| Objective:To investigate the role of p38α,p38β,p38γ and p38δ in the malignant behavior of Esophageal squamous cell carcinoma(ESCC)and its possible mechanism;and to investigate the clincopathological significance of p38β,p38γ and p38δ expression in ESCC tissues and to analyze the role of p38α,p38β,p38γ and p38δ in the proliferation,migration,invasion,cell cycle and apoptosis of ESCC cell line Eca109 cells;to analyze the tumorigenic ability of p38γ and p38δ;and to identify the specific miRNA regulating p38δ.Methods:To investigate the clinicopathological significance of p38α,p38β,p38γ and p38δexpression in ESCC and paired normal control tissues,immunohistochemistry was employed with appropriate statistical analysis methods.The clinicopathological parameters include patients ‘age,gender,clinical stage,T classification,N classification,differentiation,tumor size,gross pathology and overall survival time.Cross-table statistical analysis was used to analyze the clinical correlation between expression of p38α,p38β,p38γ and p38δ and clinicopathological parameters with the exception of overall survival.Kaplan-Meier survival curve was employed to analyze the expression of p38α,p38β,p38γ and p38δ and overall prognosis of patients with ESCC.To investigate the role of p38α,p38β,p38γ and p38δ in the proliferation,migration,invasion,cell cycle and apoptosis of ESCC cell line Eca109 cells.First of all,constructed were specific short-hairpin RNA(shRNA)interference vectors against p38α,p38β,p38γ and p38δsubtypes as well as eukaryotic over-expression vectors harboring full-length cDNA of p38α,p38β,p38γ and p38δ.Followed by transfection with Lipofectamine 2,000 intoESCC cell line Eca109 cells.Both interference and re-expression effect were evaluated using qRT-PCR and western-blot on mRNA and protein level,respectively.To evaluate the influence over proliferative,migratory and invasive variation before and after transfection with shRNA and Eukaryotic over-expression vectors into Eca109 cells,MTT,wound-healing and Transwell assays were employed.To detect the cell cycle and apoptotic variation before and after transfection with shRNA and Eukaryotic over-expression vectors into Eca109 cells,Flow Cytometry(FCM)was utilized.Given the endogenous mRNA expression level of p38γ and p38δ in Eca109 cells,to analyze the tumorigenic ability of p38γ and p38δ,established were transgenic cell line Eca109 whose basal p38γ was stably knocked down and whose endogenous p38δ was stably up-regulated.Then,to keep pure of the positive cells after transfection,all the transfectant cells were subjected to sorting using FCM.Followed by subcutaneous injection into athymic nude mice with cells after being sorted out with FCM.The tumors grown until palpable were measured both in terms of size and weight at the fourth week after injection.Results:Part I: p38αwas pronouncedly up-regulated in ESCC tissues in comparison with paired normal control tissues(P=0.000);however,there was no significant difference between p38αexpression and clinicopathological parameters,including gender(P=0.436),gender(P=1.000),clinical stage(P=0.514),T classification(P=0.429),N classification(P=0.646),differentiation degree(P=0.670),tumor size(P=0.610)and gross pathology(P=0.551).no significant association was observed between p38αexpression and overall prognosis(P > 0.05);In comparison with adjacent normal control tissue,p38βwas remarkably up-regulated in ESCC tissues(P=0.000).there was significant association between p38β expression and tummor size but no association was observed between p38βexpression and other clinicopathological parameters,including gender(P=0.730),age(P=0.294),clinical stage(P=0.118),T classification(P=0.692),N classification(P=0.109),differentiation degree(P=0.470)and gross pathology(P=0.609)whereas there was significant difference of overall prognosis between patients with high p38β expression and p38βlow expression(P < 0.05)after analysis using Kaplan-Meier survival curve;there was no markedly difference of p38γexpression between ESCC and paired normal control tissues in terms of expression.however,there was significant association between p38γexpression and clinical stage(P=0.017),N classification(P=0.008)and tumor size(P=0.017).No significant association was found between p38γ expression and gender(P=1.000),age(P=0.609),T classification(P=0.063),differentiation degree(P=0.093)and gross pathology(P=0.315).p38δwas markedly up-regulated in ESCC as compared with paired normal control(P=0.000).Similar to p38 a,there was no significant association between p38δ and clinicopathological parameters,including gender(P=1.000),age(P=0.603),clinial stage(P=0.089),T classification(P=0.646),N classification(P=0.568),differentiation degree(P=0.791)and gross pathology(P=0.575).Moreover,no significant association was discovered between p38δexpression and overall prognosis.Part II: In vitro in ESCC cell line Eca109,it was found that p38αwas pronouncedly able to promote the proliferation,migration and invasion in Eca109 cells after being knocked down;but p38β,p38γ and p38δ was remarkably capable of suppressing the proliferation,migration and invasion in Eca109 cells after being knocked down.However,all the malignant phenotypes,including proliferation,migratioin and invasion can be reversed after transfection with eukaryotic overexpression vectors harboring full-length cDNA of p38α,p38β,p38γ and p38δ.which is to say,p38αwas significantly able to suppress the proliferation,migration and invasion in Eca109 cells after being over-epxressed;whereas p38β,p38γ and p38δwas markedly capable of promoting the proliferation,migration and invasion in Eca109 cells after being re-expressed.results obtained from flow cytometry showed that knock-down of p38αcan significantly inhibit the apoptosis of Eca109,S phage was significantly increased;while,knock-down of p38β,p38γ and p38δ was able to promote apoptosis of Eca109 whose S phase was remarkably reduced.The apoptosis and cell cytle phenotypes were in contrast with Eca109 cells whose subtypes of p38 MAPK was knocked down.Part III: results from nude mice xenografted with Eca109 showed that over-expression of p38δ was able to promote the proliferation of Eca109 cells in vivo in comparison with control group;whereas knock-down of p38γ was capable of suppressing the proliferation of Eca109 cells as compared with control group,suggesting that both p38γ and p38δ were able to promote the proliferation of Eca109 cells in vivo.Conclusion:(1)Among p38α,p38β,p38γand p38δ,only there was no significant difference of p38γexpression between ESCC and paired normal control tissues,while the remaining three subtypes whose expression was significantly up-regulated in ESCC tissues in comparison with paired normal control tissues.After clinicopathological analysis it was shown that p38γwas significantly associated with lymph nodes metastasis and tumor sizewhereas p38β,p38γ and p38δ was associated with tumor progression in ESCC;(2)In vitro in ESCC cell line Eca109 cells,p38α plays an anti-oncogenic role in the malignant phenotype of Eca109 cells,whereas p38β,p38γ and p38δplay a tumor-promoting role in the malignant phenotype of Eca109 cells;(3)In xenografted nude mice,both p38γ and p38δ were able to promote the proliferation of Eca109 cells in vivo. |
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