Expression Of Gli1 And EMT-related Proteins And Its Clinical Significance In Non-small Cell Lung Cancer

Objective To investigate the expression and clinical significance of glioma cancer related gene homologous protein 1(Gli1)and EMT-related proteins Snail,E-cadherin and N-cadherin in non-small cell lung cancer(NSCLC).To study the molecular mechanism of Hedgehoog signaling pathway regulating the invasion and metastasis of non-small cell lung cancer,and to provide a new target for the diagnosis and treatment of non-small cell lung cancer.Methods 1.Immunohistochemical staining was performed to determine the expressions of Gli1,Snail,E-cadherin and N-cadherin in 67 NSCLC cases and 67 cases of normal lung tissues to explore its relationship with clinicopathological features such as clinical stage,pathological type,lymphatic metastasis and differentiation and prognosis.2.Another 20 samples of fresh NSCLC tissues and corresponding nomal lung tissues were collected to detect the m RNA level by reverse transcription polymerase chain reaction(RT-PCR).3.Based on Gli1 expression level in A549 and H1299 cells,A549 and H1299 cells were selected as in vitro models.The expression levels of Gli1 were assessed by Western blot analysis,and the results showed that A549 cells had low-level Gli1 expression,while H1299 cells presented high levels.To establish stable Gli1 knockdown or Gli1-overexpressing cells,A549 and H1299 cells were stably transfected.4.The migration of A549 and H1299 cells were determined after Gli1 transfection by the wound healing assay.The invasion of A549 and H1299 cells were detected after Gli1 transfection by Transwell experiment.5.The changes of the expression of EMT related protein Snail,E-cadherin and N-cadherin in A549 and H1299 cells were detected after Gli1 transfection by Western blot.Results1.The positive rates of Gli1,Snail,E-cadherin and N-cadherin in NSCLC were 61.2%(41/67),50.8%(34/67),56.7%(38/67),53.7%(36/67),normal lung tissues were 14.9%(10/67),8.9%(6/67),98.5%(66/67),5.9%(4/67),the statistical difference of these two sets of data is significant(P<0.05).2.The high expression of Gli1 in tumor tissues was closely related to lymph node metastasis,TNM stage and tumor differentiation(P<0.05)and not associated to gender,age,tumor size and pathological type,The expression of Gli-1 in NSCLC tissues was negatively correlated with the expression of E-cadherin(r=-0.325,P<0.05)and positively correlated with the expression of Snail and N-cadherin(r=0.379,r=0.490,P<0.05).3.RT-PCR showed that the expression level of Gli-1,Snail and N-cadherin m RNA were(0.933±0.115vs0.656±0.014vs0.255±0.078)in NSCLC cases significantly higher than that in normal lung cases(0.510±0.208 vs 0.086±0.362 vs 0.033±0.043,P<0.05),the expression level of E-cadherin m RNA was lower in tumor tissues(0.461±0.087)than in lung tissues(0.811±0.180,P<0.05).4.Patients with high expression levels of Gli1,Snail and N-cadherin had significantly worse prognosis and lower survival rate than patients with low expression(all P<0.05),and patients with low expression levels of E-cadherin had significantly better prognosis lower survival rate than patients with high expression(P<0.05).Multivariate Cox's proportional hazard regression analysis indicated that E-cadherin-negative group expression,lymph node metastasis,TNM stage and tumor differentiation were independent prognostic factors for NSCLC.5.Establish stable Gli1 knockdown H1299 cells and Gli1-overexpressing A549 cells.6.The wound healing assay results showed that Gli1 knockdown significantly decreased the abilities of H1299 cells to migrate,whereas Gli1 overexpression had an inverse effect on A549 cells.The Transwell experiment results demonstrated that Gli1 knockdown significantly decreased the abilities of H1299 cells to invade,whereas Gli1 overexpression had an inverse effect on A549 cells.7.The Western blot results showed that A549 cells overexpressing Gli1 demonstrated increased levels of the EMT-related factors Snail and N-cadherin,down-regulated the E-cadherin,In contrast,knockdown of Gli1 expression resulted in contrasting results of these genes in H1299 cells.Conclusion 1.The expression of E-cadherin in normal lung tissues was higher than that in non-small cell lung cancer tissues.The expression of Gli1,Snail,and N-cadherin in NSCLC was higher than that in normal lung tissues,and the expression of Gli1 was correlated with Snail,N-cadherin The expression was positively correlated and negatively correlated with the expression of E-cadherin,indicating that the occurrence of EMT in non-small cell lung cancer is related to the abnormal activation of the Hedgehog pathway.2.The expression of Gli1 and E-cadherin in different degree of differentiation,lymph node metastasis and clinical stage were different.The higher the degree of differentiation and clinical stage,the higher the expression of Gli1,the lower the expression of E-cadherin and lymph node metastasis.In patients with Gli1,the expression of E-cadherin was lower than that in patients without lymph node metastasis.The expression of Snail and N-cadherin is related to lymph node metastasis and clinical stages.3.Gli1 facilitates the migration and invasion of NSCLC cells,Gli1 may regulate the expression of E-cadherin and N-cadherin to promote the EMT process of NSCLC cells by regulating the expression of Snail.4.In NSCLC,the abnormal activation of Hedgehog signaling pathway has correlation with Epithelial-mesenchymal transition phenomenon happening.Detecting the expressions of Gli1 and EMT-related proteins Snail,E-cadherin and N-cadherin may give a clue on clinicopathological features and prognosis in patients with non-small cell lung cancer.