| Esophageal cancer is one of the most common gastrointestinal malignant tumors throughout the world, mainly of which is esophageal squamous cell carcinoma (ESCC). The vast majority of ESCC deaths result from carcinoma cell invasion and metastasis, so understanding the underlying mechanisms is a key to cut the carcinoma deaths and important in improving current anticancer therapy.EMT, namely epithelial-mesenchymal transition, refers to a process in which epithelial cells lose their apical-basolateral polarity and cell junctions, remodel cytoskeleton, switch from polarized epithelial phenotype to a spindle-shaped, fibroblast-like mesenchymal phenotype under some physiological or pathological factors. During the process of EMT carcinoma cells would be endowed with invasive and metastatic properties. EMT has the the following features:epithelial markers such as E-cadherin and CK are down regulated; mesenchymal cells sources of proteins expression are up regulated, such as Vimentin, a-smooth muscle actin(a-SMA) and fibronectin;cells have prominent morphological changes, switching from the cobble kind of epithelial phenotype to a spindle-shaped, fibroblast-like mesenchymal phenotype which make the cells have the ability to migrate and have mesenchymal morphology and properties, and more conducive to cells to through the extracellular matrix. EMT is a critical step in embryonic development, as well as an important mechanism in the invasion and metastasis of tumors. Signal transducers and activators of transcription 3 is a kind of protein existing in cytoplasm, coupling with tyrosine phosphorylation signal pathway.It is one of the members of signal transducers and activators of transcription (STAT) family of transcriptional factors, which acted as an acute-phase response factor to IL-6 in liver cells in 1994.A variety of cytokines such as growth factors and cancer proteins can induce Stat3 to active Stat3. Researches suggest that Stat3 is an important transcription factor in the process of EMT, but its mechanism is unclear.A Research shows that Stat3 plays an important role in EMT though regulating the expression of its downdream genes such as Snail and Twist.However, whether Stat3 plays an important role in EMT in esophageal squamous cell carcinomas (ESCC) through Snail has hardly no reported.Our group has proved that Stat3 is related with EMT in ESCC. In order to detect whether Stat3 can induce EMT through Snail, the experiment is focused on the following research.1) We used immunohistochemical method to detect the expression level of Snail in ESCC tissue, besides we analyzed the relevancy between Snail, activated Stat3 and E-cadherin, Vimentin; 2) We surveyed the levels of activated Stat3 and Snail, E-cadherin, Vimentin with RT-PCR and Western-blot in human esophageal squamous carcinoma cell line EC-1 which were transfected with pXJ40-Stat3-Flag and activated by IL-6; 3) We surveyed the levels of Snail, E-cadherin and Vimentin with RT-PCR and Western-blot after the transfection of Snail siRNA in EC-1 which expressed activated Stat3 highly and stably; 4) Inverted microscope, Boyden chamber methods and scratch assay were respectively used to detect cell invasion capacity and migration ability.Materials and methods1. Immunohistochemical method was used to detect the expression of Snail protein in 80 cases of ESCC and corresponding normal esophageal mucosa tissues adjacent to cancers.2. RT-PCR and Western-blot were adopted to examine the expressions of activated Stat3, Snail, E-cadherin and Vimentin mRNA and protein in EC-1 cells which were transfected with pXJ40-Stat3-Flag and activated by IL-6.3. Applying small interfering RNA (siRNA) technique to transfect Snail siRNA into ESCC cell line EC-1 which expressed activated Stat3 stably and highly, in order to specifically silenced Snail gene.4. RT-PCR and Western-blot were adopted to examine the expressions of Snail, E-cadherin andVimentin mRNA and protein in EC-1 cells which expressed activated Stat3 stably and highly at Oh,24h,48h and 72h of treatment of Snail siRNA.5. Boyden chamber in vitro was used to detect the invasion ability of the EC-1 cells which expressed activated Stat3 stably and highly at Oh,24h,48h and 72h of treatment of Snail siRNA.6. The scratch assay in vitro was used to detect the migration ability of the EC-1 cells which expressed activated Stat3 stably and highly at Oh,24h,48h and 72h of treatment of Snail siRNA.7. Statistical analysis:All the dates were analyzed by SPSS 17.0. Enumeration date were expressed by x±s. Using t-test and ANVOA to analyze the mean.The count information calculated the positive rate.Using Chi-square to compare the positive rates, and using Pearson's to analyze the relation of two variable groups. P values less than 0.05 were considered statistically significant.Results1. The positive rate of Snail protein were higher in carcinoma tissues(73.8%) than that in normal tissues (30.0%) P<0.01).The positive rate of Snail in deeper invasion group (79.7%) were higher than that in superficial invasion group 50.0%(P <0.05). The positive rate of expressions in gradeⅢ(95.2%) were significantly higher than that in grade I (47.4%) (P<0.01). The expressions of Snail did not have relationships with gender (P>0.05), age (.P>0.05) and lymph node metastasis (P>0.05).2. The expression of Snail protein was positively correlated to that of Vimentin protein(r=0.241,P<0.05), and was negatively correlated to that of E-cadherin protein(r=-0.301,P<0.05); the expression of activated Stat3 protein was positively correlated to that of Vimentin protein(r=0.321,P<0.05), and was negatively correlated to that of E-cadherin protein(r=-0.506,P<0.05); the expression of Snail protein was positively correlated to that of activated Stat3 (r=-0.396,P<0.01).3. RT-PCR and Western-blot results showed that the levels of activated Stat3 and Snail in the EC-1 cells which were transfected with pXJ40-Stat3-Flag and activated by IL-6 were Stat3:0.72±0.01 vs 0.72±0.03; Snail:0.60±0.02 vs 0.78±0.03, which were higher than that of the cells which were activated by IL-6 (Stat3: 0.57±0.02 vs 0.65±0.01; Snail:0.50±0.04 vs 0.70±0.02) P<0.01), the cells which were transfected with pXJ40-Stat3-Flag (Stat3:0.65±0.01 vs 0.54±0.01; Snail: 0.36±0.02 vs 0.60±0.05) (P<0.01),the cells in empty carrier group (Stat3:0.59±0.03 vs 0.52±0.04; Snail:0.36±0.03 vs 0.58±0.04) (P<0.01) and the cells in blank group (Stat3:0.57±0.04 vs 0.53±0.05; Snail:0.38±0.02 vs 0.57±0.02) (P<0.01); The level of Stat3 mRNA in the cells which were transfected with pXJ40-Stat3-Flag was higher than the cells which were activated by IL-6(.P<0.05), the cells which were transfected with pXJ40-Stat3-Flag (P<0.05) and the cells in empty carrier group (P<0.05); The level of Stat3 mRNA in the EC-1 cells which was activated by IL-6 had no statistically significant with that in empty carrier group and in blank group (P>0.05); The level of activated Stat3 protein in the EC-1 cells which was activated by IL-6 were higher than that of the cells which were transfected with pXJ40-Stat3-Flag (P<0.05),the cells in empty carrier group and blank group (P<0.05).The levels of Snail mRNA and protein in the EC-1 cells which were activated by IL-6 were higher than that of the cells which were transfected with pXJ40-Stat3-Flag(P<0.05),the cells in empty carrier group and blank group (P<0.05).4. RT-PCR and Western-blot results showed that the level of E-cadherin in the EC-1 cells which were transfected with pXJ40-Stat3-Flag and activated by IL-6 were 036±0.03 and 0.43±0.02, which were obviously lower than that of the cells which were activated by IL-6(0.42±0.03 vs 0.51±0.02) (P<0.01), the cells which were transfected with pXJ40-Stat3-Flag(0.53±0.02 vs 0.54±0.04) (P<0.01), the cells in empty carrier group (0.52±0.02 vs 0.54±0.03) (P<0.01), and the cells in blank group (0.51±0.04 vs 0.53±0.02) (P<0.01). The level of E-cadherin in the EC-1 cells which were activated by IL-6 were lower than that of the cells which were transfected with pXJ40-Stat3-Flag (P<0.05), the cells in empty carrier group and blank group (P<0.05); The level of E-cadherin had no statistically significant with the cells which were transfected with pXJ40-Stat3-Flag,the cells in empty carrier group and blank group (P>0.05).5. RT-PCR and Western-blot results showed that the level of Vimentin in the EC-1 cells which were transfected with pXJ40-Stat3-Flag and activated by IL-6 were 074±0.02and 0.74±0.04, which were obviously higher than that of the cells which were activated by IL-6(0.63±0.04 vs 0.60±0.01) (P<0.01), the cells which were transfected with pXJ40-Stat3-Flag (0.54±0.03 vs 0.52±0.02) (P<0.01), the cells in empty carrier group (0.56±0.02 vs 0.49±0.04) (P<0.01), and the cells in blank group (0.56±0.01 vs 0.50±0.03) (P<0.01). The level of Vimentin in the EC-1 cells which were activated by IL-6 were higher than that of the cells which were transfected with pXJ40-Stat3-Flag (.P>0.05), the cells in empty carrier group and blank group (P<0.05); The level of Vimentin had no statistically significant with the cells which were transfected with pXJ40-Stat3-Flag, the cells in empty carrier group and blank group (P>0.05).6. RT-PCR results showed that in the EC-1 cells which expressed Stat3 stably and highly, the expressions of Snail mRNA in Snail siRNA group were obviously lower than that in negative control group, empty carrier group and blank group in 24h,48h,72h, and the expressions of Snail mRNA decreased with different time in Snail siRNA group (24h 0.64±0.03 vs 0.67±0.05,0.67±0.05,0.69±0.02; 48h 0.53±0.03vs 0.69±0.03,0.68±0.03,0.70±0.03; 72h 0.43±0.04 vs 0.68±0.06, 0.65±0.04,0.68±0.03) (P<0.05). With the extension of the transfection time, the expressions of E-cadherin mRNA in Snail siRNA group were obviously higher than that in control groups in 24h,48h,72h,and the expressions of E-cadherin mRNA increased with different time in Snail siRNA group (24h 0.46±0.03 vs 0.39±0.06, 0.39±0.04,0.40±0.04; 48h 0.53±0.04 vs 0.40±0.03,0.38±0.03,0.39±0.03; 72h 0.62±0.02 vs 0.36±0.02,0.35±0.03,0.38±0.03) (P<0.05). With the extension of the transfection time, the expressions of Vimentin mRNA in Snail siRNA group were obviously lower than that in control groups in 24h,48h,72h (P<0.05),48h and 72hVimentin mRNA were obviously lower than 24 h (P<0.05), but there was no statistically significant in 48h and 72h (P>0.05) (24h 0.69±0.04 vs 0.77±0.05, 0.78±0.07,0.80±0.06; 48h 0.60±0.05 vs 0.79±0.03,0.79±0.04,0.77±0.03; 72h 0.60±0.06 vs 0.78±0.05,0.76±0.03,0.79±0.05)7. Western-blot results showed that in the EC-1 cells which expressed Stat3 stably and highly, the expressions of Snail protein in Snail siRNA group were lower than that in the three control groups in 24h,48h,72h,and the expressions of Snail protein decreased with different time in Snail siRNA group (24h 0.68±0.03 vs 0.73±0.03,0.73±0.02,0.72±0.04; 48h 0.61±0.03 vs 0.72±0.02,0.74±0.03,0.70±0.01; 72h 0.53±0.02 vs 0.71±0.02,0.69±0.03,0.70±0.02) (P<0.05). With the extension of the transfection time, the expressions of E-cadherin protein in Snail siRNA group were higher than that in the three control groups in 24h,48h,72h, and the expression of E-cadherin protein in Snail siRNA group in 72h was the highest (24h 0.61±0.02 vs 0.51±0.08, O.51±0.05,0.50±0.04; 48h 0.63±0.04 vs 0.48±0.04,0.54±0.03, 0.48±0.02; 72h 0.74±0.02 vs 0.52±0.04, 0.53±0.03,0.54±0.04) (P<0.05).With the extension of the transfection time, the expressions of Vimentin protein in Snail siRNA group were lower than that in the three control groups in 24h,48h,72h,and the expressions of Vimentin protein decreased with different time in Snail siRNA group (24h 0.59±0.02 vs 0.62±0.02,0.64±0.02,0.63±0.03; 48h 0.52±0.02 vs 0.64±0.02, 0.63±0.02,0.62±0.02; 72h 0.43±0.02 vs 0.63±0.01,0.64±0.04,0.60±0.02) (P<0.05).8. Boyden chamber showed that in the EC-1 cells which expressed Stat3 stably and highly, the number of cells transfected with Snail siRNA which passed through Matrigel gum decreased with the extension of the time, compared with the control groups (Oh 107.33±7.37 vs 107.67±5.51,111.67±11.02,108.50±9.16; 24h 92.67±5.46 vs1 10.66±11.37,113.23±1.15,110.33±3.06; 48h 68.33±5.69 vs 114.00±3.61, 111.34±4.93,107.00±16.82; 72h 53.67±7.77 vs 112.66±3.51,107.06±1.53, 104.33±7.09) (P<0.05).9. The scratch assay showed that in the EC-1 cells which expressed Stat3 stably and highly, the movement distance of cells transfected with Snail siRNA shrinked with the extension of the time compared with the control groups (48h 0.22±0.01vs0.31±0.02,0.29±0.03,0.30±0.01; 72h0.24±0.01 vs0.42±0.03,0.40±0.04, 0.41±0.05) (P<0.05). Conclusions1. The expressions of Snail were increased in ESCC tissue. Abnormal expression of the Snail gene had close relation to carcinogenesis of ESCC.The expression of Snail protein was negatively correlated to E-cadherin protein, and positively associated with Vimentin protein. Snail may be involved in the process of EMT in ESCC.2. The EC-1 cells which were transfected with pXJ40-Stat3-Flag and activated by IL-6 could up regulate the expression of Snail and Vimentin, and down regulate the expression of E-cadherin. Snail siRNA could inhibited the expression of Snail successfully in ESCC.At the same time it could up regulate the expression of E-cadherin, and down regulate the expression of Vimentin. EMT induced by Snail may be relevant with Stat3. Stat3 may participate in EMT through Snail in ESCC.3. Snail siRNA had significant inhibition effects on the expression of Snail mRNA and protein in the EC-1 cells which expressed active Stat3 stably and highly. It could also inhibit the invasion and movement ability of EC-1 in ESCC. The results showed that Snail siRNA in ESCC could take an active effect in preventing the malignant progress of ESCC, and Snail could be an adjunctive therapy target to ESCC. |
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