Preliminary Screening Of Drug Resistance Genes In Laryngeal Cancer Stem Cells

Objective:To understand the relationship between laryngeal cancer stem cells and chemotherapy tolerance,and selected the differential genes between stem cells and normal laryngeal carcinoma cells by using transcriptome sequencing technology.The mechanism of multidrug resistance in laryngeal cancer stem cells was preliminarily analysed.Methods:1.The expression rates of CD133 and CD44 in laryngeal squamous-cell carcinoma cell lines Hep-2 and TU177 were detected by Flow cytometry.RTCA method was used to detect the growth curve of Hep-2 and TU177 cells under the action of three kinds of chemotherapeutic drugs including cisplatin,5-FU and paclitaxel,and calculate the half maximal inhibitory concentration at different time.Comparing the relationship between the expression of CD133 and CD44 and the tolerance of chemotherapy.2.We isolated CD133+CD44+ cell populations from laryngeal squamous-cell carcinoma cell lines Hep-2 and TU177 by magnetic-activated cell sorting.QPCR method was used to detect the expression of MDR1 and MPR2 gene in CD133+CD44+ cells and parental cells.To verify the relationship between stem cells and chemotherapy resistance.3.We sequenced the CD133+CD44+ cells,CD133-CD44-cells and normal Hep-2 and TU177 cells to detect the differential genes between stem cells and normal laryngeal cancer cells,screening the genes related to drug resistance to analyze the mechanism of stem cell resistance.Results:1.The positive rate of CD44 and CD133 in Hep-2 cells was higher than that in TU177 cells.Moreover,the IC50 value of Hep2 cells at different time under three drugs was higher TU177 cells.The results showed that Hep-2 cells were more resistant than TU177 cells,and it had a significantly positive correlation with the positive expression rate of CD44 and CD133.2.The CD133+CD44+ fraction of Hep2 and TU-177 cells was as high as 80%-90%,respectively,suggesting that MACS enriched CD133+CD44+ cells efficiently.The expression of MDR1 and MRP2 were increased in different levels after sorting,indicating that the stem cells were related to the resistance of laryngeal carcinoma.3.We found 12 differential genes by transcriptome sequencing,FOS,JUN ?NR4A1 may be associated with multidrug resistance of tumor.We supposed that these three genes may play an important role in multidrug resistance of tumor by promoting tumor cell proliferation and inhibiting apoptosis.Conclusion:Laryngeal cancer stem cells were related to multidrug resistance,and may be regulated by FOS,JUN and NR4A1 genes,which need to be further verified.