RESUMEN
Objective To investigate the effect and underlying mechanism of lncRNA MEG3 on the radiosensitivity of nasopharyngeal carcinoma cells.Methods this experiment,overexpression control group,MEG3 overexpression group,miR-NC inhibition group,miR-7-5p inhibition group,overexpression control+4 Gy group,MEG3 overexpression+4 Gygroup,miR-NC inhibition+4 Gy group,miR-7-5p inhibition+4 Gy group,MEG3 overexpression + miR-NC overexpression group,MEG3 overexpression + miR-7-Sp overexpression group were established.The expression of miR-7-5p and MEG3 was detected by qRT-PCR.The radiosensitivity of nasopharyngeal carcinoma cells was measured by clone formation assay.Cell apoptosis was assessed by flow cytometry.The fluorescence activity was evaluated by dual luciferase reporter assay.Results MEG3 was lowly expressed in nasopharyngeal carcinoma tissues and cells.Overexpression of MEG3 and inhibition of miR-7-5p expression increased the radiosensitivity of nasopharyngeal carcinoma cells and promoted radiation-induced cell apoptosis.MEG3 could targetedly regulate the miR-7-5p expression.Overexpression of miR-7-5p reversed the effect of overexpression of MEG3 on the sensitization of nasopharyngeal carcinoma cells and the promotion of apoptosis induced by radiation exposure.Conclusions Overexpression of MEG3 increases the radiosensitivity of nasopharyngeal carcinoma cells and promotes radiation-induced cell apoptosis.The mechanism may be related to the down-regulation of miR-7-5p expression.
RESUMEN
This study aims to investigate the effect of down-regulation of miR-205-5p by transfection of miR-205-5p inhibitor on the sensitivity of HNE1/DDP cells to cisplatin (DDP) induced apoptosis and explore the underlying mechanism. qRT-PCR was used to detect the expression of miR-205-5p in HNE1 or HNE1/DDP cells. The expression level of miR-205-5p was analyzed after transfecting HNE1/DDP cells with miR-205-5p inhibitor. MTT assay was used to evaluate the inhibitory effect of DDP alone or in combination with miR-205-5p inhibitor on the proliferation of HNE1/DDP or HNE1 cells. Apoptosis of cells treated with miR-205-5p inhibitor alone or in combination with DDP (8 μmol·L-1) was assessed using flow cytometry with PI staining, with the nucleus was counterstained with DAPI staining. The expression of Bax, Bak, Mcl-1, or Bcl-2 was analyzed by Western blot. HNE1/DDP cells showed a high level of expression of miR-205-5p, and the expression of miR-205-5p was significantly decreased by transfection of miR-205-5p inhibitor. Down-regulation of miR-205-5p significantly increased the sensitivity of HNE1/DDP cells to DDP (P<0.05). Transfection of miR-205-5p inhibitor enhanced the sensitivity of HNE1/DDP cells to DDP induced apoptosis. Treatment of HNE1/DDP cells with miR-205-5p inhibitor combined with DDP (8 μmol·L-1) for 24 h resulted in an apoptotic rate of 28.93% ± 2.50%, significantly higher than that treated with miR-205-5p inhibitor (9.83% ± 1.31%) or DDP alone (10.83% ± 1.70%) (P<0.05). DAPI staining showed that HNE1/DDP cell nucleus became significantly condensed and fragmented in miR-205-5p inhibitor combined with DDP group. The combined group up-regulated the expression of Bax and down-regulated the expression of Bcl-2 in HNE1/DDP cells. Therefore, down-regulation of miR-205-5p can enhance the sensitivity of HNE1/DDP cells to cisplatin induced apoptosis, and the mechanism may involve up-regulation of Bax and down-regulation of Bcl-2 expression.
RESUMEN
Objective research the effects of metformin on proliferation and apoptosis in nasopharyngeal carcinoma cell CNE-1 and investigate the role of miR-let-7a、IGF-1R in it. Methods The nasopharyngeal carcinoma cell CNE-1 was treated with different concentrations of metformin for 24h, then the proliferation activity of cell was detected by CCK8 method; the apoptosis rate of cell was measured by flow cytometry; the expression levels of bcl-2、bax and IGF-1R mRNA and miR-let-7a were detected by real-time quantitative PCR; the expression level of IGF-1R protein was detected by Western blot. Results Compared with the control group, the cell proliferation activity of metformin group decreased(P<0.05), and it gradually decreased along with the increase of metformin concentration. The cell apoptosis rate of metformin group increased(P<0.05 except for the 5 mmol/L group), and it gradually increased along with the increase of metformin concentration. The expression levels of bax mRNA and miR-let-7a were up-regulated in the metformin group(P<0.05), while the expression levels of bcl-2 mRNA,IGF-1R mRNA and IGF-1R protein were decreased(P<0.05). Conclusion Metformin could inhibit the proliferation and induce apoptosis of CNE-1. The mechanism maybe related to the up-regulation of miR-let-7a and down-regulation of IGF-1R.
RESUMEN
Objective To investigate the effect of E1A gene on the radiosensitivity of human nasopharyngeal carcinoma cells and its possible mechanism. Methods The E1A gene was transfected into nasopharyngeal carcinoma CNE-2R cells by adenovirus vector. The expression of E1A gene was detected by RT-PCR. Untransfected CNE-2R cells(PBS group)and CNE-2R cells transfected with empty vector Ad-β-gal(Ad-β-gal group)and E1A(Ad-E1A group)were given 0 Gy,2 Gy,4 Gy,6 Gy,8 Gy 6 MV X-ray irradiation. The changes in radiosensitivity of CNE-2R cells were determined by colony-forming assay. Flow cytometry was used to analyze cell apoptosis in each group. The expression of NF-κB, CK2α, Bcl-2, and cleaved caspase-3 was measured by Western blot. Results RT-PCR confirmed that the E1A gene was transfected into CNE-2R cells and stably expressed. The Ad-E1A group had a significantly lower plating efficiency than the PBS group and the Ad-β-gal group(P<0.05). The Ad-E1A group had significantly lower cell survival rate at 2 Gy irradiation than the PBS group and the Ad-β-gal group(0.217 vs. 0.602, P<0.05;0.217 vs. 0.585, P<0.05). The Ad-E1A group had a significantly higher α/β value than the PBS group and the Ad-β-gal group(24.680 vs. 5.268, P<0.05;24.680 vs. 5.132, P<0.05). Flow cytometry results showed that irradiation alone could promote the apoptosis of CNE-2R cells,when combined with E1A gene,the apoptosis rate was significantly increased(P<0.05). Western blot results showed that E1A gene down-regulated the expression of NF-κB/p65,CK2α,and Bcl-2 and up-regulated the expression of cleaved caspase-3. Conclusions E1A gene can enhance the radiosensitivity of nasopharyngeal carcinoma cells by inhibiting the expression of CK2 to block the NF-κB signaling pathway and promoting cell apoptosis.
RESUMEN
objective To research whether the combination of chrysin and camptothecin can promote the apoptosis of human nasopharyngeal carcinoma cell line CNE2 and to explore the molecular mechanism of the combinative effect. Methods: CNE2 cells were pretreated with designed dose of chrysin (10|, 20, and 40 μmol/L) for 2 h, then treated with camptothecin (1 μg/mL) for 24 h. The morphologic changes were observed under inversed microscope and the cell viability was measured using MTT. The activity of caspase-3 and PARP, which was regarded as the protein marks of apoptosis, was determined by Western blotting. Then the cells were treated with chrysin for different time and the time course of apoptosis inhibitory protein, Bcl-xL was also detected using Western blotting. Results: Increases of cell death were observed in the group with combined chrysin and camptothecin, but no obvious cell death could be found in chrysin, camptothecin alone, and control groups; The data of cell viability supported this results; With the enhance of pretreatment dose of chrysin, the cell viability decreased. There were the significant differences between the combined groups and the control one (P<0.05), and between the combined groups and both the chrysin and camptothecin groups separately (P<0.05). Chromatin condensation, which was the indication of apoptosis, could be observed when the cells were stained with Hochest 33342; The proprotein of caspase-3 and PARP degraded and there were the dose-dependent and time-dependent effect. The pan-caspase inhibitor Z-VAD-fmk could inhibit the apoptosis of CNE2 cells which were treated with the combination of chrysin and camptothecin, according to the cell viability and the activation of caspase-3 and PARP; The time-dependent down-regulation in the apoptosis inhibitory protein Bcl-xL could be observed. Conclusion: The cotreatment of chrysin and camptothecin could promote the apoptosis of CNE2 and the down-regulation of apoptosis inhibitory protein Bcl-xL played an important role in the combinative effect.
RESUMEN
Objective To explore the effect of decrease of Survivin expression on the proliferation and apoptosis of the naso-pharyngeal carcinoma cells.Methods To observe the effect of decrease of Survivin expression on nasopharyngeal carcinoma cell morphology using RNAi technology combined with RT-PCR and Western blot.Cell proliferation and apoptosis were de-tected by MTT and TUNEL assay.The expression of PARP,Bcl-2 and Bax was detected by Western blot.Results RT-PCR result showed the survivin mRNA expression in Surviving-siRNA groups was downregulated (about 0.26±0.02)and the inhibition ratio was 43.7% (P<0.05).MTT result showed cell proliferation rates were significantly different between 24,48 and 72 h after transfection.The cell inhibition rates were 21.9%,37.1% and 29.6%,respectively (P<0.05).West-ern blot result showed the Survivin protein expression in Survivin-siRNA2 groups was downregalated and the relative ex-pression level was reduced by 57% (P<0.05).TUNEL assay showed that cell apoptosis rate was increased obviously in surviving-siRNA groups.The expression of PARP (89KD)and Bax wasupregulated (3.9 and 2.4 fold change)and the ex-pression of Bcl-2 was downregulated (0.3 fold change).The phosphorylation of AKT was inhibited when Survivin was down-regulated (about reduced by 5 7%).Conclusion Silencing Survivin gene by siRNA can inhibit the proliferation of Na-sopharyngeal carcinoma cells and induce apoptosis.Survivin may become a potential gene for therapy target of nasopharynge-al carcinoma.
RESUMEN
0.05).The mRNA expression of Cyclin B1 of IMRT group was significantly higher than that of ART group at each dose point(P
RESUMEN
Background and purpose:Somatostatin receptors have been found in a variety of tumors and are therefore amenable to treatment with somatostatin analogs, like octreotide. However, the study of SSTRs expression has been rarely studied in nasopharyngeal cancer.We investigated the expression of somatostatin receptors gene subtypes in human nasopharyngeal cancer cell line CNE_ 2 . Methods:We have harvested cultured human nasopharyngeal cancer cell line CNE_ 2 . Using both techniques, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical assay, we analysed mRNA of different subtypes of somatostatin receptors in human nasopharyngeal cancer cell line CNE_ 2 .Results:The positive rate of somatostatin receptors subtype SSTR_ 1 SSTR_ 2 SSTR_ 4 was manifested in the human nasopharyngeal carcinoma cell line CNE_ 2 by RT-PCR and immunohistochemical assay. According to immunohistochemical assay, SSTR_ 1 and SSTR_ 2A showed strongly positive expression and SSTR_ 3 and SSTR_ 5 negative expression,respectively.Conclusions:There are more than one SSTR subtypes expressed in the human nasopharyndeal carcinoma cell line CNE_ 2 . This study demonstrated the presence of SSTR_1,SSTR_ 2 in the human nasopharyndeal carcinoma cell line CNE_ 2 .
RESUMEN
Objective:To investigate the effects of tetrandrine(Tet) on proliferaton and apoptosis of nasopharyngeal carcinoma cell line CNE.Methods:CNE cells were divided into groups and treated by Tet at different concentrations.Inhibitory effects of tetrandrine were determined by MTT assay.The morphologic changes of CNE cells were observed by transmission electron microscope.DNA fragmentations were determined by gel electrophoresis assay.Cell apoptosis was investigated by flow cytometer.The expressions of apoptosis-related genes were detected by retrotranscriptase polymerase chain reaction(RT-PCR).Results:Tetrandrine possessed inhibitory effect on CNE cell proliferation in a concentration-dependent manner(P
RESUMEN
Objective:To explore the effect of inhibiting mdr1 by antisense technology on radiosensitivity in nasopharyngeal carcinoma cell line(CNE). Methods:There were four groups in the study:control group,lipofectin group,sense oligodexynucleotides(SODN) group,and antisense oligodexynucleotides(ASODN) group. The expression of mdr1 was detected by RT-PCR. Cellular response to irradiation was evaluated by the colony forming test. Methylguanine-DNA methyltransferase(MGMT)protein expression was determined by immunohistochemical staining. Results:ASODN group could significantly decrease the proliferation rate and downregulate the expression of MGMT of irradiated nasopharyngeal carcinoma cells(vs lipofectin group or SODN group,P
RESUMEN
Objective Radiosensitivity is highly correlated with DNA doub le stra nd breaks(DSBs). The repair of human DSB is possible mainly through nonhomologou s DNA end joining(NHEJ)pathway. This study was designed to determine the relat ionship between expression of 70Ku/ 80Ku prot ein (a modulating subunit of DNA-PK, which is an important component in NHEJ pathway) and radiosensitivity of nasoph aryngeal carcinoma cell lines. Methods Radiosensitivity parameters of the nasoph aryngeal carcinoma cell lines were obtained by the colony forming assay and radi ation dose-survival curve was done by linear quadratic model. Western blot was p erformed to determine the expression of 70Ku and 80 Ku in total extract of CNE1 a nd CNE2 at baseline level、different time after 4?Gy irradiation、12 h after di ff erent doses of irradiation. To perform a semi-quantitated assay of protein expr e ssion, the optic density (OD) value of each band was estimated by automatic imag e analysis system. Results Surviving fraction of CNE1 was higher than those of CNE2 at each dose point. Mean inactivation dose was higher in the CNE1(2.35)t han that in the CNE2(1.11), but the quantity of 70Ku/ 80Ku in either cell line was similar at both baseline and postirradiation levels. Conclusions CNE2 is mo re radiaosensitive than CNE1; as there was no marked correlation observed betwee n the quantity of Ku protein and radiosensitivity of the nasopharyngeal carcinom a cell lines. X-irradiation cannot result in any change in total quantity of Ku protein.
RESUMEN
Objective To examine the variation of protein expression in nasopharyngeal carcinoma cell lines with different biological characteristics and to identify the radiobiological associated proteins. Methods Biological characteristics of 5-8F and 6-10B were compared by flow cytometry assay after irradia- tion. The total proteins of 5-8F and 6-10B were separated by immobilized pH gradient(IPG) IEF-SDS two- dimensional gel electrophoresis technique. The differentially expressed proteins were cut from the gel and di- gested into peptides for MALDI-TOF MS and the Q-TOF mass spectrometric analysis. Identification of pro- tein was made through searching in protein sequence database. Protein expressions were examined by western blot and immunohistochemistry method. Results Nine most differentially expressed proteins between 5-8F cell and 6-10B cell were identified, p73 and CK19 expression examined by western blot were conformal with that by proteomic method, p73 expression in 5-8F cell was higher than in 6-10B cell. CK19 expression in 6- 10B cell was higher than in 5-8F cell. Conclusion Differentially expression of proteins exist in nasopha- ryngeal carcinoma cell lines with different biological characteristics. These proteins may be associated with cell radiobiological characteristic with the p73 as a potential biomarker.
RESUMEN
Objective:To investigate the mechanism of ursolic acid inducing the apoptosis of human nasopharyngeal carcinoma cell line CNE-2Z.Methods:CNE-2Z were treated by ursolic acid with different concentration.Cell proliferation was determined by MTT assay.Cell cycle and apoptosis rate were analysed by flow cytometry(FCM).The expressions of bax,bcl-2 and cox-2 were assessed by SP method of immunocytochemistry,while the expression of caspase 3 by Western blot.Results:Ursolic acid inhibited the proliferation of CNE-2Z cells and induced the apoptosis in dosage and time dependent manner.FCM showed that the cell population reduced in S phase and cell cycle was arrested in G0/G1 phase.Immunocytochemistry showed that the expression of bax was up-regulated in the apoptosis,while the expression of bcl-2 and cox-2 down-regulated.Western blot showed that the expression of caspase 3 was strengthened.Conclusion:Ursolic acid can induce apoptosis of CNE-2Z in vitro.Its molecular mechanism probably is through promoting activation of caspase3,up-regulating the expression of bax,and down-regulating the expressions of bcl-2 and cox-2.