Down-regulation Of Telomerase And Its HTERL And Inhibiting Of Cancer Cell Line Induced By Hypoxia In Nasopharyngeal Carcinoma

BackgroundHypoxia is a common phenomenon occurring in the majority of human tumors, including the nasopharyngeal carcinoma(NPC). The presence of hypoxia is significantly associated with angiogenesis, tumor growth, therapeutic resistance, metastasis and apoptosis. However the molecular mechanisms of tumor malignant transformation induced by hypoxia have not been explored. Studies have confirmed that Hypoxia-inducible factor-1(HIF-1), a central transcriptional factor for tumor growth and malignant progression.Our previous studies have demonstrated that telomerase activity and Telomerase Catalytic Subunit (hTERT) expression appears elevated in most cancer cells, including the nasopharyngeal carcinoma, which is significantly contributing to tumor unlimited proliferation, recurrence and metastasis. The present findings suggest that hypoxia promote tumor proliferation, metastasis and chemoresistance may be achieved by up-regulating the telomerase activity, and that HIF-1plays a critical role as a transcription factor. Based on these, we investigated the effects of hypoxia and HIIF1α-siRNA on telomerase and its subunits and cancer cell proliferation and drug-resistance in nasopharyngeal carcinoma cell line. Further providing new insights into tumor hypoxia to be used as a novel therapeutic target for cancer.ObjectiveTo explore the effects of hypoxia and its intervention factor on telomerase and its subunits (hTERT) and cancer cell proliferation, metastasis and drug-resistance in nasopharyngeal carcinoma cell line.Methods1. Culture of tumor cell lines and hypoxia treatmentTwo nasopharygneal carcinoma cell lines (5-8F and CNE2) were used. CNE-2and5-8F cells were cultured in RPMI1640supplemented with10%fetal bovine serum (FBS) and antibiotics (100U/mL penicillin and10μg/mL streptomycin). For normoxic condition, cell cultures were maintained in a humidified incubator at37℃in5%CO2,95%air. Cells in the logarithmic phase of growth were used for all studies described. For hypoxia treatment, cells were placed in a three gas incubator with a mixture of1%O2,5%CO2, and94%N2.2. The effects of hypoxia and its intervention factor on telomerase and its subunits in nasopharyngeal carcinoma cells2.1Effects of hypoxia on HIF1α and hTERT gene expression in nasopharyngeal carcinoma cellsFor nasopharyngeal carcinoma cell lines5-8F and CNE2were seeded onto6-well plates, subconfluent cultures were incubated under hypoxic condition for0h～72h. The cells were collected and lysed in SDS lysis buffer, protein content was determined using the BCA Protein Assay, HIF-1α and hTERT protein level at various time intervals(0、4、8、16、24、48、72h) were determined by Western-blot. Actin was used as an internal control to verify equal protein loading in each nasopharyngeal carcinoma cell line during the experiment.2.2Transient transfection of small interference RNA (siRNA) and analyze transfection efficiencyNasopharyngeal carcinoma cells were seeded at2～2.5×105cells/6-well plates (5×103cells/96-well) and grown to30%～50%confluence before transfection. Cells were transfected with double-stranded siRNAs (at final concentrations of30nM) for6h by the lipofectamine2000according to the manufacturer’s instructions. Negative-siRNA was labeled with Cy3fluorescent dye, tumor cell lines transfected with Negative-siRNA-Cy3were showed red fluorescence evaluated by Fluorescence microscopy, and transfection efficiency was detected by flow cytometry. The experiment was repeated three times.2.3Screening effective siRNA5-8F and CNE2cells were transfected with three pairs of siRNA (HIF1α-siRNA-1, HIF1αa-siRNA-2and HIF1α-siRNA-3), Negative-siRNA or lipofactamine2000, and were incubated under normoxia or hypoxic condition for48h. Real-time PCR was performed to quantify human HIFla mRNA, relative to P-actin, with three replicates of each cDNA sample. Then, the siRNA had the strongest inhibitory effect on HIF1α expression at mRNA levels was used for targeting HIF1α.2.4The effect of HIF1α-siRNA on the expression of HIF1α and hTERT in nasopharyngeal carcinoma cells5-8F and CNE2were transfected with both HIF1α-siRNA and Negative-siRNA and were incubated under normoxia or hypoxic condition for48h. The expression of HIF1α and hTERT were detected by Western-blot and Real-time PCR. The experiment was repeated three times, respectively. 3. Effect of hypoxia and HIF1α-siRNA on profiferation, metastasis and drug resistance of nasopharyngeal carcinoma cells3.1Cell cycle assayExperiments were divided into four groups:Untreated cell (normoxia), Untreated cell (hypoxia), Negative-siRNA cells (hypoxia) and HIF1α-siRNA cells (hypoxia). CNE2and5-8F cells were seeded at2～2.5×105cells/6-well plates and transfected with siRNAs according to the group of experiments, and continue maintained in a incubator for12h, then cells were incubated under hypoxic condition for36h. Cells were collected and washed in cold phosphate-buffered saline (PBS), resuspended in PBS at1×106/mL, fixed in cold ethanol (70%) overnight at37℃and washed again with cold PBS. Then cells were stained with100μL Rnase A and400^L PI with incubation at37℃for30min in the dark, and analyzed usingusing a FACS Calibur flow cytometer. The experiment was performed in duplicates and repeated twice.3.2Cell migration assayExperiments grouping and transfecting were the same as above. After36h of hypoxia incubation,5-8F and CNE2cells were harvested and resuspended with serum-free RPMI1640medium containing0.2%BSA at1.O×106cells/mL. Approximately100μl cell suspension was added into the upper chamber. As a chemo-attractant,500μL RPMI1640medium with10%FBS was added to the lower chamber. After12h of incubation, transwells were removed from24-well plates, and nonmigrated cells on the top of the trans well was scraped with a cotton swab. The cells which migrated onto the lower surface of the membrane were fixed with4%formparaaldehyde solution for20min, washed with PBS and stained with crystal violet for20min. The migrated cell was counted under a light microscope at400magnification, the number of migrated cells was expressed as the average of five randomly selected fields.3.3Cell viability assayThe chemosensitivity was evaluated using the MTT assay.5-8F and CNE2cells Cells were plated at1×104cells per well in96-well plates. Twenty-four hours after plating, cells were transfected with both HIF1α-siRNA or Negative-siRNA and were incubated under hypoxic condition for12h, experiments grouping was the same as above. Then, cells were incubated at various concentrations of5-FU (0.10.25.50.75.100.150μg/mL) or DDP (0.5.10.15.25.75.100μg/mL) for36h under normoxia or hypoxia condition,20μL of5mg/mL MTT reagent was added to each well, incubated at37℃for4h; the supernatant was removed and150mL of DMSO was used to dissolve the resultant formazan crystals. Absorption values were read at570nm using a multiscanner autoreader. SPSS13.0statistical software were used to calculate the IC50of DDP and5-Fu. Each concentration had five replicate wells and the experiment was repeated three times.4. Statistical analysisStatistics were calculated with SPSS13.0software and results were expressed as mean±sd. Statistical analysis was performed using the One-Way-ANOVA(Fisher’s exact test). P-values less than0.05were accepted as significant.Result1. The effect of hypoxia treatment on HIF1α and hTERT expression of nasopharyngeal carcinoma cellsNasopharyngeal carcinoma cell lines5-8F and CNE2were incubated under hypoxic condition for0h～72h. HIF-1α and hTERT protein levels at various time intervals were detected by Western-blot. The results showed that the HIF-1α and hTERT protein were moderate elevated in all two tnasopharyngeal carcinoma cell lines during incubation in normoxia condition. Moreover, with prolonged hypoxia time, HIF-la and hTERT protein expression reached a peak at16h and24h of5-8F, respectivel. Hypoxia up-regulated HIF-la and hTERT expression in CNE2cells, with peak induction at24h of hypoxia. HIF-la and hTERT protein levels in all two tnasopharyngeal carcinoma cell lines were then declined progressively with longer incubation time. These results indicate that hypoxia up-regulate HIF-la and hTERT expression in nasopharyngeal carcinoma cell.2. Transient transfection with siRNA-Cy3identified by flow cytometryNasopharyngeal carcinoma cells were transfected with chemically synthesized Negative-siRNA-Cy3for6h by the lipofectamine2000. After12h(within6to24h)of transfection, fluorescence microscopy showed that the transfected cells with red fluorescence. And the transfection efficiency of5-8F and CNE2were detected by flow cytometry were (98.833±1.070)%and (98.367±0.586)%, respectively.3. Effect of HIF1α-siRNA on the expression of HIF1α and hTERT in nasopharyngeal carcinoma cellsThree pairs of siRNA (HIF1α-siRNA-1, HIF1α-siRNA-2and HIF1α-siRNA-3) were transfected into5-8F and CNE2cells. The Real-time PCR results demonstrated that the HIF1a-siRNA-1had the strongest inhibitory effect on HIF1α expression at the mRNA level(P<0.05). The levels of HIF1α mRNA of5-8F and CNE2cells in HIFla-siRNA-1group were reduced by0.703±0.021and0.813±0.012, respectively, compared with untransfected cells, and a non-significant effect on HIFla expression was observed in Negative-siRNA cells (P>0.05). Therefore, HIF1α-siRNA-1was selected as effective siRNA for inhibiting HIF1α expression in5-8F and CNE2cells.Real-time PCR and Western-blot demonstrated that the levels of HIFla/hTERT mRNA and protein in HIFla-siRNA cells were significantly reduced(P<0.05), compared with untransfected cells. HIFla/hTERT mRNA and protein of5-8F cells were reduced by0.710±0.020,0.633±0.051and0.583±0.065�'�0.492±0.069 respectively; HIF1α/hTERT mRNA and protein of CNE2were cells reduced by0.799±0.023,0.677±0.067and0.649±0.193,0.746±0.089, respectively.4. Hypoxia induces cell cycle arrest and HIF1α-siRNA-targeted HIF1α induces cell cycle redistribution in nasopharyngeal carcinoma cellsTo investigate the mechanism by which HIF1α-siRNA inhibited proliferation and drug-resistance, we analyzed the effect of hypoxia and HIF1α-siRNA on cell cycle using flow cytometry. The results showed that the CNE2cell line exhibited hypoxia-associated G1/S arrest after exposure to hypoxic conditions. In comparison,5-8F cell line lacked an observable G1/S arrest (defined as an increase in the G0/G1peak with a decrease in the S peak). After silenceing HIF1α, in CNE2Cells, the ratio of G0/G1phases decreased(P<0.05), partial G0and G1phase of the cells backed into the cell cycle and G2/M phase cells increased (P<0.05), compared with untransfected cells. For5-8F cells, the S phase cells reduced(P<0.05), the G2/M phase increased(P<0.05), while no significant change in G0/G1phase. No significant difference was observed between untransfected cells and Negative-siRNA cells (P>0.05).5. Silenceing HIF1α significantly inhibited migration in nasopharyngeal carcinoma cellsHypoxia significantly promoted the migration of5-8F and CNE2cells (P<0.05). The presence of hypoxia, silencing of HIF1α, compared with untransfected group and Negative-siRNA group, the migration ability of HIF1α-siRNA group significantly decreased in both5-8F and CNE2cells (P<0.05). No difference between Negative-siRNA group and untransfection group (P>0.05).6. Down-regulation of HIF1α enhanced the chemosensitivity of nasopharyngeal carcinoma cellsNasopharyngeal carcinoma cell lines5-8F and CNE2were treated with5-FU and DDP to determine their chemosensitivity.5-FU reduced the growth of all cell lines in both, in a dose-dependent manner under normoxia condition. However, after hypoxia treament, chemosensitivity to5-FU decreased in both5-8F and CNE2(P<0.05), compared with normoxia condition. Silenceing HIF1α, the5-Fu IC50S of HIFla-siRNA cells significantly decreased in both5-8F and CNE2cell lines (P<0.05), Compared with untransfected cells and Negative-siRNA cells. As well as5-FU, chemosensitivity to DDP decreased in both5-8F and CNE2(P<0.05) after hypoxia treatment, Additionally, the chemosensitivity of5-8F and CNE2cells to DDP was significantly enhanced after transfected with HIF1α-siRNA (P<0.05), compared with untransfected cells.ConclusionOur results suggest that hypoxia seems to be promote tumor progression and resist to chemotherapy via up-regulating hTERT. Moreover, HIF1α-siRNA targeting HIF1α have been shown to inhibit the expression of hTERT and reverse drug-resistance of tumor to commonly used chemotherapy drugs,5-Fu and DDP. It is prompted that the effects of hypoxia on the biology of nasopharyngeal carcinoma may be achieved by regulating the telomerase activity and further providing new insights into tumor hypoxia to be used as a novel therapeutic target for cancer.