| Background Hypoxic microenvironment and epithelial mesenchymal transition(EMT) have close relationships with tumor invasion and metastasis, apoptosis and drug resistance. EMT is an important mechanism of hypoxia induced tumor progression and metastasis. Signal transducer and activator of transcription 3(STAT3) is a transcription factor related with tumorigenesis and tumor development. Activated phosphorylated STAT3 regulates cellular hypoxia responses involved in malignant transformation and progression with hypoxia inducible factor-1(HIF-1). Is there any hypoxia-induced EMT existing in esophageal cancer? Dose STAT3 take part in hypoxia-induced EMT as in other hypoxia responses? This study will focus on solving these problems.Part I. Expression of p STAT3, HIF-1 α and EMT of esophageal Squamous Cell Carcinoma(ESCC): the correlation and clinical significanceThe study was designed to investigate the relationship of p STAT3, HIF-1α and E-cadherin, clinical and prognosis significant of ESCC. Objective:Methods: 106 cases from ESCC patients under radical excision, were detected using immunohistochemical methods of the target protein. The clinical features and pathological characteristics, recurrence and survival time of patients were recorded and collected. p STAT3, HIF-1α, E-cadherin expression of normal esophageal epithelia and ESCC were compared and analyzed. Survival data were analyzed using Kaplan-Meier methods. Factors affecting survival were analyzed using Log-rank and Cox regression methods.Results: 1. Expression of p STAT3, HIF-1-α and E-cadherin was 71.7%(76/106), 73.6%(78/106) and 40.6%(43/106) in ESCC, and was 57.4%(58/101), 60.4%(61/101) and 100%(101/101) in normal esophageal epithelial, separately, p STAT3 and HIF-1-α expression in ESCC obviously increased than in normal esophageal epithelial, and E-cadherin expression significantly decreased(P<0.05); 2. Of ESCC, p STAT3 and HIF-1α were positive correlation(γ = 0.21), p STAT3 and E-cadherin were negative correlation(γ =-0.20), HIF-1α and E-cadherin were negative correlation(γ =-0.18), all with significant difference(P<0.05); 3. The average follow-up time was 40.12 months, the average disease-free survival(DFS) was 36.71(95%CI 32.78~40.64) months, and the average overall survival(OS) was 40.12(95%CI 36.85~43.40) months. Multivariate analysis indicated the increased stage and expression of p STAT3 were related with poor DFS and poor OS(P<0.05).Part II. Effects of hypoxia on ESCC EMTObjective: The study was designed to determine the relationship between STAT3, hypoxiaand EMT in human ESCC.Methods: ESCC cell lines TE-1 and EC-1 were used and Co Cl2 was used to mimic hypoxia condition. Changes in cell morphology were observed. Wound healing method and transwell invasion method were used to detect cell migration and invasion abilities. RT-PCR and Real-time PCR were used to detect of HIF-1α, E-cadherin, vimentin and STAT3 m RNA. Immunoblotting was used to detect the protein expression of HIF-1 α, E-cadherin, vimentin and p STAT3. Immuno fluorescence method was used to detect the expression of HIF-1α, E-cadherin and vimentin in ESCC cells.Results: 1. TE-1 and EC-1 performed EMT in hypoxia; 2. The ESCC cell migration rates were significantly increased in hypoxia than in normoxia [migration rate in hypoxia Vs normoxia 12 h, EC-1:(35.14 ± 1.45) % Vs(13.12 ± 3.25) %, TE-1:(30.65 ± 1.91) % Vs(14.51±2.17) %](P < 0.05). The invasion rates were significantly increased in hypoxia than in normoxia [invasion rate in hypoxia Vs normoxia 12 h, EC-1:(318.26±40.83) % Vs 100%, TE-1:(300.56±16.83) % Vs100%](P < 0.05); 3. In hypoxia vs normoxia, E-cadherin m RNA significantly decreased [relative m RNA level in hypoxia Vs normoxia 24 h, EC-1: 0.02(range 0.01~0.03) Vs 1, TE-1: 0.02(range 0.01~0.02) Vs 1](P < 0.05); vimentin m RNA [relative m RNA level in hypoxia Vs normoxia 24 h, EC-1: 37.07(range 35.13~39.12) Vs 1, TE-1 7.34(range 5.42~9.94) Vs1] and STAT3 m RNA [relative m RNA level in hypoxia Vs normoxia 24 h, EC-1: 31.48(range 25.90~38.26) Vs1, TE-1 17.60(range 14.68~21.12) Vs 1] increased(all P < 0.05). HIF-1-α m RNA fluctuated [relative m RNA level in hypoxia Vs normoxia 24 h, EC-1: 1.38(range 1.36~1.41) Vs 1, P=0.01; TE-1: 0.79(range 0.76~0.82) Vs 1, P=0.14]; 4. In hypoxia vs in normoxia, expression of E-cadherin protein significantly decreased [relative protein level in hypoxia Vs normoxia 24 h, EC-1:(0.77 ± 0.07) Vs1, TE-1:(0.55± 0.05) Vs 1](P < 0.05), vimentin [relative protein level in hypoxia Vs normoxia 24 h, EC-1:(1.74 ± 0.08) Vs 1, TE-1:(1.86±0.35) Vs 1], HIF-1α [relative protein level in hypoxia Vs normoxia 24 h, EC-1:(2.67 ± 0.37) Vs 1, TE-1:(2.67 ± 0.37) Vs 1] and p STAT3 [ relative protein level in hypoxia Vs normoxia 24 h, EC-1:(2.45±0.29) Vs 1, TE-1:(1.38 ± 0.22) Vs 1] significantly increased(all P < 0.05).Part III. Effects of STAT3 si RNA on hypoxia induced EMT in ESCCObjective: The study was designed to investigate the role of STAT3 on EMT induced by hypoxia in ESCC.Method: STAT3 si RNA plasmid was transfected to ESCC cells EC-1. Changes in cell morphology, cell migration and invasion in normoxia and in hypoxia conditions were observed. Protein and m RNA of HIF-1α, E-cadherin, vimentin and STAT3(p STAT3) were detected. Ch IP assay was used to detect STAT3 binding with HIF-1 α promoter.Results: 1. STAT3 si RNA inhibited STAT3 expression in ESCC effectively; 2. In normoxia condition, blank control(Mock), empty vector(NC), and STAT3 si RNA groups, the migration rates were(17.94 ± 0.62) %, and(16.28 ± 0.21) %, and(9.90 ± 0.97) %, respectively. In hypoxia, the migration rates were(33.82 ± 1.42)%, and(35.90 ± 1.51)%, and(10.77 ± 1.56)%, respectively. In normoxia condition, invasion rate of Mock, and NC, and STAT3 si RNA groups were(100.00 ± 0.00) %, and(89.65 ±6.91),(33.60 ± 4.17), respectively. In hypoxia, the invasion rates were(276.08 ± 19.88) %,(268.71 ± 21.59) % and(109.95 ± 11.96) %, respectively. STAT3 si RNA significantly inhibited the cell migration and invasion abilities both in normoxia and hypoxia(all P<0.05). 3. In normoxia and hypoxia, STAT3 si RNA inhibits vimentin, HIF1-α m RNAexpression [relative m RNA level of si RNA vs Mock in normoxia: vimentin 0.05(0.04 ~ 0.06) Vs 1, HIF1-α 0.11(0.09 ~ 0.13) Vs 1; in hypoxia: vimentin 0.31(0.23 ~ 0.42) Vs 32.25(30.48 ~ 34.13), HIF-1α 0.14(0.12 ~ 0.15) Vs 1.22(1.15 ~ 1.29)](all P < 0.05). E-cadherin m RNA level increased with STAT3 si RNA treatment [ relative m RNA level of si RNA vs Mock in normoxia: 4.43(4.18 ~ 4.70) Vs 1, in hypoxia 4.06(3.56 ~ 4.63) Vs 0.06(0.05 ~ 0.07)](all P < 0.05); 4. In normoxia and hypoxia, STAT3 si RNA inhibits vimentin and HIF1-α protein expression [relative protein level of si RNA vs Mock in normoxia: p STAT3 0.49 ± 0.06 Vs 1, HIF-1α 0.68 ± 0.06 Vs 1, vimentin 0.78 ± 0.07 Vs 1; in hypoxia: p STAT3 0.97 ± 0.09 Vs 1.46 ± 0.11, HIF-1α 1.01 ± 0.07 Vs 1.32 ± 0.06, vimentin 0.84 ± 0.10 Vs 1.76 ± 0.15](all P < 0.05). Protein of E-cadherin significantly increased [relative protein level of si RNA vs Mock in normoxia: 3.21 ± 0.16 Vs 1, in hypoxia 3.40 ± 0.37 Vs 0.81 ± 0.03](all P < 0.05); 5. Ch IP assay indicated STAT3 binds to the HIF-1α promoter.Part IV. Effect of STAT3 si RNA on hypoxia induced EMT in ESCC in vivoObjective: The study was designed to observe the hypoxia induced EMT in ESCC in vivo and to study the effects of STAT3 si RNA on it. Method: ESCC cells EC-1 xenografts in nude mice. STAT3 si RNA or control treatment were performed via tail vein injection. Tumor growth and expression of STAT3, p STAT3, HIF-1α, E-cadherin and vimentin were detected in different groups.Results: 1. In saline control group, blank vector control group and STAT3 si RNA group, the volume of xenograft tumor were(2.97 ± 0.21) × 103 mm3,(2.80 ± 0.39) × 103 mm3,(2.21 ± 0.40) × 103 mm3, respectively( P < 0.05); 2. Expression of STAT3, p STAT3, HIF-1α and vimentin decreased, butE-cadherin increased of STAT3 siRNA group than that of control groups.Conclusion 1. The expression of p STAT3, HIF-1α increased, and E-cadherin expression decreased in ESCC; expression of p STAT3, HIF-1α and E-cadherin were relevant in ESCC; p STAT3 predicts poor survival of ESCC; 2. Hypoxia promotes HIF-1α, STAT3 and EMT, and also with cell migration and invasion in ESCC; 3. STAT3 si RNA inhibits of STAT3 gene transcription and downregulated p STAT3 protein level effectively in ESCC cells. HIF-1α expression were downregulated by STAT3 si RNA and so with the hypoxia induced EMT phenomenon. STAT3 binds to HIF-1α promoter and regulates HIF-1α from gene transcription level; 4. In ESCC xenografts in nude mice, there exists EMT induced by hypoxia; Inhibiting STAT3 downregulated HIF-1 α expression and inhibits EMT in ESCC in vivo. |
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