RACK1-RNAi. (E) Statistical analysis of wound healing assay. (F) Representative images of wound healing assay performed in RACK1-overexpressed CaSki cells. (G) Wound healing assay was performed in RACK1-silenced CaSki cells. Data are shown as mean ± SD from three independent experiments. *P<0.05; **P<0.01; ***P<0.001.
Figure 3
Figure 3
Transwell invasion and migration assay and β-galactosidase staining assay between CaSki-RACK1 cells and CaSki-vector cells. (A) Transwell migration assay. (B) Transwell invasion assay. (C) Statistical analysis of cell number for Transwell invasion and migration assays. Results are presented as mean ± SD from three independent experiments. (D) mRNA expression levels of MMP-2, MMP-3, MMP-9 and MMP-10. (E and F) β-galactosidase staining and statistical analysis of cell numbers for β-galactosidase staining assay. The assay was conducted as three independent experiments. *P<0.05; **P<0.01; ***P<0.001.
Figure 4
Figure 4
Cell cycle analysis, cell apoptosis and the mitochondrial membrane potential were detected by flow cytometry in CaSki cell lines. (A) Flow cytometry analysis of cell cycle between CaSki cells transfected with pEGFP-N1 (vector) and transfected with pEGFP-N1-RACK1. For x-axis, it represents the mean fluorescence intensity (MFI) detected by flow cytometry. For y-axis, it represents cell numbers. Statistical analysis of percentages of cells at different stages (G1, G2 and S) was also performed. (B) Flow cytometry analysis of apoptosis in RACK1-overexpressed CaSki cells (upper image) and RACK1-silenced CaSki cells (lower image). Cells in the gate R3 frame represent late apoptotic cells, and in gate R5 were early apoptotic cells. Data are representative of three independent experiments. (C) The mitochondrial membrane potential (MMP) was detected by JC-1 assay. The loss of mitochondrial membrane potential was represented by the fluorescence change from red to green. Vector indicates CaSki cells transfected with the pEGFP-N1 plasmid, and RACK1 CaSki cells transfected with the pEGFP-N1-RACK1 plasmid. Data are representative of three independent experiments. *P< 0.05; **P<0.01.
Figure 5
Figure 5
Western blot analysis indicates that RACK1 overexpression affected the expression of p53, p38, p21, STAT1, NF-κB, cyclin D1 and CDK4 of cervical cancer cell in vitro. The protein expression levels of p53, p38, p21, STAT1, NF-κB P65, cyclin D1 and CDK4 in RACK1-overexpressed stable cervical cancer cell line of CaSki were analysed by western blotting. Vector, CaSki cells transfected with the pEGFP-N1 plasmid, Rack1, CaSki cells transfected with the pEGFP-N1-RACK1 plasmid.
Figure 6
Figure 6
The diagram of a potential signaling pathway that RACK1 regulates the cell proliferation and cell invasion in cervical cancer cells.
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