| BACKGROUND:Lung cancer has become one of the most common malignant tumor in the worldwide accoring to recent study.Since the mid-20th,the incidence rates of lung cancer show a rising trend in most countries and the constant increasingly year by year,which has become highest incidence malignant tumor and leading cause of cancer mortality worldwide.Lung cancer contains non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC represents 80%of lung cancer.Now the main treatment for patients with lung cancer is surgical resection,chemotherapy and radiation therapy,but curative effect is far from satisfactory,the 5-year survival rate is still less then 20%for NSCLC.At present,with the improvement of diagnostic methods and advent of targeted therapies,the survival of lung cancer patients has been extended.However,to improve survival rate by a large margin,it still depends on research about genes or molecular markers correlated with tomorigenesis and metastasis of lung cancer,and then discusses the molecular mechanisms of occurrence,development and metastasis of lung cancer,which have crucial significance on early diagnosis,treatment and evaluating the prognosis.TGF-?(transforming growth factor beta)is one kind of multifunctional cytokine,which has been shown to play an important role in growth cell.As one of the major components of TGF-P signaling pathway,TGF-?1 can inhibit the growth of normal epithelial cell in normal physiological conditions.Notably,TGF-?1 plays a dual role in carcinogenesis.During the early stage of cancer,TGF-?1 functions as a tumor suppressor by inhibiting cellular proliferation and thus promoting cellular differentiation or apoptosis.In contrast,it acts as a tumor promoter to accelerate tumor progression and metastasis during the progressive stage of cancer.Previous studies have reported that TGF-?1 were high-expression and related with poor prognosis in breast cancer,liver cancer,gastric cancer and colorectal cancer.Although the significance of TGF-?1 protein expression in lung adenocarcinoma tissues has been reported,few studies have been focused on other subtypes of NSCLC by immunohistochemical method,such as squamous cell carcinoma and large cell lung cancer.To the best of our knowledge,no studies have yet been reported about the expression of TGF-?1 in NSCLC quantitatively and in large samples.Whether the expression of TGF-?1 is associated with progression and prognosis in NSCLC needs to be studied.Lung cancer is one of the most common malignant tumor that threat to human health,and most patients died of invasion and metastasis.At present many researches suggest that epithelial-mesenchymal transition(EMT)plays an important role in the process of invasion and distant metastasis of tumor cells,which has been confirmed in many kinds of tumors.TGF-?1 act as main inducer and thus promote the tumor inva-sion and metastasis.Related researches in molecular level found that:the expression of epithelial cells marker E-cadherin was reduced or abcenced,while mesenchymal cells marker N-cadherin was unusually high expression in cancer cells that encounter EMT process.Previsou study showed that exogenous TGF-?1 induced lung adenocarcinoma cell A549 to occur EMT process,A549 cells converted into the cell phenotype similar to fibroblasts or myofibroblasts and intercellular tight junctions was damaged and space increased.TGF-?1 can obviously inhibit the expression of epithelial marker E cadherin and promote the mesenchymal marker Vimentin or N-cadherin expression,which make A549 cells become active and ultimately promote tumor invasion and metastasis.However,under the stimulation of exogenous TGF-?1,will lung adenocarcinoma cells GLC-82 occur EMT process and what is the relationship with invasive?Thus we will carry on this research.Phosphatase and tensin homology deleted on chromosome ten(PTEN)was a new cancer suppressor gene with phosphatase activity that was found in recent years,which acts on and dephosphorylation phosphatidyl inositol 3,4.5-triphosphate(PIP3)and focal adhesion kinase(FAK),thus inhibit signaling pathways transduction of PIP3 and FAK,finally induce cell apoptosis,regulate proliferation of cell negatively and inhibit the invasion and metastasis of carcinoma cells.PTEN gene mutations were found in a variety of cancers currently,which lose the ability of regulate cell growth negatively and lead to the occurrence of tumor.In addition,many studies have shown that low-expression of PTEN protein was related with the occurrence and development of lung cancer,TGF-?1 has a correlation with PTEN in gastric cancer and liver cancer,but the results were not coincident.quantitative expression characteristics and related relationship between them in non-small cell lung cancer tissue is still unclear,which is one part of our studies.In summary,we firstly use quantitive and semi-quantitive methods to analysis the relationships between TGF-?1 protein expression and clinicalpathological characteristics,thus study the prognosis significance in non-small cell lung cancer tissues,secondly explore the correlation betwwen TGF-?1 and PTEN in non-small cell lung cancer tissues;Then finally explore TGF-?1 induced epithelial-mesenchymal transition,leading to a increase of migration ability in lung adenocarcinoma cells GLC-82.OBJECTIVE:1.To analysis the quantitive expression of TGF-?1 in non-small cell lung cancer tissues,then explore its prognostic significance in non-small cell lung cancer.2.To explore the correlation between TGF-?1 and PTEN in non-small cell lung cancer tissues.3.To explore TGF-?1 induced epithelial-mesenchymal transition and promote the migration ability in lung adenocarcinoma cells GLC-82.METHODS:1.The quantitive expression of TGF-?1 and its prognostic significance in non-small cell lung cancer tissues.Immunohistochemical SP method was applied to detect the expression ofTGF-?1 in 24 cases of normal lung tissue and 194 cases of non-small cell lung cancer;By computer image analysis software,the positive unit(PU)of protein expression was tested,quantitatively analysis the expression of TGF-?1 and relationship with clinical pathological characters,and then conduct postoperative survival analysis together with clinical pathological characters and follow-up data for 115 case of non-small cell lung cancer patiens with five-year of follow-up.Semi-quantitative criteria:TGF-?1 staining intensity and the number of positive cells were estimated using a four-tiered scoring system as described previously:negative(-),no staining at all;weak(+),weak staining regardless of positive cell percentages or moderate staining of<30%of cells;moderate(+ +),moderate staining of>30%of cells or strong staining of<50%of cells;strong(+++),strong staining of>50%of cells.Low expression of TGF-?1 was defined as(-)or(+),High expression was defined as(++)or(+++).2.The quantitative expression of TGF-?1 and PTEN in non-small lung cancer tissuesImmunohistochemical study for TGF-?1 and PTEN protein using SP methods was carried out in 110 cases of non-small lung cancer tissues,analysis the association with clinicalpathological characteristics,and then explore relationship between TGF-?1 and PTEN.3.To study of TGF-?1 induces lung adenocarcinoma cells GLC-82 to epithelial-mesenchymal transition and promotes the migration ability.The study was divided into experiment group and control group.Experimental group was treated with 5 ng/ml TGF-?1 recombinant for 48 h,while we did nothing to the control group.Morphological transformation of GLC-82 cell were observed by phase contrast microscopy;Western blot and QPCR detection protein and gene expression of EMT related marker E-cadherin and Vimentin were detected through Real-time quantitative PCR and Western blot.By using Transwell experiment cell migration ability were tested.To explore the TGF-?1 inducing lung adenocarcinoma cells GLC-82 to epithelial-mesenchymal transition and promoting the migration ability4.Statistical analysisAll statistical analysis were carried out with using SPSS 16.0 statistical software package..One-way ANOVA test was used in the comparison of multiple sample averages.When the variances are heterogeneous,Dunnett's T3 test were used for multiple comparisons between groups.Otherwise,LSD test were used.Two independent-sample t-tests were used between the two sample means.Paired samples used paird samples t-test;Five-year survival rate was checked with Pearson?2 test.Survival analysis was estimated by Kaplan-Meier method with log-rank test,and Cox's proportional hazards model was for multivariate analysis.The correlation analysis was assessed using Spearman rank correlation.Statistical significance was defined as the 2-sided p-value lower than 0.05.RESULTS:1.The quantitive expression of TGF-?1 and its prognostic significance in non-small cell lung cancer and normal lung tissues.The results of Immunohistochemical method and quantitative analysis showed that there was an distinct difference about TGF-?1 protein expression in normal lung and non-small cell lung cancer tissues,and the TGF-?1 protein PU value in the non-small cell lung cancer tissues was higher than those in normal lung tissues,the difference was statistically significant(P= 0.000).Our study showed that TGF-?1 expression in non-small lung cancer tissues was correlated with lymph node metastasis and TNM stage(Both P values were 0.000,respectively),TGF-?1 protein PU value was significantly higher in patients with lymph node metastasis(15.05±4.60)than in patients without lymph node metastasis(10.58±4.50)(P =0.000),and it was higher in patients with TNM stage?-?(15.87±4.44)than in patients with TNM stage ?-?(11.55±4.75).but There was no association between TGF-?1 protein PU value and patient's gender,age,smoking history,tumor differentiation,histological subtypes and tumor location in non-small cell lung cancer(P>0.05).The prognositic significance of TGF-?1 protein expression in 115 non-small lung cancer patients with 5-year follow-up data was analyzed.The semi-quantitative analysis showed that 47 cases(40.8%)were low expression,68 cases(59.2%)were high expression.Kaplan-Meier survival analysis indicated that the prognosis in patients with high expression of TGF-?1 protein was poorer than patients with low expression,(P value is 0.000,Log rank test).Multivariate cox proportional hazards model analysis showed that the expression of TGF-?1(Relative risk = 2.565,P=0.002)and lymph node metastasis(Relative risk =1.874,P-= 0.030)could be served as independent prognostic factors in non-small cell lung cancer.2.Quantitative analysis of the expression and correlation of TGF-?1 and PTEN in non-small cell lung cancer tissuesthe PU value of TGF-?1 expressed in non-small lung cancer tissues was signicantly higher than in normal lung tissues,the difference was statistically significant(P = 0.000),TGF-?1 protein expression was associated with TNM stage and lymph node metastasis in non-small lung cancer tissues(P value were 0.000?0.002 respectively).The PU value of PTEN protein expressed in non-small lung cancer tissues was lower than in normal lung tissues(P value was 0.000),which was related with TNM stage(P value was 0.003),As the stage became advanced,the PTEN PU value decreased.Spearman relative analysis indicated that PTEN was negatively correlated with TGF-?1 expression in non-small cell lung cancer tissues(r was 0.380,P value was 0.000).3.To study of TGF-?1 induces lung adenocarcinoma cells GLC-82 to epithelial-mesenchymal transition and promotes the migration ability.Under phase contrast microscopy,After treatment with exogenous recombinant TGF-b1,we observed that the cell became elongated and displayed spindle-shape,fibroblast-like pheotype compared to the blank control group,The conection betweens cells became loose and the intercellular space were larger;The results of Western blot and QRT PCR indicated that the geme and protein expression of E-cadherin,the epithelial phenotype marker,was significantly decreased,and those of mesenchymal phenotype markers,vimentin,were greatly increased in experimental cells after adding recombinant TGF-beta 1.Transwell invasion assay found the number of cells in experimental group(68.20 + 5.49)getting through the small room was increased compared with cells in control group(36.00 + 4.38),the difference was statistically significant(t = 13.920,P value was 0.000),which indicated that TGF-beta 1 treatment enhanced significantly cell migration ability compared with the control group.Conclusions:1.TGF-(31 protein PU value was associated with TNM stages and lymph node metastases.Univariate analysis indicated that patients with high TGF-?1 protein expression carried a poor prognosis.COX multivariate analysis showed that TGF-?1 protein expression and lymph node metastases were independent prognostic factors in patients with NSCLC.Thus,TGF-?1 protein expression may be correlated to oncogenesis and serves as an independent prognostic biomarker for NSCLC.2.PTEN was correlated negatively with TGF-?1 expression in lung carcinoma.The abnormal activation of TGF-?1 protein may leads to the decrease or deletion of PTEN expression,which palys an important role in promoting the initiation and development of lung cancer.Thus combined detection of TGF-?1 and PTEN may act as one of the indicators in reflecting the biological behavior of lung cancinoma.3.TGF-B1 induces epithelial mesenchymal transition of GLC-82 cells,make morpho logical alteration from epithelial to mesenchymal cells.The expressions of epithelial cell marker E-cadherin was decreased,while the expression of mesenchymal cell marker N-cadherin were increased,which enhanced the motility and invasion ability of lung cancinoma cell. |
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