| Esophageal cancer is one of the most predominant cancers in the world, the prognosis of which is very poor. Although surgery is potential curative for early stage esophageal cancer, metastasis and recurrence is still an inevitable outcome for most patients presenting with advanced disease. Therefore, seeking new potential therapeutic targets of esophageal squamous cell carcinoma (ESCC) will help to improve the clinical outcome of patients with ESCC.Cadherins, a class of transmembrane proteins, play an important role in intercellular adhesion. E-cadherin and N-cadherin are two important factors which have been extensively studied. Previous studies have demonstrated that N-cadherin is expressed in more aggressive tumor cell lines which lack E-cadherin expression, suggesting that N-cadherin may play a pivotal role in tumor progression. Recent studies on prostate cancer and breast cancer showed that N-cadherin is associated with tumor invasion and may contribute to tumor progression. In addition, N-cadherin is also involved in angiogenesis and tumor metastasis. Beyond its role stimulating cell migration, N-cadherin also promotes cell growth and survival by suppressing apoptotic signals. Numerous studies thus support a role for N-cadherin in the suppression of apoptosis.In the present study, it is the first time to explore the expression patterns of N-cadherin and E-cadherin in ESCC, and to investigate the potential functions of N-cadherin in the proliferation, cell cycle, apoptosis and invasiveness of ESCC cell line (EC9706), discussing the roles N-cadherin plays in the development and progression of ESCC.Part I:Clinical pathological significance of E-cadherin and N-cadherin expressions in esophageal squamous cell carcinomaMethods:1. PV immunohistochemistry and RT-PCR were used to detect the expression patterns of E-cadherin and N-cadherin in 62 normal esophageal epithelium specimens, 31 adjacent atypical hyperplasia epithelium specimens and 62 esophageal squamous cell carcinoma specimens.2. Statistical treatment:Statistical analysis was made using one way ANOVA or paried-samples t test. Theχ2 test and Spearman rank correlation coefficient analysis were used to assess the univariate association between the immunohistochemical status and the clinicopathological characteristics. Results were expressed. as mean±standard deviation (SD). Statistical analyses were performed using the SPSS 13.0 package and A P value<0.05 was considered statistically significant.Results:1. The positive rates of E-cadherin protein and relative expression value of E-cadherin mRNA decreased by turns in normal esophageal epithelium, adjacent atypical hyperplasia epithelium and esophageal squamous cell carcinoma (ESCC) specimens which were 95.2%,71.0%,40.3%(P<0.05)and 0.576±0.043,0.421±0.083, 0.259±0.094 (P<0.05), respectively, while those of N-cadherin increased in sequence, which were 29.0%,61.3%,75.8%(P<0.05) and 0.154±0.019,0.461±0.024, 0.663±0.016(P<0.05).2. E-cadherin expression was negatively correlated to N-cadherin expression in ESCC (γ=-0.534, P<0.05).3. Both positive expression of N-cadherin and negative expression of E-cadherin were associated with the invasion, differentiation, and lymph node metastasis of ESCC (P<0.05), but not with sex and age (P>0.05). Part II:Effect of N-cadherin Knock-down on the Biological Behavior of EC9706 cell in vitroMethods:1. N-cadherin siRNA was transfected into EC9706 cells to inhibit the expression of N-cadherin. The effect of RNAi was detected by RT-PCR and Western blots.2. RT-PCR and Western blots were employed to examine the expression levels of the E-cadherin and MMP-9 in the transfected EC9706 cells.3. MTT assay, Flat plate colony formation assay, Flow cytometric analysis and Transwell chamber assay were employed to investigate the potential functions of N-cadherin on the proliferation, cell cycle, apoptosis and invasiveness of EC9706 cells.4. Statistical treatment:Statistical analysis was made using one way ANOVA or paried-samples t test. Results were expressed as mean±standard deviation (SD). Statistical analyses were performed using the SPSS 13.0 package and A P value <0.05 was considered statistically significant.Results:1. The expression levels of N-cadherin mRNA and protein decreased in the EC9706 cells after transfected by N-cadherin siRNA(P<0.05), which indicated that RNAi was effective.2. The expression levels of MMP-9 mRNA and protein decreased in the N-cadherin knock-down cells(P<0.05). However, it seems that N-cadherin siRNA did not affect the expression levels of E-cadherin mRNA and protein(P>0.05).3. The cell growth rate decreased significantly in the N-cadherin shRNA group, compared to that in untreated and control vector groups (P<0.05).4. The colony-forming efficiency decreased evidently in the N-cadherin shRNA group(16.42%±4.86%), compared to that in untreated(54.58%±5.54%) and control vector(57.50%±6.25%) groups (P<0.05).5. There were more cells blocked at G0/G1 phase in N-cadherin shRNA group(76.1%±1.54%) comparing with untreated(48.6%±1.43%) and control vector(50.1%±1.36%) groups(P<0.05).6. There was no prominent difference in untreated(5.13%±0.71%) and control vector(5.93%±0.80%) groups in the early apoptotic rate of EC9706 cells (P>0.05); however, the early apoptotic rate of N-cadherin shRNA group(26.37%±1.10%) versus untreated and control vector groups significantly increased (P<0.05).6. The numbers of migrated cells in the transwell chamber assay decreased evidently in the N-cadherin shRNA group(49.60±6.80), compared to that in the untreated(123.40±8.23) and control vector(126.00±10.30) groups(P<0.05).PartⅢ:Effect of N-cadherin knock-down on the tumor formation of EC9706 cells in nude miceMethods:1. EC9706 cells from untreated EC9706 cells, control vector and N-cadherin shRNA groups were inoculated (1×106 cells/animal) subcutaneously into the right flank of BalBC mice (five for each group). When tumors became palpable, their diameters were measured with a caliper each week after subcutaneous implantation, and tumor volume (mm3) and weight (g) were calculated.2. PV immunohistochemistry and Western blots were employed to examine the. expression levels of E-cadherin, N-cadherin, MMP-9 in tumor tissues.3. TUNEL method was employed to analysis the cell apoptosis of tumor tissues in 3 groups of nude mice.4. Statistical treatment:Statistical analysis was made using one way ANOVA or paried-samples t test of SPSS 13.0. Results were expressed as mean±standard deviation (SD). AP value<0.05 was considered statistically significant.Results:1. There was an obviously decrease in the volumes and weights of tumors in the N-cadherin shRNA group, compared to that in the untreated and control vector groups (P<0.05).2. E-cadherin expression was not affected, there was no difference in three groups (P>0.05), whereas, N-cadherin and MMP-9 expressions in N-cadherin shRNA group were apparently reduced, compared to that in untreated and control vector groups (P<0.05).3. The positive number of cell apoptosis obviously increased in the N-cadherin shRNA group(106.81±6.47), compared to that in untreated(51.55±4.68) and control vector(54.17±5.26) groups (P<0.05).Conclusions:1. Both positive expression of N-cadherin and negative expression of E-cadherin were associated with the invasion, differentiation, and lymph node metastasis of ESCC, suggesting that down-regulation of E-cadherin expression and up-regulation of N-cadherin expression may be involved in tumorgenesis of ESCC and reflect the invasion and metastasis of ESCC to a certain extent.2. E-cadherin expression was negatively correlated to N-cadherin expression in ESCC, suggesting that they may undergo a switch from E-to N-cadherin expression.3. Down-regulation of N-cadherin could inhibit the proliferation of EC9706 cells by arresting cell cycle at G0/G1 phase and inducing cell apoptosis.4. N-cadherin knock-down could reduce the invasiveness of EC9706 cells in vitro, by the mechanism of decreasing the MMP-9 expression. The MMP-9 reduction resulted in the less degradation of ECM, and thereby, the cancer cells were less aggressive.5. N-cadherin knock-down could inhibit the tumor formation of EC9706 cells in nude mice, which indicate that N-cadherin is an important factor in the progression and metastasis of ESCC and N-cadherin may serve as a potential molecular target for biotherapy of ESCC. |
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