Lentivirus-mediated Silencing of Rhomboid Domain Containing 1 Suppresses Tumor Growth and Induces Apoptosis in Hepatoma HepG 2 Cells

Many functionally important membrane proteins are cleaved within their transmembrane helices to become activated (Ha, 2009). This process is catalyzed by a class of highly specialized and membrane-bound proteases, named as intramembrane proteases, including metalloproteases, aspartyl proteases and the rhomboid-like family (De Strooper et al., 1998; Akiyama et al., 2004; Lemberg and Freeman, 2007). Although initially seen as purely degradation enzymes, it has become clear that they are involved in several crucial cellular and developmental processes, such as, cell signaling, apoptosis and blood clotting (Brown et al., 2000; Kroos and Yu, 2000; Weihofen and Martoglio, 2003; Wolfe and Kopan, 2004; Freeman, 2008). In the past years, a growing number of papers have been published to address the structural features, functions and mechanisms of these membrane proteins. Drosophila rhomboid-1, originally discovered as a key genetic factor in the fly epidermal growth factor receptor (EGFR) signaling pathway, is the founding member of the family (Urban et al., 2001). Subsequent researches showed that rhomboid proteases are present in most organisms. In yeast, there are two rhomboids, named Rbd1p and Rbd2p. Rbd1p is localized in the inner mitochondrial membrane and mutant cells have disrupted mitochondria membrane


Introduction
Many functionally important membrane proteins are cleaved within their transmembrane helices to become activated (Ha, 2009).This process is catalyzed by a class of highly specialized and membrane-bound proteases, named as intramembrane proteases, including metalloproteases,

Lentivirus-mediated Silencing of Rhomboid Domain Containing 1 Suppresses Tumor Growth and Induces Apoptosis in Hepatoma HepG2 Cells
Xue-Ni Liu, Zheng-Hao Tang, Yi Zhang, Qing-Chun Pan, Xiao-Hua Chen, Yong-Sheng Yu*, Guo-Qing Zang* remodeling (McQuibban et al., 2003).In parasites, a rhomboid-like protease plays an important role in host cell invasion (Howell et al., 2005).Despite the functions in lower eukaryotes, the functions of vertebrate rhomboidlike family members remain unclear.However, several investigations have provide strong evidence that rhomboid protease may play an important role in cancer cell growth and apoptosis.For example, human rhomboid family-1 (RHBDF1), a novel family member of rhomboid proteases, participates in the modulation of GPCR-mediated EGFR transactivation and disrupts growth signals in several cancer cell growth, including epithelial cancer, breast cancer, and head and neck squamous cancer cells (Yan et al., 2008;Zou et al., 2009).Rhomboid domain containing 1 (RHBDD1), an important gene highly expressed in the testis, is shown to participate in the cleavage of BIK and modulate BIK-medicated apoptosis (Wang et al., 2008).RHBDD2, markedly over-expressed in primary tumors from patients with recurrent disease, has been shown to regulate cell proliferation of breast cancer cells (Abba et al., 2009).
Consequently, to determine the potential role of RHBDD1 in human primary hepatocellular carcinoma (HCC), we studied the effect of RHBDD1 knockdown in HCC HepG2 cells via a lentivirus-mediated silencing system.Our data show that in vitro RHBDD1 silencing regulates HepG2 cell proliferation and apoptosis.

Cell culture and transfection
The human hepatoma cancer cell line HepG2 was purchased from the American Type Culture Collection (ATCC).The cells were grown in RPMI-1640 culture medium (Invitrogen, Carlsbad, CA) containing 1% penicillin/streptomycin, at 37°C with 5% CO 2 .In the following experiments, purified RHBDD1-shRNA and control-shRNA lentiviruses were applied to HepG2 cells.To monitor the infection efficiency, infected cells were collected for fluorescence microscopy and quantitative real-time PCR (qRT-PCR) assay 48 hrs after lentivirus treatment.

RNA extraction, reverse transcription and qRT-PCR
Infected (RHBDD1-shRNA and control-shRNA) HepG2 cells were collected and subjected to RNA extraction and reverse transcription.Total RNA was isolated by Trizol (Invitrogen, Carlsbad) reagent, and 2 μg total RNA was transcripted into cDNA with random primers.QRT-PCR was performed according to the ABI (Applied Biosystems, Bedford, MA, USA) manufacturer's protocols.Relative expression levels of RHBDD1 mRNA were compared to the levels of reference gene (GAPDH) by comparative cycle threshold (ct) method as fold difference = 2 -(rct of target gene-rct of reference) .Two pairs of primers were used in this experiment.
All samples were examined in triplicate.

Cell proliferation assay
Cell proliferation assay was determined by measuring GFP-positive cells using the Cellomics Array Scan VTI HCS (Thermo Fisher Scientific, Pittsburgh, PA).Briefly, HepG2 cells were infected with lentiviruses (RHBDD1-shRNA or control-shRNA) for 48 hrs, and infected cells were re-cultured in 96-well plates with a density of 2000 cells/well.Plates were read on the Thermo Scientific Cellomics ArrayScanat indicated time points for cell count.The cell count was expressed as fold changes.

Colony formation assay
HepG2 cells were cultured in 24-well plates and treated with RHBDD1-shRNA or control-shRNA lentiviruses.And 0.5 mL under layers consisting of 0.8% agar medium was prepared in 6-well plates.After 48 hrs of incubation, cells were washed, re-cultured in the prepared 6-well plates, and allowed to form natural colonies.After 14 days, cells in both groups were subjected to Giemsa staining.Firstly, cells were washed and fixed by paraformaldehyde.Then fixed cells were washed twice with PBS solution, treated with Giemsa (MBCHEM, New Jersey, USA) for 10 min, washed 3 times by ddH 2 O, and then photographed with a digital camera.The number of colonies (>50 cells/ colony) were counted.

Cell cycle analysis by flow cytometry
Flow cytometry was used to analyze the percentage of cells in each phase.In brief, HepG2 cells were synchronized by serum starvation for 24 hrs.Then cells were transfected with RHBDD1-shRNA or control-shRNA lentiviruses.After 48 hrs incubation, cells were trypsinized, washed with PBS and fixed in 70% ethanol.The fixed cells were then incubated in propidium iodide (PI, 100 μg/mL) (Sigma, USA)/PBS solution with Rnase (10 μg/mL) for at least 30 min at 37 °C in dark.PI stained cells were analyzed by a FACs caliber II sorter and Cell Quest FACS system (BD Biosciences, USA).The cell cycle distribution in G1, S or G2/M phases in both groups was determined.All samples were examined in triplicate.

Apoptosis analysis by Annexin-V
The apoptotic effect of RHBDD1 in HepG2 cells was examined by flow cytometry using Annexin V Apoptosis Detection Kit (eBioscience, San Diego, CA, USA).Firstly, HepG2 cells were infected with lentiviruses containing RHBDD1-shRNA or control-shRNA.After 72 hrs, infected cells were trypsinized, washed with D-Hanks buffer, PBS buffer and 1× binding buffer, consecutively.And cells were subjected to 1× staining buffer containing annexin V-APC for 15 min.The percentage of apoptotic cells was analyzed by FACS Calibur (BD Biosciences, USA).

Statistical analysis
The data shown are the means ± SD of three experiments.Statistical significance was estimated with Student's t-test.A P-value of less than 0.05 was considered significant in different experiments.

Suppression of RHBDD1 by siRNA in human HepG2 cells
The lentiviruses containing RHBDD1-shRNA or control-shRNA were co-transfected into HepG2 cells.After 48 hrs, these cells were examined via fluorescence microscope.As shown in Figure 1A, The GFP tag in lentiviruses enables visualization of transfection efficiency via bright (Figure 1A, left) and fluorescence (Figure 1A, right) microscope.In both groups, >80% cells were successfully infected.To confirm RHBDD1-shRNA silencing effect, cells were lysated, followed by RNA extraction, reverse transcription and qRT-PCR.Over 60% RHBDD1 mRNA was decreased after treatment using RHBDD1-shRNA as compared to that using control-shRNA (Figure 1B).Therefore, the knockdown efficiency of RHBDD1 by lentiviral system was verified.

Reduction of RHBDD1 caused growth delay of human HepG2 cells
To assess the role of RHBDD1 in the survival of human HepG2 cells, cell proliferation assays were performed to examine the growth of HepG2 cells with reduced RHBDD1 mRNA expression.As shown in Figure 2A, down-regulation of RHBDD1 resulted in significant delay of HepG2 cell growth as compared to the control-shRNA on the third day following lentivirus infection (P<0.001).On the fourth and fifth day, the growth inhibitory effect of RHBDD1-shRNA was more remarkable.
To study the role of RHBDD1 in human HepG2 cell tumorigenesis in vitro, we further evaluated the colony formation capacity of HepG2 cells in soft agar.As Figure 2B shows, reduction of RHBDD1 in HepG2 cells caused a substantial reduction in colony formation in soft agar as compared with the control group (P<0.01).The RHBDD1-shRNA treated group formed 74.0 ± 12.5 colonies; while control-shRNA formed 144.3 ± 15.6 colonies in 14 days after lentivirus treatment.Thus, the reduced RHBDD1 expression ultimately caused significant inhibition of growth in human HepG2 cells, suggesting that RHBDD1 is critical for tumorigenicity of human HepG2 cells in vitro.

Suppression of RHBDD1 induced G2/M phase arrest
In order to find out whether RHBDD1-shRNA delays HepG2 cell proliferation partly through regulation of cell cycle, we then examined cell cycle distribution by flow cytometry.Firstly, cells were infected with lentiviruses containing RHBDD1-shRNA or control-shRNA.After 48 hrs incubation, cells were harvested and analyzed by PI staining and flow cytometry.Compared with control-shRNA treated HepG2 cells, RHBDD1-shRNA treated cells showed a substantial decrease in G1-phase (P<0.001) and an increase in G2/M phase populations (P<0.001) in HepG2 cells (Figure 3).

Decreased RHBDD1 induced apoptosis
To determine the apoptotic effect of RHBDD1-shRNA on HepG2 cells, an Annexin V apoptosis assay was performed.As Figure 4 shows, more than 95% of the control-shRNA cells were viable, while less than 80% of the RHBDD1-shRNA cells were alive.The apoptotic cells in RHBDD1 inhibition group was 7.7 times that in control group (P<0.001).Our data indicated that RHBDD1 has a potential role in the regulation of apoptosis in human HepG2 cells.

Discussion
HCC is one of the most common malignancies worldwide and is estimated to cause half a million deaths annually (Zeng et al., 2012).Accumulating researches have reported that EGFR and key members in EGFR signaling are aberrantly expressed in HCC, contributing to invasion, metastasis and resistance to chemotherapy and radiotherapy (Yoneda et al., 2011;Ezzoukhry et al., 2012;Gao et al., 2012).Moreover, various sites of EGF receptor and related kinases can be targets of treatments for HCC (Desbois-Mouthon et al., 2006;Berasain et al., 2012;Sogawa et al., 2012).For example, is a chimeric monoclonal Ig G1 antibody directed against the EGFR and blocks binding of endogenous EGFR ligands (Furuse, 2008).Cetuximab alone or in combination of erlotinib, other tyrosine-kinase inhibitors and cytotoxic chemotherapy, can inhibit the proliferation of HCC cells in vivo and in vitro (Sanoff et al., 2011;Chen et al., 2012;Ezzoukhry et al., 2012).
In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands.Intriguingly, in human, members of rhomboid intramembrane proteases have also been shown to play an important role in the activation of EGFR (Zou et al., 2009;Adrain et al., 2011).More importantly, the disruption of EGFR signaling by family may lead to the alterations of the biological characteristics of cancers, including proliferation and invasion (Zou et al., 2009).However, it is still unclear whether rhomboid family is involved in HCC development.Therefore, in the present study, we try to identify the biological functions of RHBDD1, a new member of the Rhomboid family, in the proliferation and apoptosis of HepG2 cells via a lentivirus-mediated silencing system (Figure 1).
We found that lentivirus-mediated down-regulation of RHBDD1 in human hepatoma cancer cell line HepG2 cells leads to marked inhibition of cell proliferation (Figure 2).Moreover, the suppression of HepG2 cell growth may partly resulted from the dysregulation of cell cycle progressing.As Figure 3 shows, RHBDD1-shRNA induced an accumulation of HepG2 cells in G2/M phase (23.1%), as compared to control-shRNA (11.3%).We thus suspect that the inhibition of HepG2 cell growth and cell cycle delay may be triggered by EGFR related signaling pathway.
Knowing that the RHBDD1-involved proteolytic modification is upstream of the BIK protein degradation pathway and knock-down of RHBDD1 can enhance BIKmediated apoptosis, we also evaluate the apoptotic effect of RHBDD1-shRNA in HepG2 cells.Our data shows that inhibition of RHBDD1 was effective in apoptosis induction in HepG2 cells, causing an increase of the number of apoptotic cells from 2.8% in the control groups to 21.7% in the RHBDD1-shRNA group.It is conceivable that the RHBDD1 gene regulate HepG2 cell apoptosis through BIK-mediated pathway.However, the underlying mechanisms responsible for the biological function of RHBDD1 need further investigations.The EGFR is a member of a family of four closely related receptors: EGFR (ErbB-1), HER-2/neu (ErbB-2), HER-3 (ErbB-3) and HER-4 (ErbB-4).Interestingly, the expression of HER may play a role in the invasion, metastasis and progression of HCC (Bacaksiz et al., 2008;Zhang et al., 2010;Xu et al., 2011).Therefore, whether RHBDD1 participates in the above signaling pathways during HCC development remains wide open.
In conclusion, our findings strongly suggest that the RHBDD1 gene plays a critical role in the regulation of HCC cell growth and apoptosis, indicating that RHBDD1 may serve as a potential drug target for hepatoma cancer therapeutics.

Figure 1 .
Figure 1.Lentivirus-mediated Silencing of RHBDD1. A. Lentivirus infection in human hepatoma HepG2 cells.Fluorescence photomicrographs of GFP-positive cells infected by lentivirus.Pictures were taken 48 hrs after lentivirus treatment at a magnification of ×200.B. Identification of RHBDD1 knockdown efficiency using shRNA lentivirus via qRT-PCR.GAPDH was used as reference gene.*P<0.05 compared to control