| Gastric cancer(GC)is the most common gastrointestinal tumor.There are about 951,000 new cases and 723,000 deaths every year in the world.In China,the incidence of GC is very high due to special dietary habits and Helicobacter pylori infection in the population.The number of new GC cases in China accounts for about half of the new cases in the world,and most of patients have been in the middle and late stages.Despite the existence of surgery and chemotherapy,the treatment of GC often fails due to tumor metastasis and recurrence.Molecular targeted therapy has become an alternative therapeutic approach or used as a combinational therapy with standard chemotherapy agents for GC.It is of great clinical significance to find sensitive early screening markers and effective molecular therapeutic targets for the early diagnosis and treatment of GC.SOX4 is a member of Sox(Sry-related high-mobility group box)family and contains a highly conserved DNA binding high mobility group(HMG)box.SOX4 plays an important role in embryonic development,neurological development,and sex determination.Recent advance suggests an important role for SOX4 in progression of malignant tumors such as breast cancer and colon cancer.It has been reported that SOX4 is upregulated in GC and is associated with histological grade,metastasis and prognosis,however the mechanism is not clear.Taking into account that epithelial-mesenchymal transition(EMT)and stem cell-like characteristics(Stemness)play crucial roles in the metastasis and recurrence of tumors,we further explored the role of Sox4 in the progression of GC from the perspective of EMT and stemness.Part ? The expression and significance of Sox4 in GCObjectives:To detect the expression pattern of Sox4 in GC and the relationship between Sox4 and the prognosis of GC patients.To investigate the correlation between Sox4 and EMT and stemness markers.Methods:The expression of Sox4,EMT and stemness markers in 84 GC tissues and adjacent non-cancer tissues were detected by quantitative real-time PCR(qRT-PCR)and immunohistochemistry.The relationship between the expression of Sox4 and the survival time of GC patients was analyzed by Kaplan-Meier curve.The correlation of Sox4 between EMT and stemness markers were analyzed using linear correlation model according to the ?Ct value from qRT-PCR.Results:The positive staining rate of Sox4 in GC tissues(65/84 vs 19/84)and staining score(4.48 vs 2.25)were significantly higher than that in paracancerous tissues.The Kaplan-Meier survival curve confirmed that overexpression of Sox4 indicated poor prognoses in GC patients.Compared with GC patients with low Sox4 expression,the positive rate of E-cadherin was lower in GC patients with high Sox4 expression(20/65 vs 14/19),while positive rate of Vimentin(46/65 vs 6/19)and EpCAM were higher(40/65 vs 6/19).The results of qRT-PCR and correlation analysis showed that the expression of Sox4 was negatively correlated with E-cadherin(r=-0.384),and was positively correlated with Vimentin and EpCAM(r=0.674,0.433).Conclusion:Sox4 is upregulated in GC tissues and indicates a poor prognosis in GC patients.Part II Sox4 promotes EMT and stemness acquisition in GC cellsObjectives:To investigate whether Sox4 promotes the EMT and sternness acquisition of GC cells and explore the underlying mechanisms.Methods:The Sox4 overexpression vector and silencing vector was transfected into MKN28 and BGC823 cells,respectively.Transwell assays were used to detect the migration ability.qRT-PCR,Western blot and immunofluorescence were used to detect the expression of EMT molecules.Sphere formation assays were used to measure the self-renewal of GC cells,and flow cytometry was used to measure the proportion of CD44+/EpCAM+ GC cells.After Sox4 was overexpressed or silenced,the expression of EMT transcription factors Twistl,Zebl,Snaill,Snail2 and sternness transcription factors Sox2,Oct4 and Nanog were detected by qRT-PCR and Western blot.The expression level and localization of?-catenin were detected by Western blot and immunofluorescence.Finally,the nude mice xenograft model and lung metastasis model were constructed to detect the effect of Sox4 on proliferation and metastasis of GC cells in vivo.Results:Compared to MKN28-NC cells,E-cadherin of MKN28-SOX cells was downregulated while the N-cadherin and Vimentin were increased.Compared to BGC823-NC cells,E-cadherin of BGC823-sh cells was upregulated while Vimentin was downregulated.Transwell assays showed that overexpression of Sox4 enhanced the migration ability of MKN28 cells,while silencing Sox4 decreased the migration of BGC823 cells.After overexpression of Sox4,the self-renewal of MKN28 cells was enhanced and the proportion of CD44+/EpCAM+ GC cells was decreased.However,silence of Sox4 resulted in the opposite results in BGC823 cells.Further studies showed that overexpression of SOX4 upregulated the expression of EMT transcription factors Twist1 and zebl and sternness transcription factors Sox2 and Oct4,and promoted the nuclear translocation of P-catenin in MKN28 cells.The silence of Sox4 downregulated the expression of EMT transcription factors Twistl and Snail1 and stemness transcription factors Sox2 and Oct4,and inhibited the nuclear translocation of P-catenin in BGC823 cells.Finally,we showed that SOX4 promoted the lung metastasis and tumor formation ability of GC cells in nude mice.Conclusion:Sox4 significantly promoted EMT and stemness of GC cells through the upregulation of upstream transcription factors and Wnt pathway activity.Part III SOX4 mediates TGF-?1-induced EMT and sternness of GC cellsObjectives:To investigate the role of Sox4 in EMT process and stemnes,s acquisition induced by TGF-? pathway in GC cells.Methods:After MKN28-NC cells were treated with TGF-?1,the expression of SOX4 was detected by qPCR and Western blot.After BGC823-NC,BGC823-sh cells were were treated with TGF-?1,the migration ability was detected by Transwell assays,the expression of EMT molecules was detected by qRT-PCR and Western blot.The self-renewal was measured by sphere formation assays,and the proportion of CD44+/EpCAM+ GC cells was measured by flow cytometry.Results:TGF-?1 successfully activated TGF-? signal pathway in MKN28 cells and upregulated the expression of Sox4 in GC cells.Silencing Sox4 partially reversed the TGF-?1-induced EMT and sternness,reversed TGF-?1-induced downregulation of E-cadherin and upregulation of Vimentin.Conclusion:SOX4 mediates TGF-?1-induced EMT and sternness in GC cells... |
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