| Lung adenocarcinoma(LAC)is the most fetal cancer of human with the highest morbidity and mortality.Although the wide application of low dose computed tomography screening and the improvement of operation techniques have brought an increasing mortality,the average 5-year survival rate of patients with LAC was only about 30%due to early-stage metastasis and recurrence.Malignant proliferation is the mainly reason for the poor prognosis in LAC patients,and it's a multi-stage,multi-factor and multi-step procedure.To explore the mechanism of LAC malignant proliferation and search for potential molecular therapeutic targets has been an urgent task in the field of LAC research.The malignant biological behavior presented by LAC is mainly caused by uncontrolled proliferation.In normal biological individuals,cell proliferation is strictly restrained by cell cycle checkpoint to ensure the accuracy of DNA replication and chromosome redistribution.This is a precise process involving with multiple genes and proteins.Cell division cyclin 25 A(CDC25a)is a member of the specificity phosphatase family,which plays an important role in G1/S transition process of cell cycle.There has been a number of researches indicated that many nuclear transcription factors such as STAT3,FOXM1,E2F and CBP,could activate CDC25a promoter directly or indirectly,while some others such as p21,Smad3/4 could down-regulate its transcriptional activity.We assume that if there were other nuclear transcription factors could start CDC25a transcription during the occurrence and development of LAC,and further disturb cell cycle detection mechanism to accelerate malignant tumor proliferation.Human Y box-binding protein-1(YBX1),one of the current achievements of cancer research,is expected to solve this problem.YBX1,a member of the DNA-binding protein family,is an important nuclear transcription factor.It regulates genetic expression via binding the targets with Y-box sequence(5'-CTGATTGG-3')located on promoters of many genes.The main role of human YBX1 gene is mainly concentrated in the following aspect:? a vital modulator of cell cycle;?a chemotherapy drug resistance regulation factor of tumor cells,and ?a repair factor of DNA injuries.Therefore,the YBX1 mediated tumor proliferation signal transduction network might be a common and efficient terminal target for multiple anti-tumor therapies.Numerous researches showed that YBX1 directly or indirectly accelerates tumor cell cycle and suppresses the activities of many cycle suppressors such as p53,p21,p27 in breast,stomach,colon cancer,but there was no an clear mechanism of YBX1.Using bioinformatics methods,we found three reverse Y-Box sequences which could be bound by YBX1 specifically by checking the CDC25a promoter area(GenBank:AJ242714.1).We speculated that YBX1 may specifically binds to the promoter of CDC25a,and up-regulates its transcription,and further activate CDC25a signal pathway,which leads to malignant tumor proliferation.The current study made use of siRNA interference system,SDS-PAGE,flow cytometry,immunocytochemistry staining and animal models to demonstrate the relationship between YBX1 and CDC25a and reveal the mechanism of YBX1 mediating LAC proliferation via CDC25a signal pathway.Besides,an early post-operative prognostic estimation system may be set up based on YBX1/CDC25a expression to offer reliable reference on accurate treatment for LAC patients.Part 1.Relation between YBX1 and CDC25a in LAC cancer and prognostic study.Objectives Explore the expression of YBX1 and CDC25a in different LAC subtypes,and retrospectively analyze the patients'prognosis with different clinical-pathological factors,then to confirm the expression and location of YBX1 and CDC25a in various LAC cell lines and normal lung epithelium cell lines.Methods We collected 116 cases of LAC treated in the First Affiliated Hospital of Dalian Medical University during January 2008 to December 2010.The mean ages of those patients was 64 years(ranging from 45 to 86).All of the patients had completed follow-up information.The expression and location of YBX1 and CDC25a were examined using immunohistochemical staining.Western Blot and immunofluorescent staining were employed to reveal the expression and location of YBX1 and CDC25a in different LAC and normal lung epithelium cell lines.Results The expression of YBX1 has relatation with tumor size(p=0.006),TNM staging(p=0.006)and CDC25a expression(p=0.016),besides gender,age,tissue differential degree,and the IASLC/ATS/ERS classification(p>0.05).The expression of CDC25a had relevance to differentiation(p=0.007),lymph node metastasis(p=0.026),TNM staging(p=0.033)and the IASLC/ATS/ERS classification(p<0.001).Univariate analysis showed that LAC patients' overall survival rate was closely relevant to the IASLC/ATS/ERS classification(p=0.037),differentiation(p=0.001),lymph node metastasis(p=0.008),CEA concentration(p=0.045),CDC25a expression(p=0.004),YBX1 expression(p=0.044)and TNM staging(p<0.001).In the Cox proportional hazards regression model,TNM staging(HR=3.428,95%CI:1.519-7.737,p=0.003)and high CDC25a expression(HR=2.384,95%CI:1.008-7.642,p=0.048)were recognized as independent risk factors of LAC prognosis.The expression of YBX1 or CDC25a in LAC cell lines was significantly higher than that in normal lung epithelium cell line,following with obvious nuclear shifting.Conclusion The expression of YBX1 and CDC25a in LAC had a positive correlation.TNM staging and high CDC25a expression were independent risk factors of LAC prognosis.There was higher expression of YBX1 and CDC25a in LAC cell lines in vitro.Part 2.YBX1 controls the LAC proliferation via regulating CDC25a signal pathway.Objectives To explore the regulation of YBX1 on CDC25a promoter,and to reveal the mechanism of YBX1 regulating cell cycle via CDC25a pathway,then to verify the effection of YBX1 on the malignancy biological behavior in LAC.Methods Firstly,Chromatin Immunoprecipitation and fluorescence protein reporter gene assay was utilized to confirm that YBX1 could specially bind to CDC25a promoter area.Secondly,RT-PCR and Western blot were employed to learn the influence of YBX1 on CDC25a pathway at the molecular level.Finally,clone formation assay,flow cytometry,MTT(Thiazolyl Blue)and scratch test were used to find the effection of YBXl on the malignancy biological behavior of LAC.Results YBX1 bind to CDC25a promoter(-183--72)and regulate its transcriptional activity.The suppress expression of YBX1 in LAC cell lines led to down-regulated CDC25a,p-RB,cyclin D1 and Stat3(p<0.05)and an up-regulated p21 and p53 expression(p<0.05).But other proteins such as CDK2,CDK4,CDK6 and cyclinE1 were observed no significant change.In terms of cellular function,suppress expression of YBX1 caused cell cycle G1/S block(p<0.05),a reduction of colon formation(p<0.05),a boost of cell apoptosis(p<0.05),and a inhibited cell migration(p<0.05).Conclusion YBX1 specifically binds to the CDC25a promoter,and up-regulates the transcription of CDC25a.YBX1 can control cell cycle,proliferation,apoptosis and migration capability of LAC cells via CDC25a pathway.Part 3.The study of YBX1 control tumor cell proliferation in vitro.Objectives To verify siRNA YBX1 suppress the tumor growth in vitro.Methods The nude mice LAC tumor-bearing models were constructed.The siRNA YBX1 was injected into mice to observe its influence on tumor growth.Then tumor tissues were stained with HE to observe the pathological structure change.Immunohistochemistric staining was used to identify YBX1,CDC25a,Ki67 and cleave-caspase3 expression in those tumors.Results The siRNA YBX1 treated mice had smaller tumors in both size(p<0.01)and weight(p<0.01).In addition,lower expression of YBX1,CDC25a and Ki67 and higher expression of cleave-caspase3 were found in their tumor tissues.Conclusion The siRNA YBX1 can suppress the proliferation activity of nude mice-planted LAC tumors,and down-regulate YBX1,CDC25a and Ki67 expression and up-regulate cleave-caspase3 in those tumor tissues. |
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