GLUT-1-targeted Inhibition Of Laryngeal Cancer Stem Cells Enhances The Radiosensitivity Of Laryngeal Carcinoma In Vitro And In Vivo

Background:Laryngeal cancer is one of the common malignancies in the head and neck with a five-year survival rate of 72.3%.At present,the treatment of laryngeal cancer includes surgical treatment,chemoradiotherapy and comprehensive treatment.However,the therapeutic effect of laryngeal cancer is not satisfactory,and the survival rate has not improved.On the other hand,the retention of laryngeal function is the current trend in the treatment of laryngeal cancer.Radiotherapy has obvious advantages for the retention of laryngeal function.However,the existence of radiotherapy resistance limits the application of radiotherapy in the treatment of laryngeal cancer.At present,there is a lack of effective ways to increase the radiosensitivity of laryngeal cancer.We have previously found that antisense oligonucleotides(AS-ODN)and small interfering RNA(siRNA)inhibit glucose transporter-1(GLUT-1)can increase the radiosensitivity of laryngeal cancer,and at the same time found GLUT-1 is highly expressed in laryngeal cancer stem cells.Therefore,laryngeal cancer stem cells may play an important role in the radiotherapy resistance of laryngeal cancer.Objectives:This dissertation aims to use AS-ODN,siRNA to targeted inhibit GLUT-1 of laryngeal cancer stem cell,and to study the radiation resistance mechanism of laryngeal cancer stem cell in vitro and in vivo.1.Construct a radioresistant laryngeal cancer cell line,Hep-2R,and sort CD133+Hep-2R,a laryngeal cancer stem cell expressing CD 133.2.Verify the radioresistance in laryngeal cancer is associated with increased expression of GLUT-1 in laryngeal cancer stem cells,and clarify the mechanism of inhibiting GLUT-1 expression in laryngeal cancer stem cells can increase the radiosensitivity by in vitro studies.3.Verify the radioresistance in laryngeal cancer is associated with increased expression of GLUT-1 in laryngeal cancer stem cells,and clarify the mechanism of inhibiting GLUT-1 expression in laryngeal cancer stem cells can increase the radiosensitivity by in vivo studies.Methods:Firstly,radioresistance cell line Hep-2R was established by X-irradiation on laryngeal cancer cell line Hep-2.Radioresistant laryngeal cancer stem cell CD133+Hep-2R was selected by immunomagnetic sorting,and the ratio of CD133+cells was detected by flow cytometry;CD133+Hep-2R was then divided into control group CD133+Hep-2R-siRNA-NC(0 Gy;2 Gy;4 Gy;8 Gy;12 Gy)and experimental group CD133+Hep-2R-siRNA-GLUT-1(0 Gy;2 Gy;4 Gy;8 Gy;12 Gy).After transfected with GLUT-1-siRNA and dealed with X-irradiation,RT-PCR was used to detect GLUT-1 mRNA transcription level,Western blot was used to detect GLUT-1 protein expression,CCK-8 was used to detect cell proliferation and flow cytometry was used to detect the apoptosis rate,Transwell assay was used to detect cell invasion ability;Laryngeal cancer cells Hep-2,Hep-2R,CD133+-Hep-2,and CD133+-Hep-2R was injected subcutaneously into nude mice to establish a laryngeal tumor xenografts.They were randomly divided into 18 groups:Hep-2,Hep-2R,CD 13 3+-Hep-2,CD133+-Hep-2R,CD133+-Hep-2+X-ray,CD133+-Hep-2R+X-ray,CD133+-Hep-2+AS-ODN-NC,CD133+-Hep-2R +AS-ODN-NC,CD133+-Hep-2 +AS-ODN-GLUT-1,CD133+-Hep-2R +AS-ODN-GLUT-1,CD 13 3+-Hep-2+AS-ODN-GLUT-1+X-ray,CD133+-Hep-2R+AS-ODN-GLUT-1+X-ray,CD133+-Hep-2 + siRNA-NC,CD133+-Hep-2R + siRNA-NC,CD133+-Hep-2 + siRNA-GLUT-1,CD133+-Hep-2R +siRNA-GLUT-1,CD 13 3+-Hep-2+siRNA-GLUT-1+X-ray,CD 133+-Hep-2R+siRNA-GL UT-1+X-ray.Observe and record the volume and weight of the transplanted tumors,and the weight of the nude mice in each group,calculate the tumor inhibition rate.RT-PCR and Western blot were used to detect the GLUT-1 mRNA transcription and protein expression in all groups of transplanted tumor tissue.Flow cytometry was used to detect the apoptosis rate and cell cycle.Two-way ANOVA and t-test were used to statistical analysis by IBM SPSS 19.0 software,p<0.05 indicates a statistical difference.GraphPad Prism 5 software was used for plot analysis.Results:Part ?1.Hep-2R cells were successfully established.The Hep-2R and Hep-2 were adherent 24 hours later after plated.The Hep-2 cell line was more easily to form colonies than the Hep-2R cell line,and the Hep-2R cell line was slightly larger.The CCK-8 assay showed that the Hep-2 cell line entered the logarithmic growth phase on the first day after plated and reached the plateau phase on the fifth day;the Hep-2R cell line entered the logarithmic growth phase on the second day and reached the plateau phase on the 6th day after plated.The cell doubling times of Hep-2R and Hep-2 cell lines were 48.4 h and 40.7 h,respectively.The results of this experiment showed that the ?,? value of the Hep-2R cell line was significantly smaller than that of the Hep-2 cell line,and the SF2 value of the Hep-2R cell line was also increased.2.After the immunomagnetic sorting was used to separate Hep-2R cells,the CD 133 expression rate of Hep-2R cells increased from 8.42%to 90.82%.The separated CD133+Hep-2R cells are in good condition and meet the needs of follow-up experiments.Part ?1.RT-PCR and Western blot results showed that GLUT-1 mRNA transcription and protein expression were significantly decreased in CD133+Hep-2R cells after transfection with siRNA-GLUT-1.Low dose radiation such as 2Gy and 4Gy significantly increased the GLUT-1 mRNA transcription and protein expression in the control group,4Gy is more obvious;when the dose is increased to 8Gy,there is no obvious change in GLUT-1 mRNA transcription and protein expression;when the dose is further increased to 16Gy,GLUT-1 mRNA transcription and protein expression obvious reduction.In the experimental group,after X-irradiation,GLUT-1 mRNA transcription and protein expression were reduced,among which 4Gy had the smallest decrease and 16Gy had the largest decrease.2.CCK-8 results showed that the OD450 of CD133+Hep-2R cells in the experimental group after transfection with siRNA-GLUT-1 was lower than that of the control group;with the increase of X-ray dose,The decrease range of OD450 of CD133+Hep-2R cells in the control group was lower,of which 8 and 12 Gy decreased more significantly than at 2 and 4 Gy.The OD450 values of the cells in the experimental group decreased at 2,4,and 8 Gy were not significant,and the OD450 values at 12 Gy decreased significantly.3.Flow cytometry results showed that the survival rate of CD133+Hep-2R cells in the experimental group after transfection with siRNA-GLUT-1 was significantly lower than that in the control group,suggesting that the apoptosis of the cells after transfection with siRNA-GLUT-1 increased;with the dose of X-rays gradually increased,the survival rate of CD133+Hep-2R cells in the control group decreased less,with the most significant decrease at 12Gy;the survival rate of CD133+Hep-2R cells in the experimental group decreased with the X-ray dose increased,most obvious at 12Gy.4.Transwell assay to detect cell invasiveness showed that:With the increase of X-ray dose,the number of CD133+ Hep-2R cells in the control group was not significantly reduced,and the number of CD 133+ Hep-2R cells was increased when 8Gy;The number of CD133+ Hep-2R cells at 0 Gy was significantly lower than that of the control group,and decreased with the increase of the X-ray dose.Part ?1.Compared with Hep-2 group,the tumor volume significantly reduced in Hep-2R group,and significantly increased in CD133+-Hep-2 group;tumor volume in CD133+-Hep-2R group was significantly larger than Hep-2R group,significantly decreased than CD133+-Hep-2 group,and there was no significant difference with Hep-2 group.RT-PCR results showed that the GLUT-1 mRNA transcription level in the CD133+-Hep-2 group was significantly higher than Hep-2 group;the GLUT-1 mRNA transcription level in the CD133+-Hep-2R group was significantly higher than Hep-2R group.The results of flow cytometry showed that the apoptosis rate of tumor cells in Hep-2R group was significantly higher than that of Hep-2 group.The apoptosis rate of tumor cells in CD133+-Hep-2R group was significantly higher than that of CD133+-Hep-2 group.Compared with Hep-2 or Hep-2R group,the apoptosis rate of tumor cells in CD133+-Hep-2 or CD133+-Hep-2R groups was significantly reduced.2.After X-ray irradiation,the tumor volume was significantly reduced.Compared with the transfected antisense nucleotide control group,the tumor size of the transfected AS-ODN-GLUT-1 group was significantly smaller.Compared with the transfected siRNA control group,tumor size was significantly reduced in the transfected siRNA-GLUT-1 group.When X-rays were irradiated,the size of tumors in CD133+-Hep-2 group with GLUT-1 knocked down was significantly lower than that of CD133+-Hep-2R group with GLUT-1 knocked down.3.After X-ray irradiation,the tumor inhibition rate increased significantly.Compared with the transfected antisense nucleotide control group,the tumor inhibition rate of the transfected AS-ODN-GLUT-1 group was significantly increased.Compared with the transfected siRNA control group,the tumor inhibition rate of the transfected siRNA-GLUT-1 group was significantly increased.When X-irradiation was performed,the tumor suppression rate in CD133+-Hep-2 group with GLUT-1 knocked down was significantly higher than that of CD133+-Hep-2R group with GLUT-1 knocked down.4.After X-ray irradiation,the body weight of mice was significantly reduced.Compared with the transfected antisense nucleotide control group,there was no significant body weight change in the transfected AS-ODN-GLUT-1 group.There was no significant change in body weight in the transfected siRNA-GLUT-1 group compared to the transfected siRNA control group.When X-irradiated,the body weight of mice was reduced,and the body weight of mice in CD133+-Hep-2 group with GLUT-1 knocked was slightly higher than that of CD133+-Hep-2 group with GLUT-1 knocked.5.The results of RT-PCR showed that the expression of GLUT-1 mRNA in tumor tissue of Hep-2R group was significantly decreased after X-ray irradiation and transfection with AS-ODN-GLUT-1 and siRNA,compared with Hep-2 group.Compared with the Hep-2 or Hep-2R group,the expression of GLUT-1 mRNA in the tumor tissue of the CD133+-Hep-2 or CD133+-Hep-2R group was significantly increased.Compared with the X-ray-free group,the expression of GLUT-1 mRNA in the tumor tissue was significantly reduced after X-ray irradiation.Compared with transfected antisense nucleotide and siRNA control group,GLUT-1 mRNA expression in tumor tissue of AS-ODN-GLUT-1 group and siRNA group was significantly reduced;Western blot showed that after X-ray irradiation,transfection with AS-ODN-GLUT-1 and siRNA,the expression of GLUT-1 protein in tumor tissue of Hep-2R group was significantly lower than that of Hep-2 group.Compared with the Hep-2 or Hep-2R group,the expression of GLUT-1 protein in the tumor tissue of the CD133+-Hep-2 or CD133+-Hep-2R group was significantly increased.Compared with the X-ray-free group,the expression of GLUT-1 protein in the tumor tissue was significantly reduced after X-irradiation.Compared with transfected antisense nucleotide control group and siRNA group,the expression of GLUT-1 protein in tumor tissues transfected with AS-ODN-GLUT-1 group and siRNA group was significantly reduced.6.The results of flow cytometry detection of apoptosis showed that the apoptosis rate of tumor tissue increased significantly after X-ray irradiation compared with no X-ray irradiation group.Compared with the transfected antisense nucleotide and siRNA-GLUT-1 control group,the apoptotic rate of the tumor tissue in transfected with AS-ODN-GLUT-1 group and siRNA-GLUT-1 group was significantly increased.After irradiation with X-rays,the apoptosis rate of Hep-2R cells was significantly lower than that of Hep-2 cells.7.Flow cytometry analysis of cell cycle results showed that after X-irradiation,the proportion of tumor cell in G0/G1 phase was slightly increased,in S and G2/M phase was slightly decreased compared with the X-ray-free group,indicating a slight decrease in cell proliferation.Compared with the transfected antisense nucleotides and siRNA-GLUT-1 control group,the proportion of G0/G1 phase cells in the transfected AS-ODN-GLUT-1 group and siRNA-GLUT-1 group was slightly increased,the proportion of cells in S and G2/M phase decreased slightly,indicating a slight decrease in cell proliferation.When irradiated with X-rays,the Hep-2R cell group showed a slight decrease in the proportion of G0/G1 phase cells,slightly increased S and G2/M phase cells in the tumor tissue compared with the Hep-2 group,suggesting that cell proliferation was slightly increased.Conclusions:1.The radioresistance of laryngeal cancer stem cells after X-ray irradiation is further enhanced and plays an important role in the radioresistance of laryngeal cancer.2.Targeted inhibition of GLUT-1 in laryngeal cancer stem cells can increase the radiosensitivity of laryngeal cancer.