Expression Of Nestin In Gastric Adenocarcinoma And Its Relationship With Epithelial-mesenchymal Transition

Background and objectiveGastric cancer is one of the most common malignant diseases in digestive tract.And more than 95%of gastric cancer cases are adenocarcinomas.The high recurrence rates and the nodal metastasis are the main causes of patients’ death.The epithelial-mesenchymal transition(EMT)is a key programme by which epithelial cells lose their polarized,epithelial morphology and acquire a mesenchymal-like phenotype.Increasing evidence has shown that EMT is crucial for the metastatic dissemination of epithelial cancer cells,providing tumor cells with the capacity to escape from the primary site and invade the surrounding stroma.Nestin,a cytoskeleton-associated class Ⅵ intermediate filament(IF)protein,was detected in cytoplasm and nucleus.As a neuronal stem/progenitor cell marker,nestin is expressed in progenitor cells of various tissues,including brain tumors,liver cancer,osteosarcoma,pancreas cancer,rhabdomyosarcoma,malignant melanoma,lung cancer,prostate cancer and so on.Recent reports support a link between nestin and malignant characteristics,and suggest that abundant nestin expression is correlated with greater malignancy and poorer prognosis in different cancers.Data from A P Xiang suggested that the histoscore of nestin expression in high malignant GIST was2.2366,and that in low malignant GIST was 1.3783.However,in some benign tumors such as cavernous angioma,which did not express nestin.Moreover,in this study,nestin was predominantly found in the cytoplasm of GIST samples,whereas in poorly differentiated GIST tumour cells,nestin was expressed in the nucleus.Similar result was reported by Loja and Krupkova in brain tumor cells.They supposed that nestin may have a function in chromatin remodeling and gene regulation.However,little is known about the effect of this differential localization of nestin and the classical nuclear localization signals in human tumors.Kleeberge W et al.suggested that nestin was mainly expressed in the metastasizing tissues and cell lines.Knockdown of nestin with short hairpin RNAs in prostate cancer cells resulted in decreased migration and invasion.These results specify a function for nestin in cell motility and identify a novel pathway for prostate cancer metastasis.However,up to date,the specific function of nestin in the invasive and metastatic behaviors of gastric cancer cells remains poorly understood.Based on above,we performed the following experiments in three parts below.(1)Part 1:125 gastric adenocarcinoma(GAC)paraffin-embedded samples and 60 adjacent normal gastric mucosa samples were collected and serially sectioned at 3 μm thickness.Then,nestin was detected by immunohistochemistry in these tissue samples.Last,we analysed the correlation between nestin(including the different sublocalizations of nestin)and clinicopathological factors and patient survival period by statistical methods;(2)Part 2:The expression of EMT-related proteins(E-cadherin,vimentin and Snail)were investigated by immunohistochemistry.And the potential association of nestin and EMT-related proteins with clinicopathological factors and patient survival period were analysed;(3)Part 3:Knockdown of nestin with lentivirus-mediated shRNA in MKN28 and AGS cells in vitro,the changes of migration,invasion potentiality and the expression of EMT-related proteins were tested to verify the relationship of nestin and EMT.Part 1 Expression of nestin and its clinical implications in gastric adenocarcinomaMethods1.Paraffin tissue samples were collected from 125 gastric adenocarcinoma patients,the control samples(normal gastric mucosa)were collected from the 60 cases of them in the operation at a distance of 5 cm from the tumor.All of them were serially sectioned at 3 μm thickness.2.Samples from 125 patients with gastric adenocarcinoma and 60 normal gastric muosa were examined for nestin expression by immunohistochemical staining.Then,the correlation between nestin(including the different sublocalizations of nestin)and clinicopathological factors and survival outcomes were analysed.ResultsPositive nestin immunostaining was most obviously detected in the cytoplasm,nucleus or both cytoplasm and nucleus of tumor cells in GAC,which was significantly higher than that in paracancerous tissues(P=0.001).The expression of nestin in cytoplasm,nucleus were correlated with T classification and N classification(P<0.05),whereas nuclear nestin expression also was association with histologic grade(P=0.017).The patients with positive nestin expression showed a shorter survival time than those with negative nestin expression(P<0.05).Compared with the group with either cytoplasmic or nuclear nestin expression,those patients with both cytoplasmic and nuclear nestin expression had a trend of lower 5-year survival rate.N classification was an independent prognosis factor for the survival of gastric adenocarcinoma patients.Conclusions1.Expression of nestin was involved in the pathogenesis of GAC.Patients with positive nestin expression showed a shorter survival time than those with negative nestin expression.2.Nestin in the nuclei(including nestin expression in nucleus only and in both cytoplasm and nucleus)of tumor cells may stand for a more malignant grade.3.Patients with positive staining of nestin both in cytoplasm and nucleus displayed a tendency of more unfavorable prognosis than those with either cytoplasmic or nuclear nestin staining.Part 2 Association of nestin and EMT-related proteins with clinicopathological factors and patient survival period Methods1.To investigate the expression of EMT-related proteins(E-cadherin,vimentin and Snail)in gastric adenocarcinoma by immunohistochemistry.Then,analyse the potential association of EMT-related proteins and clinical features.2.To study the correlation between nestin and EMT-related proteins(E-cadherin,Vimentin,Snail)and their prognostic significance.Results1.The expression of nestin and its survival outcomes were displayed in Part 1.2.Positve membranous of E-cadherin was detected in 53.6%(67/125)of GAC and 90.0%(54/60)of normal gastric mucosa.Through the X2-test,a significant difference was displayed in these two groups(X2 =23.739,P = 0.000).Patients with positive E-cadherin showed a longer survival time(P = 0.017).3.Cytoplasmic localization of Vimentin was highly expressed in 12.8%(16/125)of tumor cases.In contrast,cytoplasmic Vimentin expression was negative in normal gastric mucosa,and mainly expressed in stromal and inflammatory cells.The difference in the Vimentin positive expression between the cancer group and the normal gastric mucosa group is significant(X2 =8.407,P = 0.004).Positive Vimentin expresstion was associated with no significant difference in outcomes,but with a trend toward reduced survival in patients with positive Vimentin expression(P = 0.077).4.In gastric cancer tissues,the percentage of Snail positive expression which was found to be present in both the cytoplasm and nucleus of tumor cells was 20.8%(26/125).And some of the tumors studied had large solid areas of malignant cells that showed no increase in Snail expression within the central region of the cell groups but a tendency for increased expression in the peripheral layers of cells.Malignant cells at the invasive edge of the tumor tended to show increased staining that was more pronounced if the invasive edge was penetrating the subserosal tissue.In normal gastric mucosa tissues,only 3.3%(2/60)cases displayed Snail positive expression.The X2-test indicates a significant difference in the Snail positive expression between the cancer group and the normal gastric mucosa group(X2 =5.543,P=0.002).Snail staining had a more unfavorable prognosis than those with negative nestin and Snail expression(P=0.001).5.Spearman correlation test suggested that the positive expression of nestin was negatively correlated with E-cadherin(r=-0.280,P=0.002)and was positively associated with Vimentin(r=0.482,P=0.000)and Snail(r=0.451,P=0.000)expression.Interestingly,the patients with cytoplasmic nestin expression was closely related to Vimentin and Snail expression(P<0.05),but there was no significant correlation with E-cadherin expression(P>0.05).The group with nuclear nestin expression was association with E-cadherin and Snail(P<0.05),whereas no statistical difference with the expression of Vimentin(P>0.05).However,a significant association was exhibited between both cytoplasmic and nuclear nestin expression with negative E-cadherin expression(r =-0.192;P =0.032),also for positive Vimentin(r = 0.427;P = 0.000)and Snail(r = 0.212;P＝0.018)expression,and patients with both cytoplasmic and nuclear nestin expression(0)displayed a lower 5-year survival rate than those with cytoplasmic nestin only(28.6%)or nuclear nestin only(30%).6.Tumor size,histologic grade,T classification,N classification,pTNM stage,Nestin,E-cadherin and Snail were analysed in Cox models to determine the independent factors for gastric adenocarcinoma patients.Only N classification might be an independent prognostic factor for human gastric adenocarcinoma after resection(RR=3.559,P=0.003).Conclusions1.Nestin was involved in regulating the EMT and malignant progression in GAC.2.Nuclear localization of nestin(including nestin expression in nucleus only and in both cytoplasm and nucleus)correlated with down-regulation of E-cadherin.So nuclear localization of nestin might be involved in regulating the expression of E-cadherin.3.Nestin,particularly,coexpression of nestin in cytoplasm and nucleus might be regarded as a valuable target for clinical therapies of GAC.Part 3 SiRNA-mediated silencing of the nestin gene suppress invasion,metastasis and the expression of EMT-related proteins of human gastric adenocarcinoma cells in vitroMethods1.To detect the expression of nestin in GAC cell lines.1)To detect the expression of nestin in GAC cell lines(MKN28,AGS,SGC7901 and MGC803)and normal gastric muosa cell GES-1 by Q-PCR,ICC and WB.2)3 pairs of siRNAs which targeting human nestin mRNA were designed and synthesized by RiboBio.Then,transfect them into MKN28 and AGS respectively.Western blot help to choose the best siRNA which can efficiently silience nestin expression.2.To study the changes of invasion,metastasis ability and the expression of EMT-related proteins of human gastric adenocarcinoma cells in vitro.The changes of migrate ability and invasion potentiality of GAC cells after siRNA transfection were detected by wound scratch assay,transwell and boyden test,respectively.Additionally,WB was performed to display the changes of EMT-related proteins expression after nestin silenced.Results1.Q-PCR、ICC、WB showed that the expression of nestin was higher expressed in GAC cell lines than in Ges-1(the normal gastric mucous cell line).Combined with the cellular activities,we finally chose the MKN28 and AGS which showed a higher nestin expression to perform the following experiments.2.MKN28 and AGS were seeded for 12h and transfected with the riboFECTTM CP Transfection Kit(166T)(RiboBio)according to the manufacturer’s instructions.WB confirmed that siRNA-002 was the best one to inhibit the nestin expression.3.Wound scratch assay and transwell test showed that siRNA-mediated silencing of the nestin gene suppress the ability of migration of MKN28 and AGS cells;Results from boyden test indicated that the number of invasion cells were largely reduced after nestin down regulation(P<0.001).Those results above suggested that the ability of invasion and metastasis of GAC cells was reduced after nestin knockdown.4.Western blot indicated that cells infected with nestin shRNA lentivirus exhibited increased E-cadherin expression but reduced Vimentin and Snail expression,which suggested that nestin might induce the EMT in GAC cells.ConclusionsSiRNA-mediated silencing of the nestin gene can suppress the invasion,metastasis and the expression of EMT-related proteins of human gastric adenocarcinoma cells in vitro.