ABSTRACT
OBJECTIVE：To study the inhibi tory effects of genistein on the growth of human nasopharyngeal carcinoma. CNE 1 cells and predict its potential target. METHODS ：CCK-8 method was used to test the effects of 0（blank control ），12.5，25，50， 100，150 µmol/L genistein on the proliferation of CNE 1 cells after treated for 24，48，72 h. Flow cytometry was carried out to detect the effects of 0（blank control ），15，30，60 µmol/L genistein on the cell cycle and ap optosis of CNE 1 cells after treated for 24 h. Scratch test was used to investigate the effects of 0（blank control ）， 10， 20， 30 µmol/L genistein on themigration ability of CNE 1 cells after treated for 24 h. High （No.18210156） throughput sequencing was conducted to discover the differential genes in CNE 1 cells after treated with 0（blankcontrol），30 µmol/L genistein for 24 h. RT-qPCR assay was adopted to verify the mRNA expression of related differential genes in above trials. RESULTS ： Compared with blank control，12.5，25，50，100，150 µmol/L genistein sho wed significant inhibitory effect on the proliferation of CNE 1 cells（P＜ 0.01），in a concentration- time-effect manner ；15，30 µmol/L genistein could arrest CNE 1 cell cycle at G 0/G1 stage（P＜0.05 or P＜ 0.01）；30，60 µmol/L could arrest CNE 1 cell cycle at G 2/M stage and promoted cell apoptosis （P＜0.05 or P＜0.01）. 10，20，30 µmol/L genistein could significantly inhibit the migration ability of CNE 1 cells（padj＜0.01）. High throughput sequencing revealed a total of 2 271 differentialgenes（P＜0.05），1 154 of which were up-regulated while 1 117 of which were down-regulated ；8 potential target genes ，including p53，p21，STC2，FGF2，CDK6，CYCLIN D ，PI3K，AKT，were screened by cell experiment. After validated by RT-qPCR assay ，mRNA expression of p53，p21，STC2，FGF2，CDK6，CYCLIN D and AKT were significantly down-regulated（P＜0.05），which consistent with the sequencing results. CONCLUSIONS ：Genistein can effectively inhibit the growth of human nasopharyngeal carcinoma CNE 1 cells，the mechanism of which may associated with inhibiting the expression of mutant gene p53，restoring the function of wild-type P 53 protein and inhibiting the activity of PI 3K/Akt pathway.
ABSTRACT
Objective:Astragalus polysaccharide (APS) was used in combination with ionizing radiation (IR) to investigate the mechanism of APS on the radiosensitivity of human nasopharyngeal narcinoma CNE-1 cells and the epithelial-mesenchymal transition (EMT). Method:Cell counting kit-8 (CCK-8) was used to detect the cytotoxicity of different concentrations of APS (0，6.25，12.5，25，50，100，200 g·L-1) on CNE-1 cells. Colony formation assay was used to calculate the survival fraction (survival fraction, SF) of CNE-1 cells treated with 12.5 g·L-1 APS combined with different radiation doses (0，2，4，6 Gy). The linear quadratic equation mathematical model (LQ) was used to draw the radiosensitivity curve according to SF value. Cell scratch and transwell chamber test were used to detect the migration and invasion ability of cells in each group. The apoptosis of cells in each group was detected by flow cytometry, Western blot was used to detect the expressions of EMT markers, apoptosis markers and protein kinase B/extracellular regulated protein kinases (Akt/ERK) pathway proteins in each group. Result:The results of colony formation assay and radiosensitivity curve showed that the combination of non-toxic dose of 12.5 g·L-1 APS and radiation dose of 4 Gy could significantly increase the radiosensitivity of CNE-1 cells. Compared with blank group and IR group, APS combined with IR could significantly inhibit the migration and invasion of CNE-1 cells (P<0.05), and increase the rate of apoptosis (P<0.05). In addition, compared with the blank group and the IR group, APS combined with IR could significantly down-regulate the expressions of N-cadherin, p-Akt and p-ERK, and significantly up-regulate the expressions of E-cadherin, Bax and Caspase-3 (P<0.05). Conclusion:APS combined with IR can inhibit the migration and invasion of CNE-1 cells, and increase the apoptosis induced by radiotherapy, which may be related to the inhibition of EMT and Akt/ERK pathway.
ABSTRACT
Aim To establish luciferase reporter gene expression cell models of CNEi-RACI-Luc2 based on the target of RAC1 promoter, and explore the application of screening anti-tumor active of rhein derivatives targeted regulating RAC1 at transcriptional level. Methods The lentiviral carrying luciferase reporter vector was designed and synthesized using RAC1 promoter se-quence, and CNE1 cells were infected with recombinant plasmid lentiviral to obtain cell lines that stably expressed firefly luciferase. Luciferase reporter assay was used to detect the luciferase luminescence value after stimulating with RAC1 activator PMA and inhibitor NSC23766 that targeted regulating the RAC1 promoter activities in cells, and RAC 1 expression was verified by Western blot. The effect of series of rhein derivatives on the lu-cifcrase activity of RAC1 promoter was observed, and RAC1 expression was determined by Western blot. Results The identification result of double enzyme digestion showed that a lentiviral expression vector carrying luciferase reporter vector rc-combined with RAC1 promoter was successfully constructed. Lcntivirus-infcctcd CNE1 cells were screened by puromycin, the CNE1-RAC1-Luc2 cells stably expressing firefly luciferase were obtained, and the Iransfection efficiency was over 90%. The RAC1 luciferase reporter assay system was sensitive to FMA and NSC23766 and consistent with the result of RAC1 protein expression by Western blot. The regulation of series of rhein derivatives to RAC1 luciferase activity of CNE1-RAC1-Luc2 cells was consistent with the results of Western blot. Conclusions The cell model of luciferase reporting system containing RAC1 promoter can be successfully constructed, which provides a practical platform for high throughput screening of RAC1-targeted drugs.
ABSTRACT
Objective To examine the expressions of multidrug resistance gene (MDR)-1 and P-glycoprotein (P-gp) in nasopharyngeal squamous cell carcinoma CNE1 cell line before and after X-ray exposure.Methods CNE1 cells were exposed to X-ray.After the irradiation,the CNE1 cells were cultured for 24 hours and tested.The mRNA expressions of MDR-1 in CNE1 cells were measured by semi-quantitative real-time polymerase chain reaction (RT-PCR) before and after X-ray exposure,and the protein expressions of P-gp in CNE1 cells were detected by Western blotting.The protein expressions of P-gp in CNE1 cells were observed by confocal microscope before and after X-ray exposure.Results The results of RT-PCR showed that the absorbance (A) values of MDR-1 mRNA in CNE1 cells were 0.17 ±0.01 and 0.34 ±0.03 before and after irradiation,and the difference was statistically significant (t =16.541,P ＜ 0.001).The results of Western blotting showed that the A values of P-gp protein in CNE1 cells were 0.02 ± 0.01 and 0.04 ± 0.01,and the difference was statistically significant (t =4.612,P =0.016).The green fluorescence intensity of P-gp protein in CNE1 cells after X-ray irradiation was higher than that before X-ray irradiation by confocal microscope.Conclusion X-ray irradiation can cause the high expressions of MDR-1 and P-gp in CNE1 cells,suggesting that X-ray irradiation can induce the occurrence of multidrug resistance in CNE1 cells.
ABSTRACT
Objective: To explore the role of histone H3 phosphorylation at serine10 (Ser10) in regulating proliferation and transformation of human nasopharyngeal carcinoma cell line CNE1. Methods: The level of histone H3 phosphorylation at Ser10 was detected by Western blotting in the presence of epidermal growth factor (EGF). The wide-type histone H3 (H3WT) and mutant histone H3 (H3S10A) recombinant expression vectors were constructed, and then were transfected into CNE1 cells. The CNE1 cells stably expressing wide-type and mutant histone H3 were screened with blasticidin, and then the expression of histone H3 and EGF-induced histone H3 phosphorylation at Ser10 were detected in transfected cells by Western blotting. Cell counting kit-8 (CCK-8) assay and soft agar assay were employed to assess the effect of over-expression of wide-type and mutant histone H3 on proliferation and colony-formation of CNE1 cells. The expression levels of c-Jun, c-Fos and Bcl-2 were detected by Western blotting, and the activator protein-1 (AP-1) transcriptional activation were examined by dual-luciferase reporter gene assay. Results: EGF induced phosphorylation of histone H3 at Ser10 in CNE1 cells. The CNE1 cells stably overexpressing histone H3WT and mutant H3S10A were successfully established. The level of histone H3 phosphorylation at Ser10 induced by EGF was greatly decreased in mutant H3S10A CNE1 cells. As compared with the mock control tranfected with an empty vector, the abilities of proliferation and colony formation were obviously promoted in H3WT-overexpressing CNE1 cells stimulated by EGF, as well as the expression levels of c-Jun and c-Fos and transcriptional activation of AP-1 were significantly increased. However, no significant changes were observed in mutant H3S10A-overexpressing CNE1 cells. Conclusion: Histone H3, especially the Ser10 motif, might have an important role in regulating EGF-induced proliferation and transformation of CNE1 cells, which is relate with promoting c-Jun and c-Fos gene transcription and thereby enhancing AP-1 activity.
ABSTRACT
1)were up-regulated in test and 184 genes(RA1.5)and down-regulation(RA
ABSTRACT
AIM: To discuss the discrepancy of microRNA (miRNA) in nasopharyngeal carcinoma (NPC) cells CNE-1 and CNE-2 on the basis of validating their different radioresistance.METHODS: Following the effect of X ray on the clones of CNE-1 and CNE-2 cells, the dose-survival curve and biological characteristics of CNE-1 and CNE-2 cells were determined by SigmaPlot software and the linear quadratic model of survival curves analysis. MicroRNAs were detected by Paraflo microfluidic microRNA chip, hybridization images collected using a laser scanner and normalizing the signals using a LOWESS filter. The relationship between the discrepancy of NPC radiosensitivity and the expression of microRNA was predicted according to Targetscan3.1 database (http:www.targetscan.org) after analyzing the data.RESULTS: Compared to CNE-2 cells, 20 microRNAs were gain-of-function and 13 microRNAs loss-of-function in CNE-1 cells among 326 detected microRNAs. 4 miRNAs that one detective value was more than 2000 and 3 folds than the other were hsa-miR-152, hsa-miR-7, hsa-miR-205 and hsa-miR-572. Data showed that radio-sensitivity was relative to the distinct discrepancy of miRNAs. CONCLUSION: The discrepancy of miRNAs is presents in different radioresistant NPC cell lines and related to radiosensitivity.