| Malignant lymphoma is a frequent malignant tumor in mankind.It affects human health seriously due to its high incidence and recurrence rate.Extranodal natural killer/T-cell lymphoma(NK/T cell lymphoma)is a major type of natural killer(NK)and T cell neoplasm and its incidence is higher in Asia and Latin America than it is in Western countries.NK/T cell lymphoma was the most prevalent subtype of lymphoma in China according to epidemiological evidence.NK/T-cell lymphoma is considered to be an aggressive disease with distinct clinical and histopathological features.Recurrence of disease and resistance to therapy maybe correlate with worse prognosis.Due to the complicated pathogenesis and clinical heterogeneity of NK/T cell lymphoma,optimal treatment modalities and prognostic factors have been difficult to determine.It is difficult to achieve the goal of individualized treatment for patient.In recent years,immunotherapy and molecular targeted therapy has become a hot spot.It is of paramount importance to search for novel therapies targeting emerging oncogenic pathways in the treatment of this type of aggressive lymphoma.MicroRNAs(miRNAs)are non-coding regulatory RNAs consisting of 22 nucleotides which regulate gene expression at the post-transcriptional level and may conceivably play a key role in tumorigenesis.Hundreds of mi RNAs have been identified in plants,animals,and viruses by molecular cloning and bioinformatic approaches.Abnormal mi RNA expression is known to occur in many cancers,including in malignant lymphomas.They play important biologic roles by regulating cells in growth,development,apoptosis,and hematopoiesis.Mi RNAs,which are upregulated in cancer cells and contribute to carcinogenesis by inhibiting tumor suppressor genes,are considered oncogenic mi RNAs(Oncomi Rs),while downregulated mi RNAs,that normally prevent cancer development by inhibiting the expression of proto-oncogenes,are known as tumor suppressor mi RNAs.Mi RNA profiling may be useful in distinguishing subtypes and developing new therapeutic strategies for lymphoma.mi R-34 a is a member of the mi R-34 family,and it is located in chromosome 1p36 encoded by its own transcript.The mi R-34 a gene promoter region contains p53-binding sites and Cp G island,and the expression of mi R-34 a is decreased due to the inactivation of the p53-binding sites and hypermethylation of the Cp G island.Mi R-34 a has been found to be decreased in a variety of solid tumors.Mi R-34 a might function as a potent tumor suppressor by controlling the expression of specific target proteins involved in cell cycle,cell aging and autophagy.However,other studies in vitro have revealed that increased expression of mi R-34 a induced by LMP-1 could promote the progression of tumors in some EBV-positive lymphomas.Therefore,mi RNA function varies among tissues of different developmental origin.In our previous studies,mi R-34 a was downregulated in the NK/T lymphoma cell lines by next-generation solexa high-throughput sequencing technology.To the best of our knowledge,the relevant report of the expression and function of mi R-34 a in NK/T-cell lymphoma has not seen in other studies at home and abroad.In the present study,we investigated mi R-34 a expression by RT-PCR in Chinese NK/T cell lymphoma and analyzed its correlation with clinicopathological features and prognostic significance.Lentiviral vectors that could increase or decrease mi RNA-34 a expression were constructed and packaged.Proliferation,apoptosis and invasion ability of cells were detected after lentivirus infection.Meanwhile,The YTS cell xenografted tumor in nude mouse models was established.Computer software,luciferase assays and western blot were performed to verify the direct target of mi R-34 a.Biological functionsof the direct target were detected too.The aim of the present study was to investigate the effect of mi R-34 a expression and find new therapeutic targeting in NK/T cell lymphoma.Main contentPart?:The expression of mi R-34 a in NK/T cell lymphoma and its correlation with clinicopathological features and prognostic significanceMethods: 1.30 cases of NK/T cell lymphoma paraffin-embedded tissues and corresponding normal tissues samples was collected and filtered.2.The expression of mi RNA-34 a in NK/T cell lymphoma tissues and control tissues were detected by RT-PCR.3.Correlation between expression level of mi R-34 a and gender,age,B symptoms,LDH level,Ann Arbor stage,IPI score were analyzed by SPASS software.4.Correlation between above clinicopathological parameters and overall survival of patients were analyzed by univariate and multivariate analyses.Results: 1.Levels of mi R-34 a expression in tumor tissues were significantly lower than in control tissues.2.Expression level of mi R-34 a in NK/T cell lymphoma tissues had correlation with age and Ann Arbor stage(P<0.05),while its expression was irrelevant to gender,B symptoms,LDH level and IPI score(P>0.05).3.mi R-34 a expression was irrelevant to overall survival of patients,and IPI score?3 was individual risk factor of poor outcome.Part ?: The effect of mi RNA-34 a on biological characteristics of NK/T lymphoma cell linesMethods: 1.The expression of mi RNA-34 a in NK/T cell lymphoma cell YTS,SNK-6 and NK cell were detected by RT-PCR.2.Pre-mi R-34 a sequence and anti-mi R-34 a sequence was synthetized and ligated into Mlu I and Not I restriction sites of p Lenti-mi R vector and p Lenti-antimi R vector to construct p Lenti-mi R-34 a and p Lenti-mi R-anti-mi R34 a.3.Co-transfection and packaging HEK-293 T cells with recombinant lentiviral particles,?8.2 and VSV-G,and determining their titers.4.Four groups were designed: blank group,negative control group,Lenti-mi R34 a and Lenti-anti-mi R34 a.Proliferation,apoptosis and invasion ability of YTS cell were detected after lentivirus infection by MTT,flow cytometry and transwell chambers experiment.Results: 1.Levels of mi R-34 a expression in YTS cell and SNK-6 cell were significantly lower than in NK cell.2.Lentiviral vectors that could increase or decrease mi RNA-34 a expression were constructed and determined by RT-PCR and sequence analysis.3.MTT assay showed that OD450 value of YTS cells at 72 h after co-transfection was decreased in the Lenti-mi RNA-34 a group and increased in the Lenti-anti-mi R34 a group when compared with control group,the difference had statistically significant(P< 0.05).4.Flow cytometry showed that the apoptosis rate in the Lenti-mi RNA-34 a group was higher than that in the other three groups,the difference had statistically significant(P< 0.05).5.Transwell invasion experiment showed that compared to control group,the cell number went through membrane was significantly reduced in the Lenti-mi RNA-34 a group and increased in the Lenti-anti-mi R34 a group(P< 0.05).Part ?:Prediction and Identification of mi RNA-34 a targetMethods: 1.The target of mi RNA-34 a was predicted from Targetscan,Pic Tar and mi Randa website.2.Construct the recombination vector pmir GLO-CD47,pmir GLO-MYB,and pmir GLO-MYC.3.To validate the target genes of mi R-34 a by dual-luciferase assays,293 T cells were transiently co-transfected with each of the reporter constructs: pmir GLO-CD47,pmir GLO-MYB,pmir GLO-MYC,and blank group,Pmir GLO-control.4.After infection of YTS cell with recombinant vectors(Lenti-mi R34 a,Lenti-anti-mi R34a),and control groups,m RNA expression and protein expression of CD47 in each group was detected by RT-PCRand Western Blotting.5.Design the sh RNA sequence targeting CD47 and transfect them in YTS cells.Use RT-PCR and Western blot todetect CD47 m RNA and protein expression.Four groups were designed: blank group,negative control group,mi R-34 a group and CD47-sh RNA group.Biological functionsof YTS cell in each group were detected after lentivirus infection by MTT,flow cytometry and transwell chambers experiment.Results: 1.CD47,MYC,MYB was identified as the potential target of mi R-34 a by using bioinformatics software.2.Lentiviral vectors pmir GLO-CD47,pmir GLO-MYB,and pmir GLO-MYC were constructed and determined by RT-PCR and sequence analysis.3.Dual-luciferase assays showed that luciferase activity in the pmir GLO-CD47 group was significantly reduced than that in the control group(P<0.05).No significant difference in luciferase activity between experimental group(pmir GLO-MYB and pmir GLO-MYC)and control group(P>0.05).It suggested that mi R-34 a could bond with 3 'UTR regions of CD47.4.Lenti-mi R34 a group,Lenti-anti-mi R34 a group,and negative control were no significant difference in the m RNA level of CD47(P>0.05).Western Blotting showed that CD47 protein expression in the Lenti-mi R34 a group was significantly lower than that in the negative control(P<0.05).5.After infection of YTS cell with CD47-sh RNA,CD47 m RNA and protein expression was significantly reduced in CD47-sh RNA group.MTT assay showed that OD450 value of YTS cells at 48 h,72h and 96 h was decreased in the mi RNA-34 a group and CD47-sh RNA group when compared with control group.Flow cytometry showed that the apoptosis rate in the mi RNA-34 a group and CD47-sh RNA group was higher than that in the control group.Transwell invasion experiment showed that compared to control group,the cell number went through membrane was significantly reduced in the the mi RNA-34 a group and CD47-sh RNA group,difference had statistically significant(P< 0.05).Part ?: The impact of mi R-34 a on the YTS cell xenografted tumor in nude mouse modelsMethod: 1.The YTS cell xenografted tumor in nude mouse models was established.Three groups were designed: negative control group,Lenti-mi R34 a and Lenti-anti-mi R34 a.To draw the tumor growth-curve of tumor volume in each groups.The mice were killed and the tumor tissue was separated at the end of the experiment.2.The pathologic features,immunohistochemical findings and EBV positivity in tumor tissue of each nude mice group were detected by HE stains,immunohistochemitry and ISH detection.3.The apoptosis in tumor tissue ofeach nude micegroup was dectected by TUNEL.Results: 1.The volume of the xenografted tumors in Lenti-mi R34 a group was significantly smaller than that in the control group and Lenti-anti-mi R34 a group,the difference was statistically significant(P< 0.05).2.The gross morphology and pathologic features in each group were similar.The immunohistochemical stains in each group showed CD3,CD56,Granzyme-B and TIA-1were positive,but CD20 negative.All cases were EBV positive by ISH detection.The expression of CD47 protein in the Lenti-anti-mi R34 a group was higher than that in the control group,but no significant difference between the two groups(P>0.05).While in the Lenti-mi R34 a group was significantly lower than in the control group,the difference was statistically significant(P< 0.05).3.TUNEL results showed the apoptosis cells of tumor tissue in Lenti-anti-mi R34 a group increased than that in the control group and Lenti-anti-mi R34 a group.The difference was statistically significant(P< 0.05).Conclusions: 1.Lower expression of mi R-34 a in both NK/T cell lymphoma cell lines and tissues implicated this mi RNA may play an important role in tumorigenesis.2.Recombinant lentiviral vectors that could increase or decrease mi RNA-34 a expression had been constructed successfully.Up-regulation of mi R-34 a in NK/T cell lymphoma in vitro could significantly inhibit cells growth,promote cells apoptosis,and reduce the invasion ability of cells.3.CD47 was identified as a direct target gene of mi R-34 a.mi R-34 a could bond with 3'UTR regions of CD47,demonstrating that CD47 protein expression was directly downregulated by mi R-34 a via the inhibition of translation.4.The NK/T cell lymphomaxenografted tumor in nude mouse models was established.The animal experiment indicated that mi R-34 a could inhibit the xenografted tumor growth and promoted cells apoptosis innude mouse models.5.Taken together,mi R-34 a had tumor-suppressive roles in NK/T cell lymphoma.Further,mi R-34 a may represent novel candidate agents for the treatment of malignant lymphoma. |
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