| Background: How to deal with the lymphatic metastasis and distant metastasis of malignant tumor is always a worldwide problem,especially for the head and neck squamous cell carcinoma---HNSCC,which has high recurrence and metastasis.HNSCC belongs to epithelial derived tumor cells,before metastasis,epithelial mesenchymal transition----EMT,is necessary to make it free from the basement membrane and can travel between tissues and more aggressive.If we can understand the mechanism of EMT and block it,then the metastasis could be hold in the early stage.TIP30 is a cancer suppressor gene associated with inhibition of tumor metastasis.Previous studies have found that TIP30 can bind nuclear transfer factor----importin-β competed with Snail,inhibiting the occurrence of tumor cell EMT.At the same time,we also found that the expression change of TIP30 in laryngeal cancer cells not only affects the transportation to the nuclear of Snail,but also changes its stability.Overexpression of TIP30 can significantly increase the degradation of Snail.So,we believe that TIP30 must also have other mechanisms to regulate the EMT process of tumor cells.Therefore,after the intervention of TIP30 expression in laryngeal cancer cells,the downstream signaling pathway of TIP30 was screened by high-throughput genome chip and biological analysis.Then we found that the low expression TIP30 could abnormally activate wnt/β-catenin signaling pathway.In HCC cells,TIP30 was also found to increase the transcription of DKK-1,a wnt receptor antagonist protein.However,the wnt/β-catenin pathway is closely related to tumor EMT,suggesting that TIP30 can regulate the activity of wnt/β-catenin pathway.Therefore,it is speculated that TIP30 may regulate the EMT process of head and neck squamous cell carcinoma by inhibiting the wnt/β-catenin pathway.To certificate the hypothesis,we first studied the relationship between TIP30 and several EMT related molecules and the metastasis of hypopharyngeal cancer through clinical case study.Subsequently,the relationship between the expression of TIP30 in the HNSCC and E-cadherin of EMT was studied in cells,tissues and animal models.Finally find,TIP30 regulate HNSCC EMT by effcet DKK1,one of Wnt/β-catenin pathway antagonist.Our study will provide potential candidates for prognosis prediction and a new way for clinical treatment in HNSCC.Part 1Objective: To find if TIP30,E-cadherin、β-catenin、ser552-p-β-catenin(as for ser552),ser675-p-β-catenin(as for ser675),p53 and p21 can predict the risk of recurrence to prove the clinical outcomes.Methods: A retrospective review of our Hospital’s patients’ database between January 2002 and september 2012 was conducted and a total of 111 patients were enrolled in the study.Patients’ clinical and oncological information was collected and survival rates were analyzed using Kaplan-Meier curves.Prognostic factors were evaluated with multivariate Cox model survival analysis.The immunohistochemistry statuses of the moleculars were tested in 111 HNSCC cases.Results : Among the 111 patients 95.5 % were males and 4.5 % were females,with an average age of 59 years.patients with stage T2 to T4 are 18.9%,63.1%and 18%,respectively.With stage N0 to N3 are 20.7%,23.4%,53.2% and 2.7%,respectively.The 5-year overall survival(OS),disease free survival(DFS)were 40.37 % and 40.19 %respectively.Multivanate Cox regression model showed significant association between 5-year overall survival rate and TIP30 TU and SER552 LN.Conclusion: TIP30 is associated with metastases suppression in hypopharygneal.Abnormally activated wnt/β-catenin signal ways may do something in hypopharygneal cancer metastates.Part 2Objective: To verify if TIP30 plays a role in HNSCC EMT.Methods: We analysised TIP30 and E-cadherin’s expression in 4 of HNSCC cell by qPCR,immunoblotting and immunofluorescence.Then their invasion function were tested.Then the proliferation ability of them were compared by CCK8 method,the migration ability was compared by scratch method,and the invasiveness of several cells was compared with the chamber migration method(transwell).Subsequently,synthesis lentivirus vectors,expression TIP30 vector(lv-tip30 and its control lv-non)and expression siRNA vector(sh-tip30 and its control sh-non),respectively.The expression vector was used to transfection the FADU and Tu212 cells,and then the overexpression of TIP30 cell line were formed screening by puromycin.HN31 and HN22 cell lines were transfected with siRNA vector,and the stable strain were obtained after screening by puromycin.The biological characteristics of them were tested again.Results: TIP30 was expressed in all 4 cell lines.The highest one in mRNA level was HN22,followed by HN31,and FADU was the lowest.The protein expression level is in accordance with the level of mRNA.The TIP30 expression of HN22 and HN31 were disturbed by lentivirus infection,and the biological activity of FADU and TU212 was changed after TIP30 was overexpressed.After interference,the expression of E-cadherin decreased and the cell proliferation,migration,invasion were enhanced.After overexpression of TIP30 the expression of E-cadherin increased,the invasiveness of FADU and TU212 decreased significantly.Conclusion: 4 HNSCC cell lines all express TIP30,and the expression of E-cadherin is similar to that of them.Intervention the expression of TIP30 by lentivirus will downregulate E-cadherin’s expression and enhance migration,invasion and proliferation,suggesting the start of EMT.Part 3Objective: To compare the oncology and metastasis of tumor cells overexpressing TIP30 and the tumor cells that interfered with TIP30 expression.Methods:(one): Lung metastasis model: 4 group of BABLc male nude mice were selected.HN31 shTIP30 and control,FADU overexpress TIP30(LVTIP30)and control were injected into the tail vein.The lung metastase was observed by living imaging every week.After 4 weeks,the mice were killed for histopathological examination.(Two): Systemic metastase models: 2 groups of BABLc male nude mice,each have two subgroup,had been injected HN31 shTIP30 and control,FADU LVTIP30 and control respectively into submental.The time of the tumorigenesis,the volume of the tumor,the general condition and the weight of mice were observed.The growth and infiltration of local tumor,and the liver and spleen and kidney metastasis examined by B-ultrasound in mice after 8 weeks.Then,killed,for histopathological examination,taking lymph node,liver,spleen and lung tissue for HE staining and immunohistochemical staining.Results:(1)Lung metastasis model: HN31 shTIP30 and HN31 shNon group: From the day 7,the lung fluorescence intensity of nude mice in shTIP30 group began to be stronger than that in shNon.The difference reached the maximum until twenty-eighth days,and the difference was significant.The FADU cells overexpressed by TIP30 did not successfully form a tumor.(2)Systemic metastase models: By the end of 8 weeks,the mice still alived in group HN31 shTIP30 is fewer than that of HN31 shNon.B ultrasound showed liver metastasis,3 cases of shTIP30 group(3/4),and HN31 shNon group 1(1/6).Dissecting nude mice,local lymph node metastasis: HN31 shTIP30 group was 4/4,HN31 shNon group 1/6.Liver,spleen,lung and other pathology,frozen section immunofluorescence staining,and paraffin section immune-histochemical staining and HE staining.HE staining indicated local lymph node metastasis: HN31 shTIP30 group was 4/4,HN31 shNon group 1/6.Liver metastases: submental group(3/4),HN31 shNon(1/6).Lung: the HN31 shTIP30 group is(2/4),and HN31 shNon is(1/6).In spleen,group HN31 shTIP30 was(2/4).HN31 shNon is(0/6).The lung tissue immunohistochemical staining result shows,after downregulated the expression of TIP30,the expression of E-cadherin decreased.Conclusion: In these two models of tumor formation,the lymph node metastasis and distant metastase rate was higher in HN31 shTIP30 than that of the control group.After interfering the expression of TIP30,it will promote the migration and metastase of the tumor.Part 4Objective:To explore the relationship between TIP30 and DKK1,the antagonist of wnt/beta-catenin pathway.Methods: The basal DKK1 in supernatants of four HNSCC were detected by ELISA.After expressing or interfering with TIP30,the cells supernatants DKK1 were detected again.After transfected with DKK1 siRNA,FADU LVTIP30 had been observed that the localization of β-catenin was changed by immunofluorescence.The cell before and after transfection was tested by Westen-blotting,for E-cadherin.Results: The expression of DKK1 was consistent with that of TIP30 in four HNSCC cells.The expression of TIP30 was changed,and the DKK1 in the cell supernatants were changed together.After FADU LVTIP30 with siRNA interferes with DKK1,β-catenin is shown to be transported into nuclear,and the E-cadherin was downregulated.Conclusion: TIP30 influences the process of EMT of HNSCC by affecting DKK1.The abnormal activation of wnt/β-catenin signal way will start EMT.TIP30 can block the wnt/β-catenin signal way by affecting DKK1,one antagonist of wnt,then suppress the mestastases of hypopharygneal.Hope this mechanism of tumor suppressed gene may be useful for clinical practice in the near future. |
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