Inhibition Of Invasion And Enhances Radiosensitivity Induced By Silencing EBV-LMP1 In NK/T Cell Lymphoma

Background and purposeExtranodal natural killer T cell lymphoma, nasal type(NKTCL)- is a kind of lymphoma with vascular central necrosis characteristic, The disease occurs mainly in East Asia and South America. EB virus(Epstein-Barr virus EBV) infection is closely related to the occurrence and development of NKTCL. Radiotherapy is the major treatment for NKTCL especially for the early stage of this malignant lymphoma.But due to the lack of biological indicators to predict the radiosensitivity of disease effectively, there are still 30% of the patients with poor treatment effect, excavation the molecular marker to predict the radiation sensitivity is worth our further research.One of EBV express products LMP1(latent membrane protein, LMP1) gene is oncogene has been confirmed, which are available in most of nasopharyngeal carcinoma, Hodgkin’s lymphoma and non-Hodgkin lymphoma expression, Therefore LMP1 has become in the study of EBV related hot topics in the study of malignant tumor, especially for the tumor lymphatic hematopoietic system research. Specifically block LMP1 EBV gene corresponds to the removal of carcinogenic factors, this treatment has become one of the related research of tumor gene therapy EBV.However, studies of this treatment in EBV-related lymphoma in particular, NKTCL less, LMP1 gene NKTCL sensitivity of its molecular mechanism of radiation is no research.In this experiment, we have chosen LMP1 as a molecular targets for genetherapy to NKTCL, explore the gene blocking technology—RNA interference to inhibit the expression of LMP1 gene to increase the sensitivity of the feasibility of radiation. We first organization NKTCL patient specimens and cell lines to detect the expression of LMP1, then build and carry LMP1 c DNA and LMP1 sh RNA recombinant lentivirus expression vector, transfection NKTCL cell lines. Learn LMP1 gene expression silencing effect on biological characteristics NKTCL proliferation, invasion and metastasis and radiation sensitivity, and finally detection of apoptosis, invasion and metastasis related molecules and DNA damage repair pathway genes, LMP1 explore the biological functions and NKTCL the molecular mechanisms that provide molecular targets for clinical diagnosis and treatment of NKTCL.Part Ⅰ: LMP1 differential expression levels of NK / T cell lymphoma and its relationship to its radiosensitivity Objective:LMP1 in NK / T cell lymphoma tissues, cells from the expression level of m RNA and protein levels and its relationship with the radiotherapy.Materials and Methods:1. We have been collected fresh tumer specimens of 40 NK / T cell lymphoma cases in the First Affiliated Hospital of Zhengzhou University,Aug.2014 – Sep.2015.The expression m RNA and protein levels of LMP1 were detdcted by RT-PCR and western blot respectively. Analyze the relationship between LMP1 expression levels and clinicopathological features and radiotherapy efficacy. Benign reactive lymphoid hyperplasia specimens of 20 cases as control.2. Expression of LMP1 in SNK-6,NKL, NK92, YTS and YT cell lines were detected by fluorescence quantitative PCR and western blot. Then 6MV-X ray dose of 0,2,4,6,8Gy were given by radiation therapy, CCK-8 method and colony formation assay to detect cell proliferation, and explore suitable radiation dose, cytology explore LMP1 and radiosensitivity relevance.Results:1.40 cases of patients, 34 cases were detected LMP1 m RNA and protein, the positive rate of 85% in this malignant lymphoma. 34 patients’ tissue were detected by RT-PCR and Western blot, NK/T cell lymphoma LMP1 m RNA relative expression and protein were significantly higher than in benign reactive lymphoid hyperplasia(F=248.367, P<0.001). Serum β2- microglobulin levels NKTCL LMPl expression,lymph node metastasis, B symptoms, in other words, β2- microglobulin abnormal lymph node metastasis, with B symptoms, high LMPl expression levels(P <0.01).Radiotherapy group LMPl expression and radiotherapy are closely related, that is worse by radiotherapy, LMP1 expression levels higher(P <0.05).2. Real-time PCR detection of EBV-LMP1 in SNK-6, NKL, NK-92,YT, YTS Cells. Expressing NKL cell as a reference group, analyzed by ANOVA results show:The expression of LMP1 was significant difference between five kinds of cell lines(F=163.374, P<0.001).LMP1 in SNK-6, YT, YTS, NK-92, NKL in descending order.Homogeneity of variance test showed that the homogeneity of variance, LSD pairwise comparison after the discovery.The result showed that LMP1 was the highest expressed in SNK-6,It was higher than YT(P<0.001), YTS(P<0.001), NK-92(P < 0.001), NKL(P<0.001) cells; LMP1 expression YTS cells is higher, and higher than YT(P<0.05), NK-92(P<0.00l), NKL(P<0.001) cells expression; expression of LMP1 in YTS is higher than NK-92(P=0.032), NKL(P=0.018), while no significant difference between the expression of LMP1 lower NKL and two NK-92 cells(P =0.748). All of the cell lines were treated with radiation dose 0,2,4,6,8Gy, CCK-8 and colony formation assay cell radiosensitivity display YT, SNK-6, YTS, NK-92, NKL’s radiosensitivity increased successively. YT, SNK-6 is not sensitive to radiotherapy,YTS moderately sensitive to radiation, and the NK-92, NKL sensitive to radiotherapy,the difference was statistically significant. We found that the higher the expression of LMP1 cells, the lower its radiosensitivity.Conclusion:LMP1 differential expression correlated with the radiosensitivity of NK/T cell lymphoma negatively.Part II: Construction of lentivirus-mediated overexpression andsilencing LMP1 stable of NK / T cell lymphoma cell lineObjective:To construct a stable high / silent LMP1 of NK/T cell lymphoma cell line by recombinant lentiviral expression vector.Materials and methods:Select the first part of the five kinds of cell lines showed moderate expression of LMP1 and has some invasive ability YTS cell lines, used lentivirus p CDH-CMVMCS-EFl-cop GFP as a carrier, connected LMP1 template PCR product was cloned,forming a high expression lentivirus vector p CDH-LMP1. Designed and synthesized three LMP1 gene sh RNA,the interference lentiviral vector of p LVX-sh RNA1 was degisted by Eo R Ⅰ and Bam H Ⅰ,We finally synthesized recombinant plasmid p LVX-LMP1-sh RNAa, p LVX-LMP1-sh RNAb, and p LVX-LMP1-sh RNAc.According to Real-Time PCR detection of LMP1-m RNA levels to screen out effective RNA interference sequences. The overexpression and interference of expression lentivirus packaging, purification, concentration and titer identified liposome transfected lymphoma cells was determined lentivirus infection efficiency lymphocytes.Results:Digestion and sequencing showed that the recombinant overexpression of LMP1 lentivirus p CDH-LMP1 and interference LMP1 expression lentivirus p LVX-LMP1-sh RNAa, b, c was built successfully, and the highest p LVX-sh RNAa interference efficiency.Select this plasmid into the next experiment. The concentrated viral titer108IFU/ml. YTS could be infected, RT-PCR detection of cell transfection efficiency relative quantitation t-test showed that the expression LMP1 in p CDH-YTS in higher than YTS/parental expression(F=79.461, P=0.006) the results have significant differences,and p LVX-sh RNA-YTS expression is lower than YTS/parental expression(F=133.23, P=0.005).Conclusion:We successfully constructed the stably overexpressing and interference LMP1 of NK/T cell lymphoma cell subline.Part III: Targeting inhibition of LMP1 inhibited the invasion abilityand inhanced radiosensitivity of NK/T cell lymphoma and mechanism analysis Objective:To investigate the lentivirus-mediated LMP1-sh RNA interference expression vector of NK/T cell lymphoma radiosensitivity and mechanisms.X-ray irradiation using stably transfected passage 6 on behalf of more than lentivirus mediated interference LMP1 expression YTS lymphocyte cell line YTS-LMP1-sh RNA, By CCK-8 assay and colony formation assay to detect the radiotherapy sensitization effect and Transwell invasion assay and cell invasion ability changes. Western blot was used to detect the invasion changes of TIMP-1, MMP-9, DNA damage repair pathway related protein Ku70 and DNA PKCs changes and apoptotic death associated protein Bcl-2 and Bax expression level changes. Analysis of the correlation mechanism of radiation enhancement and inhibition of invasion from the molecular level.Results:CCK-8 experiments show, PCDH-LMP1 transfected cell lines compared with the control group, the radiosensitivity decreased(F=16.187, P<0.001), while the LMP1-sh RNA cell lines transfected with the control group ratio, enhanced radiosensitivity(F=13.556, P <0.001). Colony formation assay showed by 0 ~ 8Gy X ray radiation, LMPl-sh RNA transfected group survival curves decreased(P<0.01).Invasive experiments have shown that simple exposure group, pure transfection sh LMP1 group, the irradiation + transfection sh LMP1 group were significantly less than the control group(P<0.01);Irradiation combined transfection sh LMP1 group than the rest of the group, cell membrane cell count were significantly reduced(P<0.01). Further analysis revealed that in addition to the Ku-70 and other selected proteins in NK / T cell lymphoma are expressed at more or less, 6Gy X irradiation can induced YTS cells apoptosis and induce the invasion associated protein, MMP-9 and associated protein decreased the expression of Bcl-2 and DNA damage and repair of DNA PKCs protein expression increased, and transfection of LMPl-sh RNA can increase radiation-induced apoptosis(P < 0.01), and can be up-regulated in YTS cells themselves and radiation induced Bax and TIMP-1 protein expression(P < 0.01), cut themselves and radiation induced MMP-9, the expression of DNA PKCs protein(P <0.01), bcl-2 protein expression level had no significant change(P > 0.05).Conclusion:The invasion ability of YTS cells can be reduced by transfection of LMP1-sh RNA, but the radiation sensitivity is enhanced due to transfection. Through the mechanism analysis, consider this result may be due to the LMP1 gene regulation of invasion metastasis, the expression of proteins related to apoptosis and DNA damage repair pathways, thereby increasing the radiation induced invasion and metastasis ability and ability to repair DNA damage and increased cell apoptosis.