The Mechanisms Of Signal Transducer And Activator Of Transcription 3 And Its Target Gene On Biological Behaviour Of Non-Small-Cell Lung Cancer

The development and progression of non-small-cell lung cancer (NSCLC) is a multifactor and multistage process involving quite some genes abnormality. Many genes and proteins were involved in the carcinogenesis and progression of lung.However, the precise mechanisms that underlie the development and progression of NSCLC are far from clear. Up to date, no biomarkers of NSCLC have been proposed.Recent studies have revealed that several genes and molecules are involved in the origin and progression of lung cancer. Signal transducer and activator of transcription 3 (STAT3) is one of the transcription factors reported to play an important role in tumor cell growth, proliferation, apoptosis, and has been classified as an oncogene.STAT3signaling pathway is activated after receiving extra cellular signals such as epidermal growth factor, cytokines. Activated STAT3 (phosphorylated-STAT3)enters cellular nucleus and binds to specific DNA promoter to regulate target genes transcription which control fundamental physiologic processes such as cell proliferation, aptosis and angiogenesis. In normal condition, activation of STAT3 signaling pathway is temporary and is controlled accurately. But with various kinds of tumor genesis signal stimulation, STAT3 presents in the cell nucleus permanently and regulates target genes transcription continuously being involved in malignant transformation of tumor cells, called constitutively activation of STAT3 signaling pathway. STAT3 signaling pathway could convert various kinds of oncogenic tyrosine kinases signals and promote tumor cell generation, invasion and metastasis by inducing transcriptional activation of downstream target genes like VEGF, VEGF-C and MUC1.Constitutive activation of STAT3 has been documented in some kinds of tumors.Resent studies show blocking activation of STAT3signaling pathway could inhibit cell growth and induce apoptosis in many different kinds of tumors. Therefore only targeting one protein-STAT3 is enough to oppose many different oncogenic or anti-oncogene in the upstream or downstream of STAT3 in tumor cells. These findings place STAT3 in a pivotal position in oncogenic tyrosine-kinase signaling, providing a compelling rationale for its suitability as molecular target for cancer therapy.So far, there are seldom reports about whether STAT3 signaling pathway is constitutively activated in NSCLC and whether activated STAT3 signaling pathway panicipates in malignant transformation and progression of STAT3. To investigate the molecular mechanism of STAT3 signaling pathway in NSCLC, the correlation between STAT3 signaling pathway and its target gene in patients with NSCLC, and the prognostic significance of STAT3 signaling pathway and its target gene in patients with NSCLC, STAT3 and its target gene VEGF, VEGF-C and MUC1were detected immunohistochemistry (IHC).We also evaluate the levels of STAT3 and p-STAT3 expression in NSCLC specimens and paracancerous normal tissues by western Blotting. STAT3 gene was silenced using STAT3siRNA to block the activation of STAT3 signaling pathway in NSCLC. And then the influence of blocking STAT3 signaling pathway on cell proliferation and apoptosis discussed This finding will provide theory bases for the opinion that STAT3 signaling pathway could be considered potential therapeutic target for NSCLC.Part I:Clinicopathological Significance of STAT3 and p-STAT3 Expression in Patients with Non-Small-Cell Lung CancerObjective:This study was to investigate the clinicopathological significance of STAT3 and p-STAT3 expression in patients with non-small-cell lung cancer (NSCLC).Methods:The subjects were 72 patients with NSCLC who underwent resection from September 2011 to December 2011.72 NSCLC specimens and 20 paracancerous normal tissue specimens were obtained from these patients. All the specimens were examined by western blot and immunohistochemistry assays to detect STAT3 expression and p-STAT3 expression. According to the clinicopathologic factors, the enumeration data was calculated me positive rate, the comparison of positive rates uses the Chi-square; measurement data was represented with averaged±standard deviation and t-rest or F-test coupled with SNK test (q-test)were used. Differences were considered significant when the P value was less than 0.05.The statistical data were obtained using an SPSS software package (SPSS 13.0 Inc., Chicago, IL, USA).Results:The positive expressions of STAT3 protein were located both in cytoplasm and in cell nucleus. And the positive expressions of p-STAT3 protein were located in cell nucleus. The expression of STAT3 protein and p-STAT3 protein in the cancerous tissue group was significantly higher than that in the paracancerous normal tissues group respectively (P<0.01, P<0.05). STAT3 protein expression was correlated with pTNM (P<0.01). p-STAT3 protein expression was correlated with pathological type (P<0.05), pN (P<0.01) and pTNM (P<0.01).Conclusions:STAT3 expression was correlated with pTNM. p- STAT3 expression was correlated with pathological type, pN and pTNM.Overexpression of STAT3 and p-STAT3 might plays an important role in carcinogenesis, invasion and metastasis of NSCLC. measurement data was represented with averaged±standard deviation and t-rest or F-test coupled with SNK test (q-test)were used. Differences were considered significant when the P value was less than 0.05.The statistical data were obtained using an SPSS software package (SPSS 13.0 Inc., Chicago, IL, USA).Results:The positive expressions of STAT3 protein were located both in cytoplasm and in cell nucleus. And the positive expressions of p-STAT3 protein were located in cell nucleus. The expression of STAT3 protein and p-STAT3 protein in the cancerous tissue group was significantly higher than that in the paracancerous normal tissues group respectively (P<0.01, P<0.05). STAT3 protein expression was correlated with pTNM (P<0.01). p-STAT3 protein expression was correlated with pathological type (P<0.05), pN (P<0.01) and pTNM (P<0.01).Conclusions:STAT3 expression was correlated with pTNM. p- STAT3 expression was correlated with pathological type, pN and pTNM.Overexpression of STAT3 and p-STAT3 might plays an important role in carcinogenesis, invasion and metastasis of NSCLC.Part Ⅱ:Correlation between STAT3 Signaling Pathway and Its Target Gene VEGF, VEGF-C and MUC1 in Patients with Non-Small-Cell Lung CancerObjective:The purpose of the present study was to investigate the correlation between STAT3 signaling pathway and its target gene VEGF, VEGF-C and MUC1 in patients with non-small-cell lung cancer (NSCLC).Methods:A total of 72 NSCLC patients were enrolled in this study at the department of Thoracic Surgery, Provincial Hospital Affiliated to Shandong University from September 2011 to December 2011. All patients underwent complete tumor resection (lobectomy or pneumonectomy) with regional lymph node dissection.72 NSCLC specimens and 20 paracancerous normal tissues were obtained from these patients. Expression levels of STAT3, p-STAT3, VEGF, VEGF-C and MUC1 in NSCLC specimens and paracancerous normal tissues were studied using immunohistochemistry. The correlation between STAT3, p-STAT3, VEGF, VEGF-C and MUC1 expression and clinicopathological factors was analyzed using Fisher’s exact probability test or x2 test. Spearman’s rank correlation analysis was used to determine the relationships among the above variables. Differences were considered significant when the P value was less than 0.05.The statistical data were obtained using an SPSS software package (SPSS 13.0 Inc., Chicago, IL, USA).Results:The positive expressions of VEGF protein were located in cytoplasm. The positive expressions of VEGF-C protein were located in cytoplasm. And the positive expressions of MUC1 protein were located in cytoplasm. The expression of VEGF, VEGF-C, and MUClin the cancerous tissue group was significantly higher than that in the paracancerous normal tissues group respectively (P<0.05, P<0.05, P<0.05).VEGF protein expression was correlated with pT (P<0.05), pN (P<0.01) and pTNM (P<0.05). VEGF-C protein expression was correlated with pN (P<0.01). And MUC1 protein expression was correlated with pT (P<0.01) and pN (P<0.01). There was a positive correlation between STAT3 protein expression and p-STAT3 protein expression in the cancerous tissue group (r=0.307, P<0.01). There was a positive correlation between STAT3 protein expression and VEGF protein expression in the cancerous tissue group (r=0.309, P<0.01).And there was a positive correlation between p-STAT3 protein expression and VEGF protein expression(r=0.381, P<0.01), and between p-STAT3 protein expression and MUC1 protein expression in the cancerous tissue group (r=0.495, P<0.01).Conclusions:VEGF protein expression was correlated with pT, pN and pTNM. VEGF-C protein expression was correlated with pN. And MUC1 protein expression was correlated with pT and pN.There was a positive correlation between STAT3 expression and p-STAT3, between STAT3 expression and VEGF expression in the cancerous tissue group. And there was a positive correlation between p-STAT3 expression and VEGF expression, between p-STAT3 expression and MUC1 expression in the cancerous tissue group.The mechanism of STAT3 signaling pathway contributing to carcinogenenisis might be due to promoting transcriptional activation of VEGF and MUC1 in NSCLC.Part Ⅲ Prognostic Significance of STAT3, p-STAT3,VEGF, VEGF-C and MUC1 Expression in Patients with Non-Small-Cell Lung CancerObjective:The purpose of the present study was to investigate the prognostic significance of STAT3, p-STAT3, VEGF, VEGF-C and MUC1 in patients with non-small-cell lung cancer (NSCLC).Methods:A total of 72 NSCLC patients were enrolled in this study at the department of Thoracic Surgery, Provincial Hospital Affiliated to Shandong University from September 2011 to December 2011. All patients underwent complete tumor resection (lobectomy or pneumonectomy) with regional lymph node dissection.72 NSCLC specimens and 20 paracancerous normal tissues were obtained from these patients. Expression levels of STAT3, p-STAT3, VEGF, VEGF-C and MUC1 in NSCLC specimens and paracancerous normal tissues were studied using immunohistochemistry. Follow-up was complete for all the 72 patients. Univariate analysis was performed by modeling Kaplan-Meier survival curves. The log-rank test was used to calculate the survival rate. Multivariate analysis was carried out by the use of the Cox proportional hazard model. Differences were considered significant when the P value was less than 0.05.The statistical data were obtained using an SPSS software package (SPSS 13.0 Inc., Chicago, IL, USA).Results:The Kaplan-Meier method indicated that the 3-year survival rates of the 72 patients were 54.2%. In univariate analysis by the log-rank test, the 3-year survival rate in patients after operation was significantly associated with pT (P<0.01), pN (P<0.01), pTNM stage (P<0.01). Moreover, the 3-year survival rate of the patients with STAT3 protein expression in NSCLC tissues was significantly lower than that of the patients without STAT3 protein expression (45.9%:100%, P<0.01). And the 3-year survival rate of the patients with p-STAT3 protein expression in NSCLC tissues was significantly lower than that of the patients without p-STAT3 protein expression (43.6%:66.7%, P<0.05).The 3-year survival rate of the patients without VEGF expression in NSCLC tissues was significantly lower than that of the patients without VEGF expression (44.4%: 70.4%, P<0.05). The 3-year survival rate of the patients with MUC1 expression in NSCLC tissues was significantly lower than that of the patients without protein expression (44.2:69.0%, P<0.05). The results of Cox regression multivariate analysis confirmed that pN was independent relevant factors. However, no statistically significant correlations with g VEGF-C expression was demonstrated for the 3-year survival rate.Conclusions:The 3-year survival rate in patients after operation was significantly associated with pT, pN, pTNM stage, STAT3 expression,p-STAT3 expression, VEGF expression, and MUC1 expression. However, pN was the independent relevant factor. NSCLC tissues was significantly lower than that of the patients without protein expression (44.2:69.0%, P<0.05). The results of Cox regression multivariate analysis confirmed that pN was independent relevant factors. However, no statistically significant correlations with g VEGF-C expression was demonstrated for the 3-year survival rate.Conclusions:The 3-year survival rate in patients after operation was significantly associated with pT, pN, pTNM stage, STAT3 expression,p-STAT3 expression, VEGF expression, and MUC1 expression. However, pN was the independent relevant factor.Part Ⅳ Blocking Constitutive Activation of STAT3 Signaling Pathway in Lung Cancer A549 Lines by STAT3siRNAObjective:The purpose of the present study was to investigate the influence of blocking STAT3 signaling pathway on cell proliferation and apoptosis by blotting STAT3 gene silenced using STAT3siRNA in lung cancer A549 lines.Method:A549 cells were transfected with STAT3siRNA by transfection reagents as transfection group; and the cells were transfected without STAT3siRNA as control group(untransfection group). After transfected with or without STAT3siRNA at 0,24, 48 and 72h, cells were collected and total RNA was isolated to measure the amount of STAT3mRNA.There protein levels were determined in cytoplasmic extracts using Western Blotting. A549 was transfected with or without STAT3siRNA, and cell viability was detected by MTT. And the cells were stained with PI and analyzed cell cycle by flow cytometry. The cells were also stained with FITC-annexjn V and PI and analyzed cell apoptosis by flow cytometry using Cell Quest acquwasition and analysis programs. The measurement data was represented with average± standard deviation and t-rest were used. Differences were considered significant when the P value was less than 0.05.The statistical data were obtained using an SPSS software package (SPSS 13.0 Inc., Chicago, IL, USA).Results:After transfected STAT3siRNA 48h, A549 cells morphology were significantly transformed. Compared with control cells, transfected cells became less confluent as some cells rounded and detached from the culture plates. The transfected cells became shrinker and rounder than control cells, and have more grains. Dead suspension cells in the medium indicates the death of cells. Transfected with siRNA caused special degradation of the STAT3 mRNA in A549cells at 0、24、48、72h. The result showed the expressions of STAT3mRNA almost was disappeared after 48h of transfection while STAT3 and p-STAT3 protein levels also gradually decreased after transfected with STAT3siRNA in different time. The A value had significant difference between cells transfected and un transfected with STAT3siRNA at 48 and 72h. After transfection with STAT3siRNA 72h, the apoptosis in A549 was higher than the apoptosis in control cells. The percentage of cells in the G0/G1 phase of A549 increased 17.8% compared with control cells. At the same time there was a decrease in the percentage of cell in the S phase.Conclusions:STAT3siRNA can block STAT3 signaling pathway on cell proliferation and apoptosis in lung cancer A549 Lines.