The Effect Of NF-κBp65 On EMT In Esophageal Squamous Cell Carcinoma

The epithelial-mesenchymal transition (EMT) is a fundamental process governing morphogenesis in multicellular organisms. This process is also reactivated in a variety of diseases including fibrosis and in the progression of carcinoma. The molecular mechanisms of EMT were primarily studied in epithelial cell lines, leading to the discovery of transduction pathways involved in the loss of epithelial cell polarity and the acquisition of a variety of mesenchymal phenotypic traits. Turning an epithelial cell into a mesenchymal cell requires alterations in morphology, cellular architecture, adhesion, and migration capacity. Commonly used molecular markers for EMT include increased expression of N-cadherin and Vimentin, nuclear localization ofβ-catenin, and decreased expression of epithelial markers such as E-cadherin and Cytokeratin.Nuclear transcription factor-kappa B (NF-κB) is activated in many human tumors. The NF-κB family can form various homo- or hetero-dimers. However, the most studied from is a heterodimer of the p50 and p65 submits predominant in many kinds of cells. Constitutively activated NF-κB has been implicated in survival, adherence, transformation and proliferation of tumor cells. Activation of NF-κB has been demonstrated in many cancers including hepatocellular, colonic and cervical cancers.Studies show that NF-κB plays an essential role in the induction of EMT in mammary epithelial cells. Loss of NF-κB activity abrogated protection of these cells from TGF-β-induced apoptosis, but also actively suppressed an EMT gene program, thus blocking EMT. In contrast, gain of NF-κB activity induced EMT in these cells in the absence of TGF-β. Moreover, NF-κB also plays an essential role in maintaining the mesenchymal state subsequent to EMT, as its inhibition caused reversal of EMT.Esophageal cancer is one of the most frequently diagnosed cancers ranking the sixth most common cause of death from cancers in the world. In this study, to detect whether EMT occurs in esophageal squamous cell carcinoma (ESCC), paraffin-embedded biopsies from 100 esophageal squamous cell carcinoma cases and ESCC cell line EC9706 were tested for Vimentin and E-cadherin proteins expression. In vitro studies, antisense oligodeoxynucleotides of NF-κBp65 were used to inhibit the NF-κB signaling pathway in ESCC cell line EC9706. To judge the relationship between NF-κB signaling pathway and the EMT, we detected the changes of Vimentin and E-cadherin mRNA and protein expressions. The study also showed that the antisense oligodeoxynucleotides suppressed the EC9706 cells proliferation, promoted the cells apoptosis and decreased the migration capacity of EC9706 cells. Our findings will provide theory bases for the opinion that NF-κB signaling pathway could be considered potential therapeutic target for ESCC, and offer the experiment basis for antisense oligodeoxynucleotides acting as gene agent and offer theory basis for gene therapy on ESCC.Methods:1. Paraffin-embedded biopsies from 100 esophageal squamous cell carcinoma cases were tested for Vimentin and E-cadherin proteins expression by immunohistochemistry(IHC).2. In the EC9706 cells, detect the NF-KBp65, E-cadherin and Vimentin expressions in mRNA and protein level by RT-PCR method, immunocytochemistry(ICC) and flow cytometry(FCM) methods.3. Designed antisense oligodeoxynucleotides (ASODN) according to the sequence of NF-κBp65 transcription initiation region, also synthesis the sense oligodeoxynucleotide (SODN) and nonsense oligodeoxynucleotide (NSODN). The ODNs were transfected into the EC9706 cells with the help of positive ion polymer.3.1 After the transfection, we observed the appearance change of EC9706 cells.3.2 72 hours after the transfection, using the immunocytochernistry and flow cytometry to detect the expressions of E-cadherin Vimentin and NF-κBp65 proteins. And their changes in mRNA level were detected by RT-PCR.3.3 The mobility and invasion of EC9706 were evaluated by wound-healing assay.4. The proliferation of EC9706 cells were observed by MTT assay; cell cycle and apoptosis were determined by flow cytometry.5. Statistical analysis: All the dates were analyzed by SPSS 13.0 statistical package, the count information calculated the positive rate, and enumeration dates are expressed by standard deviation [x|-±s]. The comparison of positive rates uses the Chi-square, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable groups is analyzed by the Kendall correlation analysis. The level of significant difference isα=0.05.Result:1. The positive rates of E-cadherin protein were significantly higher in normal mucosa tissues (85/100) than in cancer tissues (43/100)(P<0.01). The positive rates of Vimentin protein were significantly lower in normal mucosa tissues(0/20) than in cancer tissues (23/100) (P<0.05) . There were negative correlations between the positive rate of E-cadherin protein expression and that of Vimentin protein expression in cancer tissues.2. In the EC9706 cells, NF-κBp65, E-cadherin and Vimentin were expressed in mRNA (NF-KBp65: 0.448±0.045; E-cadherin: 0.218±0.065; Vimentin: 0.893±0.089)and protein level: NF-κBp65(ICC: 17.15±1.78%; FCM: 15.76%) E-cadherin (ICC: 14.44±1.65%; FCM: 12.22%) and Vimentin (ICC: 19.52±1.07%; FCM: 17.76%) . 3. After the transfection for 72h, the NF-κBp65 mRNA level of ASODN group(0.135±0.060) was obviously lower than the EC9706 group (0.448±0.045 ) (P<0.01). The result of immunocytochemistry and flow cytometry showed the positive rate of NF-KBp65 (ICC: 11.35±1.32%; FCM: 11.78%) protein expression in ASODN group was depressed compared with the EC9706 cell(ICC: 17.15±1.78%; FCM: 15.76%) (P<0.01) .4. Compared with the other control group (E-cadherin: 0.218±0.065; Vimentin: 0.893±0.089 ) , the E-cadherin mRNA level in the ASODN group (0.360±0.084 ) was higher (P<0.01) , and the Vimentin mRNA (0.753±0.066) was lower(P<0.01) .Using the immunocytochemistry and flow cytometry to detect the protein expression of E-cadherin and Vimentin in these groups, we got the similar result.5. By the wound-healing assay, the migration capacity of cells in ASODN group(48h: 0.32±0.06,72h: 0.45±0.08)was weaker than the EC9706(48h: 0.69±0.12, 72h: 0.81±0.11) (P<0.01) .6. In the ASODN group, the apoptosis rate( 1.38% ) was obviously higher than those in the other groups (0.96% ) (P<0.01). There were more G1 phase cells (78.5%) in the ASODN group (P<0.01) .Conclusion:1. Compared with normal mucous membrane tissue, the E-cadherin protein expression was lower in esophageal squamous carcinoma. And the Vimentin expression was higher. The result showed that the epithelial-mesenchymal transition (EMT) may exist in carcinogenesis and development of esophageal squamous cell carcinoma.2. The NF-KBp65 ASODN can be used as an experimental tool with which to suppress specific p65 expression.3. In the EC9706 cells transfected with NF-κBp65 ASODN, the expressions of E-cadherin increased in mRNA and protein level. And the expressions of Vimentin decreased. The migration capacity of cells in ASODN group was depressed. Our studies showed that the NF-κBp65 ASODN could participate the EMT progress in ESCC cell line EC9706.4. Transfection of NF-κBp65 ASODN leads to suppression of proliferation, induction G1 arrest and induction of apoptosis in ESCC cell line EC9706.