| BackgroundEsophageal cancer is one of the most common digestive tract malignant tumors, and it accounts for about2%of all malignant tumors. It ranks eighth in order of occurrence and its mortality is ranked sixth in the world. Esophageal cancer pathology type mainly include esophageal squamous cell carcinoma(ESCC) and esophageal adenocarcinoma(EADC), esophageal cancer also has the characteristic of regional distribution, most of the esophageal cancer type in Asian countries is squamous cell carcinoma, and China is one of the countries with high incidence of esophageal cancer in the world, about half of the patients died of esophageal cancer each year were Chinese people. About90%of esophageal cancer in our country is squamous cell carcinoma. Middle thoracic esophagus is the predilection site of esophageal cancer, followed by the lower thoracic esophagus, and the risk of esophageal cancer in the upper thoracic esophagus is less. Along with the development of the social science, the treatment of esophageal carcinoma has developed into an integrated treatment model which is to the surgery treat primarily combined with radiotherapy and chemotherapy and so on. In our country, surgical operation is still the main treatment of resectable esophageal cancer, in recent years, along with the improvement of the diagnosis and operation technique, with the application of preoperative neoadjuvant chemotherapy, coupled with the regular radiotherapy and chemotherapy after operation, the life quality of patients has been improved to a certain extent. But the effect of treatment of esophageal carcinoma is still not satisfactory, the overall5-year survival rate of the patients with ESCC after operation is only20%-30%. The structure of the esophagus is different from other digestive tracts, because there are lots of lymph-vessel in mucoderm and muscularis mucosae.-Therefore, lymph node metastasis of esophageal cancer can occur even in the early T stage of esophageal cancer. To the patients with complete resection of esophageal cancer, there are still about27%of them appeared lymphatic metastatic recurrence within2-3years.So, how to improve the early diagnosis rate of esophageal cancer and how to choose the effective treatment is very important to improve the prognosis of patients with esophageal cancer, which need us to further explore the specific mechanisms of esophageal cancer occurrence, development, invasion and metastasis. With the continuous understanding of the molecular biology of esophageal cancer, many studies have proved that the occurrence and development of esophageal cancer is a multiple step complex pathological process correlated with many factors. Many specific gene changes in different cells and stages play very important roles in this process. Nowadays, the exact mechanism of esophageal cancer is still not very clear, but the molecular biomarkers associated with esophageal cancer have already been the research focus of esophageal cancer for a long time. In addition to realize the early diagnosis through routine health examination as possible, to study the mechanisms of esophageal cancer and find more effective tumor marker are more important to the diagnosis and treatment of esophageal cancer. The prognosis of patients with esophageal cancer after surgery is poor, if we could find a reliable molecular biological marker which could predict the prognosis of esophageal cancer, it will be very helpful to the clinical work, especially for the patients who would have worse prognosises.Collagen type I is a kind of collagen in human tissue, it is also the most widely distributed collagen in human body. Many studies have shown that collagen type I highly expressed in a variety of tumors, such as pancreatic cancer, breast cancer, prostate cancer, brain cancer. And collagen type I was more active in squamous cell carcinoma than in adenocarcinoma. Collagen type I may play an important role in the tumor occurrence and development process. Research also confirmed that TGF-β1could stimulate collagen type I generation.In recent years, many studies have found that transforming growth factor β1(TGF-β1) was closely correlated with tumors, it had been observed high expression in a variety of tumors, such as bladder cancer, colorectal cancer, renal carcinoma, ovarian cancer, lymphoma. TGF-β1is a member of the TGF family, its mechanism is not only complex, but also contradiction. During the early stage of cancer occurrence, TGF-β1can inhibit cell proliferation, initiate cell differentiation, induce cell apoptosis; but when cells undergo malignant transformation, TGF-β1can also promote the invasion and metastasis of tumor cells.So far, there are no reports about whether collagen type I is highly expressed in ESCC and its relationship with clinicopathological features of ESCC at home and abroad. There have already been some reports about TGF-β1and ESCC, but the research results are not consistent, some results were even opposite completely. And whether there was correlation between the expression of TGF-β1and collagen type I in ESCC is still not very clear. The aim of this study was to detect the expression of collagen type I and TGF-β1in ESCC, and further investigate the correlation between collagen type I and TGF-β1with biological behavior and prognosis of ESCC. In this study, Immunohistochemistry(IHC), Western-blot and quantitative real-time polymerase chain reaction(qRT-PCR) assays were used to detect the expression of collagen type I and TGF-β1. The findings of this study will provide theoretical bases for the further exploring of pathogenesis of esophageal cancer and searching for the ideal target for gene therapy of esophageal cancer.Part one:The expression of collagen type I and TGF-β1in esophageal squamous cell carcinoma and the relationship with clinicopathological characteristicsObjective:The occurrence and the development of esophageal cancer were complex pathological processes involved multiple factors and steps, the aim of this study was to detect the expression of collagen type I and TGF-β1in ESCC tissues and investigate the relationship between collagen type I, TGF-β1and the clinicopathological characteristics of ESCC, further explore the possible mechanism of esophageal cancer.Methods:From March2010to January2011, we collected82specimens of ESCC patients underwent the complete resection of esophageal carcinoma in the Department of thoracic surgery provincial hospital affiliated to Shandong University, including82cases of ESCC and20cases adjacent normal esophageal tissues selected randomly. The expression of collagen type I and TGF-β1protein were detected by IHC and western-blot assays. Real-time fluorescence quantitative PCR was used to detect the expression of collagen type I and TGF-β1mRNA. SPSS16.0software package was used to analyze the results. The clinicopathological characteristics and experimental results were included into the database. Enumeration data was calculated by positive rate, and compared by chi-square test; measurement data was represent with average±standard deviation, the difference of groups were compared by t-test or F-test, and SNK test for comparing the difference of pairwise groups. The relationship between the expressions of collagen type I and TGF-β1was analyzed by Spearman’s rank or Person method. P<0.05was considered statistically significant.Results:The positive expression of collagen type I was brown stained in cytoplasm by IHC, and the expression of collagen type I in ESCC tissues was significantly higher than in adjacent normal esophageal tissues (P=0.000). Collagen type I expression was correlated with depth of invasion(pT), lymph node metastasis and pTNM stage(P=0.030, P=0.044, P=0.034); The positive expression of TGF-β1was brown stained in cytoplasm or cell membrane by IHC, and the expression of TGF-β1in ESCC tissues was significantly higher than in adjacent normal esophageal tissues(P=0.000). TGF-β1expression was correlated with depth of invasion(pT), lymph node metastasis and pTNM stage(P=0.014, P=0.033, P=0.017); And there was correlation between collagen type I and TGF-β1expression in ESCC(r=0.743, P=0.000).Western-blot results showed that the expression level of collage type I in ESCC was significantly higher than in adjacent normal tissues(P=0.000), however, it was only correlated with the depth of invasion (pT) and pTNM stage (P=0.000, P=0.000), The difference between tumor invasion(pT) groups were significant(P<0.05), and pⅠ group and p II and pⅢ group were significantly different (P<0.05), but no significant differences between p II and pⅢ groups(P>0.05); the expression level of TGF-β1in ESCC was significantly higher than in adjacent normal tissues(P=0.000), it was correlated with the depth of invasion(pT), lymph node metastasis and pTNM stage(P=0.000, P=0.043, P=0.000), The difference between tumor invasion(pT) groups were significant(P<0.05), and pⅠ group and p II and pⅢ group were significantly different(P<0.05), but no significant differences between p II and pⅢ groups(P>0.05); There was correlation between collagen type I and TGF-β1expression in ESCC(r=0.743, P=0.000). Results of real-time fluorescent quantitative PCR were that the expression of collage type I mRNA in ESCC tissues was significantly higher than in paracancerous normal esophageal tissues(P=0.000). The expression of Collagen type I mRNA in ESCC was correlated with depth of invasion (pT), lymph node metastasis and pTNM stage(P=0.000, P=0.030, P=0.000). The difference between tumor invasion(pT) groups were significant(P<0.05), and pⅠ group and p II and pⅢ group were significantly different (P<0.05), but no significant differences between p Ⅱ and pⅢ groups too(P>0.05); The expression of TGF-β1mRNA in ESCC tissues was significantly higher than in paracancerous normal esophageal tissue(P=0.000). The expression of TGF-β1mRNA in ESCC was correlated with depth of invasion (pT), lymph node metastasis and pTNM stage(P=0.000, P=0.001, P=0.000). The difference between tumor invasion(pT) and pTNM stage groups were significant(P<0.05); And there was also correlation between collagen type I and TGF-β1mRNA expression in ESCC(r=0.636, P=0.000).Conclusion:There were high expression of collagen type I and TGF-β1in ESCC tissue, and there was correlation between collagen type I and TGF-β1expression. There expression were also correlated with depth of invasion(pT), lymph node metastasis and pTNM stage. Overexpression of collagen type I and TGF-β1might play important roles during the process of occurrence, development, invasion and metastasis of ESCC. Part two:The relationship between collagen type I and TGF-β1and the prognosis of esophageal squamous cell carcinomaObjective:Esophageal cancer was a malignant tumor with high incidence, mortality and poor prognosis. We always hope to find some biological indicators which could predict the prognosis of esophageal carcinoma. This study aimed to investigate the relationship between collagen type I and TGF-β1and ESCC prognosisMethods:From March2010to January2011,82patients with ESCC underwent the complete resection of esophageal carcinoma in the Department of thoracic surgery, provincial hospital affiliated to Shandong University were included into this study. The expression of collage type I and TGF-β1in ESCC tissues was detected by IHC method. Follow-up was completed in the all82patients. SPSS16.0software package for statistical analysis, the clinicopathological characteristics and IHC results were included into the database. Kaplan-Meier method was used to calculate the survival rate, and the difference of survival rates were compared by Log-rank test. Cox-regression analysis was applied to determine the independent risk factors of ESCC prognosis. P<0.05was considered statistically significant.Results:The3-year survival rate of82patients in this study was53.7%calculated by Kaplan-Meier. In univariate analysis, the3-year survival rate of the patients with ESCC after surgery was significantly correlated with depth of invasion(pT), lymph node metastasis, pTNM stage, and the expression of collagen type I and TGF-β1. The3-years survival rate of patients with different invasion depth(pT) and TNM stage were difference significantly(P=0.005, P=0.000); The3-years survival rate of patients with lymph node metastasis was significantly lower than patients without lymph node metastasis(P=0.000). The3-years survival rate of patients with positive collagen type I expression was significantly lower than patients with negative collagen type I expression(P=0.008). The3-years survival rate of patients with positive TGF-β1expression was significantly lower than patients with negative collagen type I expression(P=0.001). Cox-regression multivariate analysis showed that only the depth of tumor invasion(pT)(P=0.047) and lymph node metastasis(P=0.007) were independent risk factors of3-year survival rate of ESCC.Conclusion:Depth of tumor invasion(pT), lymph node metastasis, pTNM stage, collagen type I and TGF-β1were correlated with3-year survival rate of patients with ESCC after surgery. However, only the depth of tumor invasion(pT) and lymph node metastasis were independent risk factors3-year survival rate of ESCC. Part three:The effect of TGF-β1on the expression of collagen type I in Eca109cellsObjective:To determine whether TGF-β1could effect the expression of collagen type I in esophageal cancer109(Eca109) cells cultured the in vitro, and further explore the mechanisms of TGF-β1effecting the synthesis of collagen type I in esophageal squamous cell carcinoma.Methods:Cultured Eca109cells in vitro, and5ng/ml TGF-β1were used to stimulate the cultured Eca109cells0,12,24and48hours. Eca109cells were also stimulated by TGF-β1with different concentrations Ong/ml, lng/ml,5ng/ml and lOng/ml. RNA interference was used to silence Smad4gene, we designed Smad4siRNA squence, and Lipofecamine2000transfection was applied, to silence Smad4gene in Eca109cell lines, then stimulated by TGF-β1with certain concentration. The expressions of collagen type I in Eca109cells were detected by qRT-PCR and Western-blot assays. SPSS16.0software package was used to analyze the results, P<0.05was considered statistically significant.Results:qRT-PCR results showed that the differences of collagen type I mRNA expression in Eca109cells after TGF-β1stimulation with different time were statistically significant(P<0.05). The expression of collagen type I mRNA with12,24and48hours TGF-β1stimulation were significantly higher than0hour(P<0.05), and the expression of collagen type I mRNA with24and48hours stimulation were significantly higher than12hours(P<0.05), but there was no significant difference between24and48hours groups(P>0.05). The differences of collagen type I mRNA expression in Eca109cells after TGF-β1stimulation with different concentration were statistically significant(P<0.05). The expression of collagen type I mRNA was increased with the increase of TGF-β1concentration. Western-blot results showed that the differences of collagen type I expression in Eca109cells after TGF-β1stimulation with different time were statistically significant(P<0.05). The expression of collagen type I with12,24and48hours TGF-β1stimulation were significantly higher than0hour(P<0.05), and the expression of collagen type I with24and48hours stimulation were significantly higher than12hours(P<0.05), but there was no significant difference between the collagen type I with24and48hours(P>0.05). The differences of collagen type I expression in Eca109cells after TGF-β1stimulation with different concentration were statistically significant(P<0.05). The expression of collagen type I was increased with the increase of TGF-β1concentration. After the silence of Smad4, the expression of collagne type I was significantly reduced, and the effect of TGF-β1upregulating collagen type I in Eca109cell was also disappeared.Conclusion:TGF-β1can promote the synthesis of collagen type I in esophageal squamous carcinoma cells through downstream Smad signaling pathway, it may play an important role in the invasion and metastasis process in esophageal carcinoma. |
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