| Objective:To explore the expression, biological function and molecular mechanism of notch1 in Xuanwei female lung cancer by clinical investigation and basic researchs in vitro and in vivo.Methods:1. Studies on the expression and clinical significance of notch1 in Xuanwei female lung cancer patients:Comparision of notchl expression in Xuanwei female lung cancer, non-Xuanwei female lung cancer and benign lung lesions was performed by immunohistochemical staining, after which,54 cases of Xuanwei female lung cancer tissue and the corresponding distal normal lung tissue samples were freezed in liquid nitrogen and fixed with formaldehyde separately. Immunohistochemical techniques were applied to detect their notchl protein expression, combing with clinical pathological features to perform the Kaplan-Meier univariate survival analysis and multivariate Cox survival analysis.2. Influence of notchl gene inhibition on biological characteristics of Xuanwei female lung cancer cell XWLC-05 and its mechanism research:We transfect siNotchl by liposomes into Xuanwei female lung cancer cell line XWLC-05 to inhibit the expression of Notchl gene. Then cells morphology, ability of proliferation, migration, caspase-3 expression, cell cycle and apoptosis rate were identified by transmission electron microscope, MTT assay, transwell assay and flow cytometry, respectively. At the same time Notchl NICD expression recombinant plasmid pIRES2-EGFP-Notchl was constructed to reverse the RNAi effects and above biological characteristics of XWLC-05 cells were analyzed. In-depth mechanism study of Notchl gene was performed by processing pIRES2-EGFP-Notchl transfected cells with and the apoptosis-correlation items were studied.3. To compare the the expression of Notchl between primary tumor and corresponding brain metastases of Xuanwei female lung adenocarcinoma cancer.Immunohistochemical staining was used to compare Notchl expression in Xuanwei female lung adenocarcinoma cancer primary tumor (20 cases) and corresponding brain metastases (20 cases).Results:1. Studies on the expression and clinical significance of notchl in Xuanwei female lung cancer patients:The expression of Notchl protein in female lung cancer tissues was significantly higher than the expression in benign lung lesions (P<0.05); There were 70.37% and 69.44% positive staining of Notchl in Xuanwei and non-Xuanwei female lung cancer tissues, respectively, which had no significant difference between positive expression rate (P>0.05).Immunohistochemical staining showed that Notchl protein mainly expressed in cytoplasm. There were 70.37%(38/54) positive expression cases in cancer tissues and only 25.9%(14/54) in matched normal tissues (P=0.000); There were significant difference in the expression of Notchl protein among patients with different histological types (P=0.000), whether or not had lymph node metastasis (P=0.007) and the degree of tumor differentiation (P=0.000).The survival analysis by Kaplan-Meier method showed that the median survival time of Notchl positive-expression and negative-expression patients was 27 months and 38 months, respectively (P=0.041).Patients with lymph node metastasis or high pathological grade had significant shorter median survival time than those without lymph node metastasis (P=0.026) or low pathological grade (P=0.000);Cox regression survival analysis showed that pathological grade was a significant independent factor affecting prognosis.2. Influence of notch1 gene inhibition on biological characteristics of Xuanwei female lung cancer cell XWLC-05 and its mechanism research:SiNotchl can effectively inhibit the expression of Notchl gene in XWLC-05 cells and induce cell apoptosis (P<0.01). Recombinant plasmid pIRES2-EGFP-Notchl was successfully constructed. The Notchl protein of XWLC-05 cells were significantly down-regulated (P<0.01) and the caspase-3 protein were significantly up-regulated (P<0.01) 48 h and 72h after inhibiting the expression of Notchl gene by recombinant plasmid siNotchl when compared with non-transfected cells. The apoptosis morphological changes were observed by microscope and the cell proliferation, migration and metastasis ability were significantly reduced after inhibiting the Notchl gene expression (P<0.01). FCM showed higher apoptosis rate in siNotchl transfected cells (P<0.01). The function changes caused by Notchl targeted inhibition were reversed after transfecting of pIRES2-EGFP-Notchl plasmid and the cells apoptosis correspondly decreased.3. To compare the the expression of Notchl between primary tumor andcorresponding brain metastases of Xuanwei female lung adenocarcinoma cancer. There were 65% positive staining of Notch1 in Xuanwei female lung adenocarcinoma cancer primary tumor, respectively, There were 85% positive staining of Notchl in corresponding brain metastases of Xuanwei female lung cancer, Brain metastases showed significantly higher immunohistochemical expression of Notch1 than primary tumors did (P<0.05)Conclusions:1. There was abnormally up-regulated expression of Notchl gene at transcription in Xuanwei female lung cancer tissues.2. Notchl expression level combining pathological grade can be used as an assessment and prognosis-estimation reference for Xuanwei female lung cancer patients.3. Notchl may promote the progression of Xuanwei female lung cancer by enhancing cell proliferation ability and inhibiting the caspase-3 dependent apoptosis pathway.4. Notchl gene was closely related to the biological behavior in Xuanwei female lung cancer brain metastasis tissues. |
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