Investigation Of MHC-Ⅰ Expression On Peripheral Blood Mononuclear Cells From Esophageal Cancer Patients And Pilot Studies Of Its Associated Mechanisms

Esophageal cancer (EC) is a common digestive tract malignant cancer and the sixth most common cause of cancer-related death, the majority of which is squamous cell carcinoma. There are approximately 300,000 people dying of this disease every year1. China is one of the high incidence countries in the world, and number of death is about 150,000 people per year2. Surgery is the standard treatment for localized and resectable EC, but patients often experience distant metastasis or local recurrence even after curative operation. Furthermore, combing preoperative chemotherapy and/or radiotherapy with surgery have often shown no significant survival benefit3-5. Consequently, long-term outcome remains unfavorable and overall 5-year survival after surgery is only 25-40%. However,5-year survival of early cases exceeds 90%6. Thus, to improve patients’ prognosis, novel strategies need to be established for early diagnose.MHC-I molecules are cell surface glycoproteins that play critical roles in the regulation of immune responses. These molecules are expressed on the surface of all nucleated cells, necessary for the presentation of peptide antigens to CTLs7 and for the immune regulatory activity exerted by NK cells8. Under physiological conditions, the expression of MHC-I might be regulated in a tissue-specific, differentiation-dependent, cell cycle-controlled manner and modulated by the microenvironment, thereby altering the repertoire of T cell epitopes presented. MHC-I molecules in esophageal squamous cell carcinoma (ESCC) cells are often down-regulated at both the protein and mRNA levels, which is considered as an early event in carcinogenesis9. Many factors have the ability to reduce the expression of MHC-I on tumor surface. For instance, hypermethylation of the promoter regions is a major mechanism of transcription inactivation of MHC-I genes10. Expression levels of MHC-I molecules on EC cells are related to lymph nodes metastasis and patient’s survival11. Furthermore, polymorphisms of MHC-A and -B genes affect the genetic susceptibility to EC12. However, invasive surgery or biopsy is necessary in order to examine the MHC-I expression on EC cell surface. Thus, the expression of MHC-I molecules on tumor cells can not meet the need of early diagnosis of EC. MHC-I molecules are plentiful on the surface of peripheral blood mononuclear cells (PBMCs), but their clinical significance in tumor progression remains unclear.Chapter I. Investigation of MHC-I expression on PBMCs in esophageal cancer patientsObjective: To compare the expression of MHC-I on PBMCs at mRNA and protein levels in the serum of patients with esophageal cancer, patients with benign esophageal diseases and healthy individuals, and analyse the correlations of MHC-I with tumor stage, advance and metastatis, then investigate the relationship of MHC-I on PBMCs with clinicopathological characteristics of esophageal cancer.Materials and Methodes:ESCC patients who were histopathological confirmed in Qilu Hospital of Shandong University, Jinan, China (June 2007 to July 2010) were enrolled in this study. None of them had been treated with radiotherapy, chemotherapy prior to sampling. A total of 58 EC patients (23 females and 35 males, age range 56-69 years, median age 63.2 years) were involved in this study. According to the tumor, node and metastasis (TNM) classification,21 patients had tumors of histological grades I and II, and 37 grades III and IV. Expression levels of MHC-I protein and mRNA on PBMCs were determined by flow cytometry and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), respectively.Results:(1) The expression level of MHC-I mRNA on PBMCS of EC patients, benign disease patients and healthy volunteers was 0.48±0.18,0.85±0.33 and 1.09±0.38, respectively, which showed that MHC-I mRNA on PBMCS was significantly down-regulated in EC patients (P<0.05). And this down-regulation was more significant in EC patients with stage III and IV (P<0.01).(2) MHC-I expression on PBMCs from EC patients, benign disease patients and healthy volunteers was 75.14±38.26,115.14±33.32 and 142.58±43.77, respectively. The decreased expression of MHC-I protein on PBMCs surface was similar to that of MHC-I mRNA, with more significant than that of MHC-I mRNA.(3) The decreased MHC-I expression on PBMCs of EC patients was not influenced by age and gender, but in EC patients, this parameter was reduced significantly (p<0.05), especially in stage III and IV EC patients (p<0.01). Furthermore, MHC-I on PBMCs in EC patients with lymph node metastasis was lower than that with no lymph node metasis.(4) There was no relationship between MHC-I on PBMCs with invasive depth and differentiation degree of EC.Conclusions: MHC on PBMCs was not influenced by age and gender of patients, and had no relationship with invasion and EC differentiation, but had a negative correlation with clinical stage of EC, and was downregulated at early stage of EC. Thus, MHC-I on PBMCs of EC patients might be used as a potential biomarker of EC, and useful to early diagnosis of EC.Chapter II. Investigation of MHC-I expression on PBMCs in individuals from high-incidence region of esophageal cancerObject: To explore the relevant influencing factors in region of high-incidence esophageal cancer, and combine with detections of serum tumor markers and the MHC-I expression in peripheral blood. The present study made an evaluation of MHC -I as an indicator to reflect host immune status.Method:By the method of populmion-based case-control study, the epidemiological data and blood samples was collected from the residents aged range 35 to 70 in Dawen River Basin as region of high incidence of esophageal cancer. To make the gender, age, living conditions matched, we selected the local villages in Jinan as a normal control group.(1) All participants in the investigation were asked to sign an informed consent before the epidemiological survey, which mainly include basic information, impact factors etc.(2) Single- and multi-factors analysises were used to analysis the relationship between exposure factors and esophageal cancer.(3) The soil and water samples collected from the regions of high-incidence esophageal cancer and normal control group were for Trace elements detection.(4) The population in region of high-incidence esophageal cancer were divided into high-risk group (TC-12-positive) and low-risk group (TC-12 negative) according to result of serum tumor marker detection system (TC-12).(5) MHC-I expression on peripheral blood mononuclear cell surface in region of high incidence (including high-risk groups and low-risk group)and normal control using flow cytometryResult:(1)Single-factors analysis:According to single-factors analysis with P<0.05, the results indicated that lower income, more indoor cooking fumes, removed some food mildew before eating, tasted salty, dry and hard eating, self-made tobacco, alcohol>500 ml/day, drinking alcohol with xihu, poor self-regulation emotional, difficult to adapt to enyironmental, and family history of cancer had statistically significants, All these described above may be the risk factors. While the regular consumption of Congj iang Suan and the tea inspection may be the protective factors for esophageal cancer(P<0.05).(2) Multi-factors analysis:The results in multi-factors analysis(P<0.05)were enter the regression model for Logistic regression analysis according to sle=0.3 and sls=0.2. The results showed that drinking tea may be a protective factors for esophageal cancer,while dry and hard eating, Self-made tobacco,alcohol consumption of>500ml/day, family history of cancer may be risk factors for esophageal cancer.(3)Compared with non-high incidence, high incidence of on-site river, ground water and soil content of total chromium exceeded, the difference was statistically significant (P<0.05)(4) With the increase of age, percentages of T lymphocytes and NK cell did not show a statistically significant difference. In the same age group, there were no significant changes of percentages of T lymphocytes and NK cells between high incidence villagers and normal controls.(5) With the increase of age, there was no significant difference in the expression of MHC class I on T lymphocytes and NK cells (P>0.05). Gender had no effect on the expression of MHC class I on T lymphocytes and NK cells(P>0.05). In the same group by age or gender, there were no significant differences between high incidence villagers and normal controls.(6) Compared with normal controls, MHC class I on T lymphocytes in Lijiadian villagers were significantly lower (P<0.05). The levels of MHC class I on NK cells was even lower (P<0.001). In high incidence villagers, subjects were divided into two groups according to the C-12 levels. Expression of MHC class I on T lymphocytes and NK cells in high-risk groups were significantly lower than in the low-risk group (P<0.05).(7) By the post-data analysis showed that:in 26 cases of tumor markers in serum (TC-12) positive individuals, there were 22 cases of individual exposure to esophageal cancer related risk factors simultaneously, which explains a number of risk factors for esophageal cancer may affect tumor serum markers for abnormal expression, and further lead to reduce the expression of MHC-I in PBMCs.Conclusion:To make an evaluation of MHC-I as an indicator to reflect host immune status. MHC-I expression on peripheral blood mononuclear cell surface may provide accurate non-invasive early detection methods for esophageal cancer screening large populations, and will substantially improve the early diagnosis of esophageal cancer. Chapter III Pilot studies of the mechanisms of MHC-I changes on PBMCs in esophageal cancer patientsObject:(1) To measure the expression of cytokines secreted by Thl/Th2 lymphocytes in the serum of EC patients, in an attempt to investigate the relationship between cytokines and MHC-I molecules on the surface of PBMCs.(2) To observe the effect of culture supernant of EC cell line on MHC-I molecules expression on PBMCs surface.(3) To oberve the changes of T lymphocytes biological behaviors after MHC-I molecules were inhibited by RNAi technique.Methods:(1) Serum from EC patients, patients with benign esophageal cancer diseases and healthy individuals was collected and double antibodies sandwich ELISA were used to measure the expression levels of IL-2, IFN-y, IL-4 and IL-10.(2) PBMCs from healthy individuals were co-cultured with supernants from EC9706, or with EC9706, respectively, and MHC-I on PBMCs surface was measured by FACS.(3) PBMCs were collected, and mRNA was extracted to detect the changes of APM mRNA.(4) Using RNAi technique, MHC-I molecules were inhibited by inhibiting the expression of (32m. Then combining cell death rate and transfection rate, the optimal transfection concentration of siRNA was determined between 10 and 200 nM. After successfully inhibited, biological behaviors of T lymphocytes, such as proliferation and apoptosis, were measured.Results:(1) In the serum of EC patients, IL-2 and IFN-γwere down-regulated, and IL-4 and IL-10 were up-regulated. The ratio of Thl type cytokines and Th2 type cytokines had a negative correlation with the expression of MHC-I molecules on the surface of PBMCs.(2) EC9706 and its cultured supernant had the same ability to reduce the expreesion of MHC-I on the surface of PBMCs.(3) EC9706 could reduce the expression of TAP 1 and LMP2 Mrna (p<0.05), but had no effect on TAP2 and LMP7. (4) The optimal concentration ofβ2m to interfere with the expression of MHC-I molecules on the surface of PBMCs was 100nM. At the moment, transfection rate was 59.01%, and cell survival rate was 62.17%. Activation, proliferation of T lymphocytes were inhibited, and apoptosis was promoted after MHC-I on the surface of PBMCs was successfully inhibited.Conclusions:(1) Then levels of Thl type cytokines weakened and Th2 type cytokines enhanced. That is, Th1/Th2 shift happened in EC.(2) Cytokines secreted by EC played a major role in inhibiting MHC-I on PBMCs.(3) TAP1 and LMP2 were involved in the inhibiting role of MHC-I on PBMCs by EC, but nor TAP2 and LMP7.(4) Downregulation of MHC-I molecules on T lymphocytes could weaken the activation and proliferation, and increase the apoptosis.Creative Points in This Study1. To clarify the changes of MHC-I on PBMCs in EC for the first time, and put forward the hypothesis that this parameter may be a potential marker for the early diagnosis of EC.2. Thl/Th2 shift in EC could inhibit the expression of MHC-I on PBMCs, and TAP1 and LMP2 were involved in this process.3. Downregulation of MHC-I on T lymphocyte surface could inhibit immune activities of T lymphocytes.