A Study Of The Relationshi Pbetween Expression Features Of P53-induced Protein Phosphatase 1(Wip1) And Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer related mortality in the world. He Jie academician reported the top 10 tumor morbidity and mortalityin of China in 2012, and lung cancer ranked the first.and approximately 80% of cases are non-small cell lung cancer(NSCLC). Despite recent advances in the treatment of NSCLC, significant work remains to reduce morbidity and improve survival for NSCLC patients,the 5-year survival rate of lung cancer is about 15%. Therefore, the research on mechanism of the occurrence and development of NSCLC and screening effective therapeutic targets and treatment, has been a huge challenge in cancer research.Wild type p53 induced phosphatase 1(Wip)1 is located in the chromosome 17 q23 / q24 area, belongs to the the family of serine/threonine protein phosphatase PP2C(PP2C type protein phosphatase), codes by PPM1 D, Wip1 protein molecular weight was about 61 kd, composed of 605 amino acids,As a negative regulator of p53, Wild-type p53-induced phosphatase 1(Wip1) can promote cancer progression in a p53 dependent or independent manner. In recent years, Wip1 amplification was founded in lung cancer, breast cancer, pancreatic cancer, bladder cancer, liver cancer, meninges tumor and neuroblastoma cell line. In addition, many human primary tumor(e.g., neuroblastoma, ovarian clear cell carcinoma, breast cancer, pancreatic adenocarcinoma) contains Wip1 gene amplification and express overexpression of Wip1 protein, and is associated with poor prognosis. Naoyuki Satoh, reported that Wip1 overexpression was founded in patients with primary lung adenocarcinoma and significantly associated with tumor progression and clinical prognosis. This study intends to explored the clinical pathological significance, prognostic value and its mechanism of action in NSCLC and offer further theoretical and experimental basis for targeted therapy of NSCLC. Part I The clinical pathological significance and prognostic value of Wip1 in NSCLCObjective: To explore pathological significance and prognostic value of Wip1 in NSCLC.Methods: The expression of Wip1 was measured by immunohistochemistry in paraffin section of NSCLC. The associations between Wip1 expression and categorical variables were analyzed by Pearson’s χ2 test; excluding age, which was analyzed by Student’s t-test. The survival curve evaluated by the Kaplan-Meier method and the statistical significance between survival curves assessed using the log-rank test. OS was determined from the date of surgery to the date of mortality. Significant variables from the univariate analysis analyzed using the Cox proportional hazard model. P<0.05 indicated a statistically significant difference and all tests were two-sided.Results:1 The positive expression of Wip1 was observed in cell cytoplasm and nucleus with pale yellow, brown-yellow or brownness. The positive rate of Wip1(69.2%) was higher than control group(χ2=19.114, P=0.000).2 There was no statistically significant difference between the Wip1 positive expression group and Wip1 protein negative expression in the age of non-small cell lung cancer tissue(P = 0.268).In the group of Wip1 protein positive expression in non-small cell lung cancer tissue, men accounted for 70.4%(57/81), women accounted for 29.6%(24/81), in the group of Wip1 protein negative expression, men accounted for 83.3%(30/36), women accounted for 16.7%(6/36), there was no statistically significant difference by chi-square test(χ2=2.197,P=0.138).The rate of Wip1 positive expression in lung adenocarcinoma tissues, squamous cell carcinoma and other types of non-small cell lung cancer accounted for lung adenocarcinoma tissues,lung squamous cell carcinoma and other types of non-small cell lung cancer were 88.7%(47/53), 56.8%(25/44), and 45%(9/20), The rate of Wip1 expression in lung adenocarcinoma was higher than squamous cell carcinoma and other types of non-small cell lung cancer using chi-square test(χ2=18.106,P<0.001).In the group of Wip1 protein positive expression in non-small cell lung cancer tissue, the high degree of pathological differentiation group accounted for 29.6%(24/81), while the group of low degree of differentiation was 70.4%(57/81), in the group of Wip1 protein negative expression, the high degree of pathological differentiation group accounted for 19.4%(7/36), low differentiation group was 80.6%(29/36), Wip1 protein expression in non-small cell lung cancer tissues had no correlation with the degree of differentiation using chi-square test, there was no statistically significant difference(χ2=1.328,P=0.249).In the group of Wip1 protein positive expression in non-small cell lung cancer tissue, the rate of tumor size ≤3 cm cases accounted for 14.8%(12/81), tumor size > 3 cm cases accounted for 85.2%(69/81), in the group of Wip1 protein negative expression, the rate of tumor size ≤3 cm cases accounted for 38.9%(14/36), tumor volume > 3 cm cases was 61.1%(22/36), Wip1 protein expression in non-small cell lung cancer tissues was associated with tumor size using chi-square test, the difference was statistically significant(χ2=8.357,P=0.004).In the group of Wip1 protein positive expression in Non-small cell lung cancer tissue, the rate of no lymph node metastasis cases accounted for 55.6%(45/81), lymph node metastasis was 44.4%(36/81), in the group of Wip1 protein negative expression, the rate of no lymph node metastas cases accounted for 69.4%(25/36), lymph node metastasis was 30.6%(11/36), using chi-square test to the statistics, Wip1 protein expression in non-small cell lung cancer tissues had no correlation with lymph node metastasis using chi-square test,, there was no statistically significant difference(χ2=2.000,P=0.157).In the group of Wip1 protein positive expression in non-small cell lung cancer tissue, the rate of clinical stage I- II cases was 55.6%(45/81), III- IV accounted for 44.4%(36/81), in the group of Wip1 protein negative expression,therate of clinical stage I- II cases was 75.0%(27/36), III- IV accounted for 25.0%(9/36), Wip1 protein expression in non-small cell lung cancer tissues was associated with clinical stage using chi-square test, the difference was statistically significant(χ2=3.98,P=0.046).3 Single-factor Log-rank testing analysis shows that Wip1 expression is related to poor prognosis of patients, and that the overall survival of the Wip1 positive expression grou Pis lower than that of the Wip1 negative expression group. Further Cox regression model analysis shows that the relative risk of the high Wip1 expression is 5.096(95% of the confidence interval=1.501-17.303), that of the clinical staging is 0.159(95% of the confidence interval =0.037-0.680).Summary:1 The expression of Wip1 increased in NSCLC tissues.2 The positive expression of Wip1 related to the pathologic type, tumor size and clinical stages.3 The overall survival in positive expression of Wip1 group was lower than that of Wip1 negative expression group. Over-expression of Wip1 was a risk factor of poor prognosis of NSCLC. Part II The Inhibitory Effect of Wip1-si RNA on Cell Proliferation in NSCLC CellsObjectives: To explore the inhibitory effect and mechanisms of Wip1-si RNA on cell proliferation in A549(p53+) and NCI-H1299(p53-) cells.Methods: The gene silencing of Wip1 was used by RNA interference technology and screened the effective si RNA by real-time PCR. Cell proliferation and cell cycle were analyzed by MTT method and flow cytometry. The expression of proliferating cell nuclear antigen(PCNA), p21 and p27 were measured by Western blot.Result:1 Treatment with three Wip1-si RNA(40 n M) for 48 hours, the m RNA and protein level of Wip1 were all reduced, among which Wip1-si RNA-3 had the highest silencing effect of above 90%, while the blank control group and negative transfection group have no significant change.2 Compared with the negative transfection group, gene silencing of Wip1 had a significant inhibitory effect on proliferation in A549 and NCI-H1299 cells, while there is no significant difference in cell survival between the blank control group and negative transfection group.3 Transfection with Wip1-si RNA inhibited the cell cycles of A549 and NCI-H1299 cells, with the S-phase decreased and G0/G1-phase increased.4 The expression of PCNA was inhibited by transfection with Wip1-si RNA, accompanied by an up-regulation of p21 and p27 in A549 and NCI-H1299 cells.Summary:1 The m RNA and protein level were decreased specifically and effectively by Wip1-si RNA in A549 and NCI-H1299 cells.2 The cell proliferation of NSCLC cells were inhibited by Wip1-si RNA.3 The cell cycle of NSCLC cells were suppressed by Wip1-si RNA.4 The expression of PCNA was reduced by Wip10 si RNA, accompanied by an up-regulation of p21 and p27 in A549 and NCI-H1299 cells. Part III The Inhibitory Effect of Wip1-si RNA on Cell Migration and Invasion in NSCLC CellsObjectives: To explore the inhibitory effect and mechanisms of Wip1-si RNA on cell migration and invasion in A549(p53+) and NCI-H1299(p53-) cells.Methods:Cell migration and invasion were measured by cell wound scratch assays and Transwell assays. The expression of matrix metalloprotease(MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase(TIMP)-2 were analyzed by Western blot.Results:1 After transfecting with Wip1- si RNA for 48 hours, compared with non-Target-si RNA and the control group, the transmembrane migration activity were significantly inhibited in A549 and NCI-H1299 cells.2 Compared to non-Target-si RNA group, the invasion ability was significantly decreased by treatment with Wip1-si RNA for 48 h.3 Transfection with Wip1-si RNA inhibited the expression of MMP-2 and MMP-9, with an increasing expression of TIMP2 compared with control group and non-target-si RNA group.Summary:1 The cell migration was reduced by treatment with Wip1-si RNA.2 The cell invasion was inhibited by treatment with Wip1-si RNA.3 Transfection with Wip1-si RNA shown an up-regulation of TIMP2 and an down-regulation of MMP-2 and MMp-9.Conclusion:1 The overexpression of Wip1 is related with pathologic type, tumor size and clinical stages, which show a potential regulator and predictor in NSCLC.2 Gene silencing of Wip1 can inhibit cell proliferation by down-regulation of PCNA and promoting the expression of p21 and p27 in NSCLC cells.3 The migration and invasion of NSCLC cells are inhibited by Wip1-si RNA via regulation of MMP-2, MMP-9 and TIMP2.