| Part Ⅰ TAZ expression and subcellular localization in normal cervical and squamous cell carcinoma cells and its significanceOBJECTIVE: To compare the expression and subcellular localization of TAZ in normal cervical tissues and cervical squamous cell carcinoma cells,and to analyze the relationship between TAZ protein expression and clinical and pathological features of cervical squamous cell carcinoma.METHODS:(1)Patients with cervical squamous cell carcinoma who underwent surgery in the Department of Gynecology,Affiliated Hospital of Southwestern Medical University from January 2014 to December 2016 were selected as Han subjects.Postoperative pathological examination was confirmed as cervical squamous.All patients did not receive radiotherapy,chemotherapy and other anti-tumor treatments before surgery,and the clinical and pathological data were collected,a total of 218 cases.In the same period,normal cervical specimens of uterine fibroids undergoing hysterectomy(screening for cervical cancer by routine methods,including Pap smear,visual inspection of acetic acid white eyes,high-risk HPV test without abnormality)were used as controls.Immunohistochemistry and immunofluorescence were used to determine the expression level and subcellular localization of TAZ protein in control and cervical squamous cell carcinoma tissues.The clinicopathological features of cervical squamous cell carcinoma were analyzed by including age,clinical stage,lymph node metastasis,as well relationship between cell differentiation and other levels of TAZ expression and subcellular localization.(2)Fresh normal cervical tissue and cervical squamous cell carcinoma were collected.The nucleus and cytoplasm were separated,and the expression levels of TAZ protein in the nucleus and cytoplasm of the two tissue cells were detected by Western blotting.RESULTS:(1)The expression level of TAZ in 212 specimens of cervical squamous cell carcinoma(6 of 218 specimens failed to be detached or stained)showed that 85.85%(182/212)of squamous cell carcinoma specimens TAZ Positive staining,positive expression was mainly in the cytoplasm;TAZ nuclear expression was observed only35.85%(76/212).There was no significant difference in the expression of TAZ by analyzing immunoreactive score(IRS)between different ages and different FIGO stages(P>0.05).The proportions of IRS>4 in different degrees of differentiation,ie,poor differentiated,moderatel differentiated and highly differentiated tissue cells were 10.61%,31.08% and 47.22,respectively.%;the proportion of TAZ expression IRS>4 in tissue cells with lymph node metastasis and no lymph node metastasis was 13.11% and37.09%,respectively,and the difference was statistically significant(P<0.05).Thirty-three cases of normal cervical tissues showed TAZ expression in cytoplasm and nucleus.The expression of TAZ was particularly prominent in normal basal cells of cervical tissues.(2)Western blot results showed that the expression level of TAZ in the cytoplasm and nucleus of normal cervical tissue cells was higher than that of cervical squamous cell carcinoma cells,especially in the nucleus;cervical squamous cells The expression level of TAZ in the cytoplasm of cancer cells was significantly higher than that of the nucleus,and the difference was significant(P<0.05).CONCLUSIONS: Collectively,there is a correlation between the degree of tumor differentiation and the presence or absence of lymph node metastasis.TAZ protein is dominantly expressed in both cytoplasm and nucleus of normal cervical tissue cells.However,the expression level of TAZ protein in cervical squamous cell carcinoma cells is low,especially in low or even loss of expression in the nucleus.The results indicate that TAZ expression level and subcellular localization may be related to the occurrence,development and prognosis of cervical squamous cell carcinoma.Part Ⅱ: Analysis of the relationship between the subcellular localization of TAZ and the integrin β1-Src signaling pathwayOBJECTIVE: To determine the expression level of TAZ protein and the relationship between subcellular localization and integrin β1-Src signaling pathway in normal cervical tissue cells and cervical squamous cell carcinoma cells.METHODS:(1)30 cases of normal cervical specimens with uterine hysterectomy,and 30 cases of cervical squamous cell carcinoma were selected with postsurgery,respectively.Expression of integrin β1,Src,and TAZ as well co-expression were performed by immunofluorescence staining in normal cervical tissue and cervical squamous cell carcinoma.Western blot was used to detect the expression of integrin β1 protein and Src protein in fresh normal cervical tissues and cervical squamous cell carcinoma tissues.(2)Human cervical immortalized squamous cells(Ect1/E6E7)and human cervical epithelial carcinoma cells(ME180)were cultured in vitro,and ME180 cells were treated with Src family kinase inhibitor Su6656.DMSO was used as control.The cytosolic and nuclear extracts were obtained,and TAZ expression were detected by Western blot.Furthermore,Ect1/E6E7 and ME 180 cells were transfected with control siRNA or TAZ siRNA,and the expression difference of integrin β1 and Src protein was evaluated by Western blot.RESULTS:(1)The expression levels of integrin β1 protein and Src protein in cervical squamous cell carcinoma were higher than those in normal cervical tissues.Compared with normal cervical tissue,TAZ protein was significantly reduced in cervical squamous cell carcinoma tissue cells.Co-expression of integrin β1 protein,and Src,and TAZ proteins was significantly reduced or absent in cervical squamous cell carcinoma tissue cells.(2)After treatment with Src family kinase inhibitor Su6656 in ME180 cells,the expression level of cytoplasmic TAZ protein decreased,while the expression level of TAZ in nuclear fraction increased.Conversely,TAZ was highly expressed in cytoplasm,and low level in nucleus from DMSO control group.Transfection with TAZ siRNA revealed that the expression of integrin β1 and Src was increased in Ect1/E6E7 and ME 180 cells compared with control siRNA,repecectively.CONCLUSIONS: There is a close relationship between TAZ,integrinβ1 and Src in cervical squamous cell carcinoma.The expression and subcellular localization of TAZ in cervical squamous cell carcinoma may be related to the activation of integrin β1-Src signaling pathway. |
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