Effects of PLCE1 Gene Silencing by RNA Interference on Cell Cycling and Apoptosis in Esophageal Carcinoma Cells

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers wordwide. Although great efforts have been made in the diagnosis and surgical treatment, the survial rate of ESCC remains poor (Chen et al., 2013). Therefore, it is necessary to study the molecular mechanisms of the development of ESCC. PLCE1 belongs to the phospholipase family that catalyzes the hydrolysis of polyphosphoinositides such as phosphatidylinositol-4, 5-bisphosphate (PtdIns (4, 5) P2) to generate the second messengers Ins (1, 4, 5) P3 and diacylglycerol. These products initiate a cascade of intracellular responses that result in cell growth and differentiation and gene expression. Wang found that PLCE1 is correlated with the occurrence and development of ESCC (Wang et al., 2010). In this study, we designed PLCE1-special small interfering RNA and transfected it to EC9706 cells to investigat the effects of PLCE1 gene silencing on the cell cycle and apoptosis of ESCC and indicate its important role in the development of ESCC.


Introduction
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers wordwide. Although great efforts have been made in the diagnosis and surgical treatment, the survial rate of ESCC remains poor . Therefore, it is necessary to study the molecular mechanisms of the development of ESCC. PLCE1 belongs to the phospholipase family that catalyzes the hydrolysis of polyphosphoinositides such as phosphatidylinositol-4, 5-bisphosphate (PtdIns (4, 5) P2) to generate the second messengers Ins (1, 4, 5) P3 and diacylglycerol. These products initiate a cascade of intracellular responses that result in cell growth and differentiation and gene expression. Wang found that PLCE1 is correlated with the occurrence and development of ESCC (Wang et al., 2010).
In this study, we designed PLCE1-special small interfering RNA and transfected it to EC9706 cells to investigat the effects of PLCE1 gene silencing on the cell cycle and apoptosis of ESCC and indicate its important role in the development of ESCC.

Cell lines and culture conditions
The human ESCC cell lines EC9706 were purchased from the Tumor Cell Bank of Chinese Academy of Sciences, and cultured in RPMI1640 and supplemented with 10% fetal bovine serum (FBS, Sigma, USA). enriched atmoshere and routinely sub-cultured every 2 d by trypsinization. Then cells were preserved with RPMI 1640 free of serum or antibiotics and prepared for transfection assays.

Immunohistochemistry
40 cases esophageal cancer tissue from resected specimens and matched noncancerous were obtained from Peace Hospital Attached to Changzhi Medical College between 2011 and 2013. Histological tumor typing was carried out on the basis of biopsies or resected specimens in the Department of Pathology of the same hospital. dewaxed using xylene and rehydrated in graded alcohols. peroxidase activity was blocked by 3% hydrogen peroxide for 15min at room temperature. The sections were incubated overnight with primary antibody anti PLCE1 antibodies (IRDye conjugated donkey anti rabbit IgG). After further washing in PBS, the sections were incubated with 3, 30-diaminobenzidine dilute, lightly counterstained with hematoxylin, and observed under a photomicroscope.

RT-PCR analysis of PLCE1 mRNA levels
After being transfected with PLCE1 siRNA for 48h, cell were harvested for reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from EC9706 cells (negative control group, untreated group, PLCE1-siRNA group) using RT-PCR kit (Solarbio Biotechnology, Beijing, China). All the RNA preparations had an optical density OD 260 :OD 280 ratio of 1.9-2.0. kit (Solarbio Biotechnology, Beijing, China). Primers for PLCE1 were designed as follows: forward primer, 5' -TAACTGACGATGAGATGGCAAC-3'; reverse primer, 5'-TGTTTGAAGAAGACAGCACAGG-3'. 5'-CATCCTCACCCTGAAGTACCCC-3'; reverse primer, 5'-AGCCTGGATGCAACGTACATG-3'. The PCR (35 cycles) was conducted in a Mastercycler thermal cycler (Eppendorf). Each cycle included denaturation period was 3 minutes, and the final extension was 5minutes. The PCR products were electrophoresed on 1.5% agarose gels and bands were visualized by ethidium bromide staining and the results of gel electrophoresis. To quantify difference in gene expression, the relative gene expression of PLCE1 was analyzed by Quantity one 4.5.0 control. The PLCE1 mRNA level was evaluated by the

Cell cycle analysis
Flow cytometric analysis was carried out as described previous (Shi et al., 2009). Cells were cultured in serumfree medium for 24h to synchronize the cells, then trypsinized and suspended in phosphate buffered saline 0.1% Triton X-100 (Sigma) and 2 mg DNase-free RNase (Sigma) at room temperature. Results were analyzed using Multicycle-DNA Cell Cycle Analyzed Software.

Detection of apoptosis through fluorescein annexin V-FITC/PI double labeling
3 infected and mock-infected cells were harvested through 0.25% trypsinization, after washing with ice-cold were analyzed by FACS (Becton Dickinson) using Cell Quest Research Software (Becton Dickinson).

Protein extraction and Western blot
After being transfected with PLCE1 siRNA for 48h, cell were harvested for western blot analysis for detection of PLCE1. Cells were washed twice with ice-cold PBS, and collected cells were lysed in buffer (50 mmol Tris-HCl, pH 7.5, 150 mmol NaCl, 1mmol EDTA, 1% Triton X-100, 1 mmol NaF, 1 mmol activated Na 3 VO 4 , 0.1% sodium dodecyl sulfate and protease inhibitors. Protein concentration was measured using Bradford reagent (Sigma) and bovine serum albumin as standard, then separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to Membranes were blocked with 5% nonfat milk in Tris buffered Saline with Tween 20 (TBST, 50 mmol Tris-HCL, PH7. 6, 150 mmol NaCl, 0.1% Tween 20) for 2h at room temperature, and subsequently incubated with primary Following a wash with TBST, the membranes were incubated with IRDye conjugated secondary antibody (goat anti mouse, 1:10000, goat anti rabbit, 1:10000) for 2h at room temperature, then the membranes were washed with TBST, the protein quantity was analyzed by Odyssey bands were taken as loading control. The target protein expression was evaluated by the relative intensity ratio of

Statistical analysis
The results are expressed as the mean±SEM. All date were analyzed by SPSS 18.0 statistical software (SPSS, Chicago, IL). Differences between means of two groups were tested by paired-samples T test and between multiple groups were tested by Analysis of Variance (ANOVA). P<0.05.

Immunohistochemical analysis of PLCE1
Immunohistochemiscal staining was used to examine the expression of PLCE1 protein in 40 cases of esophageal cancer tissues and matched noncancerous. Positive granules were mainly distributed over cytoplasm of tumor cells,the positive granules were stained brown orange (Figure 1). The level of PLCE1 protein in esophageal tissue, with statistical significance (38.40±7.47 vs 5.56±0.31, F=24.43, P<0.05).

Successful transfection of small interfering RNA
We have successfully transfected PLCE1 siRNA into EC9706 cells via lipofectamine and observed by fluorescent microscopy (Figure 2). EC9706 cells in negative control group and PLCE1 siRNA group were after 6-12 hours were determined through the observation occurred at 48 hours. The cells that had clear structure and

RT-PCR analysis of PLCE1 mRNA levels among different groups
Expression level of the PLCE1 mRNA 48h posttransfection of PLCE1 siRNA revealed a significant decrease in mRNA level as compared with the negative control group and mock group as shown in (Figure 3). The difference between mock group and PLCE1 siRNA group vs 0.856±0.002, T=66.454, P=0.000) Cell cycle analysis results suggested that the down-regulation of PLCE1 can entering into the S phase as shown in Table 1 and Figure   G1 phase and decreased the cells in S phase accordingly (P<0.05).

PLCE1 gene silencing induces cell death by apoptosis
Apoptosis induced by PLCE1 gene sliencing was assay. The percentage of apoptosis in PLCE1-siRNA group was higer than that of the mock and the scrambled P<0.01). Results showed that down-regulating PLCE1 could promote the apoptosis of EC9706 cells ( Figure 5).

Western blot analysis of PLCE1 protein
The PLCE1 protein was expressed in all three groups of the EC9706 cells. The optical density value for the bands for each of the three groups was compared with cyclinD1 in PLCE1-siRNA group was lower than the other two groups ( Figure 6A), and the difference was P<0.05). The expression of apoptosis related protein caspase-3 in PLCE1-siRNA group was higher than the other two groups ( Figure 6B), and the difference was P<0.05). The results showed that PLCE1 gene silencing could inhibit the expression of the cyclinD1 while promote the caspase-3.

Was Transfected in EC9706 Cells.
Carcinogenesis is a multistage process, which involves various molecular events related to the fundamental alterations in cell physiology. It is very important to explore the molecular mechanism. In 2010, by genomewide association study (GWAS) of 25,000 patients for the susceptibility loci to esophageal squamous C20orf54 as the susceptibility loci to ESCC (Wang et al., 2010). Our previous study showed that the functional SNP squamous cell carcinoma (Ji et al., 2011). PLCE1 showed tissue. Some studies found that PLCE1 is correlated with the occurrence and development of gastric carcinoma (Luo et al., 2011), colorectal carcinoma (Danielsen et al., 2011), small intestine cancer (Li et al., 2009), head and neck squamous cell carcinoma (Bourguignon et al., 2006) and skin cancer (Bai et al., 2004).
PLCE1 has a Ras-associating domain at its C terminus and a CDC25-like domain at its N terminus. CDC25 domain is a key regulators in response to cell cycle checkpoints, plays a very important role in the The role of PLCE1 in ESCC remains unclear. PLCE1 encodes a enzyme that produces two vital intracellular second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which induce activation of protein kinase C and mobilization of Ca 2+ from intracellular stores,respectively. Protein kinase C is an important signaling molecule in mediating tumor cell adhesion,movement,and invasion,and a PKC suppressor has displayed anti-invasion characteristics in vitro and in vivo experiments. As a special domain of PLCE1, the  CDC25-like domain could change the molecular structure of Ras family via acting on guanosine triphosphate signaling pathways (Cui et al., 2013) and then regulate cell apoptosis and growth, promotes cell proliferation, differentiation and even malignant change (Perrin et al., 2013). Ada-Nguema AS (Ada-Nguema et al., 2006) found mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion, because R-Ras was cocorollary is that PLCE1 may lead to tumor formation by a chain of active reaction like regulating cell proliferation and differentiation, affecting the cell movement and cytoskeleton (Lad et al., 2006;Ou et al., 2010). The development of research on RNA interference encourage us to further study on ESCC mechanism. Li (Li et al., 2013) found that HDAC6 siRNA can effectively downregulate the expression of HDAC6 mRNA and protein in EC9706 cells. Down-regulation of HDAC6 expression can obviously inhibit cell proliferation, arrest We designed PLCE1-special siRNA (small interfering RNA) to investigate the effects of PLCE1 (phospholipase of esophageal squamous carcinoma cells (ESCC), and attempt to disclose the role of PLCE1 in ESCC.
ESCC has a striking geographic distribution worldwide, with higher prevalence in some areas of China. Taihang mountain of China is is one of high incidence district of esophageal cancer. Peace Hospital Attached to Changzhi Medical College is located in the taihang mountain. Clinical specimens include esophageal squamous carcinoma tissues and adjacent normal tissues were taken from our surgery department. Immunohistochemical experiment results showed that the level of PLCE1 protein that in normal tissue, suggesting PLCE1 is associated with development and progression of ESCC. RT-PCR result showed that the expression level of the PLCE1 mRNA 48h decrease in mRNA level as compared with the negative control group and mock group. Flow cytometry revealed that the down-regulation of PLCE1 can arrested cell cycle than that of the control group. Western blot showed that the expression of apoptosis related protein caspase-3 in PLCE1-siRNA group was higher than the other two groups and the expression of cell cycle related protein cyclinD1 in PLCE1-siRNA group was lower than the other two groups,the down-regulation of PLCE1 could inhibit the expression of the cyclinD1 while promote the caspase-3. Cyclin D1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle (Ferrandiz complexes and hyperphosphorylated retinoblastoma protein (Rb) (Takahashi et al., 2007). Our study showed that PLCE1 gene silencing by RNAi results in decreased expression of cylclin D1 and blocked transition from G1 to S phase.
Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. Caspase-3 is a member of the caspase (cysteine aspartic proteinases) family of enzymes, which are the major inducers of apoptosis. Caspase-3 is activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways (Huang et al., 2014). One signaling event is the introduction of granzyme B, which can activate initiator caspases, into cells targeted for apoptosis by killer T cells. This extrinsic activation then triggers the hallmark caspase cascade characteristic of the apoptotic pathway, in which caspase-3 plays a dominant role. Our experiment showed that PLCE1 gene silencing by RNAi results in increased expression of caspase-3,and then promotes the apoptosis of ESCC. Flow cytometry results have proved this.
In summary,our study revealed that PLCE1 gene is closely associated with development and progression of ESCC. PLCE1 could promote cell proliferation by up-regulating the expression of Cyclin D1,and decrease apoptosis by down-regulating the expression of caspase-3.