| Objective:The aim of this study is to investigate the expression of human telomerase reverse transcriptase（hTERT）m RNA and protein in laryngeal cancer stem cells,and to observe the proliferation and differentiation activity of laryngeal cancer stem cells after down-regulation of hTERT expression.To investigate the expression of telomerase hTERT in laryngeal cancer stem cells and its role in maintaining the activity of laryngeal cancer stem cells.Methods:1.Human laryngeal carcinoma Hep-2 cells were cultured at 37°C in a 5%CO2 cell incubator.The proliferation of laryngeal cancer Hep-2 cells treated with different concentrations of CDDP（1,2,3,4,5μg/ml）was detected by cell counting kit-8（CCK-8）,and the optimal time and concentration of CDDP were selected.2.CDDP chemotherapy was used to enrich laryngeal cancer Hep-2 cells to obtain laryngeal cancer CD133+cells,which were verified by fluorescence activated cell sorter（FACS）.western blot（WB）and quantitative real time-PCR（qRT-PCR）were used to detect the relative expression of hTERT gene m RNA and protein in laryngeal cancer Hep-2 group and CD133+group,respectively.Telomerase activity was detected by enzyme linked immunosorbent assay（ELISA）.3.The hTERT gene silencing lentivirus plasmid was constructed and packaged into virus.The virus was infected into CD133+cells of laryngeal cancer to obtain hTERT-sh RNA cells,and then the empty virus was infected into CD133+cells of laryngeal cancer to obtain NC-sh RNA cells.CCK-8 method was used to screen the best action time points in CD133+group,CD133++NC group,CD133++sh-hTERT 1 group,CD133++sh-hTERT 2 group,and CD133++sh-hTERT 3 group.WB and qRT-PCR were used to detect the expression of hTERT protein and m RNA in each group,and the best silencing sequence was selected.4.The telomerase activity of CD133+group,CD133++NC group and CD133++sh-hTERT group was compared,and the cell cycle and apoptosis of each group were detected by flow cytometer（FCM）.Cell scratch test and transwell assay were used to detect the effect of CD133++sh-hTERT on the migration and invasion ability of laryngeal cancer stem cells in vitro.Results:1.The IC50 of CDDP on laryngeal cancer Hep-2 cells was 5.154μg/ml at 12h,3.431μg/ml at 24h and 2.979μg/ml at 36h after treatment with different concentrations of CDDP.The IC50 of 36h was taken for subsequent experiments.2.FCM test results showed that the proportion of stem cells in laryngeal cancer Hep-2 cells increased from 0.03%before chemotherapy to 11.92%;Compared with Hep-2 group,the expression of hTERT protein and m RNA was increased（all P<0.01）,and the telomerase activity（0.212±0.053）in the Hep-2 group was significantly lower than that in the CD133+group（0.529±0.009）,P<0.001.3.CD133+cells transfected with hTERT lentivirus,CD133+group,CD133++NC group,CD133++sh-hTERT 1 group,CD133++sh-hTERT 2 group and CD133++sh-hTERT 3 group showed the most significant inhibition at 48 hours after intervention;The expression of CD133++sh-hTERT protein and m RNA was significantly lower than that of CD133+group and CD133++NC group,all P<0.01;WB and qRT-PCR results showed that the hTERT gene silencing effect in CD133++sh-hTERT 2 group was significant.4.ELISA kit showed that the relative content of hTERT in CD133+group,CD133++NC group and CD133++sh-hTERT group was（0.505±0.010）,（0.514±0.007）and（0.328±0.013）,respectively.The results indicated that the telomerase activity in CD133++sh-hTERT group was decreased（P<0.01）;FCM results showed that the apoptosis rates of the three groups were 2.21%,2.92%and 25.35%,respectively.The percentage of G0-G1 phase in CD133+group,CD133++NC group and CD133++sh-hTERT group was 62.11%,63.99%and 55.72%,respectively.Wound healing assay showed that knockdown of hTERT significantly inhibited the migration of CD133+cells（P<0.01）;Transwell assay confirmed that low expression of hTERT inhibited the invasion ability of laryngeal cancer stem cells.Conclusion:CD133+cells were successfully enriched by CDDP chemotherapy,and verified by FACS,CD133+cells showed the characteristics of stem cells.The expression of hTERT protein,m RNA and telomerase activity in CD133+cells were significantly higher than those in Hep-2 cells.Reducing the expression of hTERT in CD133+cells of laryngeal cancer can promote the apoptosis of CD133+cells,reduce the proportion of cells arrested in G0-G1 phase,and inhibit the migration and invasion of CD133+cells.Studies have confirmed that hTERT regulates the stemness of laryngeal cancer stem cells,which lays a foundation for targeted killing of laryngeal cancer stem cells.hTERT gene is expected to become a new target for laryngeal cancer stem cell therapy. |
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