| Laryngeal squamous cell carcinoma is one of the most common head and neck neoplasms.Due to the highly invasive and metastatic nature of intermediate and advanced laryngeal carcinoma,the recurrence and mortality rate is still high,and there is no obvious improvement of general prognosis over the past three decades.PDCD4(Programmed cell death 4),a newly discovered tumor suppressor gene,was reported to display low or no expression in a variety of solid malignant tumors.Epithelial mesenchymal Transition(EMT)renders epithelial cells the mesenchymal cell phenotype and makes the tumor cells more invasive and metastatic.At present,few studies about PDCD4 controlling EMT were carried out in laryngeal carcinoma.In our study,the relation between the expression feature of PDCD4 and the expression of EMT associated protein in laryngeal carcinoma tissues and cells has been studied using gene silencing and over expression in order to define the function of PDCD4 on laryngeal carcinoma.The further study on the mechanism underlying how PDCD4 controls EMT and promotes laryngeal carcinoma cell invasion and metastasis may provide a new way of thinking in the research on molecular mechanism of the tumorigenesis and development of laryngeal carcinoma.Part ?:Expression of PDCD4 and EMT related proteins in laryngeal squamous cell carcinomaObjective:To investigate the expression of PDCD4 and EMT-related proteins in laryngeal squamous cell carcinoma tissues,and to explore the relationship between PDCD4 and EMT-related proteins and clinical pathology features,and the correlation between PDCD4 and EMT-related protein expression.The expression of PDCD4 gene in different laryngeal carcinoma cell lines was detected.Methods:1.The expression of PDCD4,E-cadherin and N-cadherin in laryngeal carcinoma and adjacent normal laryngeal tissues were detected by immunohistochemical SP method and their relationship with clinicopathological features.The correlation between PDCD4 and EMT-related proteins E-cadherin and N-cadherin expression was analyzed by statistic method.2.The expression of PDCD4 in human laryngeal carcinoma cell line Hep-2 and SNU-899 was detected by fluorescence quantitative PCR,which laid a foundation for further study on the biological function and molecular mechanism of PDCD4 gene in laryngeal carcinoma.Results:1.Compared with adjacent normal tissues,the expression of PDCD4 and E-cadherin in laryngeal squamous cell carcinoma was significantly decreased and the expression of N-cadherin was significantly increased.The expression of PDCD4,E-cadherin and N-cadherin was not associated with age,sex and clinical type of laryngeal squamous cell carcinoma,which was related to pathological grade,cervical lymph node metastasis and clinical stage.2.There was a positive correlation between PDCD4 protein and E-cadherin protein expression in laryngeal squamous cell carcinoma,and there was a negative correlation between PDCD4 and N-cadherrin protein expression.3.The expression of PDCD4 m RNA was down-regulated in human laryngeal carcinoma cell line,and the expression of Hep-2 cell line was higher than that of human laryngeal carcinoma SNU-899 cell line.Conclusion:PDCD4 in laryngeal squamous cell carcinoma is closely related to the clinicopathological features of tumor,which can be used as a molecular marker to judge the clinical pathology of tumor.PDCD4 and EMT there is a certain correlation.The expression of PDCD4 m RNA in human laryngeal carcinoma Hep-2 cell line was higher than that in human laryngeal carcinoma SNU-899 cell line.Part?:Effects of PDCD4 gene silencing on biological behavior and EMT-related proteins in laryngeal carcinoma Hep-2 cells in vitroObjective: To investigate the effect of stable PDCD4 gene silencing on the cell biology of cycle,proliferation,apoptosis,migration and invasion of laryngeal carcinoma cell and its effect on epithelial mesenchymal transition.Methods: 1.Screening and construction of Hep-2 cell lines with stable silencing PDCD4 gene.Real-time PCR was used to detect the changes of PDCD4 m RNA after sh RNA-PDCD4 transfection of Hep-2 cells,and analyzing the sh RNA interference efficiency,the sh RNAs with the highest silencing efficiency were screened out finally.Western blot analysis verified that the protein expression of PDCD4 after sh RNA-PDCD4 transfected Hep-2 cells.2.The proliferation of Hep-2 cells was detected by MTT assay.The cell clone formation ability was detected by plate clone formation assay.The effect of PDCD4 gene silencing on Hep-2 cell cycle and apoptosis was detected by flow cytometry.Transwell assay was used to detect the effect of PDCD4 gene silencing on the migration and invasion of Hep-2 cells.3.Western blot was used to detect the expression of E-cadherin and N-cadherin in Hep-2 cells transfected with sh RNA-PDCD4.Results: 1.To screen and construct polyclonal Hep-2 cells(PDCD4-sh RNA8653 / Hep-2)with stable PDCD4 gene silencing,which was confirmed at gene and protein level.2.The results of MTT assay showed that the proliferation ability of laryngeal carcinoma cell line Hep G2 cells was significantly increased compared with the control group after PDCD4 gene silencing.The results of platelet cloning showed that the cell clone formation ability of laryngeal carcinoma Hep-2 was significantly enhanced compared with the control group after PDCD4 gene silencing.The results of flow cytometry showed that the proportion of Hep-2 cells in G2 phase was significantly higher than that in control group after PDCD4 gene silencing,and the proportion of apoptosis was significantly decreased.Transwell results showed that a significant increase in Hep-2 cell migration and invasion after PDCD4 gene silencing.3.Western blot showed that the expression of E-cadherin protein was significantly decreased and the expression of N-cadherin protein was significantly increased after PDCD4-sh RNA8653 transfection into Hep-2 cells.Conclusion: Hep-2 cell line with stable PDCD4 gene silencing was successfully constructed.Silencing the expression of PDCD4 gene in Hep-2 cells,which promotes the proliferation of laryngeal cancer cells,inhibits its apoptosis,promotes the migration and invasion of laryngeal cancer cells;inhibits the expression of epithelial markers,promotes the expression of mesenchymal markers,promotes Hep-2 cell epithelial mesenchymal transition.Part ?: Effects of PDCD4 overexpression on biological behavior and EMT-related proteins in laryngeal carcinoma SNU-899 cells in vitroObjective: To investigate the effect of PDCD4 overexpression on cell cycle,proliferation,apoptosis,migration and invasion of SNU-899 laryngeal carcinoma cells and the influence of markers on epithelial mesenchymal transition.Methods: 1.Construction of PDCD4 overexpressing lentiviral vectors GV358-PDCD4 and GV358-PDCD4-NC.LV-PDCD4,LV-NC lentivirus concentrated solution were prepared,Infected SNU-899 cells laryngeal carcinoma SNU-899 cells,and overexpression and negative control SNU-899 cell lines were established.Real-time PCR was used to detect the changes of PDCD4 m RNA in SNU-899 cells.The expression of PDCD4 protein was detected by Western blot after overexpression of SNU-899 cells.2.MTT assay was used to detect the proliferation capacity changes of PDCD4 overexpression in SNU-899 cells.Plate cloning assay was used to detect cell cloning ability.The effect of PDCD4 gene overexpression on the cell cycle and apoptosis of SNU-899 cells was detected by flow cytometry.Transwell assay was used to detect the effect of PDCD4 overexpression on the migration and invasion of SNU-899 cells.3.The expression of E-cadherin and N-cadherin in PDCD4 overexpressing SNU-899 cells was detected by Western blot.Results: 1.PDCD4 overexpression of lentiviral vector was successfully constructed,and the SNU-899 cell line with PDCD4 gene overexpression was successfully established,the result was tested at the gene and protein level.2.The results of MTT assay showed that the proliferation ability of laryngeal carcinoma cell line SNU-899 was significantly decreased after PDCD4 overexpression compared with the control group.The results of platelet cloning showed that the cell clone formation ability of laryngeal carcinoma cell line SNU-899 was significantly reduced after PDCD4 gene overexpression compared with the control group.Flow cytometry showed that the proportion of G2 phase cells in SNU-899 cells was decreased after PDCD4 overexpression,and the proportion of S phase and G1 phase cells was significantly increased.The percentage of cells in the SNU-899 cells was significantly higher than that in the control group.The results of Transwell showed that the migration and invasion of SNU-899 cells were significantly decreased after overexpression of PDCD4 gene.3.Western blot showed that the expression of E-cadherin protein was significantly increased and the expression of N-cadherin protein was significantly decreased after PDCD4 gene expression in SNU-899 cells was overexpressed.Conclusion: The laryngeal carcinoma SNU-899 cell line over expressing PDCD4 gene was constructed successfully.After Stabilized expression PDCD4 gene on SNU-899 cells,Cell proliferation was inhibited,apoptosis was promoted,the cell migration and invasion were inhibited,the expression of epithelial markers was increased,the expression of mesenchymal markers was decreased,and the epithelial transformation of SNU-899 cells was reversed.Part ? Tumorigenesis experiments of sh RNA-PDCD4 / Hep-2 cells in nude miceObjective: To observe the effect of PDCD4 gene silencing on the tumor growth in nude mice,and to verify the biological effect of Sh RNA silencing Hep-2 cells PDCD4 gene.Methods: 1.Establish laryngeal cancer model in nude mice:10 mice were randomly divided into two groups,empty vector NC group and PDCD4 gene silencing sh RNA-PDCD4 group.Hep-2 cells were cultured in vitro to prepare cell suspension.Sh RNA-NC / Hep-2 cells and sh RNA-PDCD4 / Hep-2 cell suspension were injected subcutaneously in the right axillary axis of nude mice.2.Tumor observation: observe the state of mice and transplanted tumor occurrence and growth.The time of tumor formation was recorded and the tumor was measured once every 3 days.The volume growth curve of nude mice was drawn.3.After feeding for 30 days,the animals were sacrificed.Removed the tumor specimens and took pictures,weighed cancer tissue block,cut 2/3 for frozen in the-80 ? refrigerator.The rest made into wax pieces.4.HE staining and immunohistochemical staining.Results: 1.Nude mice growth status : sh RNA-NC group and sh RNA-PDCD4 group were respectively formed Tumor on the 12 th day and 9th day after transplantation.Sh RNA-PDCD4 group grow rapider than sh RNA-NC group.Growth rate was significantly faster than the control group.The expression of PDCD4 gene can significantly promote the tumorigenic ability and tumor growth capacity of Hep-2 cells in nude mice transplanted tumor model.2.Two groups of nude mice were sacrificed and the subcutaneous tumor was completely cut and weighed.The weight of sh RNA-PDCD4 group was significantly higher than the empty vector sh RNA-NC group.3.HE staining showed that the tumor tissue had the characteristic of cancer cells.4.Immunohistochemical staining showed that the expression of PDCD4 and E-cadherin protein was significantly decreased,but the expression of N-cadherin protein was significantly increased in sh RNA-PDCD4 group compared with the control group.Conclusion: 1.Successfully established laryngeal carcinoma Hep-2 cells transplanted tumor model in nude mice.2.In vivo experiments confirmed that after silencing PDCD4 gene,the growth capacity of laryngeal carcinoma Hep-2 cells was significantly improved and leading tumor cell phenotype epithelial transformation in vivo.Part ?: The mechanism Study of PDCD4 regulating the transformation of epithelium in laryngeal carcinomaObjective: To investigate the mechanism of PDCD4 regulating the transformation of epithelial mesenchymal in laryngeal carcinoma Hep-2 cells.Methods: 1.The expression of p-STAT3 and ?-catenin in Hep-2 cells after PDCD4 gene silencing was detected by Western blot.2.The expression of mi R-21 was detected by Taqman real-time RT-PCR.Results: The expression of ?-catenin protein,p-STAT3 protein,mi R-21 in Hep-2 cell PDCD4 gene was significantly increased after PDCD4 gene silencing.Conclusion: In the laryngeal carcinoma Hep-2 cells,Silencing of PDCD4 gene expression can lead to promote cell epithelial mesenchymal transformation,part of the mechanisms achieved by STAT3 / mi R-21 pathway and activating ?-catenin signaling pathway. |
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