Investigation Of The Relationship Between Expression Of E-cadherin And Chemotherapy Effect And Its Influence On Chemo-sensitivity In Gastric Cancer

Gastric cancer is a common malignant tumor in China, and its morbidity andmortality is still at a high level, seems to be an upward trend in the incidence ofgastric cancer in rural areas and some cities. Because symptoms of gastric cancer andbenign stomach disease is difficult to distinguish, the early gastric cancer is not easyto be found, and usually diagnosed in late and unresectable stage, thus chemotherapyhas become the most important method of non-surgical treatment.In recent years,despite the progress, but gastric cancer chemotherapy efficiency is low, the majorityof patients can not benefit from it. Therefore, predict gastric cancer response tochemotherapy, individualized choose effective strategies to help improve the survivalof groups of patients with gastric cancer marker studies can provide useful help forclinical decision making.The tumor markers achieved rapid development in recent years, related to avariety of marker expression in gastric cancer and gastric cancer chemotherapyefficacy. E-cadherin is a key to the epithelial cell adhesion molecule, its abnormalexpression is closely related to the invasion and metastasis of gastric cancer, may beinvolved in the occurrence of drug resistance, and its abnormal expression may berelated to the sensitivity of tumor cells to chemotherapy.In this study, usingimmunohistochemical methods to detect the expression of E-cadherin in42cases of gastric carcinoma, observed objective response in patients with FOLFOX4chemotherapy, made analysis of E-cadherin positive expression with chemotherapyefficacy, clinical pathological features and survival correlation.E-cadherin become a protein of molecular weight of80kDa after degradation,which was later found to be soluble E-cadherin (sE-cad). This protein can be releasedinto the culture medium, inhibit the function of E-cadherin affects cell growth andmetastasis characteristics and chemosensitivity in serum and urine of cancer patients,is able to detect high levels of sE-cad expression tumor cells,showing that sE-cad andtumorigenesis are closely related.In this study, before chemotherapy with FOLFOX4chemotherapy efficacy and survival with chemotherapy in the first eight weeks ofgastric cancer patients serum sE-cad expression levels were detected by ELISA.RNAi is a new technology developed a method for inhibiting gene expressionusing RNA mediated specifically block and reduce the expression of the target genein recent years, which is not only easy to operate, but the effect is good and stable,and has been widely used in molecular biology study, which has become a good toolto study the role of the gene in the tumor. We observed lentiviral siRNA-mediatedinhibition of E-cadherin expression in gastric cancer SGC-7901cells, cell apoptosisinduced by5-FU before and after the suppression and the possible molecularmechanisms.This study includes the following four parts：Part I：The expression of E-cadhein in tissues and sE-cad inserum in patients with advanced gastric cancerMethods1. The expression of E-cadherin protein in gastric cancer tissues was detected byimmunohistochemistry.2. The levels of sEC in serum of gastric cancer patients were tested by enzymelinked immunosorbent assay(ELISA).3. The correlations between the clinical features of patients and the expression of E-cadherin in the gastric cancer tissue were analyzed．4. The correlations between the clinical features of patients and the expression ofsE-cad in serum of gastric cancer patients were analyzed．5. The correlations between expression of E-cadherin in tissues and sE-cad in serumof gastric cancer patients were analyzed．6. Statistical analysis were performed using the SPSS17.0package．Measurementdata using t test, using χ2test count data. All tests of statistical significance weretwo-sided and P<0.05was considered as statistically significant difference．Results1. E-cadherin main positive expression in gastric cancer cell membrane, positive ratefor42.86%.2. E-cadherin positive expression was associated with age, pathologic gradesignificantly, but not with sex, clinical stage, primary tumor site.3. Prior to chemotherapy in patients with gastric cancer serum sE-cad levelssubstantially higher than the healthy control group.4. Expression of serum sE-cad levels and clinical stage, pathological grade, gender,age, primary tumor site were not significantly related.5. Expression of E-cadherin protein expression in gastric cancer tissue and serumsE-cad showed no significant correaltion.Part II: Relationship between E-cadherin expression in humangastric cancer and chemotherapy tumor responseMethods1. The expression of E-cadherin protein in gastric cancer tissues was detected byimmunohistochemistry.2. Chemotherapy was performed for42patients with gastric cancer using threecycles of FOLFOX4regimen, tumor response adverse effect were observed, andsurvival was also followed up.3. Comparative analysis of E-cadherin positive rate correlation with chemotherapy. 4. Comparative analysis of E-cadherin positive relations with the PFS and OS.5. Statistical analysis were performed using the SPSS17.0package．Using χ2testcount data, measurement data using t test. Using Kaplan-Meier method forsurvival analysis. All tests of statistical significance were two-sided and P<0.05was considered as statistically significant difference．Results1. E-cadherin positive expression appears significant correlation with highereffective rates of chemotherapy by FOLFOX4regimen.2. Expression of E-cadherin-positive patients appear good median PFS comparingwith E-cadherin-negative ones, but no significant difference.3. Expression of E-cadherin-positive patients appear good median OS comparingwith E-cadherin-negative ones, but no significant difference.Part Ⅲ: Relationship between sE-cad expression in serum ofgastric cancer patients and chemotherapy tumor responseMethods1. ELISA was used to determinate serum levels of sE-cad expression in gastriccancer patients before chemotherapy and at8th week.2. Chemotherapy was performed for42patients with gastric cancer using threecycles of FOLFOX4regimen, tumor response adverse effect were observed, andsurvival was also followed up.3. Comparative analysis of serum expression and Clinical pathological features ofsE-cad before and after chemotherapy.4. Comparative analysis of changes of serum sE-cad and PFS and OS before andafter chemotherapy.5. Statistical analysis were performed using the SPSS17.0package．Using χ2testcount data, measurement data using t test, including paired sample t test andindependent sample t test. Using Kaplan-Meier method for survival analysis. Alltests of statistical significance were two-sided and P<0.05was considered as statistically significant difference．Results1. Before and after chemotherapy compared to the8th week, serum levels of sE-cadexpression did not show significant change.2. sE-cad changes before and after chemotherapy in patients with Ⅱ/Ⅲ stage weresignificantly different.3. Effective group expression of serum sE-cad levels declined significantly afterchemotherapy compared with before chemotherapy, progress group performanceto higher serum levels of sE-cad expression after chemotherapy compared withbefore chemotherapy.4. Proportions of sEC levels decline or increase between effective group afterchemotherapy and invalid group showed significantly difference.5. sE-cad expression before chemotherapy between valid group and invalid groupshowed significant statistical differences.6. Serum levels of sE-cad expression in patients with PFS≥4.6months droppedsignificantly after chemotherapy, PFS<4.6months in patients with no significantchange.7. Serum levels of sE-cad expression in patients with OS≥9.6months droppedsignificantly after chemotherapy, PFS<4.6months in patients with no significantchange.Part IV: Lentivirus-mediated E-cadherin gene silencing effecton the proliferation and apoptosis of gastric cancer cell lineSGC-7901Methods1. Lentivirus-mediated siRNA-CDH1infected gastric cancer cell line SGC-7901,RT-PCR and Western blot was used to detect E-cadherin in mRNA and proteinexpression.2. MTT was used to detect the cell proliferation and inhibition rates of SGC-7901 cell before and after E-cadherin down-regulated.3. Flow cytometry was employed to observe the cell cycle and apoptosis rate beforeand after E-cadherin down-regulated.4. ELISA was used to determinate cell culture medium levels of sE-cad expressionbefore and after E-cadherin down-regulated.5. Statistical analysis were performed using the SPSS17.0package. Measurementdata was analyzed using t test. All tests of statistical significance were two-sidedand P<0.05was considered as statistically significant difference．Results1. Lentivirus-mediated E-cadherin interference could inhibit E-cadherin gene andprotein expression in gastric cancer cell line SGC-7901.2. Down-regulation of E-cadherin significantly decreased inhibition rates of5-FU onSGC-7901cell, effective inhibiting concentration increased.3.5-FU inducing apoptosis rate of SGC-7901cell decreased after Down-regulationof E-cadherin by lentivirus-mediated E-cadherin interference.4. Increasing expression of bcl-2mRNA and decreasing expression of bax mRNAwere found in SGC-7901cell after down-regulation of E-cadherin with nosignificant difference, which reached a significant difference when5-FU inducingapoptosis.5. sE-cad expression level decreased in cell culture medium after E-cadherin wasdown-regulated.CONCLUSIONS1. E-cadherin positive expression was associated with age, pathologic gradesignificantly, but not with sex, clinical stage, primary tumor site.2. Serum sE-cad levels expressed higher in gastric cancer group than the healthycontrol group, with no significant correlation with the clinical features of patients.3. Expression of E-cadherin protein expression in gastric cancer tissue and serumsE-cad showed no significant correaltion.4. E-cadherin expression and chemotherapy associated with chemotherapy significantly higher effective rates of e-cadherin expression, with no significantassociation with median PFS and OS comparing with E-cadherin-negative one.5. Effective group expression of serum sE-cad levels declined significantly afterchemotherapy compared with before chemotherapy, progress group performanceto higher serum levels of sE-cad expression after chemotherapy compared withbefore chemotherapy. Higher sE-cad expression before chemotherapy showedbetter effect and good survival.6. Down-regulation of E-cadherin significantly decreased inhibition rates of5-FU onSGC-7901cell, effective inhibiting concentration increased.7.5-FU inducing apoptosis rate of SGC-7901cell decreased after Down-regulationof E-cadherin by lentivirus-mediated E-cadherin interference, with an increasingexpression of bcl-2mRNA and decreasing expression of bax mRNA.8. sE-cad expression level decreased in cell culture medium after E-cadherin wasdown-regulated.