Twist On The Effects And Mechanism Of The Radiosensitivity Of A549 Cells

Part One:Expression and clinical significance of Twist and VEGF protein in non-small cell lung cancerObjective:To investigate the expression and clinical significance of Twist and VEGF in non-small cell lung cancer(NSCLC), explore the correlation of their expressions and the influence of them on the prognosis.Methods:The expression was detected in 61 cases of NSCLC, adjacent normal lung tissue, and 13 cases of benign lung diseases by immunohistochemical staining(SP).Results:The positive expression rates of Twist and VEGF in NSCLC were 73.8%and 77.0%, respectively, which were remarkably higher than those in adjacent normal lung tissue and benign lung diseases(P< 0.05). The expression of Twist and VEGF was closely associated with lymph node metastasis(P<0.05), but not correlated with age, sex, pathology, degree of differentiation and staging (P>0.05). There was a significant correlation between the expression of Twist and VEGF in NSCLC(r=0.738,P<0.01). The 1-year disease free survival (DSF) of Twist positive and negative expression was 73.3%and 87.5%(P>0.05), and that in VEGF positive and negative expression was 74.5%and 85.7%(P>0.05).Conclusion:Patients with high expression of Twist and VEGF were prone to have lymph node metastasis in NSCLC. Twist and VEGF may collaboratively regulate invasion and metastasis process in NSCLC. Though the 1-year DSF of Twist and VEGF positive patients was lower, it had no statistical significance. This may be concerned with the small number of cases(P>0.05). Part Two:Effects of Twist-shRNA on biological behaviors and radiosensitization of A549 lung cancer cells and molecular mechanismsObjective:To investigate the effects of Twist-shRNA on biological behaviors and radiosensitization of A549 lung cancer cells and molecular mechanisms.Methods:Viral particles that express short hairpinRNA(shRNA) targeting Twist (positive RNAi viral particle) or express shRNA that does not match any known human coding mRNA(negative RNAi viral particle) was constructed, and transfected into A549 cells. Twist expression of A549 cells(control group), negative RNAi A549 cells(negative control group,NC group) and positive RNAi A549 cells(positive group) were evaluated by western blot. MTT assays were used to examine cell viability before radiation and clonogenic survival assays were used to assess radiosensitivity of the three groups of cells. Flow cytometry was used to assess cell cycle and the percentage of apoptotic cells. To investigate the migration and invasion ability, transwell chamber assays were used. And at last western blot was used to detect p53, p21, Bax, Bcl-2 protein expression of three groups after radiation.Results:Transfection of A549 lung cancer cells with shRNA against Twist specifically reduced Twist expression. The inhibition of Twist has no significant influence on A549 cell viability, cell cycle and cell apoptosis. Twist depletion significantly increased radiosensitivity of A549 cells. The SER was 1.31 compared with NC group. Twist depletion led to cell cycle arrest in G0/G1 phase(82.00%±2.72%) compared with the control group(63.66%±2.47%) and NC group(69.43%±0.32%), (P<0.05). Moreover, Twist depletion significantly increased the apoptosis percentage of A549 cells (13.88%±0.16%) compared with the control group(6.31%±0.57%) and NC group(7.37%±0.67%), (P< 0.05). Furthermore, Twist depletion decreased the migration and invasion of A549 cells after radiation. In addition, Twist depletion with radiation increased the level of p53, Bax, p21 and down-regulated Bcl-2 expression.Conclusion:The inhibition of Twist had no significant influence on A549 cell viability, cell cycle and cell apoptosis. Twist depletion significantly sensitized A549 cells to radiation. Twist depletion also decreased the migration and invasion of A549 cells after radiation. Furthermore, the inhibition of Twist by shRNA greatly increased radiation-induced G0/G1 phase arrest and cell apoptosis involving in increasing expression of tumour suppressor p53, p21, Bax and decreasing expression of Bcl-2 in A549 lung cancer cells.