EZH2Overexpression Promotes Cell Invasion And Metastasis Of Nasopharyngeal Carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is one of the commonest carinomas with a high rate of malignant phenotype in Southern China and Southeast Asia. Unlike other head and neck malignancies, NPC is notorious for its geographic pattern, familial aggreation of incidence and marked tendency to highly metastatic nature. Although excellent local control can be achieved with advances in concurrent chemora-diotherapy, but a five-year survival rate did not increase significantly. The leading cause of death in NPC patients is distant metastasis. Therefore, it is of great clinical value to further understand the molecular mechanisms of NPC metastasis as well as novel therapeutic strategies.Our previous studies found that the growth inhibitory effect of miR-26a was mediated by repressing EZH2(polycomb group protein enhancer of zeste homolog2) in NPC cells. Meanwhile, the results of RT-PCR showed that EZH2was overexpressed in NPC cells and tissues, especially in5-8F cells with metastatic capacity and metastatic NPC tissues. All these analyses suggest that EZH2is a candidate oncogene to promote NPC metastasis.EZH2, a member of the polycomb group (PcG) of genes, has been associated with embryonic stem cell maintenance, cell differentiation, tumour stem cell self-renewal and oncogenic fuctions. EZH2is located in human chromosome7q35, where CGH analysis showed that this chromosome band might relate to metastasis of NPC. EZH2is the catalytic subunit of the polycomb repressive complex2(PRC2). EZH2appears to mediate transcriptional silencing by either methylating lysine27in histone H3(H3K27) or recruiting DNA methyltransferases to its target genes that catalyze de novo DNA methylation. EZH2is overexpressed in a number of epithelial and hematological malignancies and its overexpression has been associated with poor prognosis in prostate and breast cancers patients, thereby contributing to the proliferative and metastatic capacity of tumor cells.Epithelial mesenchymal transformation (EMT) refers to the transfer of epithelial cells to fibroblasts or mesenchymal cells morphologically and the aquirement of metastatic ability. EMT endows cells with migratory and invasive properties, induces stem cell properties, prevents apoptosis and senescence, and contributes to immunosuppression. EMT also contributes to tissue repair, but it can adversely cause organ fibrosis and promote carcinoma progression through a variety of mechanisms. EMT plays crucial roles in cancer progression, especially epithelial cancer invade and metastasis. However, the epigenetic regulator EZH2modulates its gene silencing effect on the dowmstream genes including E-cadherin to improve EMT in squamous cell carcinoma. Gastric cancer cells transfected with EZH2siRNA showed translocation from the nucleus to the membrane which associated with EMT. Base on our surprised findings, we want to know whether EZH2is correlated with EMT in NPC or not.Our study aimed to validate the expressions of EZH2in multiplticity NPC tissue and cell lines, especially in5-8F and6-10B by immunohistochemistry, RT-PCR and western blot. We explicit the effects of EZH2silence or overexpression on invasion and metastasis in vivo and in vitro. We want to know whether EZH2promotes metastasis of NPC by inducing EMT. In this part, we detect the expression of EZH2deregultion on EMT-related markers by RT-PCR and western blot.Materials and Methods1. Cell culture An immortalized nasopharyngeal epithelial cell NP69was cultured in Keratinocyte-SFM supplemented with bovine pituitary extract as described previously. The human NPC cell lines5-8F,6-10B, CNE1, CNE2and HONE1were culture in RPMI-1640supplemented with10%fetal bovin serum and1%penicillin-streptomycinin. HEK293FT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). All cells were cultured in a humidified atmosphere of95%air and5%CO2at37℃.2. Clinical specimensPrimary NPC paraffin specimens and normal paraffin of thenaspharynx were obtained from Nanfang Hospital (Southern Medical University, Guangzhou, China). Both tumor and normal tissue were histologically confirmed by H&E (hematoxylin and eosin) staining. There were97NPC specimens with5years follow-up periods were classified as undifferentiated nonkeratinizing type. None of the NPC patients had received preoperative radiation or chemotherapy before diagnosis. Informed conset was obtained from each patient, and the research protocols were approved by the Ethics Committee of Nanfang Hospital.3. ImmunohistochemistryParaffin sections of specimens prepared for detection were stained by SP method, and the results of IHC were observed by light microscope and5fields of vision were counted according to semiquantitative scales. The degree of immunohistochemistry of EZH2was categorized as follows:high reaxtivity, more than50%of cells showing intense immunohistochemistry in their nucleus; low reactivity,50%of fewer cells showing intense immunohistochemistry in their nucleus. The mean percentage of positive of positive tumor cells was determined in at least five areas at high power field.4. Production and purification of lentivirusThe plVTHM/shEZH2and plVTHM/EZH2lentiviral vectors for shRNA and cDNA delivery of EZH2were gifts of Dr. Chen from Chinese University of HongKong. The transfer vector and the packing plasmids were co-transfected into293FT cells using lipofectamineTM2000. The supernatant was harvested48-72h posttransfection. Then the viral particles were concentracted by ultractrifugation. The packaged lentiviruses were named LV/EZH2and LV/shEZH2, respectively. The empty lentiviral vector LV/control was used as a control. The titration of lentiviral suspension was calculated by making a tenfold serial dilution.5. Cell transfectionThe packaged lentivirus were used to infect NPC cell lines. At48h transfection, the cells were observed using a fluorescence microscope to detect green fluorescent protein (GFP). Following fluorescence-activated cell sorting, the GFP-positive cells were isolated, and the cell lines were established, which cound stably silence EZH2and overexpress EZH2respectively.6. Real-time PCRTotal RNA were extract from cells and tissues. The RNA was reversely transcribed into cDNA. Real-time PCR was perfomed using SYBR Green PCR master mix. All samples were normalized to internal controls and fold changs were calculated through relative quantification (2-ΔΔCt).7. Western blotProtiens were extracted with RIPA buffer and quantified using BCA protein assay reagent. Western blot analysis was performed with50μg of protein on10%SDS-PAGE. After fractionation, protein were transferred to polyvinylidened fluoride (PVDF) membrane electrophoretically. Then, the membrane was incubated with antibodies and detected by chemiluminescence using Bio-Rad ChemiDoc XRS+Imaging System. The intensity of protein fragments was quantified with the Image Lab.8. Cell biology experiments(1) Wound-healing assay:Cells were at a density of5×105cells per6-well plates. After12h of incubation, a wound was created using an aeosol P200pipette tip, and photomicrographs were taken immediately (time0h) and thereafter24h and48h to observed the effects on wound healing.(2) Invasion Chamber assay:Cells after24h serum-free medium incubation were trypsinized, and1×105cells in200μl of serum-free medium were added into the upper chamber. In the lower chamber,600μl of medium containing10%fetal bovine serum were placed. The cells were allowed to migrate though the intermediate membrane for24h at37℃. Membranes were then fixed with4%paraformaldehyde, stained with5%crystal violet, and quantified in microscopy.9. In vivo metastasis experimentNude mice (BALB/C nu/nu,4-6weeks old,18-24g in weight) were maintained under specific pathogen-free conditions. The nude mice were established by either direct injection of NPC1×106cells under the liver capsule. The mice were sacrificed on day24, and tissues of liver and lung were collected for pathologic analysis.10. The expression of EMT-related markersThe expression of epithelial markers (E-cadherin and CK18) and mesenchymal markers (β-catenin and N-cadherin) in5-8F/control,5-8F/shEZH2and6-10B/control,6-10B/EZH2cells were detected by quantitive real-time PCR and western blot.Results1. The expression of EZH2in NPC cellsA panal of human NPC cell lines (5-8F,6-10B, HONE1, CNE1, CNE2) was firstly analyzeed to quantitate the expression level of EZH2. The result of RT-PCR showed that the expression of EZH2was increased in all fine NPC cell lines, compared with the immortalized nontumorigenic cell line NP69(F=707.589, P=0.000). And the expression of EZH2was higher in5-8F cell line than6-10B. The results were similar with western blot.2. The expression of EZH2in NPC tissues(1) The results of immunohistochemistry showed that the positive expression of EZH2were mainly localized in the nucleus. Based on semi-quantitative method, the positive expression rate of EZH2in NPC tissues was88.2%, while in chronic nasopharyngitis tissues only8.7%, which had a significant difference (P=0.000).(2) According to the expression of EZH2, all NPC specimens were divided into two groups, high expression and low expression. After statistical analysis, there was no correlation between EZH2expression and the clinical data (sex, age)(P>0.05). Interestingly, there was a significant correlation between EZH2expression and T stage, N stage, M stage classification (P<0.05).(3) Kaplan-Meier survival analysis of97NPC specimens with5years follow-up periods showed that the higher expressed patients had the worse prognosis (P=0.000).(4) All variables that significantly affected survival in univariate analysis were introduced into a Cox proportional-hazard model. At the end of the stepwise process, M stage and EZH2expression maintained its independent prognostic influence on survival (P<0.05).3. The effects of EZH2on the metastasis ability of NPC cells in vivo and in vitro(1) The establishment of NPC cell lines with stable EZH2over-expression or knockdownFollowing fluorescence activated cell sorting, the GFP positive cells were isolated, cell lines5-8F/shEZH2were established to stable knockdown EZH2and6-10B/EZH2with EZH2overexpression stably.(2) The effects of EZH2knock-down on cell migration and invasionThe results of wound healing assay indicated that cells with EZH2knock-down were less motile in actin-based active movement. Further study by matrigel invasion assay showed that the invasive capacity of EZH2knock-down was significantly inhibited as reflected by less penetrating cells.(3) EZH2over-expression promoted NPC cell lines migration and invasion in vitroThe invasion ability of6-10B cell line has increased5.49times after EZH2overexpression.(4) EZH2knock-down inhibited NPC metastasis in vivoThe nude mice were divided by two treatment groups (n=5/group) with the primary tumors being established by either direct injection of NPC cells under the liver capsule. Liver parenchyma invasion in5-8F/control group. The liver of5-8F/shEZH2group was found isolated metasstic nodule in HE assay. The number of metasstic nodules in live and lung of5-8F/control were more than that of5-8F/shEZH2group. 4. The effect of EZH2deregulation on EMT-related markers(1) EZH2knock-down could increase the expression of epithelial markers E-cadherin and Keratin18by1.83-fold and1.61-fold respectively, and downregulate the expression of mesenchymal markers β-catenin and N-cadherin by42.7%and41.5%respectively in mRNA, similar with protein levels.(2) Opposite results were obtained by RT-PCR and Western blot analysis after EZH2overexpression.Conclusions1. EZH2is overexpression in NPC cells and tissues. There is a significant correlation between EZH2expression and the clinical features and prognosis of NPC patients. Therefore, EZH2overexpression is expected to be a potential prognostic marker for NPC.2. EZH2can promote the migration, invasion and metastasis ability of NPC cells in vitro and in vivo, which functions as a metastasis promoter gene in NPC.3. EZH2significantly stimulates metastasis of NPC which mediated by inducing EMT.