Prognostic Significance Of Claudin-1 And Cyclin B1 Protein Expression In Patients With Hypopharyngeal Squamous Cell Carcinoma

Hypopharyngeal squamous cell cancer (HSCC) is relatively rare in the head and neck malignant tumors, more than 90% of it is squamous cell carcinoma. Although the incidence of HSCC is low, but it is one of the high degree of malignant tumor, its specific etiology is unclear. The vast majority of patients with HSCC have a long history of smoking and drinking, so we considerate that it may be closely associated with long-term smoking and drinking. In the clinic patients with HSCC main show pharynx unwell, pharynx foreign sensation, sore throat, swallowing pain, neck bag piece etc. The terminal clinical features can appear hoarseness, difficulty swallowing or breathing difficulties etc. Due to atypical early symptoms, and hypopharynx with rich lymphatic network, most patients with HSCC in the clinic have been already late, assaulted and often occurred near neck lymph node metastasis. At present the most take surgical treatment with radiation therapy or chemical comprehensive treatment methods. But in recent years HSCC disease-free survival in patients is still poor, abroad reported 5-year survival rate is only 30%.And early discovery and treatment will be effective to improve the prognosis of patients with HSCC. Therefore looking for some ideal tumor markers will help us understand the biological characteristics about HSCC, thus to formulate individualized treatment plan, improve the prognosis of patients with HSCC.Tight junction (TJ) exists between epithelial and endothelial cells and functions as a barrier to maintain a steady cellular state and cell polarity. Claudin proteins are major barrier proteins whose numbers and distribution structures directly affects TJ structure and function within the cell membrane. In various tumors, abnormal claudin expression alters TJ structure and function, causing a disruption in cell polarity and decrease in cell adhesion, and encourages tumor cell invasive and metastatic potentials. Of the claudin proteins, claudin-1 has been found to be expressed at different levels in different cancers. It is reported that claudin-1 is over expressed and promotes tumor development in renal cell carcinoma, ovarian cancer, gastric adenocarcinoma and colorectal cancer. However, in breast cancer and lung adenocarcinoma, studies have found that claudin-1 expression is significantly reduced. These expressional differences have been confirmed and found to closely correlate to tumor occurrence and development.A normal cell cycle is regulated by specific cyclins and cyclin-dependent kinases and exhibits an orderly start and finish. Malignant cells are characterized by uncontrolled proliferation due to a loss of cell cycle regulation. Cyclin Bl is a classic cell cycle protein involved in cell cycle checkpoint control, promotion of the G2/M phase transformation and acceleration of the cell cycle process. Furthermore, studies have shown that cyclin B1 is overexpressed in many malignancies, such as in breast cancer, gastric cancer, early non-small cell lung cancer, colorectal cancer and prostate cancer. Moreover, uncontrolled cyclin B1 expression is closely related to the transformation and malignant proliferation of tumor cells, with overexpression correlated to prognosis in esophageal cancer, tongue cancer and lung cancer. These finding suggest that cyclin B1 may be a useful tumor diagnostic marker.The critical characteristic of tumor is infiltration, proliferation and metastasis. Tumor metastasis is a complex process that is influenced by many factors and comprises three steps:adhesion, degradation, and migration. The most common tumor metastatic pathways are vascular and lymphatic. It has been confirmed that vascular and lymphatic generation are very important in tumor growth, invasion, and metastasis. Studies on tumor vessel metastasis have made substantial advances; lymphatic vessel growth also plays an important role in tumor metastasis. Tumor cells in primary tumors grow quickly, and pull away from the original site, infiltration, going to the nearby micro lymphatic vessels, along the micro lymphatic vessel to the target node, proliferate in the target node, and eventually form metastatic lymph nodes. With the discovery of the specific antibody to lymphatic endothelial cells, lymphatic micro vessels as the channel for invasive tumor metastasis have received increasing attention. Micro-lymphatic vessel density (MLVD) has become the most important factor for accessing malignant tumor metastasis.In conclusion, any possible correlations between HSCC and claudin-1 and cylin B1 expression as it relates to clinical stage, pathological grade and prognosis remain to be examined. It is not clear whether the claudin-1 protein is associated with the formation of the micro lymphatic vessels in HSCC. In this study, the expression of claudin-1 and cyclinBl in HSCC were determined by immunohistochemistry and real-time quantitative PCR. The relationship between the clinical pathological characteristics and prognosis of patients was analyzed. Micro lymphatic vessel density was detected by D2-40 and determined by immunohistochemistry.The relationship between claudin-1 protein and the formation of the micro lymphatic vessels in HSCC was detected. In order to provide molecular biological Indicators for the early diagnosis and prognosis of HSCC, provide a research direction for the treatment of tumor targeted therapy.At the same time, the effect of claudin-1 on the proliferation and invasion of FaDu cells was observed in vitro, and further studied the biological characteristics of claudin-1 cells.The first part Prognostic significance of claudin-1 and cyclinBl protein expression in patients with hypopharyngeal squamous cell carcinomaAlthough HSCC incidences are low, the primary tumor location tends to be hidden and difficult to locate, with most patients diagnosed late when a tumor near a neck lymph node has metastasized. Most patients still result in poor prognosis.Claudin-1 has been found to be expressed at different levels in different cancers(overexpressed or lowexpressed). These expressional differences have been confirmed and found to closely correlate to tumor occurrence and development. CyclinBl is a classic cell cycle protein. It is found that uncontrolled cyclin B1 expression is closely related to the transformation and malignant proliferation of tumor cells. The expressions of claudin-1 and cyclinB1 protein in hypopharyngeal squamous cell carcinoma are few reported. So we first studied the expression of claudin-1 and cyclinBl in HSCC, and to explore the relationship between them and the clinical pathological data and prognosis of patients. Objective and methods:1.97 cases with complete clinical pathological data of the patients with HSCC were selected as the research object, and 90 cases of adjacent normal mucosa tissues as the control group. The expression of cyclinB1 and Claudin-1 in HSCC and normal mucosa were analyzed by immunohistochemistry, and the relationship between the expression and clinical pathological parameters and prognosis of patients were analyzed.2. With RT-PCR,30 cases of carcinoma tissues and adjacent normal mucosa adjacent to HSCC were selected to investigate the expression in mRNA level and quantitative analysis of claudin-1 and cyclinBl, and to explore the correlation between them.Results:1.Both claudin-1 and cyclinBl were highly expressed in HSCC. Claudin-1 protein expression was correlated with tumor differentiation and lymph node metastasis. CyclinBl protein expression was correlated with tumor differentiation. Kaplan-Meier analysis showed that claudin-1 was associated with patient survival(P=0.003). The relationship between claudin-1 and cyclinBl was significantly positive correlation.2. The expression levels of claudin-1mRNA and cyclinBlmRNA in the carcinoma tissues were significantly higher than those in the adjacent tissues. Spearman rank analysis results show that the relationship between claudin-1mRNA and cyclinBlmRNA in tumor tissues and adjacent tissues was significantly positive correlation.Conclusion:1.Claudin-1 was highly expressed in HSCC, and related with the differentiation of tumor and lymph node metastasis, and was associated with the survival rate of patients. Claudin-1 low expression had a higher survival rate.2.CyclinB1 was highly expressed in HSCC, and related to the differentiation degree of the tumor, and had nothing to do with the survival rate of patients.3. The expression of claudin-1 and cyclinBl in HSCC had a significant positive correlation.The second part Relationship between claudin-1 protein and micro vessel density in hypopharyngeal squamous cell carcinomaThe location of hypopharyngeal squamous cell carcinoma is concealed. The hypopharynx contains a rich network of lymphatic vessels, which facilitates cervical lymph node metastasis. The cervical lymph node metastasis is easy to occur in early stage, and lymph node metastasis rate of N1-N3 in clinical stage is 65%-80%. At present, cervical lymph node metastasis is one of the main reasons for the failure of the treatment of HSCC, and the important factor that affect the prognosis of patients. The important characteristics of malignant tumors include invasion and metastasis, but lymphatic metastasis is one of the most common and the most important way of metastasis.At present, it is considered that the formation of lymphatic vessels plays an important role in the growth, invasion and metastasis of tumor. Micro-lymphatic vessel density (MLVD) has become the most important factor for accessing malignant tumor metastasis. Recent studies have found that D2-40 is the most specific marker of lymphatic vessels, and is used widely for studying the generation of tumor lymphatic vessels.In our previous studies, the expression of claudin-1 protein in HSCC was related to the lymph node metastasis. However, it is not clear whether the claudin-1 protein is associated with the formation of the micro lymphatic vessel in HSCC. It is not clear whether the lymph nodes are generated by the micro lymphatic vessels. In this study, we studied the relationship between claudin-1 protein and the formation of lymphatic vessels in the hypopharyngeal squamous cell carcinoma, and to explore the mechanism of lymph node metastasis in the hypopharyngeal squamous cell carcinoma.Objective and methods:97 cases with complete clinical pathological data of the patients with HSCC were selected as the research object, and 90 cases of adjacent normal mucosa tissues as the control group. The expression of claudin-1 and MLVD in HSCC and normal mucosa were analyzed by immunohistochemistry, and the relationship between the expression and clinical pathological parameters were analyzed. The correlation between claudin-1 and the expression of the MLVD in the squamous cell carcinoma and normal mucosa tissues was analyzed, and the mechanism of lymph node metastasis in HSCC was di scussed.Results:1. In HSCC, the number of micro lymphatic vessels (MLVD)in the peripheral area of the cancer nests was significantly higher than that of MLVD in the central area of the cancer nests, and there was significant difference between the two groups (P<0.05). The difference was not statistically significant (P>0.05) between the MLVD in the central area of the cancer nests and the adjacent normal mucosa.2. There was no statistical significance (P>0.05) in the MLVD in the central area of the cancer nests and the clinical indicators of the patients. MLVD in the peripheral area of the cancer nests were significantly correlated with the histopathological grading, clinical stage and lymph node metastasis, and the analysis was statistically significant (P<0.05).3. There was no significant difference in MLVD between claudin-1 positive expression and the negative group in the central area of cancer nests. There was significant difference (P<0,05) in MLVD between claudin-1 positive expression and the negative group in the peripheral area of cancer nests. Correlation analysis showed that the expression of claudin-1 was positively correlated with the MLVD in the peripheral area of HSCC.Conclusion:l.High expression of lymphatic vessel density in the peripheral area of the nest of the HSCC was positively correlated with lymph nodge metastasis (P<0.05). It is suggested that the lymphatic vessels in the surrounding areas of cancer nests were functional, and the tumor may be transferred to a local lymph node by these lymphatic vessels.2. The micro lymphatic vessels density between claudin-1 positive expression and negative expression in the peripheral area of the cancer nests were different. It is suggested that high expression of claudin-1 may induce tumor lymphatic vessel formation,thus promote lymph node metastasis.The third part Effect of silent claudin-1 on cell proliferation and invasion in vitroWe found that claudin-1 was highly expressed in HSCC, and related to the differentiation and lymph node metastasis, and the content of claudin-1mRNA in the carcinoma cells was increased.The promote function of claudin-1 in tumor cell proliferation and local invasion and metastasis in HSCC was considered. So we through in vitro experiments, observed FaDu cell proliferation and invasion ability by silence claudin-1 in FaDu cell line. To further study the biological characteristics of claudin-1. Objective and methods:Claudin-1 was silenced by the transfection of human pharyngeal carcinoma cell line (FaDu) with lipofectamine 2000. Using cell culture technique, performe the proliferation and invasion experiments.Results:1. FaDu cells were recovery and cultured, the cell proliferation activity was good. The cells appeared adherent after 12 hours in inoculation medium.2.Transfection efficiency was about 60-80%. Blot Western test results confirmed that:the effect of FaDu-siRNA instantaneous silence is very good.3. After silence claudin-1,FaDu-siRNA (silent group), FaDu-control (negative control group) and FaDu untreated group, three groups of cells appeared different degree of proliferation.After 48 hours, the cell proliferation was significantly affected by the silent group and the negative control group or the untreated group. Silent group, the proliferation ability was decreased, The difference was statistically significant (P<0.01). While there was no significant difference between the negative control group and the untreated group (P>0.05).4. Invasion experiments showed that the number of cells in the chamber, silent group, negative control group and blank control group respectively for 15 ± 6.45,47 ± 6.36,50 ± 8.73, the difference is statistically significant (P<0.001). After silence claudin-1,cell invasion ability decreased significantly. FaDu-control group as well as FaDu untreated group have stronger ability to attack than the FaDu-siRNA group. Conclusion:Claudin-1 can promote the proliferation, adhesion and invasion of the carcinoma cells in vitro.