| According to histocytological characteristics,Lauren divided gastric cancer into intestinal type and diffused type.Intestinal type gastric cancer(ITGC),including papillary adenocarcinoma and tubular adenocarcinoma,is derived from intestinal metaplasia and well differentiated with characteristics of adenosine formation columnar cells,the brush border of free margin similar to intestinal absorptive cell,some cancer cells resembling goblet cell of intestinal metaplasia,and para-carcinoma mucosa accompanied with extensively atrophic gastritis and intestinal metaplasia.Gross morphology of most ITGC is fungating type.Diffused type gastric cancer(DTGC),including undifferentiated carcinoma and mucinous carcinoma,is derived from lamina propria and poor differentiated with characteristics of none adenosine formation,diffused small round cells,poor adhesion between cells and para-carcinoma mucosa without or with only small patches of atrophic gastritis and intestinal metaplasia.Pathogenesis and metastatic mechanism of ITGC and DTGC is different.To clarify detailed molecular pathway is very important for precise therapy,controlling illness and improving prognosis.Epithelial-mesenchymal transition(EMT)is a process where epithelial cells are transformed into cells with mesenchymal phenotypes with enhanced invasive and migratory abilities,characterized by downregulation of epithelial marker of E-cadherin and upregulation of mesenchymal markers including N-cadherin,Fibronectin and Vimentin.Reprogramming of energy metabolism is regarded as the seventh hallmarker of cancer.The most distinctive metabolic phenotype of tumor cells is the Warburg effect.Pyruvate dehydrogenase kinase 1(PDK1),served as inhibitor of pyruvate dehydrogenase,can promote liver metastasis in breast cancer by increasing conversion of glucose-derived pyruvate into lactate.In view of the importance of making full use of Warburg effect to decrease lactate production in tumor microenvironment,it is indispensable to elucidate driver genes in upstream and downstream signal pathway of lactate for more precise and efficacious treatment.Lactate dehydrogenase A(LDH-A)is crucial rate-limiting enzyme responsible for catalyzing pyruvate into lactate irreversibly,which is the final step of the aerobic glycolysis.Sonveaux not only found that lactate is the bedrock of symbiotic relationship between glycolytic and oxidative tumor cells during the course of energy metabolites,but also demonstrated that targeting lactate-fueled respiration can specifically kill hypoxic tumor cells in mice.Considering hypoxia is a significant inducer of gastric cancer EMT,it is therefore reasonable to deduce that LDH-A knockdown in tumors could downregulate EMT.We detected EMT-related factors including Snail?ZEB1?ZEB2?Vimentin.N-cadherin and E-cadherin with the purpose of selecting the factor with the most remarkable change.Part ? LDH-A promotes malignant progression via upregualtion of ZEB2 in intestinal-type gastric cancerObjectives:To investigate if LDH-A can promote invasion and migration of ITGC cells and the related mechanism.Methods:1.Two ITGC cell lines with higher LDH-A expression were selected from MKN28,SGC7901,BGC823 and GES,according to westernblot results.2.Westernblot was applied to detect LDH-A expression after three siRNAs transfection.The siRNA showing the best inhibition of LDH-A expression in cell lines was chosen.3.We detected the mRNA expression of EMT-related genes by real-time RT-PCR 48 hours after transient infection achieved by the siRNA selected from experiment mentioned above.4.We used westernblot to detect the protein expression of genes exhibiting significant change at mRNA level.5.Migration assay and matrigel invasion assay were performed to detect the migratory and invasive ability,respectively.Results:1.Westernblot results indicated that SGC7901 and BGC823 showed higher levels of LDH-A expression.2.Compared with siRNA506 and siRNA980,the siRNA376 exhibited strong suppression of LDH-A expression in both BGC823 and SGC7901 cells and was therefore selected for subsequent experiment.3.To explore whether LDH-A promotes the metastasis of ITGC cells by EMT,we detected the mRNA expression of EMT-related genes including ZEB2,Snail,ZEB1,E-cadherin,N-cadherin,Fibronectin and Vimentin.Real-time RT-PCR results indicated that 48 h after LDH-A knockdown in SGC7901 cells,mRNA expression of ZEB2 and Vimentin was significantly decreased(P<0.001),coupled with remarkable upregulation of epithelial marker E-cadherin(P<0.001).The downregulation of ZEB2 and Vimentin and upregulation of E-cadherin at the protein level was confirmed by westernblot.4.Real-time RT-PCR analysis revealed that 48h after LDH-A knockdown in BGC823 cells,mRNA expression of ZEB2,Vimentin(P<0.001)and Fibronectin(P<0.01)was remarkably decreased,coupled with the upregualtion of epithelial marker E-cadherin(P<0.001).The downregulation of ZEB2 and Vimentin and upregulation of E-cadherin at the protein level was confirmed by westernblot.5.LDH-A knockdown significantly decreased migratory and invasive ability in both SGC7901 and BGC823 cells.Conclusions:LDH-A promotes migratory and invasive ability of ITGC cells via activation of ZEB2,suggesting a possible correlation between LDH-A/ZEB2 and ITGC prognosis.Part ? The relationship of LDH-A/ZEB2 coexpression with ITGC prognosisObjectives:To investigate the expression of LDH-A and ZEB2 in intestinal-type gastric cancer,the association of LDH-A with ZEB2,Vimentin,clinicopathological parameters,and the correlation between combined expression of LDH-A and ZEB2 and overall survival(OS).Methods:According to immunohistochemistry outcome,we performed statistical analysis of 371 ITGC specimens.All patients had follow-up information for 5 years.The follow-up deadline was March 2010.Survival analysis of OS was assessed.Results:1.A comparative analysis of immunohistochemistry(IHC)results of 371 ITGC specimens indicated that both LDH-A and ZEB2 were differentially upregulated in ITGC tissues compared to adjacent non-cancerous tissues(P<0.001).2.The increased LDH-A expression was significantly related to positive ZEB2(P=0.003),Vimentin(P=0.024),invasion depth(P<0.001)and regional lymph node metastases(P<0.001).We did not find any significant correlations between LDH-A expression and other clinicopathologic variables,including patient age,sex,tumor size and differentiation.3.Univariate analysis indicated that OS of patients with positive LDH-A expression was significantly worse(P<0.001,Log-rank test),so is that of patients with positive ZEB2 expression.The ITGC patients with both LDH-A and ZEB2 positive expression exhibited poorer OS.4.The multivariate Cox regression model indicated that high expression of ZEB2 is an independent prognostic marker for OS(P=0.003).Conclusions:The close relationship between LDH-A and ZEB2 was verified in ITGC specimens.The combination expression of LDH-A and ZEB2 exhibited poorer OS.Thus,the association of LDH-A and ZEB2 provides a potential therapeutic strategy for ITGC. |
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