| BACKGROUNDNasopharyngeal carcinoma(NPC)is one of the most common malignancies of head and neck in China,whose morbidity is only second to thyroid cancer.Its epidemiological characteristic is unique geographical distribution,which frequently occurs in areas such as South China and Southeast Asia.Radiotherapy is a major treatment as well as an essential component of curative treatment for NPC.At present,NPC has achieved a better survival prognosis.Nonetheless,over 20%NPC patients develop recurrence or distant metastasis after standard treatment.Radioresistance is considered as the major obstacle for the effective treatment of NPC,but the specific mechanism of radioresistance remains unclear.More and more studies have shown that radioresistance in cancers might be related to cancer stem cells(CSCs).CSCs are the rare cancer cells with out-of-control proliferation,self-renewal capacity,multilineage differentiation and stem cell traits,which are resistant to radiotherapy and other anti-cancer treatments.Previously,some scholars discovered that,the radioresistant esophageal cancer cells obtained through fractional irradiation had displayed CSC traits and high level telomerase activity.Telomerase is a ribonucleoproein complex enzyme,which is involved in the telomere maintenance mechanism of about 90%human cancers.Human telomerase reverse transcriptase(hTERT)is the essential catalytic subunit that maintains telomerase activity,and its expression is consistent with telomerase activity.hTERT and telomerase play crucial roles in maintaining the biological traits of CSCs,which are also suggested to participate in regulating cancer radiosensitivity.Our previous studies indicated that,the radioresistant NPC cell line CNE-2R over-expressed CD 166 protein,which was considered as a potential stem marker.To sum up,we speculated that CNE-2R cells might also possess the CSC-like traits,and expression high level telomerase activity and hTERT.So far,few reports are available regarding the CSC-like traits of NPC radioresistant cells,and the improvement of their radiosensitivity based on their CSC traits.Moreover,it remains unclear about whether the NPC radioresistant cells display high level telomerase activity and high hTERT expression,whether the CSC-like traits can be regulated based on the target of hTERT to improve the radiosensitivity of the radioresistant NPC,and whether there is a deeper mechanism.Taken together,we believe that it is necessary to intensively explore and verify the proposed hypothesis.OBJECTIVEFirstly,a series of experiments were carried out to verify that the radioresistant NPC cell line CNE-2R constructed previously by our research group displayed the CSC-like traits,high level telomerase activity and high hTERT expression.Subsequently,hTERT expression in CNE-2R cells was silenced through the lentivirus-mediated RNA interference technique,and changes in the CSC-like traits and radiosensitivity of CNE-2R cells were observed through experiments in vitro and in vivo.Finally,the potential signaling pathway between hTERT and the CSC-like traits were further investigated,so as to determine the way of hTERT in regulating the CSC-like traits and eventually affect the radiosensitivity of CNE-2R cells.The final results of this study might provide some new thinking for searching for radiosensitization therapy targeting CSCs in NPC.METHODS1.The relative mRNA expression of stem cell-related genes,?-catenin and hTERT genes in the radioresistant NPC cell line CNE-2R and the parent radiosensitive cell line CNE-2 were detected through qPCR.Meanwhile,expression of the stem cell-related proteins,P-catenin and hTERT proteins in CNE-2R and CNE-2 cells was also detected through Western blot assay.The proportions of the label-retaining cells(LRCs)in CNE-2R and CNE-2 cells were detected by immunocytochemistry,and the telomerase activity in each group was detected through PCR-ELISA.The expression rates of CD133 in CNE-2R and CNE-2 cells were detected through flow cytometry.Then,the CNE-2R-CD133+cells and CNE-2R-CD133-cells were isolated through magnetic activated cell sorting(MACS),and it was verified through CCK-8 assay,sphere formation assay,and tumorigenicity test in nude mice that,CNE-2R-CD133+cells had displayed the CSC traits.2.The RNA interfering lentivirus vector of hTERT gene was designed and constructed.The hTERT interfering lentivirus was transfected into the CNE-2R cells to obtain the hTERT-shRNA cells,and the empty vector was used to infect CNE-2R cells to obtain the NC-shRNA cells.Then,the transfection efficiency was detected by flow cytometry.At the same time,the hTERT mRNA and protein expression in the transfected cells was detected by qPCR and Western blot assay,so as to verify the silencing effect,and the cell line with stably silenced hTERT was eventually constructed.3.The telomerase activity in CNE-2R cells before and after hTERT silencing was detected through PCR-ELISA.Then,the radiosensitivity in vitro was detected through colony formation assay,CCK-8 assay and flow cytometry;subsequently,the influence of silencing hTERT on the radiosensitivity in vivo was observed by constructing the nude mice xenograft model.The expression levels of hTERT protein,stem cell-related proteins and ?-catenin protein in vivo/in vitro were detected through Western blot and immunohistochemistry,while the apoptotic index in the xenograft was detected through TUNEL assay.4.The hTERT over-expressed lentivirus was used to transfect CNE-2R cells,and the hTERT mRNA and protein expression in the transfected cells were detected through qPCR and Western blot assay.Then,cells were cultured in the complete medium containing the Wnt/?-catenin signaling pathway inhibitor XAV-939 and agonist SKL2001.Later,the protein expression of hTERT,?-catenin and stem cell-related proteins was detected through Western blot assay,in order to understand the relationship between hTERT and the Wnt/?-catenin signaling pathway,as well as the influence on the stem cell-related proteins expression.Afterwards,immunofluorescence was utilized to detect the ?-catenin protein distribution in nucleus and cytoplasm after hTERT silence or over-expression.Co-immunoprecipitation assay was performed to detect whether there was direct interaction between hTERT and ?-catenin protein.Telomerase activity in each group was detected by PCR-ELISA,and the radiosensitivity of cells in each group was examined by CCK-8 assay.RESULTS1.The relative mRNA expression of stem cell-related genes,?-catenin and hTERT gene in CNE-2R cells were higher than those in CNE-2 cells(P<0.05).Similarly,the expression of stem cell-related proteins,?-catenin and hTERT protein in CNE-2R cells were remarkably higher than those in CNE-2 cells.The proportions of LRCs in CNE-2R cells and CNE-2 cells were(3.10±0.63%)vs(0.40±0.35%),respectively(P<0.001).The telomerase activity of CNE-2R cells(1.618±0.022)was significantly higher than that in CNE-2 cells(1.344±0.006)(P<0.05).Flow cytometry results indicated that,the CD 133 positive rates in CNE-2R cells and CNE-2 cells were(2.49±0.56%)vs(0.76±0.25%),respectively(P=0.008).Besides,the in vitro proliferation capacity,in vivo tumorigenicity,and telomerase activity in CNE-2R-CD133?cells were evidently higher than those in CNE-2R-CD133-and CNE-2R cells(P<0.05).2.Flow cytometry results indicated that,the lentivirus infection rates in hTERT-shRNA cells and NC-shRNA cells were as high as 90%.Fluorescent quantitative PCR results revealed that,the relative hTERT mRNA expression in hTERT-shRNA cells(0.164±0.023)was significantly lower than those in NC-shRNA cells(1.207±0.054)and CNE-2R cells(1.000±0.041)(P<0.001).Similarly,Western blot results also suggested that,the hTERT protein expression in hTERT-shRNA cells was notably down-regulated.3.Results of colony formation assay indicated that,the survival fraction of hTERT-shRNA cells at each irradiation dose was lower than those of CNE-2R and NC-shRNA cells.The sensitive enhancement ratio of hTERT-shRNA cells to CNE-2R cells was 1.23>1,revealing that hTERT-shRNA cells were more sensitive to radiation.CCK-8 results also indicated that,hTERT-shRNA cells were more sensitive than CNE-2R cells to radiation.Flow cytometry results suggested that,the apoptosis rate of hTERT-shRNA cells was slightly increased under irradiation at 0 Gy;typically,the apoptotic rates of CNE-2R cells,NC-shRNA cells and hTERT-shRNA cells were(4.82±0.73%)vs(4.85±0.35%)vs(6.25±0.38%),respectively(P=0.023).Under irradiation at 4 Gy,the apoptotic rates of the three groups of cells were(12.10±1.14%)vs(12.71±0.74%)vs(19.03±0.43%),respectively(P<0.001).Differences in the apoptotic rate of CNE-2R cells and NC-shRNA cells before and after irradiation were not statistically significant(P>0.05).PCR-ELISA results indicated that,the telomerase activity of hTERT-shRNA cells was(1.263±0.024),which was markedly lower than those in NC-shRNA cells(1.629±0.007)and CNE-2R cells(1.618±0.022)(P<0.001).Results of xenograft model suggested that,the xenograft growth in hTERT-shRNA group was slightly suppressed under irradiation at 0 Gy;the xenograft growth in 3 groups were suppressed after irradiation at 8 Gy,but the hTERT-shRNA group was subject to more obvious suppression.TUNEL assay indicated that the apoptosis index in vivo were consistent with the in vitro cell apoptosis rates.Results of Western blot and immunohistochemistry suggested that,silencing hTERT could evidently down-regulate the stem cell-related proteins expression both in vivo and in vitro.Besides,immunohistochemical results also discovered that,the ?-catenin protein in hTERT-shRNA group was mainly expressed in the cytoplasm and cell membrane,while that could also be expressed in some nuclei in CNE-2R and NC groups.4.Western blot results suggested that,hTERT could positively regulate the expression of stem cell-related proteins,but it had no obvious influence of the expression of Wnt3a and total ?-catenin proteins.Further detection indicated that,silencing hTERT could remarkably reduce the expression of nuclear-?-catenin protein,while over-expression of hTERT would up-regulate the nuclear-?-catenin protein expression.In addition,suppressing or activating the Wnt/?-catenin signaling pathway could positively regulate the expression of hTERT and stem cell-related proteins.In co-immunoprecipitation assay,the?-catenin/hTERT protein complex could be detected in CNE-2R cells,suggesting the direct interaction between ?-catenin and the hTERT protein.Additionally,PCR-ELISA results revealed that,silencing hTERT or suppressing the Wnt/?-catenin signaling pathway could reduce the telomerase activity in CNE-2R cells;meanwhile,the reduced telomerase activity after suppressing the Wnt/?-catenin signaling pathway could be reversed by the over-expression of hTERT.Similarly,results of CCK-8 assay suggested that,silencing hTERT or suppressing the Wnt/?-catenin signaling pathway could enhance the radiosensitivity of CNE-2R cells;meanwhile,when the Wnt/?-catenin signaling pathway was suppressed,the radioresistance could be reversed through the over-expression of hTERT.CONCLUSIONSThis study has firstly verified that the radioresistant NPC cell line CNE-2R constructed previously through fractional irradiation has displayed the CSC-like traits,as well as high level telomerase activity and high hTERT expression.Then,CNE-2R cell line with stably hTERT silencing was successfully constructed through the lentivirus-mediated RNA interference technique and it was discovered that silencing hTERT could enhance the radiosensitivity of CNE-2R both in vivo and in vitro;at the same time,it could down-regulate the telomerase activity in CNE-2R cells and suppress their CSC-like traits.Further research indicated that,there was a positive feedback loop between hTERT and the Wnt/?-catenin signaling pathway in CNE-2R cells,which could regulate the telomerase activity and CSC-like traits in CNE-2R cells,thus regulating the radiosensitivity of CNE-2R cells.Therefore,interfering with hTERT expression and blocking the Wnt/p-catenin signal transduction might be a promising strategy to specifically target the radioresistant NPC cells with CSC-like traits,which would provide a new thinking for studying the radiosensitization therapy for the radioresistant NPC. |
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