| BackgroundEsophageal carcinoma is one of the most predominant digestive cancers and is a big threat to human life and health. Esophageal carcinoma includes two main types, squamous cell carcinoma and adenocarcinoma. Unlike in the west, esophageal squamous cell carcinoma(ESCC) is predominant in China. In recent years the early diagnosis and treatment may improve the prognosis, however, the five-year survival rate of patients is still very low, and tumor invasion and metastasis is the major cause of recurrence and mortality in cancer patients. Therefore, a better understanding for the molecular mechanism of tumor invasion and metastasis and discovery of a more effective target for anticancer therapy help to prolong the survival and improve the life quality of patients with esophageal carcinoma.The epithelial-mesenchymal transition(EMT) was initially identified as a foundational process during embryonic development, tissue remodeling and wound healing. More recently, this transition was shown to play a critical role in tumor invasion and metastasis. During EMT, cancer cells undergo a decrease in the expression of epithelial markers like E-cadherin and an increase in the expression of mesenchymal markers like vimentin, as well as a loss of cell adhesion and a gain of cell migration, this contributing to tumor cells invasion and metastasis. EMT is mediated by key transcription factors, including Twist, Snail and zinc finger E-box-binding homeobox(ZEB). ZEB family are of two members, ZEB1 and ZEB2, the function of which is binding to the E-box within E-cadherin gene promoter via the zinc finger structure and suppressing the expression of E-cadherin directly, and further controlling EMT process.Signal transducers and activators of transcription 3(STAT3) is a kind of acute phase response factor(APRF) from interleukin 6(IL-6) signaling transduction process. In the majority of human cancers, STAT3 has been proven constitutively activated and associated with tumor cell proliferation, differentiation, apoptosis, angiogenesis, invasion, metastasis and immune response. More recently, STAT3 signaling pathway controls tumor invasion and metastasis via EMT. In particular, active STAT3 promotes the progression, invasion and metastasis of cancers by modulating EMT-related transcription factors(Twist, Snail, ZEB, etc), inducing EMT phenotype, and influencing cell biological characteristics.However, until now there has no related report about the role and mechanism of STAT3/ZEB1/E-cadherin signaling axis in EMT of ESCC. ObjectiveTo study the role and mechanism of STAT3/ZEB1/E-cadherin signaling axis in EMT of ESCC, we firstly investigate the expression of p-STAT3, E-cadherin, Vimentin and ZEB1 in human ESCC tissues, and analyze the relationship of p-STAT3 with E-cadherin, Vimentin and ZEB1 expression and tumor invasion and metastasis. Secondly, we investigate the effect of STAT3 activation/inhibition by IL-6/STAT3 sh RNA in ESCC cells on the expression of E-cadherin, Vimentin and ZEB1 and cell invasion and metastasis ability. Thirdly, we analyze whether STAT3-induced EMT through ZEB1 by using ZEB1 sh RNA. Fourthly, we detect the binding site of STAT3 and ZEB1 promoter by chromatin immunoprecipitation(Ch IP) and gene sequencing. Finally, we observe the influence of STAT3/ZEB1/E-cadherin signaling axis blockade by STAT3 sh RNA and ZEB1 sh RNA on the growth and EMT of xenograft tumors with ESCC in nude mice in vivo.Part I The significance of STAT3 and EMT-related factors inhuman esophageal squamous cell carcinoma tissues Methods1. The expression of p-STAT3, ZEB1, E-cadherin and Vimentin protein was detected by immunohistochemistry in 90 cases of human ESCC tissues and corresponding normal esophageal mucosa tissues.2. The correlation of p-STAT3, ZEB1, E-cadherin and Vimentin with clinicopathological features including gender, age, histological grade, invasion depth, lymph node metastasis and TNM staging were analyzed in ESCC tissues. Furthermore, the correlation of p-STAT3 protein expression with E-cadherin, Vimentin and ZEB1 were analyzed in ESCC tissues. Results1. In 90 cases of ESCC tissues, the positive rate of p-STAT3 protein(54.44%) was significantly higher than that in the corresponding normal esophageal mucosa tissues(27.78%, P<0.01), and positively correlated with tumor invasion depth(superficial vs deep: 31.82% vs 61.76%), lymph node metastasis(no vs yes: 46.97% vs 75.00%) and TNM staging(I vs II vs III: 34.78% vs 52.00% vs 66.67%)(P<0.05), but not correlated with gender, age and tumor histological grade(P>0.05).2. In 90 cases of ESCC tissues, the positive rate of ZEB1 protein(62.22%) was significantly higher than that in the corresponding normal esophageal mucosa tissues(23.33%, P<0.01), and positively correlated with tumor invasion depth(superficial vs deep: 40.91% vs 69.12%) and lymph node metastasis(no vs yes: 56.06% vs 79.17%)(P<0.05), but not correlated with gender, age and tumor histological grade and TNM staging(P>0.05).3. In 90 cases of ESCC tissues, the positive rate of E-cadherin protein(38.89%) was significantly lower than that in the corresponding normal esophageal mucosa tissues(100.0%, P<0.01), and negatively correlated with tumor invasion depth(superficial vs deep: 68.18% vs 29.41%), lymph node metastasis(no vs yes: 46.97% vs 16.67%) and TNM staging(I vs II vs III: 69.57% vs 28.00% vs 28.57%)(P<0.01), but not correlated with gender, age and tumor histological grade(P>0.05).4. In 90 cases of ESCC tissues, the positive rate of Vimentin protein(33.33%) was significantly higher than that in the corresponding normal esophageal mucosa tissues(0.00%, P<0.01), and positively correlated with tumor lymph node metastasis(no vs yes: 24.24% vs 58.33%)(P<0.01), correlated with tumor histological grade(I vs II vs III: 23.08% vs 44.90% vs 13.33%)(P<0.05), but not correlated with gender, age and tumor invasion depth and TNM staging(P>0.05).5. In ESCC tissues, E-cadherin protein expression was negatively correlated with Vimentin(?s=–0.226, P<0.05); p-STAT3 protein expression was negatively correlated with E-cadherin(?s=–0.277, P<0.01), but positively correlated with Vimentin(?s=0.221, P<0.05); ZEB1 protein expression was negatively correlated with E-cadherin(?s=–0.507, P<0.01), but not correlated with Vimentin(?s=0.065, P>0.05); p-STAT3 protein expression was positively correlated with ZEB1(?s=0.300, P<0.01). Part II The effect of STAT3/ZEB1/E-cadherin regulation on EMTand invasion and metastasis in human esophageal squamous cellcarcinoma cells Methods1. The expression of STAT3 m RNA and p-STAT3 protein were detected by real-time PCR and western blot in EC-1, Eca109, EC9706, TE-1 ESCC cells and normal esophageal epithelial cells(NEECs).2. After TE-1 cells with low STAT3 expression were treated with IL-6, the m RNA and protein levels of STAT3, E-cadherin, Vimentin and ZEB1 were detected by real-time PCR and western blot, the morphological alterations of cells were observed under an inverted phase contrast microscope, and cell motility and invasiveness were detected by Matrigel invasion assay and scratch wound healing assay.3. After EC-1 and Eca109 cells with high STAT3 expression were stably transfected with STAT3 sh RNA, the m RNA and protein levels of STAT3, E-cadherin, Vimentin and ZEB1 were detected by real-time PCR and western blot, the morphological alterations of cells were observed under an inverted phase contrast microscope, and cell motility and invasiveness were detected by Matrigel invasion assay and scratch wound healing assay.4. After EC-1 and Eca109 cells with high STAT3 expression were stably transfected with ZEB1 sh RNA, the m RNA and protein levels of ZEB1, E-cadherin, and Vimentin were detected by real-time PCR and western blot, cell motility and invasiveness were detected by Matrigel invasion assay and scratch wound healing assay.5. After EC-1 and Eca109 cells with stable ZEB1 sh RNA transfection were treated with IL-6, the m RNA and protein levels of E-cadherin and Vimentin were detected by real-time PCR and western blot, cell motility and invasiveness were detected by Matrigel invasion assay and scratch wound healing assay.6. The binding site of p-STAT3 and ZEB1 promoter was detected by Ch IP and analyzed by gene sequencing. Results1. The expression of STAT3 m RNA and p-STAT3 protein in ESCC cells including EC-1(5.71±0.45, 0.68±0.09), Eca109(5.11±0.33, 0.65±0.06), EC9706(5.47±0.38, 0.46±0.07) and TE-1(4.46±0.33, 0.37±0.06) were significantly higher than those in NECCs(1.00±0.00, 0.19±0.08)(P<0.01).2. After TE-1 cells with low STAT3 expression were treated with IL-6, compared with blank control cells, STAT3 Mrna(2.75±0.36 vs 1.00±0.00) and p-STAT3 protein(0.56±0.09 vs 0.35±0.07) were significantly activated(P<0.05), and moreover, cells underwent EMT morphological alterations, E-cadherin m RNA(0.51±0.08 vs 1.00±0.00) and protein(0.38±0.10 vs 0.59±0.09) were significantly downregulated(P<0.05), Vimentin m RNA(2.48±0.35 vs 1.00±0.00) and protein(0.46±0.08 vs 0.28±0.08) were significantly upregulated(P<0.05), ZEB1 m RNA(2.31±0.36 vs 1.00±0.00) and protein(0.48±0.09 vs 0.25±0.06) were significantly upregulated(P<0.05), cell invasiveness(100±10 vs 77±8) and motility(0.55±0.10 vs 0.32±0.06) were significantly enhanced(P<0.05).3. After EC-1 and Eca109 cells with high STAT3 expression were stably transfected with STAT3 sh RNA, compared with negative control(nonsense sequence) cells and blank control cells, STAT3 m RNA(EC-1: 0.26±0.05 vs 0.91±0.07 vs 1.00±0.00, Eca109: 0.28±0.08 vs 0.90±0.07 vs 1.00±0.00) and p-STAT3 protein(EC-1: 0.25±0.07 vs 0.46±0.08 vs 0.52±0.07, Eca109: 0.38±0.08 vs 0.61±0.07 vs 0.56±0.07) were significantly inhibited(P<0.05), and moreover, cells underwent mesenchymal-to-epithelial morphological alterations, E-cadherin m RNA(EC-1: 2.10±0.12 vs 0.96±0.08 vs 1.00±0.00, Eca109: 2.13±0.10 vs 0.93±0.07 vs 1.00±0.00) and protein(EC-1: 0.67±0.07 vs 0.47±0.08 vs 0.48±0.06, Eca109: 0.69±0.08 vs 0.49±0.05 vs 0.49±0.06) were significantly upregulated(P<0.05), Vimentin m RNA(EC-1: 0.80±0.06 vs 1.04±0.10 vs 1.00±0.00, Eca109: 0.70±0.06 vs 0.95±0.07 vs 1.00±0.00) and protein(EC-1: 0.42±0.05 vs 0.65±0.08 vs 0.63±0.09, Eca109: 0.43±0.07 vs 0.62±0.08 vs 0.61±0.10) were significantly downregulated(P<0.05), ZEB1 m RNA(EC-1: 0.60±0.08 vs 0.92±0.06 vs 1.00±0.00, Eca109: 0.56±0.09 vs 0.90±0.06 vs 1.00±0.00) and protein(EC-1: 0.23±0.07 vs 0.48±0.08 vs 0.52±0.04, Eca109: 0.54±0.08 vs 0.60±0.07 vs 0.58±0.06) were significantly downregulated(P<0.05), cell invasiveness(EC-1: 74±8 vs 97±11 vs 102±14, Eca109: 63±9 vs 86±10 vs 90±10) and motility(EC-1: 0.41±0.08 vs 0.71±0.11 vs 0.76±0.06, Eca109: 0.43±0.08 vs 0.77±0.10 vs 0.68±0.08) were significantly reduced(P<0.05).4. After EC-1 and Eca109 cells with high STAT3 expression were stably transfected with ZEB1 sh RNA, compared with negative control(nonsense sequence) cells and blank control cells, ZEB1 m RNA(EC-1: 0.33±0.07 vs 1.01±0.08 vs 1.00±0.00, Eca109: 0.32±0.05 vs 0.93±0.05 vs 1.00±0.00) and protein(EC-1: 0.25±0.06 vs 0.61±0.08 vs 0.56±0.09, Eca109: 0.36±0.05 vs 0.61±0.07 vs 0.55±0.06) were significantly inhibited(P<0.05), and moreover, E-cadherin m RNA(EC-1: 1.96±0.26 vs 0.96±0.06 vs 1.00±0.00, Eca109: 2.13±0.10 vs 0.93±0.07 vs 1.00±0.00) and protein(EC-1: 0.61±0.06 vs 0.46±0.07 vs 0.48±0.06, Eca109: 0.67±0.06 vs 0.47±0.07 vs 0.49±0.06) were significantly upregulated(P<0.05), Vimentin m RNA(EC-1: 0.79±0.06 vs 1.04±0.09 vs 1.00±0.00, Eca109: 0.71±0.03 vs 0.95±0.07 vs 1.00±0.00) and protein(EC-1: 0.37±0.08 vs 0.56±0.06 vs 0.60±0.06, Eca109: 0.43±0.07 vs 0.54±0.05 vs 0.64±0.07) were significantly downregulated(P<0.05), cell invasiveness(EC-1: 63±7 vs 97±11 vs 102±14, Eca109: 64±8 vs 86±10 vs 90±10) and motility(EC-1: 0.36±0.08 vs 0.71±0.11 vs 0.76±0.06, Eca109: 0.44±0.10 vs 0.77±0.10 vs 0.68±0.08) were significantly reduced(P<0.05).5. After EC-1 and Eca109 cells with stable ZEB1 transfection were treated with IL-6, compared with negative control(nonsense sequence) cells and blank control cells, E-cadherin m RNA(EC-1: 1.78±0.10 vs 1.03±0.06 vs 1.00±0.00, Eca109: 1.87±0.13 vs 0.97±0.09 vs 1.00±0.00) and protein(EC-1: 0.57±0.06 vs 0.34±0.07 vs 0.27±0.07, Eca109: 0.59±0.07 vs 0.41±0.07 vs 0.32±0.06) were not significantly downregulated(P<0.05), Vimentin m RNA(EC-1: 0.76±0.07 vs 0.96±0.08 vs 1.00±0.00, Eca109: 0.73±0.05 vs 0.98±0.05 vs 1.00±0.00) and protein(EC-1: 0.40±0.07 vs 0.63±0.06 vs 0.70±0.06, Eca109: 0.44±0.08 vs 0.68±0.08 vs 0.70±0.07) were not significantly upregulated(P<0.05), cell invasiveness(EC-1: 72±8 vs 107±12 vs 112±13, Eca109: 77±8 vs 99±8 vs 99±9) and motility(EC-1: 0.57±0.11 vs 0.82±0.10 vs 0.86±0.07, Eca109: 0.53±0.11 vs 0.81±0.09 vs 0.86±0.08) were not significantly enhanced(P<0.05).6. By Ch IP and gene sequencing, the binding sites of p-STAT3 and ZEB1 promoter were located in 2339-2527, 3306-3586, 2566-2965 and 3622-3970 of ZEB1 gene in EC-1 cells, and in 3306-3586, 2566-2965 and 3622-3970 in Eca109 cells.Part III The influence of STAT3/ZEB1/E-cadherin blockade on cellproliferation and EMT of xenograft tumors with esophagealsquamous cell carcinoma in nude mice Methods1. In order to establish xenograft tumor models of ESCC, EC-1 cells with STAT3 sh RNA stable transfection, EC-1 cells with ZEB1 sh RNA stable transfection, EC-1 cells with nonsense sequence sh RNA stable transfection, and blank control EC-1 cells were subcutaneously injected into the armpit of nude mice, respectively, with seven in every cells group.2. The volume of xenograft tumors were measured every four days and the growth curve were produced.3. At the end of the experiment, 5 mice were selected in every group. The selected nude mice were executed and the tumors were removed, photographed and measured for volume and weight.4. The histological characteristics of xenograft tumors were observed using HE sections. The m RNA and protein levels of STAT3, E-cadherin, Vimentin and ZEB1 were detected by real-time PCR and immunohistochemistry. Results1. In comparison to negative control group and blank control group, the volume(mm3)(775±315 vs 1543±182 vs 1507±206) and weight(g)(0.61±0.09 vs 1.32±0.23 vs 1.54±0.31) of xenograft tumors were significantly reduced in EC-1 cells stably transfected with STAT3 sh RNA(P<0.01).2. In comparison to negative control group and blank control group, the volume(mm3)(933±55 vs 1543±182 vs 1507±206) and weight(g)(0.72±0.07 vs 1.32±0.23 vs 1.54±0.31) of xenograft tumors were significantly reduced in EC-1 cells stably transfected with ZEB1 sh RNA(P<0.01).3. In comparison to negative control group and blank control group, the expression of STAT3 m RNA(0.34±0.09 vs 0.93±0.11 vs 1.00±0.00) and p-STAT3 protein(positive cell numbers/200 cells)(96±11 vs 133±17 vs 139±14) were significantly inhibited(P<0.05) in EC-1 cells stably transfected with STAT3 sh RNA, and moreover, cells underwent mesenchymal-to-epithelial morphological alterations, E-cadherin m RNA(1.55±0.19 vs 0.93±0.07 vs 1.00±0.00) and protein(110±11 vs 84±9 vs 91±9) were significantly upregulated(P<0.05), Vimentin m RNA(0.78±0.05 vs 0.91±0.05 vs 1.00±0.00) were significantly downregulated(P<0.05), but Vimentin protein(107±14 vs 127±17 vs 125±19) were not significantly downregulated(P>0.05).4. In comparison to negative control group and blank control group, the expression of ZEB1 m RNA(0.43±0.07 vs 0.88±0.10 vs 1.00±0.00) and protein(91±17 vs 115±16 vs 113±12) were significantly inhibited(P<0.05) in EC-1 cells stably transfected with STAT3 sh RNA, E-cadherin m RNA(1.32±0.14 vs 0.93±0.07 vs 1.00±0.00) and protein(108±14 vs 84±9 vs 91±9) were significantly upregulated(P<0.05), Vimentin m RNA(0.81±0.05 vs 0.91±0.05 vs 1.00±0.00) were significantly downregulated(P<0.05), but Vimentin protein(107±15 vs 127±17 vs 125±19) were not significantly downregulated(P>0.05). Conclusions1. In human ESCC tissues, STAT3 was positively correlated with ZEB1 expression, and moreover, with EMT and tumor invasion and metastasis, demonstrating STAT3 play a pivotal role in the EMT process and the invasion and metastasis of ESCC via ZEB1.2. In human ESCC cells, the binding sites were existed between STAT3 and ZEB1 promoter, STAT3 activation/blockade could promote/inhibit the invasion and metastasis of ESCC cells directly by regulating EMT via ZEB1, supposing STAT3/ZEB1/E-cadherin signaling axis could act as an effective index in EMT and the invasion and metastasis of ESCC.3. STAT3 sh RNA and ZEB1 sh RNA could inhibit STAT3/ZEB1/E-cadherin signaling axis effectively,reverse EMT phenotype and repress proliferation in xenograft tumors with ESCC in nude mice. STAT3/ZEB1/E-cadherin signaling axis could be used as a potential target for molecular target therapy to ESCC. |
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