| BackgroundNasopharyngeal carcinoma(NPC) is one of the most common malignant tumors in Southern China,especially in Guangdong Province. So far the etiology and pathogenesis mechanism of NPC has not yet been elucidated thoroughly. Recent studies suggest that environmental factors, genetic susceptibility and Epstein-Barr virus play roles in the development of NPC.Although the 5-year survival rate of NPC has been greatly improved through comprehensive treatment such as radiotherapy and chemotherapy, but long-term prognosis remains poor. About 55% of the NPC metastasis and recurrence occur in 5 years after treatment. Many patients show chemotherapy resistance.Cancer stem cell (CSC) theory suggests that CSC is related to tumor recurrence and metastasis as well as chemotherapy-related tolerance. Since the existence of cancer stem cells the solid tumors can not be eradicated. Although the size of metastatic tumor can be reduced after treatment, it can relapse quickly. Research showed that CSC is a small part of cell subsets which have high proliferative capacity, self-renewal and multi-directional differentiation potential, and they have the characteristics of resistance to chemotherapy. These features are similar to ordinary stem cells and embryonic stem cells. Under normal circumstances the differentiation and self-renew of stem cells are regulated restrictly by signal transduction pathways. Once the signal transduction pathway components are abnormal or destroyed, the cells differentiate abnormally, grow unlimitedly. Thus CSCs occur and they can produce tumor. Research showed that the tumor cells, stem cells and CSCs have signaling pathways in common, such as Wnt, Notch, SHH, BMP, PI3K/Akt, Bmil signal pathway.In this study nasopharyngeal cancer stem cells were evaluated by analyzing the number of side population (SP) cells. Wnt/β-catenin signaling pathway exists not only in normal stem cells, playing an important role in maintaining cell self-renewal and inhibiting cell differentiation, proliferation, migration and apoptosis, but may also controls immature cancer stem cells in drug resistance and tumor recurrence. Recent studies found thatβ-catenin signaling is important in the maintenance of CSC phenotype. Knocking down ofβ-catenin gene led to loss of the CSC phenotype and tumor degeneration. However the Wnt/β-catenin signaling pathway is not required in normal epithelial cells. This difference may be used to kill CSC directly and cure the cancer as a result. Nasopharyngeal carcinoma is a kind of malignant tumor occurred in nasopharyngeal epithelium. In this study we investigated the role of Wnt/β-catenin signaling pathway on CSC in NPC. We found that silencing the expression of P-catenin gene resulted in elimination of CSC properties and inhibition of NPC in vitro and in vivo. Our studies may provide novel approach for molecular targeted therapy for patients with NPC.ObjectiveTo build a cell model for delineating the role of Wnt/β-catenin signaling pathway on CSC in NPC by establishing a stable NPC cell line in which the expression ofβ-catenin gene has been inhibited stably. New targets for gene therapy of NPC may be found through this way.Methods1. Establish cell lines of CNE-2 in which the expression ofβ-catenin gene had been inhibited stably. We have generated two pairs of lentivirus expression vectors of shRNA targeting P-catenin CTNNB1 gene with RNAi technology, and then infected human nasopharyngeal carcinoma cell line CNE-2 with viruses. Effective silencing of the expression of P-catenin protein in CNE-2 has been validated by Western blot analyses.2. Detect the effect ofβ-catenin on CSC in NPC1) Tested the capability of the tumor cells proliferation and invasion with MTT and Transwell assays after P-catenin has been interfered.2) Monitored the expression levels of a variety of stem cell markers in cells with Western blot analyses and immunofluorecent staining.3) Examined cell cycle and SP cell ratio with FACS.3. in vivo experimentsP-catenin gene-silenced nasopharyngeal carcinoma cells were inoculated subcutaneously into nude mice. The tumor volumes were measured and the growth curves were determined. Tumor tissues were collected ans subjected to HE staining and IHC analyses for the expression of P-catenin and stem cell markers.Results1. After shRNA lentivirus interference vector targetingβ-catenin was constructed, we established CNE-2 cells withβ-catenin silenced by stable RNA interference. Two stably cell clones pLKO.l-sh-β-catenin and pLVTHM-sh-β-catenin were established. And cell clones of pLKO.l-sh-ctrl was utilized as a negative control.2. Western blot showed pLKO.l-sh-β-catenin group had higher inhibitory ratio ofβ-catenin protein,and pLKO.l-sh-β-catenin was chosen for functional exploration.3.Compared to CNE-2 untreated and pLKO.l-sh-ctrl groups, both twoβ-catenin silenced groups showed to be inhibited markedly in MTT cell proliferation assay (P＜0.001) and Transwell migration assay (P< 0.001)4.Western blot showed that the total intracellularβ-catenin,c-myc Oct3/4 and Vimentin protein decreased in pLKO.l-sh-β-catenin group,but E-cadherin protein increased in the same group. Immunohistochemistry staining showed that the expression ofβ-catenin,C-myc,Nanog,OCT3/4,Sox2,Vimentin and EpCAM protein decreased in pLKO.l-sh-β-catenin group.5. Cell cycle analysis showed that the G1 phase population increased (P=0.001,P=0.007) and S phase population decreased significantly (P=0.001,P=0.002) in pLKO.l-sh-p-catenin group in comparison to the untreated and pLKO.l-sh-ctrl groups. SP cell ratio of pLKO.l-sh-β-catenin group decreased.6. In vivo subcutaneous tumorigenesity in nude mice showed that tumor volume of pLKO.l-sh-β-catenin group was significantly decreased comparing with CNE-2 untreated and pLKO.l-sh-ctrl groups (F= 66.432, P=0.000)ConclusionIn this research two P-catenin/CTNNBl shRNA constructs were created successfully by using lentiviral vector pLKO.l. Compared with untreated cells or cells infected with control shRNA lentivirus, infection of NPC cells with pLKO.lβ-catenin shRNA resulted in markedly reduction ofβ-catenin protein. Cellular effects ofβ-catenin knockdown in NPC cells include inhibition of proliferation, migration, and cell cycle progression. Furthermore, silencingβ-catenin reduced expression of cancer stem cell markers such as c-myc, Nanog, OCT3/4 and Sox2 as evaluated with immunoblot analysis and immunofluorencent staining and decreased side population as measured by FACS. Importantly, tumorigenicity of NPC cells infected with pLKO.lβ-catenin shRNA was significantly decreased in a xenograft model of nude mice. Taken together, these results demonstrated thatβ-catenin is important in the maintenance of CSC phenotypes and silencing the expression ofβ-catenin gene is able to inhibit NPC in vitro and in vivo. |
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