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

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.

liver, ovarian, and thyriod cancers 14,15 . Lamprey is one of the most ancient vertebrates alive today, which makes it an excellent model for the study of vertebrate evolution, embryo development 16,17 , and the origin of adaptive immunity. It is also considered as a bridge that connects the invertebrates with the vertebrates. In contrast to the extensive studies of PHB2 from the mammalian, little work has been done on the PHB2 from Lampetra morri (L. morri). Until 2015, Li et al. 18 firstly identified recombinant Lm-PHB2 (rLm-PHB2) protein could significantly enhance the H 2 O 2 -induced oxidative stress tolerance in Chang liver (CHL) cells. In this study, we found other functions of Lm-PHB2 that induces G2/M phase arrest in HeLa cells.
The eukaryotic cell cycle is tightly regulated and encompasses checkpoints in each of its different phases 19 . Cellular checkpoint control is pivotal in minimizing DNA damage accumulation and ensuring genomic integrity during cell cycle progression, that degregulation and resulting DNA damage have been implicated in many diseases, including cancer and neurodegenerative disorders 20 . Research conducted during the last two decades supports that nuclear cytoplasmic cycling of important G2 checkpoint proteins-such as cyclin-dependent kinase1 (CDK1), Cyclin B1, Wee1 kinase (Wee1), polo-like kinase 1 (PLK1) and cell division control protein 25C (CDC25C) is a key mechanism of G2 checkpoint regulation 21,22 .
In this study, Lm-PHB2 was found to inhibit the proliferation of HeLa cells through down-regulating the expression level of CCNB1, CDC25C, Wee1 and PLK1. Similarly, phosphorylation level of CCNB1, CDC25C and CDK1 has been inhibited, then leading to G2/M phase arrest and cell apoptosis, suggesting that Lm-PHB2 may have therapeutic value in the treatment of cervical cancer.

Lm-PHB2 inhibits HeLa cells proliferation.
To investigate the anti-proliferative effect of Lm-PHB2, two HeLa cell lines were treated with various concentrations of rLm-PHB2 for different periods of time followed by cell viability assay. Exogenous rLm-PHB2 protein with His-tag was mostly localized in cytoplasm in HeLa and C33A cells (Figs 1A, S1). In both HeLa and HeLa299 cells lines, the untreated cells (PBS-treatment only) increased their number at a faster rate than their rLm-PHB2-treated counterparts, and this was evident in all three time points measured ( Fig. 1 B,C). In both cases, reduction in cell growth caused by PHB2 was significant for all concentrations of PHB2 tested after 48 h of treatment, except for those treated with 0.625 μM rLm-PHB2 proteins. However, in the case of 72 h treatment, all concentrations of PHB2 caused significant reduction.
Similar result was obtained when these two cells lines were transfected with a plasmid containing Lm-PHB2 (pEGFP-N1-Lm-PHB2) instead of being treated with the protein (Fig. 2A). In addition, GFP-fused Lm-PHB2 could be expressed and was mostly localized in nucleus, a small amount in the cytoplasm and mitochondria in HeLa and C33A cells (Figs 2B, S2). Cells that were transfected with pEGFP-N1-Lm-PHB2 showed significantly Lm-PHB2 induced G2/M phase cell cycle arrest. In order to better understand the mechanism of growth inhibition exerted by Lm-PHB2 on HeLa cells, the percentage of cells cycle in the presence of rLm-PHB2 protein was analyzed using double staining with both APC-BrdU and PI. The results showed that rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase (Fig. 3). In addition, the results of PI staining only showed that compared to the PBS-treated group, the percentage of G2/M phase cells in Lm-PHB2-treated groups was increased, and in an Lm-PHB2 concentration-dependent manner (Supplementary 3). Similarly, HeLa cells transfected with pEGFP-N1-Lm-PHB2 also exhibited significant increases in the proportion of G2/M phase cells compared to those transfected with pEGFP-N1 (Fig. 4). Thus, Lm-PHB2 appeared to inhibit the growth of HeLa cells by inducing cell cycle arrest at the G2/M phase. Since it was easier to observe the experimental phenomena in HeLa cells than in HeLa 229 cells, subsequent experiments therefore focused on HeLa cells only.
Lm-PHB2 induces cell cycle arrest through down-regulating the expression and phosphorylation level of cell cycle proteins. To examine the possible mechanism associated with G2/M phase arrest induced by Lm-PHB2, the effect of Lm-PHB2 on the expression of G2/M phase proteins expression and phosphorylation level was investigated. HeLa cells were transfected with pEGFP-N1-Lm-PHB2 or pEGFP-N1 for 48 h, and the transcript and protein levels of these genes were quantified by real-time PCR and western blot, respectively. Cells transfected with pEGFP-N1-Lm-PHB2 showed significantly reduced expression of CDC25C and CCNB1, both at the transcript (Fig. 5A) and protein (Fig. 5B) levels compared to cells transfected with pEGFP-N1. As for CDC25A, CDK2 and CDC2, no change in either transcript or protein level was found between cells transfected with pEGFP-N1-Lm-PHB2 and pEGFP-N1. Subsequently, according to western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1, PLK1, p-CDC25C, p-CCNB1 and p-CDK1 in HeLa cells. These

Discussion
Lamprey PHB2 induces HeLa cells apoptosis. Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are the two highly homologous subunits of the eukaryotic mitochondrial PHB complex 21 . A large number of studies have shown that prohibitin can translocate into the nucleus or the mitochondria under apoptotic signals and the subcellular shuttling of prohibitin is necessary for apoptosis process. In the nucleus, PHB regulates transcriptional activation and cell cycle. At the mitochondrial inner membrane that implicated in mitochondrial genome stabilization, mitochondrial morphology, oxidative stress, and apoptosis 16,22 . Moreover, increased membrane localization of PHBs and the PHB1/c-Raf complex in activated hepatic stellate cells may promote Tan IIA-induced apoptosis 8,23 . To our knowledge, there has been no report concerning lamprey PHB2 in cervical cancer. Recently, lampreys are considered to be the most scientifically accessible model of the remaining jawless vertebrates. In the present study, lamprey PHB2 was found to inhibit the proliferation of HeLa cells through arresting cell cycle transition. The results we obtained before showed the cell cycle arrest induced by Lm-PHB2 was not creative by protein antigenicity against human. We also found the number of S phase cells decreased after treatment with rLm-PHB2 (Supplementary 3). It is difficult that this altered G1/S phase state is caused by which rLm-PHB2 functioned against G1/S and/or G2/M transition. So the alteration of G1/S and/or G2/M transition was examined using double staining with both APC-BrdU and PI after HeLa cells treated with rLm-PHB2. This result revealed that rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase (Fig. 3).
Lamprey PHB2 arresting the cell cycle at the G2/M phase. Cell-cycle checkpoints at the G2/M as well as G1/S phases are critical for maintaining DNA integrity and regulating the passage of cells through the cell cycle 24 . It is well known that the loss of these checkpoints can lead to the transition and progression of cancer cells. CDC25C is responsible for stimulating and maintaining the complexes CCNB1-CDK1 activation that ultimately determines to pass the G2 checkpoint 25 . PLK1 also promotes G2/M transition progression through affecting the subcellular localization of CDK1 regulatory checkpoint nodes. Cytoplasmic activity of PKL1 is also required to prime the CCNB1-CDK1 complex for nuclear localization by phosphorylation of CCNB1. Moreover, Wee1 also regulate G2/M phase transition and cell mitosis through phosphorylation of CDK1 and down-regulation of CDC2 kinase activity 26 . This means that when the expression level of CCNB1, CDC25C, CDK1 and PLK1 is reduced, the abilities in tumor cells proliferation could be inhibited at the same time. According to expression level analysis of cell cycle proteins, lamprey PHB2 was found to reduce the expression level of CCNB1, CDC25C, Wee1, CDK1 and PLK1 in HeLa cells, which indicated that lamprey PHB2 could arrest the G2/M phase Similarly, phosphorylation level of CCNB1, CDC25C and CDK1 has been down-regulated in HeLa cells. Activated PLK1 could phosphorylate CDC25C on Ser216 and CCNB1 on serine residues and promote cytoplasmic-nuclear translocation of CDC25C 27 . In the nucleus, Wee1 could phosphorylate CDK1 on Tyr15 and phosphorylated CDK1 inactivates the kinase and thus induces G2/M phase transition arrest 28,29 . Above results indicated that lamprey PHB2 could induce cell cycle arrest not only through inhibiting expression level of CCNB1, CDC25C, Wee1, CDK1 and PLK1, but also through down-regulation phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. In order to meet the criterion of genetic engineering drugs, overcoming antigenicity against human and removing the His-tag from rLm-PHB2 would provide the opportunity for the application of rLm-PHB2 as a potential anti-tumor drug in the future clinical studies.  Cell culture. HeLa cell lines were from stocks preserved in our laboratory. The cells were grown in DMEM medium supplemented with 10 % fetal bovine serum and in a 37 °C humidified incubator with 5 % CO 2 . The cells were grown to 70 % confluence and then harvested by digestion with trypsin-EDTA, and further plated in 6-well (2 × 10 5 cells/well) or 96-well plates (1 × 10 4 cells/well) for subsequent experiments.

Materials
Immunofluorescence. The HeLa cells (1 × 10 5 ) were cultured on slides in 24-well plates for 24 h, and then treated with 10 μM rLm-PHB2 or 10 μM Bovine Serum Albumin (BSA) for 24 h. After rinsing with PBS, the HeLa cells were fixed with 4 % paraformaldehyde for 15 min. 1 % Triton X-100 treated with Hela cells for 15 min, and then blocked with BSA and incubated with His-tag antibodies (1:1000) for 3 h at room temperature. Secondary antibodies (1:5000) incubated for 40 min. Subsequently, the HeLa cells were washed with PBS twice, and then stained with Mitotracker (Green) 3 min at room temperature in the dark. After rinsing with PBS twice, stained with Hoechst 33258 for 3 min at room temperature in the dark. After washing with PBS twice, a laser scanning confocal microscopy was used to observe the HeLa cells at 630× magnification.  HeLa cells were grown in 6-well plates (2 × 10 5 cells/well) for 12 h, and then transfected with pEGFP-N1 or pEGFP-N1-Lm-PHB2 plasmid for 24 h. After removing the medium, the adherent cells were digested with ethylene diaminetetraacetic acid (EDTA) free trypsin (HyClone, USA) and harvested by centrifugation. The cells were Cell transfection. Transfection using Translipid Transfection Reagent (TransGen Biotech, China) was performed according to the manufacturer's instructions on cells that were at least 70 % confluent. After 24 h, the cells were washed with PBS, and the medium was replaced with fresh, normal growth medium with or without 250 mM H 2 O 2 for 3 h for subsequent MTT assays, qRT-PCR and western blotting.

Quantitative real-time PCR (qRT-PCR).
HeLa cells were transfected with the pEGFP-N1 or pEGFP-N1-Lm-PHB2 for 36 h and total RNA was then isolated from the cells using RNAiso Plus (TAKARA, China). The RNA was subjected to reverse transcription using the PrimeScript TM RT reagent Kit with gDNA Eraser (TAKARA, China). Quantitative RT-PCR was performed with the SYBR Premix ExTaq TM II Kit (TAKARA, China) according to the manufacturer's protocol using GAPDH as an internal control. Quantitative real-time PCR used primers used are listed in Table 1.
The cells were subsequently transfacted with pEGFP-N1 or pEGFP-N1-Lm-PHB2 plasmids for 24 h at 37 °C. After that, the cells were harvested by centrifugation and lysed in cell lysis buffer containing 0.2 mM pheylmethanesulfonyl fluoride (PMSF). The cell lysate was then centrifuged at 5000 × g for 15 min and the supernatant was retained. Total protein concentration in the supernatant was measured by the BCA Kit using BSA as a standard. The samples was then subjected to SDS-PAGE using 12 % gel. After electrophoresis, the proteins in the gel were transferred to a polyvinylidene difuoride (PVDF) membrane. The membrane was blocked with 5 % non-fat dairy milk in TBST buffer (20 mM Tris-HCl (pH 8.0)/150 mM NaCl/0.05 % Tween-20) for 2 h, and then incubated with the appropriate primary antibody at room temperature for 5 h. Antibody of CDC25A, CDK2, CDC25C, CCNB1, CDC2, p-CDK1, PLK1, Wee1 and GAPDH was used at 1:500 dilution, whereas antibody of p-CCNB1 and p-CDC25C was used at 1:1000 dilution. After incubation with the primary antibody, the blot was washed five times with TBST buffer followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at 1:5000 dilution for 1 h at room temperature. The blot was again washed five times with TBST buffer and finally visualized with the BeyoECL Plus detection Kit. Specific protein bands detected via immunoblot analysis were quantified via densitometry (Gel-Pro analyzed 4). The scanned image was inverted to measure the gray value of a specific protein band. The numerical value was recorded as an gray value. The histograms were shown to indicate the changes of the relative cell cycle proteins level in HeLa cells after transfacted with pEGPF-N1 or pEGFP-N1-Lm-PHB2 plasmids control with GAPDH. Statistical analysis. Statistics' t-test was used to analyze differences between test groups and control groups.
Statistical significance was considered at the P < 0.05 and or P < 0.01 level.
Data availability. All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Raw datasets generated are available from the corresponding author on reasonable request.