| Object ives:STAT3is an important member of signal transducor and activator of transcription factor family (Signal transducor and activator of transcription, STATs). It is activated by growth factors and cytokines and may regulate cell transformation, inhibit apoptosis and cell proliferation and differentiation, and related with tumor development. Recent studies indicate that abnormal activation of STAT3is related with a variety of malignant tumors, such as head and neck squamous cell carcinoma, breast cancer, prostate cancer, malignant lymphoma, colorectal and gastric cancer. Our study aimed to observe the expression of phosphorylated signal transducer and activator of transcription factor in non-small cell lung cancer (NSCLC) and to investigate the correlation between expression and clinical and pathological features, such as gender, age, tumor size, and so on as well as the relation of expression and prognosis. Moreover, to explore the role of STAT3in cell growth, proliferation in lung caner cells.Methods:1. One hundred and twenty seven paraffin-embedded non-small cell lung cancer tissues (100adenocarcinoma and27squamous cell carcinoma) and56normal lung samples were obtained. Immunohistochemistry was performed to detect the expression of p-STAT3in tumor tissue and normal lung tissues. Comparative analysis of clinical data was performed to observe the relationship between p-STAT3expression and EGFR mutation status, age, gender, clinical stage and other clinical and pathological features. And further survival analysis was performed to observe relationship between expression and prognosis.2. Construct lentiviral vectors of STAT3shRNA. pLKO-STAT3-shRNA lentivirus was produced and infected lung cancer cell A549. After puromycin selection, stable knockdown cell clones were identified and the expression of STAT3protein was detected by western-blotting analysis. Cell proliferation rate was investigated by MTT assay.Results:1. The expression of p-STAT3was mainly localized in the nuclear of tumor cells in lung cancer cells and high expression in tumor tissue, the positive rate was64.6%(82/127). While in the normal tissues, the positive rate was37.5%(21/56). The difference was statistically significant (P=0.001). The expression level was related with EGFR mutation status, gender, smoking history, clinical stage and lymph node metastasis (P<0.05), while not with age, tumor size, pathological type (P>0.05). Survival analysis showed that the overall survival time was significantly shortened in the p-STAT3high expressed group (P=0.048).2. Sequencing of STAT3shRNA lentiviral vectors confirmed the correct insertion of STAT3RNAi sequence into pLKO lentivirus vector.3. Cell infection and Puromycin selection. Poromycin selection results showed that cells in the blank control died while the cells in infected cells had apparent cell colonies with a large number of cells.4. Identification of STAT3expression. The western-blot analysis results showed that STAT3expression was significantly decreased in pLKO-STAT3-shRNA group while maintained unchanged in blank control group.5. Detection of cell proliferation by MTT assay. PLKO-STAT3-shRNA lentivirus infected A549cells grew much more slowly when compared with blank control group (P<0.05).6. Plate colony forming assay. The results showed that colony-forming rate in shRNA-STAT3group (15.18±2.33)%was less than that in blank control group (26.34±2.17)%(P<0.05).Conclusion:Phosphorylated signal transduction transcriptional activation of factor3(p-STAT3) was highly expressed in non-small cell lung cancer. The expression of p-STAT3was correlated with clinical stage, lymph node metastasis and prognosis. The expression level was related with EGFR mutation status indicated that it may be used to predict EGFR-TKI efficiency. Silencing STAT3expression inhibited cell growth and proliferation, and which may provide a new target for targeted therapy. |
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