| Objective: Studys on different expression of miR-155in NK/T celllymphoma in peripheral blood and in SNK-6cell line, to research thecorrelation with the clinical features of NK/T cell lymphoma, as well as thepossible mechanism of regulation.In order to explore the new diagnosticmethods and prognosis factor of NK/T cell lymphoma.Methods:1Patients: the blood samples were collected at the Fourth Hospital ofHebei Medical University from NK/T cell lymphoma patients between2013and Marcy2014. Clinical data is relatively completed; All patients signed theinformed consent. Normal human peripheral blood from Hebei MedicalUniversity. Human NK/T cell lymphoma cell line SNK-6was given in theFirst Affiliated Hospital of Zhengzhou University.2Cell culture of Human NK/T cell lymphoma cell line SNK-6: Thehuman NK/T cell lymphoma cell line SNK-6cells were maintained inRPMI1640,containing15%AB plasma,1000u/ml interleukin-2. SNK-6cellswere cultured at37℃in a humidified of5%CO2atmosphere. The cells werepassaged every two or three days. All experiments were using logarithmicallygrowing cells, and cells’ viability≥95%tested by trypan blue staining.3MicroRNA extraction: Genomic miRNAs were extracted by miRNAExtraction Kit from the blood of NK/T cell lymphoma patients, normal peopleand SNK-6cells, extraction of total RNA SNK-6cell lines by Trizolmethodand, then the miRNAs and total RNA were frozen immediately inrefrigerator until used.4PCR (polymerase chain reaction): The target gene Fragment was amplified by PCR, and analysis of miR-155according to the results of PCRamplification products.5After blocking of SNK-6cells in miR-155using siRNA, to detect thealteration of cell proliferation by MTT assay.SNK-6cells were divided into untreated group, blocking of homologysiRNA control group and blocking of miR-155experimental group. ThesiRNA control group and experimental group were added to siRNA-sequencehomologyof/EndoFectionTMcomplex,siRNA-155/EndoFectionTMcomplex,after48hours, the cells were collected,and cells were seeded in96-well plates at2×105/ml concentration,with200μl of air volume. Each group has3complexspace. Measure the absorbance of each well with MTT test. Keep cells in wellsfor24hours before MTT assay. Four hours before the ending point, added20μl MTT (5mg/ml) in each well. After centrifuging of the plate, discardingthe supernatants, adding150μl DMSO to each well, shaking the plate until theprecipitate dissolved. Lastly, measure each well’s absorbance at490nm withelisa reader.6After blocking of SNK-6cells in miR-155, to detect the effects of miR-155closure on NF-κ B expression level of mRNA by Realtime-PCR.SNK-6cells were divided into untreated group, blocking of homologysiRNA control group and blocking of miR-155experimental group. ThesiRNA control group and experimental group were added to siRNA-sequencehomologyof/EndoFectionTMcomplex,siRNA-155/EndoFectionTMcomplex,after48hours,total protein were collected from each group,100μg protein fromeach group were loaded into SDS gel and electrophoresis. After bloting intofilm, NF-κB（p65）protein were detected with antibodies.7Statistical analysis: All statistical analyses were performed by usingSPSS17.0statistical software. P <0.05, was considered statistically significant.If the experimental data meet the normality distribution, using the t test; if not,using non parametric test of rank transformation, and the patients and thenormal expression of miR-155in peripheral blood of the differences werestatistically analyzed. By using χ2test and multivariate analysis, to analysis the expression of miR-155and The clinical features of NK/T cell lymphomapatients.Results:1In all research objects, the expression level of miR-155in NK/T celllymphoma patients was higher than that of normal control group in peripheralblood, it shows that miR-155is high abnormal expression in patients withNK/T cell lymphoma.2In human NK/T cell lymphoma cell line SNK-6cells,miR-155is highabnormal expression.3Analysis of the frequency distribution of miR-155in the clinicalfeatures of NK/T cell lymphoma, the results show that miR-155is related withstaging of patients, bone marrow involvement, the value of Ki-67(%) and theNKPI score. Namely the expression level of miR-155is higher, the greater thechance of bone marrow infiltration, staging of the later, more disease riskfactors.Because the above index are associated with the prognosis of patientswith NK/T cell lymphoma, it shows that miR-155may affect the prognosis ofthe patients.4Because Ki-67(%) is an indicator of the proliferative activity of tumorcells, therefore, miR-155may be associated with tumor cell proliferation.AftermiR-155cloure, MTT results show that closed miR-155cell survival ratedecreased significantly, it proves that miR-155is related to the proliferativeactivity of tumor cells.5After blocking of SNK-6cells in miR-155, the expression of NF-κ BmRNA is down-regulation by Realtime-PCR method. It suggests that miR-155is involved in the pathogenesis of NK/T cell lymphoma associated with NF-κB signal pathway.Conclusions:1MiR-155is abnormal high expression in NK/T cell lymphoma, and itinvolves in the occurrence and development of NK/T cell lymphoma.2MiR-155is associated with NK/T cell lymphoma cell proliferation.3MiR-155may be related with prognosis of NK/T cell lymphoma. 4One of the pathogenesis of miR-155in NK/T cell lymphoma is NF-κ Bsignal pathway. |
PDF Full Text Request