Study On The Association Of SALL4 Expression With The Biological Behaviors And Its Role In The Radiosensitivity Of Nasopharyngeal Carcinoma

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The association of SALL4 expression with the clinic-pathological characteristics of nasopharyngeal carcinoma patients Objective: To detect the expression level of SALL4 in tissues of nasopharyngeal carcinoma(NPC)patients,and investigate its association with the clinic-pathological characteristics of NPC patients.Methods: Tissue samples from NPC patients and nasal polyps patients were collected at Tongji Hospital of Tongji Medical College,Huazhong University of Science and Technology between Jan 2015 and Aug 2018.The diagnosis of NPC for each patient was confirmed by two independent histopathologists.Then all NPC patients were classified according to the eighth edition of the American Joint Committee on Cancer(AJCC)Cancer Staging Manual.The tissue specimens embedded in paraffin were sliced into 5?m-thick sections and all slides were stain for SALL4.Then SALL4 expression was scored according to the staining scope and intensity.T-test and Chi-square test were used to analyze the relationship between SALL4 expression and clinic-pathological features.Results: Tissue samples from 10 nasal polyp patients were collected.Detectable tissue samples from 131 NPC patients who had complete clinical information were collected.None of the NPC patients received any medical interventions prior to biopsy.The age range was 16 to 74 years old,and about 79% of patients was under the age of 60.The sex ratio was about 2:1;66% of patients was male and 34% was female.Cancerous tissues displayed a relatively high level of SALL4 compared to non-cancerous tissues(5.80±0.284 vs.0±0,p<0.001).Patients in T3-4 classification showed higher score of SALL4 expression,compare with those in T1-2 classification(6.47±0.347 vs.4.53±0.441,p=0.001).Moreover,patients in stage III-IV had higher score of SALL4 expression than that in stage I-II(6.10±0.310 vs.4.05±0.575,p=0.011).SALL4 expression was not associated with patient age,gender,N classification,M classification,clinical stage or histologic subtype,but correlated to T classification(p=0.030).NPC patients in T3-4 classification showed a higher proportion of high expression of SALL4(66.3 vs.46.7%,p=0.030).Conclusion: SALL4 expression of NPC tissues was greatly higher than that of non-cancerous tissues.High expression level of SALL4 was remarkably associated with the advanced stage cancers,which might be a poor prognosis bio-marker of NPC patients.Part ? The association of SALL4 expression with the epithelial–mesenchymal transition(EMT)and the cancer stem cell-like(CSC)of nasopharyngeal carcinoma cellsObjective: To explore the association of SALL4 expression with the epithelial–mesenchymal transition(EMT)and the cancer stem cell-like(CSC)of nasopharyngeal carcinoma(NPC)cells.Methods: The acquired radioresistant CNE2 cell line(CNE2R)was generated from a poorly-differentiated CNE2 cell line.We used lentivirus-mediated systems to generate SALL4 silencing CNE2 and CNE2 R cell lines and SALL4 stably expressing CNE2 cell line.An inverted microscope was used to observe and capture the changes of cell morphology.Then,we applied wound healing assay and transwell assay to detect the migration and invasion capabilities of NPC cells in vitro,respectively.Stem cell sphere formation assay and cell counting kit-8(CCK-8)assay were recruited to detect the sphere formation ability and cell proliferation ability.Finally,the expressions of SALL4,E-cadherin,N-cadherin,Vimentin,SOX2,OCT4,Nanog,ALDH and Bmi1 were assessed by Western Blot assay.Results: The acquired radioresistant CNE2 R was successfully generated from CNE2 cell line.Gain of mesenchymal phenotypes(pseudopodia formation and broadening in the intercellular space)were found in CNE2-SALL4 cells.Relatively less mesenchymal phenotypes were observed in CNE2-sh SALL4 and CNE2R-sh SALL4 cells compared with the control groups.Cell migration and invasion capabilities were much higher in CNE2-SALL4 cells compared with that in CNE2-CTL cells,and these capabilities were significantly inhibited by silencing SALL4 in CNE2-sh SALL4 and CNE2R-sh SALL4 cells.Moreover,CNE2-SALL4 formed significant colonospheres,compared with the control cells.And knockdown of SALL4 impeded the sphere-formation capabilities in CNE2-sh SALL4 and CNE2R-sh SALL4 cells.The number of spheres was rose in CNE2-SALL4 cells while reduced in CNE2-sh SALL4 and CNE2R-sh SALL4 cells.Cell proliferation dramatically increased in CNE2-SALL4 cells,but reduced in both CNE2-sh SALL4 and CNE2R-sh SALL4 cells.Overexpression of SALL4 leaded to reduced expression of E-cadherin,but increased expression of N-cadherin and Vimentin,and CSC bio-markers(SOX2,OCT4,Nanog,ALDH and Bmi1).While,the opposite results of Western Blot were observed in the SALL4 groups.Conclusion: SALL4 expression is associated with the induction of epithelial–mesenchymal transitionand the maintainess of cancer stem cell-like of nasopharyngeal carcinoma cells.Part ? SALL4 induces radioresistance in nasopharyngeal carcinoma via the ATM/Chk2/p53 pathwayObjective: This study aimed to explore the association of SALL4 expression with radioresistance of nasopharyngeal carcinoma(NPC),and the underlying molecular mechanisms.Methods: Colony formation assay was used to detect the radioresistance.To search the mechanisms involved in SALL4-induced radioresistance,cell immunofluorescence assay was used to detect the expression of phosphorylated histone H2AX(?-H2AX).Flow cytometry analyses were applied to explore the apoptosis rate and the cell cycle.The expression levels of related proteins were assessed by Western Blot.The xenograft model was used to confirm the observation of SALL4 overexpression inducing radioresistance and SALL4 silencing reversing radioresistance of NPC cells in vivo.Results: SALL4 overexpression induced radioresistance and decreased radiation-induced DNA damage,apoptosis and G2/M arrest in CNE2 cells.Overexpression of SALL4 increased expression of p-ATM,p-Chk2,p-p53,and anti-apoptosis protein Bcl-2,while the expression of proapoptosis proteins were declined.On the other hand,inhibition of SALL4 sensitized cells to radiation both in vitro and in vivo.Furthermore,SALL4 silencing increased radiation-induced DNA damage,apoptosis and G2/M arrest in CNE2 and CNE2 R cells.Moreover,knockdown of SALL4 impaired expression of p-ATM,p-Chk2,p-p53,and anti-apoptosis protein Bcl-2,while proapoptosis proteins were up-regulated.The CNE2R-sh SALL4 tumors were much smaller,and CNE2-SALL4 tumors were larger.The tumor inhibition rates(TWI%)of irradiation was relatively higher in CNE2R-sh SALL4 cells than that in CNE2 R cells,besides,the TWI% of irradiation was significantly lower in CNE2-SALL4 cells than that in CNE2 cells.Conclusion: SALL4 could induce radioresistance via ATM/Chk2/p53 pathway and its downstream proteins related to apoptosis.Targeting SALL4 might be a promising approach for the development of novel radiosensitizing therapeutic agents for radioresistant NPC patients.