ZEB2Can Inhibit Both Cell Proliferation And Apoptosis As Well As Induce EMT And Be Involved In Maintaining Cancer Stem Cell Like Features In Nasopharyngeal Carcinoma

BackgroundNasopharyngeal carcinoma is a kind of epithelial origin in nasopharyngeal mucosa malignant tumors and has obvious regional aggregation phenomena. High incidence of nasopharyngeal carcinoma occurs in Southern China area, especially in Guangdong Province, so that the nasopharyngeal carcinoma still was called as "Guangdong cancer". Nasopharyngeal carcinoma has high degree of malignancy, and is apt to early metastasis and recurrence. The complicated pathological process of nasopharyngel carcinoma includes multiple pathways, genetic variation, risk factors include EB virus, family history of carcinoma, pickled food, smoke, dust in the residential environment, work environment, exposure to dust smoke and chemical vapor exposure, otolaryngology medical history and so on. Tumorogenesis of nasopharyngeal carcinoma involves many genes and a lot of signaling pathways, activation of oncogenes and inactivation of tumor suppressor genes. All above of these are the molecular basis of the occurrence and development of nasopharyngeal carcinoma. Therefore, we design this project to explore the function of ZEB2gene in nasopharyngeal carcinoma and cell transduction pathway correlated with ZEB2. Our study will contribute to reveal the molecular mechanism of metastasis and recurrence in nasopharyngeal carcinoma.ZEB2also called SIP1; SIP-1; ZFHX1B; HSPC082; SMADIP1encoded by ZFHX1B gene on chromosome2q22contains9exons and8introns, and its CDS area was3572bp. ZEB2is a member of the zinc finger E-box-binding protein family and contains2zinc finger cluster and variable sequence cluster link two zinc finger. The zinc finger structure in N terminal and in c terminal both can independently bind the5’-CACCT(G) sequences of target genes. ZEB2is an indispensible transcription factor during embryonic development. The first report aout ZEB2is that ZEB2mRNA was found in Africa Xenopus embryos, and subsequent studies have revealed the roles of ZEB2in early embryonic development. ZEB2begins to express in the early stage of nervous system development and is activated in neurulation. In human and mouse species, ZEB2mRNA begins to express in the early development stage of peripheral nervous system and central nervous system. In mouse embryos, ZEB2is expressed in the notochord, somites, limb, neural crest, the brain and spinal cord and expressed in neural tissues of mature rats and of fetal elder than25weeks. In non-neuronal tissues such as the thymus, heart, liver, skeletal muscle, kidney and bladder, expression of ZEB2could be detected. All above of these showed that ZEB2plays an important role in the regulation of neuronal function. Mutation of ZEB2gene can cause Hirschsprung disease also called congenital megacolon and Mowat-Wilson syndrome, the latter mainly characterized by mental retardation, developmental disorders, seizures, microcephaly and dysgenesis of the corpus callosum.Initial studies suggested that ZEB2is localized in the nucleus, but recent research shows that ZEB2is expressed in both cytoplasm and cell nucleus. It is involved in cell growth regulation, differentiation, apoptosis, embryo development and inflammation.The ZEB family is involved in tumor progression and may have clinical significance. In ovarian cancer, ZEB2mRNA expression was higher in effusions compared to other sites; in carcinoma of the exudate volume is higher than that in primary; In breast cancer and oral squamous carcinoma, the subset of patients with postulated ZEB2-induced repression of E-cadherin had a significantly worse outcome; In gastric cancer, ZEB2overexpression could not be linked to downregulated E-cadherin in diffuse-type tumors, but was found to be involved in the pathogenesis of intestinal-type gastric carcinoma. It was firstly found that ZEB2expression was high in four tumorigenic glioma cell lines but low in two nontumorigenic glioma cell lines; Knockdown of ZEB2expression statistically significantly inhibited cell migration and invasion of tumorigenic glioma cells; ZEB2maybe could be used as a potential therapeutic and diagnostic target for gliomas; Consideration of the expression levels of potential EMT markers including ZEB2in renal cell carcinoma specimens, in addition to coventional prognostic parameters, contributes to the accurate prediction of disease recurrence after radical nephrectomy for localized RCC; The expression of ZEB2is significantly associated with tumor growth and poor prognoses in NSCLC and EMT might be activated via ZEB2expression and result in accelerate tumor growth and poor survival in NSCLC. Overexpression of ZEB2at the invasion front correlates with tumor progression and predicts cancer-specific survival in primary colorectal cancer. Therefore, ZEB2may be interesting as biomarker and potential target for treatment of colorectal cancer. ZEB2is involved in the progression of pancreatic cancer and plays a role in mediating signal transduction from collagen type I to downregulate E-cadherin expression; Some findings provide a basis for the concept that cytoplasimic ZEB2expressed by peritumoral liver tissues can predict the postoperative survival of patients with hepatocelluar carcinoma. The combined cytoplasmic ZEB2prognostic model may become a useful tool for identifying patients with different clinical outcomes.The role of ZEB2in nasopharyngeal is still unknown.ObjectiveTo identify ZEB2expression characteristics in nasopharyngeal carcinoma tissues and cell lines respectively and then make sure the roles of ZEB2in regulation of cell proliferation, apoptosis, survival, invasion,metastasis and sternness and then to vertify the molecular mechanisms of all above of these roles.Contents and methods 1. To identify ZEB2expression characteristics in nasopharyngeal carcinoma tissues and cell lines respectivelyThe mRNA levels and protein levels of ZEB2expression were respectively detected by Real-time Q-PCR and Western blot in nasopharyngeal carcinoma cell lines compared to NP-69; The ZEB2protein expression in nasopharyngeal carcinoma tissues compared to chronic nasopharyngitis cases were analyzed by IHC. ZEB2mRNA expressions in III-IV stage compared to I-II grade was analyzed by real-time Q-PCR.2. To make sure the effect of ZEB2on cell proliferation and identify the molecular mechanisms in nasopharyngeal carcinoma in vitro1) Knowdown of ZEB2expression was performed by using small RNA interference and the efficiency of it was measured by real-time quantitative PCR, Western blot and immunofluorescence separately;2) Percentage of cells in different phases of cell cycle in5-8F and SUNE-1cell lines after treated with NC and si-ZEB2separately detected by FACS.3) Identification of percentage of cells in S phase after cells were treated by NC and si-ZEB2.4) The change of some proteins related to cell cycle were identified by western blot in5-8F and SUNE-1cell lines after treated with si-NC and si-ZEB2001separately3. To find the influence of ZEB2on cell apoptosis and its molecular mechanisms1) Apoptotic percentage of cells with different treatment was detected by FACS in5-8F and SUNE-1cell lines respectively.2) The change of FoxO1expression after downregulation of ZEB2was identified by immunofluorescence.3) The change of some proteins related to apoptosis after cells were treated by si-NC and siRNA respectively in5-8F and SUNE-1cell lines was identified by western blot.4) Changes in drug dose-response after downregulation of ZEB2were identified by MTT assay.4. To study the impact of ZEB2on metastasis and invasion and its molecular mechanisms1) Transwell and Boyden assay in vitro were performed after reducing ZEB2expression.2) The change of some proteins related to EMT after cells were treated by si-NC and siRNA respectively in5-8F and SUNE-1cell lines was dentified by western blot.5. To study the impact of ZEB2on stemness and its molecular mechanisms1) After extract RNAs and proteins from tumor spheres and adherent cells, Western blot and Q-PCR were performed to identify the expression of ZEB2and some proteins related to sternness;2) Percentage of side population was measured by FACS after reducing ZEB2expression;3) Changes of some proteins related to stemness were identified by Western blot;4) MicroRNA chip was performed after downregulation of ZEB2expression;Results1. ZEB2expression in nasopharyngeal carcinoma cell lines5-8F, CNE2, SUNE-1is far more than in NP69; ZEB2expression in nasopharyngeal carcinoma is higher than in nasopharyngitis specimen and the expression level of ZEB2is significantly positive associated with clinical stages, N and M stage and vascular invasion and negative associated with overall survival time. ZEB2expression in stage III-IV cases was significantly higher compared to stage I-II specimen in nasopharyngeal carcinoma.1) ZEB2expression in8nasopharyngeal carcinoma cell lines and nasopharyngeal permanent epithelial cell line named NP-69was measured by real-time quantitative PCR. Single factor variance analysis showed that ZEB2expression in8nasopharyngeal cell lines has significant difference (F=58.779, P<0.001). LSD multiple comparison between found ZEB2expression has obvious differences between5-8F, SUNE-1, CNE-2and NP69(P<0.001).2) Detection of ZEB2expression in8nasopharyngeal carcinoma cell lines and NP69was identified by Western blot with GAPDH as loading control. The result showed that ZEB2expression in5-8F, CNE-2,6-10B, SUNE-1and HNE-1is high expression compared to NP69.3) Immunohistochemistry results showed ZEB2was mainly expressed in the cytoplasm and partially in the nuclear. Expression of ZEB2in nasopharyngeal carcinoma tissues was significantly increased compared to nasopharyngitis tissues (χ2=4.517, P=0.038). ZEB2expression has no significant difference between patients with different gender, age, histological type and T stage (P=0.572,0.510,0.766,0.101). The expression level of ZEB2is significantly positive associated with clinical stages (WHO I-II stage/WHO III-IV stage, P=0.001), N stage (P=0.002) and M stage (P=0.144) and vascular invasion (P=0.04)4) Univariate analysis shows that high ZEB2expression in patients is related to poor prognosis (P=0.001). In addition, overall survival time of patients with nasopharyngeal carcinoma was associated with N stage (P=0.014), M stage (P=0.000) and clinical stage (P=0.005). Multivariate survival analysis showed that, the expression of ZEB2(P=0.012) and M staging (P=0.000) can independently affect the prognosis of patients, and N stage (P=0.706) and clinical stage (P=0.113) were not independent factors. The results of Cox regression showed that the expression of ZEB2is negative associated with overall survival time of patients (p=0.001),5) Real-time Q-PCR results showed ZEB2expression in clinical stage III-IV cases was significantly higher compared to stage I-II specimen in nasopharyngeal carcinoma(P<0.001).2. ZEB2can inhibits cell proliferation in NPC1) Efficiency of siRNAs which transiently transfected5-8F and SUNE-1was detected by real time Q-PCR after24,48and72hours separately. The siRNA-001interference fragment after48h has the highest efficient (>70%, P<0.001).2) Cell cycle results identified by FACS showed percentage of cells in G1phase decreased significantly (P=0.034and0.037, respectively) in5-8F and SUNE-lafter reducing ZEB2expression and percentage in S phase was significantly higher (P=0.001and0.019, respectively) but percentage in G2phase had no distinct change (P=0.189and0.116). 3) EDU proliferation assay showed that cells in S phase were significantly increased after downregulation of ZEB2expression in SUNE-1and5-8F cell lines separately (P=0.001and P<0.001).4) Western Blot results showed that CCND1, phospho-Rb expression raised after reducing ZEB2expression;3. ZEB2inhibits cell apoptosis in NPC cells.1) Proportion of early apoptotic cells identified by FACS after downregulation of ZEB2expression was significantly higher in5-8F and SUNE-1separately (P=0.035and0.001respectively);2) Immunofluorescence results showed expression level of FOXO1rised after knockdown of ZEB2;3) Western Blot results showed β-catenin was down regulated after knockdown of ZEB2but sFRPl expression levels was raised. After reducing expression of β-catenin in HNE-1cell line, FOXO1and Bim expression both were raised but phosphor-FOXO1had no change. Meanwhile, Western blot results of overexpression of P-catenin in6-10B cell line were consistent with that of downregulation of β-catenin.4) After overexpression of FOXO1by lentivirus, Western Blot results showed Bim expression levels rised after overexpression of β-catenin. All above of these indicated that ZEB2maybe inhibits cell apoptosis through β-catenin/FOXO1/Bim pathway.4. ZEB2induces EMT in NPC cells1) Down regulation of ZEB2can dramatically reduce5-8F and SUNE-1cell migration in vitro. After cells were treated by siRNA001for48h, the number of passed transwell membrane cells was much fewer than that of control cells;2) Downregulation of ZEB2significantly reduced5-8F and SUNE-1cell invasion in vitro. After cells were treated by siRNA001for48h, the number of passed Boyden membrane cells was much fewer than that of control cells in5-8F and SUNE-1cell lines separately;3) Western Blot results after downregulation of ZEB2showed that E-Ca rised and N-Ca, MMP-2, Snail and Vimentin expression were lower compared to negative control sample. After reducing expression of β-catenin in HNE-1cell line, E-Ca expression was raised and N-Ca,Vimentin, Snail expression was downregulation. Meanwhile, Western blot results of overexpression of β-catenin in6-10B cell line were consistent with that of downregulation of β-catenin.5. ZEB2is involved in maintaining cancer stem cell like features.1) Western Blot results showed that Nanog, OCT-4, β-catenin, ZEB2expression increased in tumor sphere cells compared to adherent cells.2) Western blot results showed that after down-regulation of ZEB2expression, Nanog, SOX-2, OCT-4expression reduced.3) Percentage of SP in si-ZEB2was fewer than that in si-NC;4) After reducing β-catenin expression, OCT-4, Nanog, SOX-2expression reduced. It suggested that ZEB2may be involved in maintaining "stemness" through activating the canonical Wnt/β-catenin pathway5) MicroRNAs chip detected that expression of miR-200a, miR-200b, miR-200c, miR-203increased after reducing ZEB2expression;6) Q-PCR showed that ZEB2expression was more higher in tumor sphere cells compared to adherent cells and miR-203expression in tumor spheres was less than in adherent cells.ConclusionZEB2expression in nasopharyngeal carcinoma is higher than in nasopharyngitis specimen and the expression level of ZEB2is significantly positive associated with clinical stages, N and M stage and vascular invasion and negative associated with overall survival time. ZEB2expression in stage III-IV cases was significantly higher compared to stage I-II specimen in nasopharyngeal carcinoma. ZEB2gene can arrest NPC cells in G1phase, because ZEB2can inhibit CCND1so that inhibit cell proliferation. ZEB2can both inhibit cell apoptosis throuthβ-catenin/FOXO1/Bim pathway. Finally, ZEB2can induce EMT in NPC cells by activating canonical Wnt/β-catenin pathway.ZEB2may be involved in maintaining stem cell like features through activating canonical Wnt/β-catenin pathway and inhibiting miR-203.