Expression Correlation And Mechanism Of Prognosis In Patients With Primary Orbital VCP MALT Lymphoma

Objective: To study the correlation of the valosin-containing protein (VCP) expression level and the prognosis of primary orbital MALT lymphoma patient, and the influence of VCP to the apoptosis and proliferation of human lymphoma cells.Methods:1. VCP expression in58samples from primary orbital MALT lymphoma patients was determined by immunohistochemisty using monoclonal antibody of VCP. The correlation between VCP expression level and prognosis was clarified with the statistical analysis. We calculated the recurrence rate and survival rate, and drawed the survival curve using the Kaplan-Meier method. We calculated the difference between the groups using the log-rank method. P values lower than0.05were considered statistically significant.2. According to the results of immunohistochemistry, which was VCP protein expression level, we chose10cases orbital tumor paraffin tissue (5cases×2groups) of primary orbital MALT lymphoma patients. Extract the total RNA, and compare the difference of VCP gene expression level between the2groups using the RT-qPCR.3. Immune cell chemical dyeing method was used to detect VCP protein expression of human lymphoma cell line Raji cell and Daudi cell. We chose VCP as gene target and established the effective method of RNA interference to the human lymphoma cell line Raji cell, using the specific, efficient and convenient operation of RNAi technology. We used lipidosome to mediate gene si-VCP transfection Raji cell. Then real-time quantitative PCR and western blot were used to assess the silencing effect of VCP for Raji cells gene from the mRNA level and protein level.4. Detected the change of apoptosis rate of human lymphoma Raji cell which the VCP gene was silenced using the flow cytometry.5. Detected the change of proliferation of human lymphoma Raji cell which the VCP gene was silenced using the MTS method.Results:1. According to the follow-up visit of all patients in this study, we found that13/58(22.4%) patients suffered from tumor recurrence. The mean value of recurrence time was39.5months (range from10to56months).8/58(13.8%) patients died of tumor,4/58(6.9%) patients died of other causes unrelated to the tumor. As shown in table1, the5-year disease-free and overall survival rates were70.7%and79.3%, respectively. The mean value of5-year disease-free survival was54.7months (range from10to60months). The mean value of overall survival in the series was57.2months (range from24to60months).2. It was found that the percentage of VCP positive cells in samples of primary orbital MALT lymphoma was ranging from32%to95%. The samples were divided into two groups (level1and level2) according to the median value (45%) of the percentage of VCP positive cells. It was found that the expression level of VCP was significantly correlated with the recurrence (P=0.003) and the tumor size (P=0.008). There was no observed correlation between the expression level of VCP and other clinical features. Meanwhile, the5-year disease-free and overall survival rate of patients of level1was significantly better than that of level2(P=0.001; P=0.032).3. In the orbital tumor tissue of10primary orbital MALT lymphoma patients, the expression level of VCP mRNA was consistent with the protein expression level of VCP. According to the results of immunohistochemical the VCP mRNA expression in tumor tissue of level2group was significantly higher than that of level1group (P=0.009).4. VCP protein expression of human lymphoma cell line Raji cell and Daudi cell was stronge. After the use of liposomes for cell transfection, VCP mRNA and protein expression level were restrained65%and57%respectively. The gene silencing was effective.5. Flow cytometry showed that the cell apoptosis enhanced obviously after the VCP gene was silence in human lymphoma cell line Raji cell.6. MTS method showed that cell growth was significantly suppressed after the VCP gene was silence in human lymphoma cell line Raji cell.Conclusion:1. VCP may serve as a novel prognostic marker for prediction of prognosis of patients with primary orbital MALT lymphoma.2. The suppressive effection of Si-VCP is obvious to the human lymphoma Raji cells. VCP mRNA expression level decreased obviously, which made the amount of VCP protein expression decreased obviously.3. The VCP gene silencing which was used lipidosome mediated gene si-VCP transfection can obviously promote the training of lymphoma cells apoptosis and reduce the cell proliferation ability. VCP plays an important role in the process of apoptosis and proliferation in human lymphoma cells. VCP might create new therapeutic targets to primary orbital MALT lymphoma.