Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins

Abstract

Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

Figure 1

Effect of purified rLm-PHB2 on the proliferation of human cervical cancer cells. (A) Confocal microscopic images of rLm-PHB2 protein entering into HeLa cells and localizing in cytoplasm. rLm-PHB2 culture 24 h were immunostained with His-tag antibody (Red). Mitochondria were stained with MitoTracker (Green), and nucleus were stained with Hoechst33258 (bule). Scale bar, 10 μm. (B,C) HeLa and HeLa 229 cells were treated with PBS or different concentrations of purified Lm-PHB2 (0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM and 10.0 μM) at 37 °C for 24 h, 48 h or 72 h and cell viability was determined by the MTT assay. Data are the means ± SDs from three experiments, each carried out in triplicate. ‘*’ and ‘**’ indicate significantly different from control cells (not treated with Lm-PHB2) at the P < 0.05 and P < 0.01 levels, respectively.

Figure 2

Effect of overexpression of Lm-PHB2 on the proliferation of human cervical cancer cells. (A)Transfer efficiency of HeLa cells after transfected with pEGFP-N1-Lm-PHB2 or pEGFP-N1 at 37 °C for 24 h. Scale bar, 100 μm. (B) Subcellular organelles localization of Lm-PHB2 protein in HeLa cells visualized by confocal microscopy with Hoechst33258 (blue, nucleus), GFP (green, Lm-PHB2), and MitoTracker Red (red, mitochondria). Scale bar, 10 μm. (C,D) HeLa and HeLa 229 cells were transfected with pEGFP-N1-Lm-PHB2 or pEGFP-N1 at 37 °C for 24 h, 36 h, 48 h, 60 h or 72 h and the viability of the cells was determined by MTT assay. Data are the means ± SDs from three experiments, each carried out in triplicate.‘*’and ‘**’ indicate significantly different from cells transfected with pEGFP-N1 at the P < 0.05 and P < 0.01 levels, respectively.

Figure 3

Effect of Lm-PHB2 on the cell cycle. The double staining with both APC-BrdU and PI for detection of G1/S and/or G2/M cell cycle alteration after HeLa cells treated with rLm-PHB2 protein and PBS treatment as control.

Figure 4

Effects of Lm-PHB2 on the proportion of cells in different phases of the cell cycle. HeLa cells were transfected with pEGFP-N1 or pEGFP-N1-Lm-PHB2 at 37 °C for 36 h followed by PI staining and flow cytometry analysis. The histogram shows the percentage of G2/M phase cells in both groups. Data are the means ± SDs from three experiments. ‘*’ indicates significantly different from PBS-treated cells at the P < 0.05 level.

Figure 5

Lm-PHB2 induces cell cycle arrest through down-regulating the expression and phosphorylation level of cell cycle proteins. (A,B) Changes in relative expression levels of cell cycle proteins determined by qRT-PCR and western blot. (C) The histogram in B compares the intensities of the various bands shown in the blot. Data in the histograms are the means ± SDs from three determinations. ‘*’ and ‘**’ respectively indicates significantly different from PBS-treated cells at the P < 0.05 or P < 0.01 level.