Chloride currents activated by cisplatin in poorly differentiated naso-pharyngeal carcinoma cells are not Ca2+-activated chloride currents

AIM：To investigate the type of chloride channel activated by cisplatin in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells). METHODS：The technique of whole-cell patch-clamp was used to investigate the role of Ca2+ in the activation of cisplatin-activated chloride currents and to analyze the effect of hypertonic stress on these currents in CNE-2Z cells. RESULTS：Chloride currents were induced when the cells were exposed to the calcium-free cisplatin solution, showing the similar density to the currents induced by cisplatin with the presence of extracellular calcium. However, the latency and the peak time of cisplatin-activated currents in the absence of extracellular calcium were prolonged. The activation of cisplatin-activated chloride currents was insensitive to the depletion of intra- and extracellular calcium. Calcium channel antagonist nifedipine had no effect on the cisplatin-activated chloride currents, while hypertonic solution completely inhibited those currents. CONCLUSION：The cisplatin-activated chloride currents are independent on intra/extracellular calcium. The chloride channels activated by cisplatin are not calcium-activated chloride channels, but are probably volume-sensitive chloride channels.     

Molecular mechanism underlining ethanol-induced chloride currents in nasopharyngeal carcinoma cells

AIM：To study the effects and mechanisms of ethanol on chloride channels in poorly differentiated nasopharyngeal carcinoma CNE-2Z cells. METHODS：The effect of ethanol on the cell growth was analyzed by MTT assay. The technique of whole-cell patch-clamp was used to detect the chloride current. The characteristics of the chloride current were analyzed by using the chloride channel blockers. The siRNA technique was used to analyze the molecular basis of the ethanol-sensitive chloride channels. RESULTS：Under isotonic conditions, the background current was weak and stable. Ethanol at concentrations of 0.17~170 mmol/L activated a chloride current in a concentration-dependent manner (an inverted U-shape), with a maximum effect at the concentration of 17 mmol/L. The currents showed obviously outward rectification and were susceptible to extracellular hypertonicity and the chloride channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). ClC-3 siRNA obviously decreased the currents activated by ethanol. CONCLUSION：Extracellular ethanol induces chloride currents through activating the ClC-3 chloride channels.     

Effects of tamoxifen on volume-activated Cl- currents of nasopharyngeal carcinoma cells at different stages of the cell cycle

AIM:To investigate the inhibitory effects of Cl- channel blocker, tamoxifen, on volume-activated Cl- currents of nasopharyngeal carcinoma cells (CNE-2Z cells) in G₁ and S phases. METHODS:Highly synchronous cells in G₁ phase and S phase were obtained by the serum starvation and the double-block techniques. The whole-cell patch clamp technique was used to observe the effects of tamoxifen on volume-activated Cl- currents and to analyze the anion permeability of volume-activated Cl- channels. RESULTS:47% hypotonic stimulation activated a Cl- current in the nasopharngeal carcinoma cells at the cell cycle stage G₁ phase and S phase. Tamoxifen at concentration of 10 to 30 μmol/L completely inhibited the current. However, the time needed to completely inhibit the current was dose-dependent and was different between G₁ phase and S phase. The time needed to completely inhibit the current was shorter in G₁ cells than that in S phase cells. The anion permeability sequence of the volume-activated Cl- channel was I->Cl->gluconate in both G₁ phase and S phase cells. The permeability of G₁ phase cells to I- was higher than that in S phase cells, but to gluconate was lower than that in S phase cells. CONCLUSIONS:The density of the volume-activated Cl- current, the anion permeability of the channel and the sensitivity of the current to tamoxifen were different between the CNE-2Z cells in G₁ phase and those in S phase. The results suggest that the expression of tamoxifen-sensitive, volume-activated chloride channels is differentiated at different stages of the cell cycle.     

Bufalin activates chloride channels in poorly differentiated nasopharyngeal carcinoma cells

AIM: To investigate the activation of chloride channels induced by bufalin and the properties of the channels in poorly differentiated nasopharyngeal carcinoma (CNE-2Z) cells. METHODS: The technique of whole-cell patch clamp was used to record the chloride currents and to analyze the characteristics of the currents in CNE-2Z cells.RESULTS: A chloride current was slowly activated by extracellular application of bufalin (1 μmol/L). The activation of the current was slower than that of the volume-activated chloride current, with an activation latency of(12.1±6.4) min. The reversal potential of the current was close to the calculated Cl^- equilibrium potential (E_Cl =-0.9 mV). The chloride current was outward-rectified and did not show significant time-dependent or voltage-dependent inactivation. The chloride channel blocker tamoxifen completely inhibited the outward and inward currents. The current was also completely inhibited by extra-cellular application of 47% hypertonic solution. CONCLUSION: Bufalin activates chloride channels and induces a chloride current in CNE-2Z cells. Compared with the volume-activated chloride current in CNE-2Z cells, the activation latency of the bufalin-induced current is longer and the outward rectification is more obvious.     

Osmolarity,cell volume and proliferation in nasopharyngeal carcinoma cells

AIM: To investigate the relationship between osmolarity, cell volume and cell proliferation in nasopharyngeal carcinoma cells. METHODS: MTT method was applied to detect the proliferation ability of the poorly-differentiated nasopharyngeal carcinoma cell (CNE-2Z) under various osmolarity conditions. The flow cytometry was used to analyse cell cycle distribution. Cell volume was obtained by the image analysis of living cells and cell viability was determined by the trypan blue assay. RESULTS: Cultivation of cells under the hypertonic conditions of 370 and 440 mOsmol/L increased cell volume by 8.7% and 27.8% and facilitated cell proliferation by 22.2% and 33.9%, respectively. However, hypotonic incubation of cells with osmolarity of 160 and 230 mOsmol/L decreased cell volume by 12.8% and 4.1% and inhibited cell proliferation by 34.0% and 15.6%, respectively. Cell volume was positively correlated with cell proliferation rate. Long-term cultivation of cells under anisotonic conditions did not significantly alter cell cycle distribution, but hypotonic cultivation decreased cell viability. CONCLUSION: Proliferation of nasopharyngeal carcinoma cells was closely correlated with the osmolarity of culture medium and cell volume. Hypotonic cultivation may inhibit cell proliferation by decreasing cell volume to facilitate cell death mechanisms.     

Calreticulin promotes migration and invasion of nasopharyngeal carcinoma cells by inducing cell EMT

AIM:To observe the expression of calreticulin (CRT) in nasopharyngeal carcinoma tissues, analyze the significance of clinical pathology and the influence on epithelial-mesencymal transition (EMT) of CNE2 cells. METHODS:The expression of calreticulin was detected by immunohistochemistry in 52 nasopharyngeal carcinoma and 57 nasopharyngeal benign tissues, and the significance of clinical pathology was evaluated. The calreticulin gene-specific small interfering RNA was constructed, and then was transfected into the NPC cell line CNE2 using the cationic liposome method. The effect of CRT on the morphological changes of the CNE2 cells was observed under light microscope. The effect of CRT on the cell migration and invasion abilities of the CNE2 cells was detected by Transwell migration and invasion assays. The expression of EMT-related proteins E-cadherin, vimentin, transforming growth factor (TGF)-β and matrix metalloproteinase (MMP)-9 in the CNE2 cells was determined by Western blot. RESULTS:The positive expression rate of CRT in the benign lesion tissues was 19.29% (11/57), which was significantly increased in the nasopharyngeal carcinoma tissues as 82.69% (43/52). The expression rate of CRT was positively correlated with the stage of nasopharyngeal carcinoma and lymph node metastasis (P<0.05). Knockdown of CRT expression made the CNE2 cells showing a spindle shape to a flat, cobblestone-like epithelial state change, arranged more compact, and the migration and invasion abilities were significantly decreased (P<0.05). Knockdown of CRT expression resulted in significant increase in the protein expression of E-cadhe-rin, and the decreases in the protein expression of vimentin, TGF-β and MMP-9 in the CNE2 cells (P<0.05). CONCLUSION:Calreticulin expression in nasopharyngeal carcinoma is significantly higher and positively correlated with nasopharyngeal carcinoma stage and lymph node metastasis. Calreticulin promotes cell migration and invasion of nasopharyngeal carcinoma CNE2 cells by inducing EMT.     

Effect of tetrandrine on radiosensitivity of nasopharyngeal carcinoma cells

AIM: To investigate the mechanism of the radiosensitizing effect of maximum non-cytotoxic doses of tetrandrine (Tet) on nasopharyngeal carcinoma cell lines CNE1 and CNE2.METHODS: The cells were treated with ma-ximum non-cytotoxic doses of Tet (for CNE1 cells at 1.5 μmol/L and for CNE2 cells at 1.8 μmol/L), irradiation at 4 Gy, or combination of irradiation and maximum non-cytotoxic doses of Tet. The cell cycle distribution was analyzed by flow cytometry. The protein levels of γ-H2AX, cleaved caspase-3, p-CDC25C, CDK1, p-CDK1, cyclin B1, ERK and p-ERK were determined by Western blot.RESULTS: The expression of γ-H2AX was increased in CNE1 cells and CNE2 cells after combined treatment with irradiation and maximum non-cytotoxic doses of Tet. The percentages of CNE1 cells and CNE2 cells at G₂/M phase in irradiation group were (18.09±0.42)% and (18.48±1.32)%, respectively, which were decreased to (15.88±1.04)% and (13.80±0.82)% in combined treatment group, respectively (P<0.05). Combined treatment enhanced the increase in the protein level of cleaved caspase-3 caused by irradiation. The protein levels of p-CDC25C and p-CDK1 were increased in a dose-dependent manner by Tet treatment (P<0.05), while the expression of CDK1 showed no difference among different doses of Tet treatments. The protein levels of p-CDC25C, p-CDK1 and CDK1 showed no difference after the treatment with maximum non-cytotoxic doses of Tet. The combined treatment with irradiation and the maximum non-cytotoxic doses of Tet decreased the protein levels of p-CDC25C and p-CDK1 (P<0.05), increased the expression of cyclin B1, and had no influence on the expression of CDK1 (P<0.05). The combined treatment resulted in an increase in the protein level of p-ERK1 (P<0.05).CONCLUSION: The maximum non-cytotoxic doses of Tet enhance the DNA damage and apoptosis in CNE1 cells and CNE2 cells caused by irradiation, and the mechanism might be associated with ending of G₂/M arrest via activation of ERK/CDC25C/CDK1/cyclin B1 pathways.     

Volume-activated chloride currents in highly differentiated nasophyaryngeal carcinoma CNE-1 cells

AIM: To characterize the induction of volume-activated chloride currents by extracellular hypotonic stress. METHODS: Whole cell recording was used to detect the Cl- currents. The properties of the currents were clarified by applying the blockers of chloride channel, substitution of anions and changing cell volume. RESULTS: Under isotonic conditions, background currents were tiny and stable. However, large currents were induced rapidly by perfusing the cells with 47% hypotonic solution. The currents showed weak outward rectification. The permeability sequence of the 4 anions was I->Br- >Cl->gluconate. Chloride channel blockers, NPPB and ATP, inhibited the currents reversibly. Moreover, the inhibition of ATP on the outward currents was stronger than that on the inward currents. The currents were sensitive to the changing of cell volume, which can be activated by cell swelling and be inhibited by cell shrinkage. CONCLUSION: Extracellular hypotonicity induces chloride currents, which are sensitive to the changing of cell volume and play an important role in cell volume regulation.     

X-ray ionizing radiation induces epithelial-mesenchymal transition in human nasopharyngeal carcinoma CNE-2 cells

AIM:To investigate the effect of X-ray ionizing radiation on epithelial-mesenchymal transition (EMT) in human nasopharyngeal carcinoma CNE-2 cells and its involved potential signaling pathway. METHODS:The nasopharyngeal carcinoma CNE-2 cells were irradiated with different doses (0 Gy, 2 Gy, 4 Gy and 8 Gy) of X-ray. The morphological changes of the cells were observed under inverted microscope after 24 h. The migration and invasion abilities were detected by wound healing and Transwell assays. The mRNA and protein levels of E-cadherin, N-cadherin and vimentin in nasopharyngeal carcinoma CNE-2 cells were determined by real-time PCR and Western blot, respectively. The protein levels of Akt and p-Akt were detected by Western blot. RESULTS:After X-ray irradiation, the CNE-2 cells exhibited typical ‘cobblestone’ or spindle-like shape, with extended pseudopodia and dilated intercellular space. The invasiveness and metastatic abilities of the CNE-2 cells were enhanced (P<0.01). The mRNA and protein expression levels of E-cadherin were significantly decreased (P<0.01), while the mRNA and protein expression levels of N-cadherin and vimentin were markedly increased after irradiation as compared with the control group (no irradiation) (P<0.05). The protein level of p-Akt was significantly enhanced (P<0.01), while the protein level of Akt showed little change after irradiation. CONCLUSION:X-ray ionizing radiation induces EMT in nasopharyngeal carcinoma CNE-2 cells, which may be related to the activation of PI3K/Akt signaling pathway.     

Role of Cl- in regulatory volume decrease of nasopharyngeal carcinoma cells

AIM:To clarify the role of Cl^- in regulatory volume decrease (RVD) of nasopharyngeal carcinoma cells (CNE-2Z).METHODS:Analysis of living cell images was used to detect the volume changes following exposure to hypotonic solution. Iron replacement and block of iron channels were also applied in the present study. RESULTS:Extracelluar hypotonic treatment made the cells swell and induced RVD. The RVD was correlated positively to the swelling in the range of 160-230 mOsmol/L. Substitution of gluconate for Cl^- in perfusing solutions markedly increased RVD. Depletion of cellular Cl^- abolished, and chloride channel blockers inhibited RVD. CONCLUSION:Cl^- is the key iron to establish the RVD in CNE-2Z cells. Activation of Cl^- channels and Cl^- efflux are the major mechanisms of RVD.     

Relationship between radioresistance and microRNA in the same genetic background nasopharyngeal carcinoma cells

AIM:To discuss the relationship between the discrepancy of microRNA (miRNA) and radioresistance in nasopharyngeal carcinoma (NPC) cells CNE-2R and CNE-2 on the basis of validating their different radioresistance.METHODS:Following the exposure of X ray on the clones of CNE-2R and CNE-2 cells, the dose-survival curve and biological characteristics of CNE-2R and CNE-2 cells were determined by SigmaPlot software and the linear quadratic model of survival curve analysis.MicroRNAs were detected by μParafloTM microfluidic chip, hybridization images collected by a laser scanner (GenePix 4000B, Molecular Device) and the signals normalized by a LOWESS filter.The relationship between the discrepancy of NPC radioresistance and the expression of miRNA was predicted according to Targetscan3.1 database (http://www.targetscan.org) after analyzing the data.RESULTS:Compared to CNE-2 cells, 37 miRNAs were gain-of-function and 29 miRNAs were loss-of- function in CNE-2R cells among 719 detected miRNAs.12 miRNAs that detective value was more than 2 000 and 2 folds than the other were hsa-miR-200b, hsa-miR-224, hsa-miR-26b, hsa-miR-125a-5p, hsa-miR-205, hsa-let-7e, hsa-let-7g, hsa-miR-19b, hsa-miR-24, hsa-miR-103, hsa-miR-106b and hsa-miR-93.Data showed that the distinct discrepancy of miRNAs was related to radioresistance.CONCLUSION:The discrepancy of miRNAs is present in different radioresistant NPC cell lines and related to radioresistance.     

Effect of transketolase-like protein 1 on proliferation of human nasopharyngeal carcinoma cells

AIM: To investigate the effect of transketolase-like protein 1 (TKTL1) on proliferation of human nasopharyngeal carcinoma cells in vitro. METHODS: The siRNA against TKTL1 mRNA was constructed and transfected into human nasopharyngeal carcinoma cells (CNE cell line). The activity of transketolase was detected before and after RNA interference.Real-time PCR was used to determine the mRNA expression of transketolase (TKT) gene family in the CNE cells.Flow cytometry and MTT test were used to detect the effect of anti-TKTL1 siRNA on cell proliferation and cell cycle in the CNE cells. RESULTS: The total transketolase activity was significantly decreased in the CNE cells transfected with siRNA TKTL1 construct compared with the cells transfected with control vector or untransfected CNE cells. No significant difference in the expression level of TKT and TKTL2 gene between the CNE cells transfected with siRNA TKTL1 construct and the cells transfected with control vector or untransfected CNE cells was observed (P>0.05). However, the expression level of TKTL1 gene was significantly downregulated in the CNE cells transfected with siRNA TKTL1 construct compared with the cells transfected with control vector.Cancer cells were arrested in G0/G1 phase, and cancer cell proliferation was significantly inhibited in the CNE cells transfected with siRNA TKTL1 construct. CONCLUSION: TKTL1 plays an important role in the total transketolase activity and cell proliferation of human nasopharyngeal carcinoma. TKTL1 may be considered as a potential target for novel anti-cancer therapy.     

Inhibitory effect of JIP on AP-1 activity induced by LMP1 in nasopharyngeal carcinoma cells and its mechanism

AIM: To investigate the mechanism of the AP-1 signal transduction pathway inhibited by JIP in nasopharyngeal carcinoma cells. METHODS: AP-1 activity was triggered by Dox-induced LMP1 expression in Tet-on-LMP1-HNE₂ cells (L7). The retention of phospho-JNK in the cytoplasm caused by JIP was examined with immunofluroscence assay. RESULTS: 24 h after transfection of L7 cells with the JIP expression plasmid, the translocation of activated JNK was inhibited, which resulted in the retention of phospho-JNK in the cytoplasm and down-regulation of the AP-1 activity. CONCLUSION: JIP down-regulates the activity of AP-1 through the inhibition of the translocation of JNK.     

Effects of aspirin on apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM: To investigate the effects of aspirin on the apoptosis of nasopharyngeal carcinoma cell lines CNE2R and CNE2 with different radioresistance and its potential mechanism. METHODS: The effects of aspirin on the cell viability, apoptosis, and protein levels of procaspase-3, cleaved caspase-3, procaspase-9, procaspase-12, PARP and cleaved PARP, PI3K p110α, Akt, Bcl-2, Bax and p27 in the CNE2R cells and CNE2 cells were detected by the methods of MTT assay, flow cytometry and Western blot. RESULTS: Aspirin inhibited the viability of homologous nasopharyngeal carcinoma cell lines CNE2 and CNE2R (with the IC₅₀ to CNE2 cells of 6.18, 3.92 and 3.06 mmol/L for 24 h, 48 h and 72 h, respectively; and with the IC₅₀ to CNE2R cells of 7.05, 3.90 and 2.20 mmol/L for 24 h, 48 h and 72 h, respectively). After treated with aspirin for 48 h, the apoptotic rate of CNE2R cells was higher than that of CNE2 cells (P<0.05). After treated with aspirin for 48 h, the protein levels of procaspase-3, procaspase-9, procaspase-12 and PARP were decreased, the protein levels of cleaved caspase-3, cleaved PARP and p27 were increased, and the protein levels of PI3K p110α, Akt and Bcl-2/Bax were decreased. CONCLUSION: Aspirin inhibits the viability of homologous nasopharyngeal carcinoma cell lines CNE2R and CNE2 with different radioresistance. Aspirin also induces the apoptosis of CNE2 and CNE2R cells, which is more effective in CNE2R cells. The underlying mechanisms may be involved in affecting PI3K/Akt signaling pathway, Bcl-2/Bax and p27 expression.     

Inhibition of cell proliferation and arrest of cell cycle progression by blocking Cl- channels in nasopharyngeal carcinoma cells

AIM: The roles of Cl^-channels in regulatory volume decrease (RVD), cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells (CNE-2Z) were investigated. METHODS: Image analysis of living cells was used to detect the volume changes following exposure to hypotonic solutions. Cell viability was determined by the trypan blue assay. MTT method was applied to detected cell proliferation. The effect of the blocker on the cell cycle distribution was monitored by the flow cytometry. RESULTS: 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) inhibited RVD and cell proliferation in a dose-dependent manner. NPPB at the concentration of 100 μmol/L arrested cells in G₁ phase (G₁ population increased from 54% to 71% at 48 h after treatments), but did not significantly alter cell viability. CONCLUSION: Block of chloride channels suppressed cell proliferation by arresting cells in G₁ phase. The results suggest that activation of Cl^-channels and RVD is necessary for facilitating cells to proceed to the S phase from G₁ phase and maintaining cell proliferation.     

Efficacy of cytokine-induced killer cell transfusion combined with chemotherapy using gemcitabine and cisplatin for nasopharyngeal carcinoma with hepatic or pulmonary metastasis

AIM: To evaluate the recent and long-term efficacy of cytokine-induced killer(CIK) cell and transfusion plus chemotherapy with gemcitabine and cisplatin(GP) for patients of nasopharyngeal carcinoma(NPC) with hepatic or pulmonary metastasis.METHODS: From Aug. 2007 to Jul. 2008, 30 patients of nasopharyngeal carcinoma with hepatic or pulmonary metastasis were enrolled in the study and randomly divided into 3 groups. Ten patients(CIK+GP group) received CIK cell transfusion plus GP chemotherapy for 4 cycles. Another 10 patients(GP group)received the regimen of GP chemotherapy alone. The other 10 patients as well as 10 healthy volunteers did not receive any anti-cancer treatment. The early response, the changes of serum EBV-DNA by quantitative PCR detection and distribution of lympholeukocyte subset in the patients of the 3 groups were determined before and after treatment. The long-term effects were observed in a follow-up process of 2 years. RESULTS: The recent complete and partial response(CR+PR) rates were 90%, 70% and 10% in CIK+GP group, GP group and patient control group, respectively, and obviously statistical differences among these 3 groups were observed. The proportion of CD3⁺ lymphocytes in NPC patients with hepatic or pulmonary metastasis were obviously lower than that in healthy volunteer. It also descended after CIK+GP/GP therapy, but partly recovered in CIK+GP group. Furthermore, no statistical difference of CD4⁺/CD8⁺ ratio among NPC patients with hepatic or pulmonary metastasis and volunteers was found. The ratio of CD4⁺/CD8⁺ in CIK+GP group increased, but decreased in GP group after treatment. In the 2-year follow-up, the overall survival rate was 60.0%, 40.0% and 20.0% in CIK+GP, GP and control group,respectively. The Kaplan-Meier survival curves showed statistical difference. CONCLUSION: The recent and long-term effects of CIK cell transfusion combined with GP chemotherapy for patients of NPC with hepatic or pulmonary metastasis have been confirmed. The chemotherapy and biotherapy of CIK+GP improve the prognosis with synergistic effects, possibly owing to the changed proportion of CD3⁺and ratio of CD4⁺/CD8⁺.     

Inhibitory role of epigallocatechin-3-gallate in proliferation of human nasopharyngeal carcinoma cells by targeting P53/miR-34a

AIM: To study the effect of epigallocatechin-3-gallate(EGCG) on the proliferation of human nasopharyngeal carcinoma(NPC) cells, and to explore its mechanism by targeting miR-34a.METHODS: Nasopharyngeal carcinoma CNE-2Z cells were treated with various concentrations of EGCG. The ability of cell proliferation was detected by CCK-8 assay, 5-ethynyl-2-deoxyuridine(EdU) incorporation assay and colony-forming assay. The cell cycle distributions were analyzed by flow cytometry. The protein levels of P53 and Notch1 were detected by Western blot. The expression of miR-34a and Notch1 mRNA was measured by real-time PCR.RESULTS: EGCG effectively inhibited the proliferation and colony formation of CNE-2Z cells in a dose-dependent manner, which was related to its induction of cell cycle arrest at G₀/G₁ phase. The expression of P53 and miR-34a in CNE-2Z cells was significantly increased after treated with EGCG, while the expression of Notch1 at mRNA and protein levels was markedly suppressed.CONCLUSION: EGCG induces cell cycle arrest and suppresses cell proliferation by regulating the P53/miR-34a/Notch1 pathway in NPC cells.     

siRNA targeting TRIM29 gene induces apoptosis of nasopharyngeal carcinoma cells by inhibiting PI3K/AKT signaling pathway

AIM:To investigate the effect of TRIM29 gene expression silencing on the apoptosis and PI3K/AKT signaling pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were divided into blank group, negative control (NC) group (transfected negative control siRNA) and si-TRIM29 group (transfected TRIM29 specific siRNA). The viability of the 5-8F cells transfected with si-TRIM29 for 0~96 h was measured by CCK-8 assay. The apoptotic rate and the protein levels of TRIM29, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, t-AKT and p-AKT in the 5-8F cells transfected with si-TRIM29 for 48 h were determined by flow cytometry and Western blot, respectively. PI3K/AKT signal specific inhibitor LY294002 at 10 μmol/L and si-TRIM29 alone or in combination were treated with the 5-8F cells, and the cells were divided into blank group, LY294002 group and LY294002+si-TRIM29 group. The apoptotic rates in the 3 groups were detected by flow cytometry. RESULTS:The protein expression of TRIM29 in the 5-8F cells transfected with TRIM29 siRNA was significantly lower than that in blank group (P<0.05). Compared with blank group, the cell viability was significantly decreased, the apoptotic rate was significantly increased, the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax were significantly increased, and the protein levels of Bcl-2 and p-AKT were significantly decreased in si-TRIM29 group (P<0.05). The apoptotic rate in LY294002 group was higher than that in blank group, while that in LY294002+si-TRIM29 group was even higher than that in LY294002 group (P<0.05). CONCLUSION:Silencing of TRIM29 gene expression induces apoptosis of nasopharyngeal carcinoma 5-8F cells by inhibiting PI3K/AKT signaling pathway.     

Inhibition of miR-9 expression suppresses proliferation,invasion and migration of nasopharyngeal carcinoma cells

AIM: To investigate the effects of down-regulated miR-9 expression on the proliferation, invasion and migration of nasopharyngeal carcinoma (NPC) cells. METHODS: Human NPC CNE1 and CNE2 cells were transfected with the inhibitor of miR-9 by Lipofectamine to down-regulate the expression of miR-9, and the cells transfected with an inhibitor control were also set up. The cell proliferation and cell cycle were evaluated by CCK-8 assay and flow cytometry. The cell invasion and migration abilities were detected by Transwell invasion and wound-healing assays. Immunoblotting was applied to analyze the levels of the proteins. RESULTS: Compared with control group, inhibition of miR-9 expression in the NPC cells by transfection of the miR-9 inhibitor significantly decreased the proliferation ability (P<0.05). The percentages of the cells in G0/G1 phase ［CNE2: (57.96±1.39)% vs (47.93±1.76)%, P<0.05; CNE1: (51.24±0.88)% vs (48.29±0.39)%, P<0.05］ were significantly increased. The migration distances ［CNE2: (186.50±7.94)μm vs (247.56±15.56)μm, P<0.05; CNE1: (139.06±16.73)μm vs (230.66±14.27)μm, P<0.01］ and the invasion ability of the CNE2 cells (43.00±3.17 vs 65.80±5.20, P<0.01) were also significantly inhibited. Moreover, the tumor cells transfected with the inhibitors produced lower β-catenin. CONCLUSION: Inhibition of miR-9 expression suppresses the proliferation, invasion and migration of nasopharyngeal carcinoma cells.