Expression Of HES5 In Lung Adenocarcinoma Cancer And The Effect On Cell Proliferation

Primary bronchogenic carcinoma is one of the most commonly malignant tumors.Because of smoking,environmental pollution and so on,the incidence of lung cancer was increasing year by year in our country.Because of the hidden process and lack of methods for early diagnosis,patients who have been diagnosed were with advance stage and the prognosis was poor.The formation of lung cancer was related with the changes of multiple gene and signal pathways.Therefore,to study the related genes and their mechanisms in the process of carcinogenesis and progression of lung cancer is very important.HES5,which locates on the chromosomal region 1p36,belongs to the basic helix-loophelix(b HLH)superfamily.It's a downstream molecule and effector of mammalian Notch pathway.As a transcription factor,HES5 binds to a consensus sequence of gene promoter called an N-box(CACNAG)and regulates target genetranscription.HES5 plays an important role in regulating differentiation in various tissues.Recent studies have found that HES5 was involved in prompting the tumor cellular proliferation in many kinds of cancer.However,the role and mechanism of HES5 palys in non small cell lung cancer(NSCLC)are unclear.In this study we will investigate the expression and clinical significance of HES5 in lung adenocarcinoma and its effects on the proliferation of lung adenocarcinoma cells,and the mechanism of HES5 will also be discussed.Part? Expression of HES5 in lung adenocarcinoma and its relationships with Ki67,clinicopathological features and prognosisObjective:To study the expression of HES5 protein in lung adenocarcinoma and explore the correlations of HES5 expresion with clinicopathological parameters and prognosis at the tissue level.Methods:1.Eight pairs of adenocarcinoma tissues of lung adenocarcinoma and adjacent nontumorous tissues samples from patients underwent surgery was collected.Western Blot was used to detect the expression of HES5 protein in the lung adenocarcinoma tissues and adjacent non-tumorous tissues.2.One hundred and fourteen lung adenocarcinoma sections were from patients who underwent surgery between 2005 and 2009 at Department of Pathology,Affiliated Hospital of Nantong University.We detected expression of HES5 and Ki67 by immunohistochemistry in lung adenocarcinoma,and analyzed the relationships between HES5 expression and clinicalpathologic factors.The influence of HES5 on prognosis were tested by Kaplan–Meier analysis.Results:1.The expression of HES5 protein between eight paired lung adenocarcinoma tissues and the adjacent non-tumorous tissues was investigated by Western Blot.The expression of HES5 protein was significantly upregulated in most lung adenocarcinoma tissues than the adjacent non-tumor ones(P<0.05).2.The expression of HES5 protein in lung adenocarcinoma tissues was expressed mainly in nucleus and cytoplasm,while Ki67 mainly located in the nucleus by immunohistochemistry.Next,we evaluated the relationships between clinicopathological factors and HES5 expression.The expression of HES5 was significantly related with tumor size,lymph node metastasis,clinical stage and histological differentiation(P<0.05).Furthermore,we found that there was a positive relation between the expression HES5 and Ki67 by Spearman rank correlation test(r=0.591,P<0.01).Kaplan-Meier analysis was used to calculate the association between the expression of HES5 and patients' survival.The survival curves showed that lung adenocarcinoma patients with high HES5 expression had significantly decreased overall survival,compared with those with low HES5 expression(P <0.05).Cox proportional hazards model indicated that HES5 expression,as well as Ki67 and histological differentiation,were independent prognostic indicators for the patients with lung cancer.Conclusions:HES5 was up-regulated in lung adenocarcinoma tissue and the expression of HES5 was positively associated with clinical stage,histological differentiation,Ki67 and tumor sizes.The high expression of HES5 suggested poor prognosis.Part ? Effect of HES5 expression on proliferation of human lung adenocarcinoma A549 cellsObjective:To analyze the effects of HES5 on proliferation of the lung adenocarcinoma cells at the cellular level.Methods:1.The expression of HES5 protein in three human lung adenocarcinoma cell lines including A549,H1299 and Spca-1 was detected by Western Blot(WB).2.The lung adenocarcinoma cells with HES5 high expression cultured with basic serum-free condition,and then given the restoration of serum.The cell cycle progression was analyzed by flow cytometry analysis,and the expression of HES5,Cyclin D1 and PCNA during this process were detectd by Western Blot.3.The lung adenocarcinoma cells were transfected with si RNA target HES5 by liposome transfection method.Then,the lung cancer cell line with HES5 gene silencing was established.Cell cycle distribution and apoptosis were analysed by flow cytometry cell and and Annexin V-FITC/7-ADD assay respectively.The proliferation and colony formation of the cell lines were detected by Cell Counting Kit-8(CCK-8)assay and colony formation assay.Results:1.We examined the expression of HES5 protein in three human lung adenocarcinoma cell lines,and found that the expression of HES5 protein in A549 cell line was high.2.A549 cells were treated by serum starved and then recovered serum refeeding.We analyzed the cell cycle progression using flow cytometry.A549 cells were arrested in G0/G1 phase.Upon serum addition,A549 cells were released from the G0/G1 phase and gradually entered into S phases.We also found that the expressions of PCNA and cell cycle regulator cyclin D1 were increased after serum stimulation in A549 cells.Meanwhile,the protein level of HES5 was also upregulated.3.We transfected A549 cells with HES5-si RNA,and the flow cytometric analysis of the cell cycle showed an increase of cell number in the G0/G1 phase and a decrease of cell number in the S phase.It indicates that downregulation of HES5 could slow down the NSCLC cell cycle.We also found that knockdown of HES5 can decrease the cell proliferation,and the rate of colony formation was significantly attenuated in knockdown of HES5 cells.Furthermore,the Annexin V-FITC/7-ADD assay also showed significant increase of cell death after HES5 knockdown.At the same time,the expression of Cyclin D1 and PCNA also declined with the down-regulation of HES5 expression.Conclusions:HES5 was involved in A549 cell cycle progression and proliferation.Knockdown of HES5 could delay the proliferation,induce an increase of cellular apoptosis and reduce colony formation.Part ? Study on the role of HES5 in STAT3 pathwayObjective:To investigate the possible mechanism of the interaction between HES5 and the STAT3 pathway at the cellular level.Methods:1.The interaction between HES5 and STAT3 pathway was detected by immunoprecipitation assay in non-transfected A549 cells.2.The expressions of STAT3 and p-STAT3 were analyzed by Western Blot,and the downstream molecules of STAT3 such as Cyclin D1 and Bcl-xl were detected in A549 cells which were transfected with si RNA target HES5.Results:Immunoprecipitation assay indicated that HES5 directly interacted with STAT3 in A549 cells.In addition,knockdown of HES5 resulted in inhibited phosphorylation of STAT3 and decreased expression of p-STAT3 and STAT3.We also observed that knockdown of HES5 decreased the expression of the downstream genes of STAT3 such as cyclin D1 and Bcl-x L in A549 cells.Conclusions:HES5 could interacted with STAT3,and knockdown of HES5 could inhibit the activity of STAT3 and decrease the expression of the downstream targets of STAT3 and p-STAT3 in A549 cells.HES5 may influence the proliferation process through the activation of STAT3 in human lung adenocarcinoma A549 cells.