| BankgroundLaryngeal squamous cell carcinoma(LSCC)is one of the highest incidence of head and neck malignant tumors,also is the second high incidence of respiratory tract cancer.LSCC is generally staged according to the tumor size(T),vervical lymph node metastasis(N)and distal metastasis(M).Carcinoma in situ of the patients with lymphatic metastasis directly affect the treatment and postoperative survival,the rates of neck lymph node metastasis of LSCC are about:T1 of 6-25%,30-70%T2,T3 and T465%to 80%.Generally,the factors of poor prognosis of LSCC include high T classification,neck lymph node metastasis and the distal metastasis,etc.However,there is still a lack of relevant literature reports for the influence factors of clinical prognosis of laryngeal squamous cell carcinoma.Therefore,the first part of rearsch is aimed to study the effect of different factors on the prognosis of LSCC.Glucose is the main sources of energy for proliferation,differentiation,invasion and metastasis of tumor cell.The uptake of glucose mainly depends on glucosetransporter(Glut),and the Glut-1 is the main carrier of cells to absorb glucose.Compared with normal cells,tumor cells themselves prefer to using the glycolytic pathway in the anaerobic environment to produce energy,leading to the higher expression of Glut-1 in the malignant tumor cells compare to normal cells.Therefore,as an important target of malignant tumor gene therapy,Glut-1 is becoming a hot spot in recent years in the field of cancer research.There is a close relation in Glut-1 and the growth and migration of tumor cells.Numerous studies have reported that inhibition of the expression of Glut-1 could effectively inhibit proliferation and migration,inducing cell cycle arrest and apoptosis of malignant tumor cells.However,the influence of Glut-1 on growth and migration in laryngeal cancer cells and its molecular mechanism rarely been reported.Therefore,the second part of study was aim to investigate the influence of knockdown the expression of Gult-1 on the proliferation,apoptosis and migration of laryngeal Hep-2 cells and its molecular mechanisms.Autophagy plays an important role in tumor cell growth and migration,and is a process that eukaryotic cells degrade damaged proteins or organelles and recycling,which is import to tumor cell growth and migration and plays a dual role in promoting cell survival and inducing cell death.The certain degree of autophagy can promote tumor growth and invasion,but continuous autophagy can activate tumor cell self degradation and lead to cell death.Beclinl is a protein which is involved in autophagy,and is one of the most important markers of autophagy.Research proved that regulating tumor cell protein and gene expression of Glut-1 could regulate cell growth and migration throught affecting cell autophagy pathways.However,the relevant literatures which are about the effect of Glut-1 on the autophagy in laryngeal cancer cells and its mechanism is still lacked.Therefore,the role of research in laryngeal cancer autophagy Gult-1 and its recovery effect on cell growth and migration become one of the main purpose of this study.The microenvironment of tumor growth plays an important role in the occurrence,development,treatment,resistance and recurrence of tumor,especially the hypoxic microenvironment is closely related to tumor treatment,resistance and recurrence,the lack of oxygen induced neovascularization and increased glucose uptake and metabolism in solid tumors is an important cause of tumor resistance to radiotherapy and chemotherapy.In the absence of hypoxia,the expression of HIF-1 alpha increased dramatically,activating large amounts of mRNA in Glut-1 and mass production of its products,Overexpression of Glut-1 can transport more glucose to meet the needs of malignant cells,high metabolic rate and fast growth,blocking some one of the important links plays an important role in the elimination of tumor resistance to treatment and recurrence.However,there is few study on the relationship between Glut-1 and chemotherapy resistance in laryngeal cancer.Therefore,the fourth part of this study is to understand the relationship between Glut-1 expression and tumor resistance in laryngeal carcinoma,and Glut-1 expression was blocked by RNA interference in human laryngeal carcinoma Hep-2 cells,to clarify the changes of Glut-1 expression in laryngeal carcinoma cells and the effect of energy supply to improve the possible mechanism of chemotherapy sensitivity of laryngeal cancer cells.Methods1.The first time of laryngeal squamous cell carcinoma of the 71 patients in january 2013 to december 2013 in our hospital were selected for the research.The gender,age,T stage,N stage,tumor location,tumor stage,pathological differentiation and treatments were analyesed for the study of the affectors of prognosis of patients.2.The inbition of Glut-1 expression was developed by short hairpin RNA(shRNA)of laryngeal cancer Hep-2 cells.The effects of cell proliferation,apoptosis and migration were observated,and its mechanisms were studied.3.The promotion of cell autophagy was implementated by rapamycin or overexpression of Beclinl,and the influences of proliferation,apoptosis,and migration of Hep-2 cells were studied.The inhibition of cell autophagy was implementated by konckdown the Beclin1,and cells proliferation,apoptosis,and migration of Hep-2 cells were also studied for the observation of the recovery effect of growth and migration in the laryngeal cancer cells that was knock down the Glut-1 and explore the role of autophagy.4.Screening effective Glut-1 interference fragments siRNA for Hep-2 cells and the effective cisplatin concentration on the function of Hep-2 cells;The effect of Glut-1 interference on cisplatin sensitivity of Hep-2 cells was investigated by RNA interference,and the possible mechanism of Glut-1 expression changes on chemosensitivity of Hep-2 cells was investigated.Results1.Survival analysis showed that age on the survival of patients with laryngeal squamous cell carcinoma(P>0.05)were ignored.The supraglottic type on the prognosis of laryngeal squamous cell carcinoma of the tumor is poorer than the glottis type.Patiants with higher tumor T stage showed lower survival rate,suggesting the greater of the tumor indicating the worse prognosis(P<0.01).The survival rate of patients with no lymph node metastasis(NO)was significantly higher than that with lymph node metastasis(N1,N2),suggesting the lymph node metastasis of laryngeal squamous cell carcinoma indicating the poor prognosis(P<0.01).Patients with higher tumor staging showed the higher mortality rate,the survival rate of patients with stage IV cancer was only 46.7%,suggesting the higher tumor staging indicating the poor prognosis(P=0.000).Laryngeal squamous cell carcinoma of the degree of differentiation and treatment had no significant effect on prognosis of patients.COX proportional hazards model regression analysis showed that age and treatment were only two variables influence on prognosis of patients with significant statistical significance(P<0.05).2.Knockdown of Glut-1 significant inhibited the expression of cell cycle related proteins cyclinD1(27%)and cell proliferation related protein c-myc(29%),thus inhibiting proliferation activity of Hep-2(There were about 87.4%,73.2%and 55.8%in 24h,48h and 72h respectively).Knockdown of Glut-1 could promote the apoptosis in Hep-2 cells(The apoptosis rate of shRNA Glut-1,Control and NC group were about 25.9%,3.2%and 25.9%,respectively),through inhibiting apoptosis protein Bcl-2(about 23.2%)and promotion of the cleaved Caspase3(2.45 times).Knockdown of Glut-1 inhibited migration(about 57.6%)in laryngeal cancer cells by upregulation of the expression of E-cadherin(2.1 times)and downregulation of the expression of N-cadherin that were involved in cell migration.3.Knockdown of Glut-1 resulted in the promotion of cell autophagy.The revulsant of autophagy rapamycin and over expression of Beclin1 could significantly induce cell autophagy occurs.The induction of autophagy could further promote the autophagy levels in cells with Glut-1 knockdown.On the basis of knockdown of Glut-1,the induction of autophagy might further reduce cell proliferation rates,which were related to the decrease of the expression of cyclin-D1 and c-myc.Cell apoptosis were further promoted in cells of induction of autophagy based on the knockdown of Glut-1,through the upregulation the expression of cleaved-caspase3 and downregulated the expression of Bcl-2.However,cell migrations were inhibitied in cells of induction of autophagy based on the knockdown of the Glut-1,by the upregulation of the expression of E-cadherin and downregulation of the expression of N-cadherin.Conversely,Knockdown of Beclin1 could significantly inhibit cell autophagy and reverse the increase of cell autophagy levels induced by Glut-1 knockdown.Additionally,knockdown of Beclin1 could reverse the decrease of the proliferation rate in cells which was suffered Glut-1 knockdown,and could inhibit the apoptosis in cells which was Glut-1 knockdown by the upregulation of the expression Bcl-2.Lastly,inhibition of autophagy could reverse the cell migrations which was inhihited by Glut-1 knockdown.4.In the 4 groups of RNA interference suppression Hep-2 cell Gult-1 expression group,siRNA726 group was the best inhibitory effect,and simultaneously inhibited the MRP1 gene;The effect of 3 different cisplatin concentrations on the function of laryngeal carcinoma Hep-2 cells is that the concentration of 3ug/mL is the best way to inhibit proliferation and promote apoptosis in Hep-2 cells;Inhibition of Glut-1 expression by RNA interference in Hep-2 cells can inhibit the expression of MRP1 gene,Cisplatin can upregulate the the expression of MRP 1 gene after Glut-1 expression inhibited by interference of RNA in Hep2 cells,and the former is more important than the latter.Conclusion1.Age,N stage,tumor location,tumor staging,treatment and other factors may have effects on the prognosis of patients with laryngeal squamous cell carcinoma.2.Gult-1 knockdown could suppress the proliferation of Hep-2 cells,promote Hep-2 cells apoptosis,inhibit the migration of laryngeal cancer cells.3.The shRNAGlut-1 and inducing autophagy can affect laryngeal cancer Hep-2 cell growth and migration,and shRNA Glut-1 could regulated by influencing the autophagy which related to cell growth and migration.4.RNA interference can inhibit the expression of MRP1 after Glut-1 inhibition,inhibition of the Glut-1 gene may reduce the resistance to chemotherapy by inhibiting the MRP1 gene. |
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