| BackgroundAt present, malignant lymphoma is one of the fastest growing cancers, which is a serious damage to human health. NK/T lymphoma is a subtype of non-Hodgkin’s lymphom, and the incidence rate is in the first place of T-cell lymphomas. The incidence rate of China is much higher than Western countries. WHO has made NK/T lymphoma as an independent type among the lymphoid hematopoietic tissue tumors in 2001. The etiology and pathogenesis of malignant lymphoma is not entirely clear, and generally considered to be the result of interaction of multiple factors, such as viral infections, some physical and chemical stimuli, and immune defection.Traditional therapy methods include chemotherapy, radiotherapy, immunother-apy and biological therapy. Some subtypes of lymphoma patients in the early stage have better curative effect, but there are still some types of lymphoma are hard to get effective treatment or a relapse after treatment. The current study suggests that there is important relation between the biological characteristics, such as lymphoma recurrence, drug resistance, with some key target genes or small molecular markers. Therefore, the basic and clinical research of lymphoma, which provides effective targets and biomarkers for clinical diagnosis, is of significance.In recent years, it has been found that a number of miRNA are associated closely with mammalian hematopoietic cell differentiation, hematopoiesis and hematologic diseases. Recent studies have shown that specific miRNA also plays an important regulatory function in T and B cells development process. In addition, miRNA is also related with lymphoma. It has been found that in diffuse large B-cell lymphoma there is the coordination between the miRNA-17-92 family and cancer gene C-myc, which can induce the lymphoma formation.The miRNA-17-92 family can regulate tumor suppressor gene PTEN and pro-apoptotic protein Bim and induce lymphoma proliferative diseases and autoimmune diseases formation.In recent years, many studies have confirmed that miRNA-155 has a wide variety of biological functions:differentiation processes of hematopoietic cell, the regulation of inflammatory cytokines and immune response, highly expression in various tumors, downregulation of other miRNAs directly or indirectly in promoting the development of tumors. The expression level of miRNA-155 in patients with lymphoma is 10-30 times more than the normal. However, the expression quantities of miRNA-155 are not the same in different lymphoma cells and all of these have indicated that miRNA-155 may have a certain correlation in the differentiation of lymphoma.At present, there are many reports about the mechanism of miRNA-155 in gastric cancer, breast cancer, cervical cancer, leukemia and B-cell lymphomas, but less in other types such as T cells and NK cell lymphomas.In this study, we do a high-throughput small RNA deep sequencing of NK/T cell lymphoma cell lines YTS, SNK-6 and as normal NK cells through high-throughput sequencing technology, We analysis miRNA gene expression of these cells by this detection. Meanwhile, we screen and establish the cell lines of endogenous high expression and low expression of microRNA-155, study the biological characteristics of these cell lines, and analysis high-throughput detection of bioinformatics data. This study evaluate the affections of miRNA-155 on biological characteristics of lymphoma cell, reveal the molecular mechanisms of miRNA-155 in lymphoma cells, and provide new treatment targets for lymphoma basic and clinical research, as well as provide new effective methods on the studies of miRNA in cancer.Main contentChapter I:Small RNA sequencing and identification of miRNA-155 expression in NK/T cell lymphoma cell linesMethod:1. Total RNA of normal NK cells, SNK-6 cells and YTS were extracted; RNA was purified and monitored so that the extracted RNA could meet the sequencing requirements to build databases. The solexa high-throughput sequencing technol-ogy of BGI was used on the cell line of small RNA sequencing.2. Bioinformatics analysis of sequencing results:first, we made a basic data assessment and treatment for all the Raw data measured in small RNA; second, we did the further advanced bioinformatics analysis, which include:genome alignment; known microRNAs appraisal and statistical information, expression spectrum information; repeat sequence aligment; Genbank aligment and Rfam aligment; exons and introns aligment; small RNA classification annotations; candidate microRNAs prediction; microRNAs variance analysis; clustering analysis; the miRNA target genes prediction.3. In order to further characterize the reliability of high-throughput sequencing, we used Real Time PCR to quantize the miRNA-34a, miRNA-34b, miRNA-34c, miRNA-155, miRNA-21 and miRNA-221 of these cell lines.Results:1. By Solexa sequencing technology, NK/T cell lymphoma cell line:YTS, SNK-6 and normal NK cells be done the high-throughput small RNA deep sequencing and got 7476202,6903218 and 8458056 clean reads respectively and the vast majority concentrated sequence were 18-24nt. Through comparative analysis of bioinformatics,194,221 and 276 known miRNAs were identified in normal NK cells, SNK-6 cells and YTS cells respectively and also 42,72 and 161 new miRNAs were found.2. Through the difference comparison analysis, cluster analysis and further biological information analysis of miRNA in SNK-6, YTS and normal NK cells, it was found that the 25 candidate miRNAs were upregulated and 12 downregulated in SNK-6 cell line, and 30 candidate miRNAs were upregulated and 12 downregulated in YTS cell line.3. By Real Time PCR to further identify some of microRNA expression, the expression of most microRNA was consistent with the result of high-throughput sequencing, but some were not. Meanwhile, it was found that miRNA-155 was significantly higher in SNK-6 cells than NK cells, and even have significant differences compared with YTS cells. It’s shown hat miRNA-155 may play an important role in SNK-6 cells.Chapter Ⅱ:The Expression and Clinical Significance of microRNA-155 in NK/T-cell lymphoma patientsMethod:1. Specimen collection and preparation:Admitted lymphoma patients, including 20 NK/T cell lymphoma patients and 15 healthy people were from Department of Oncology, the First Affiliated Hospital of Zhengzhou University, in 2011.9-2013.1. Their peripheral blood serum was separated before each chemotherapy or initial treatment. All patients signed an informed consent. Patient datas were collected include name, gender, age, symptoms start date, diagnosis date, start date of treatment, starting position, stage inspection methods, with or without B symptoms, bone marrow condition, Ann Arbor stage, number of involved lymph nodes, extranodal involvement part number, PS score, ALB, LDH, serum Hb, WBC count (before treatment, treatment options, recent treatment), long-term survival and so on. The patients, whose clinical pathological or data were incomplete, were excluded from this study. Healthy controls must exclude chronic infection or autoimmune disease. All pathological specimens were validated by three experienced pathologists according to WHO classification standard.2. The serum total RNAs were extracted with the use of Shanghai Novland blood (serum/plasma) total RNA extraction kit.3. The miRNA-155 of blood serum in lymphoma patients and normal was quantitatively detected and its expression in the lymphoma and normal serum was analyzed by the way of Real Time PCR.Results:1. miRNA-155 was significantly higher in NK/T cell lymphoma patients than the normal control serum, suggesting that miRNA-155 may be associated with the developments of NK/T cell lymphoma.2. The expression of miRNA-155 in Lymphoma serum was irrelevant to the stage, B symptoms, LDH and β32-MG levels. miRNA-155 in chemotherapy ineffective group was significantly higher than that of effective group, suggesting that miRNA-155 may be used as a potential molecular markers to determine therapeutic efficacy.Chapter III:The effection of miRNA-155 on biological characteristics of the NK/T lymphoma cell linesMethod:1. The expression miRNA-155 in lymphoma cell lines detected by Real Time PCR: Total RNAs of the lymphoma cell lines (human NK/T cell lymphoma cell lines YTS and SNK-6, human T-cell lymphoma cell line Jurkat and human follicular B-cell lymphoma cell line DOHH2) were extracted, miRNA-155 and U6 were reverse transcript, and quantitative analysis were performed by Real Time PCR using TaKaRa Fluorescent Quantitative Real Time PCR Kit.2. Construction of eukaryotic expression vector: The oligonucleotide fragments of miRNA-155 and anti-miRNA-155 were anneal, and stored at-20℃. Annealing fragments and pEGFP-C3 digested by ScaI and XhoI were legated to construct expression vector of anti-miRNA-155 and miRNA-155. Bgl Ⅱ was used to identify the correct clones, and then the correct clones were sequenced.3. Lentiviral vector construction and packaging: PEGFP-miRNA-155, pEGFP-anti-miRNA-155 and pLL3.7 were digested by NheI and EcoRI to construct lentiviral vector. High purity without endotoxin lentivirus expression plasmid and its helper packaging plasmids were puritied. Lentiviruses were packaged by co-transfecting 293T cells with Lipofectamine 2000 and the above-mentioned plasmid DNAs.48h later, the lentiviruses (Lenti(-), Lenti-miRNA-155 and Lenti-anti-miRNA-155) were collected and concentrated and purified by ultracentrifugation. Lentivirus titers were determinated by doubling dilution method based on the green fluorescent protein expression of lentiviral.4. Infection of lymphoma cells with the recombinant lentivirus: Lentiviruses were diluted with serum-free medium solution, and infected lymphoma cells SNK-6 with different MOI. Observe fluorescence expression 4 days later, and calculate fluorescent cells each 100 cells. This was to evaluate lentivirus efficiency of infection to lymphoma cell line.5. Detect the expression quantity of miRNA-155 in SNK-6 lymphoma cells after be infected by recombinant lentivirus infection: Recombinant lentivirus (Lenti(-), Lenti-miRNA-155 and lenti-anti-miRNA-155) (MOI= 100) infected lymphoma cells SNK-6. Total RNAs were extracted after 48h, reverse transcript miRNA-155 and U6. Detect the expression of miRNA-155 using quantitative PCR method.6. CCK-8 detection miRNA-155 effect on lymphocyte growth proliferation: Recombinant lentivirus (MOI=100) infected SNK-6 cells, and divided into four groups:control group (PBS), negative control group (Lenti (-)), miRNA-155 overexpression group (Lenti-miRNA-155), miRNA-155 inhibition expression group (Lenti-anti-miRNA-155). After 72h, CCK8 detect cell proliferation of SNK-6 lymphoma cell.7. Detection apoptosis by Annexin V/PI double staining: Recombinant lentivirus (MOI= 100) infected SNK-6 cells, and divided into four groups:control group (PBS), negative control group (Lenti (-)), miRNA-155 overexpression gourp (Lenti-miRNA-155), miRNA-155 inhibition expression group (Lenti-anti-miRNA-155). After 48h, the cells were collected and stained by Annexin V/PI double staining reagents for flow cytometric analysis.8. MiRNA-155 effects on arsenic trioxide(As2O3) cytotoxicity lymphoma cell line: Recombinant lentivirus (MOI=100) infected SNK-6 cells, and divided into five groups:control group (PBS), As2O3 group、negative control group (Lenti(-)), AS2O3+ miRNA-155 overexpression group (Lenti-miRNA-155), As2O3+miRNA-155 inhibition expression group (Lenti-anti-miRNA-155). After 48h, the cells were collected, and stained by Annexin V/PI double staining reagent for flow cytometry analysis.9. miRNA-155 lymphoma cell lines in the target gene detection: Recombinant lentivirus (MOI= 100) infected SNK-6 cells, and divided into four groups:control group (PBS), negative control group (Lenti (-)), miRNA-155 overexpression gourp (Lenti-miRNA-155), miRNA-155 inhibition expression group (Lenti-anti-miRNA-155). After 48h, total protein of the cells were collected, and target gene expression was detected by Western Blot.Results:1. The results showed that relative expression levels of miRNA-155 in four kinds of lymphoma cell lines(YTS, SNK-6, Jurkat cells and DOHH2) were 7.47±2.25, 78.96±18.80,17.84±4.09 and 1 (DOHH2 as the reference). The expression level of SNK-6 cells was significantly higher than the other cell lines (p< 0.05), DOHH2 cells were the lowest relative expression among these cells (p< 0.05). Relative expression levels of Jurkat cells were significantly higher than YTS and DOHH2 cells (p< 0.05).2. The eukaryotic expression vectors were digested by BglⅡ, and the results show that synthetic fragment of miRNA-155 and anti-miRNA-155 was inserted into pEGFP-C3. The sequencing results were analyses by MegAlign software. The comparison results showed that sequences of miRNA-155 and anti-miRNA-155 were consistent with the design and synthesis. These indicated that eukaryotic expression vector was constructed correctly. XhoI digestion results were also consistent with the designed, suggesting that recombination lentiviral vector was successfully constructed.3. The lentiviral expression plasmid Lenti(-), Lenti-miRNA-155, Lenti-anti-miRNA-155 and the lentiviral packaging plasmid pCMV-dR8.2, pCMV-VSV-G were co-transfected into 293T cells. After 48h, the Lentivirus (Lenti (-), Lenti-miRNA-155, and Lenti-anti-miRNA-155) were collected, concentrated and purified by ultracentrifugation. The virus titers were detected by dilution method, using the green fluorescent protein gene expression in the infection cell. The titer of the virus was concentrated up to 109 IFU/ml or more. Three kinds of recomb-ineant lentivirus Lenti (-), Lenti-miRNA-155 and Lenti-anti-miRNA-155 were: 109.13 ± 0.15, 109.11 ± 0.24 and 109.15 ± 0.12IFU/ml,and there was no significant difference between them (P> 0.05).4. Recombinant Lentiviral infection efficiency were gradually increased with MOI, low infection efficiency< 10% when MOI<10; MOI=10, the infection efficiency can be increased to more than 30%; while, the infection efficiency increases to more than 60% when MOI> 10. SNK-6 infection rates of Lenti(-), Lenti-miRNA-155, and Lenti-anti-miRNA-155 were 72.09±2.96%,72.36±2.39% and 72.89± 5.65% when MOI=100, and no significant difference between them (P> 0.05).5. Real Time PCR detected of the expression of miRNA-155 in SNK-6 cells before and after was infected by recombinant lentivirus Lenti (-), Lenti-miRNA-155, and Lenti-anti-miRNA-155. The expression of miRNA-155 showed no significant difference (P> 0.05) between recombinant lentivirus Lenti(-) infection group and no infection group. After infection by Lenti-miRNA-155, miRNA-155 exp-ression was significantly higher than the negative control group (P< 0.05), and the relative expression level was 4.33±1.245. MiRNA-155 expression was significa-ntly decreased (P< 0.05) than the negative group, and the relative expression level of miRNA-155 was 0.37±0.052.6. By CCK8 cell activity assay, we had detected the proliferation activity of SNK-6 cell was infected or not infected by Lenti (-), Lenti-miRNA-155, and Lenti-anti-miRNA-155. The results showed that the proliferation activities of SNK-6 cell of each group (negative control group, miRNA-155 overexpression group, and miRNA-155 inhibition expression group) were respectively:0.7828±0.043, 0.857±0.050 and 0.533±0.052. miRNA-155 overexpression group had no signi-ficant difference with negative control group (P> 0.05), but cell proliferation activity of miRNA-155 inhibition expression group was significantly decreased than the other two groups (P< 0.05).7. Annexin V/PI double staining to detect apoptosis. Lentivirus infected SNK-6 lymphoma cell lines, and then the cells apoptosis rate was detected by the Annexin V/PI double staining assay. Proportion of apoptotic cells rates were: (11.2±1.24)%, (12.3±1.67)% and (63.2±5.78)%. The results showed that miRNA-155 expression inhibition group (Lenti-anti-miRNA-155) promoted the SNK-6 cell apopotosis obviously.8. Flow cytometry analysis showed that the ratios of apoptotic cells of blank group, arsenic trioxide group, negative control group, As2O3 combined with miRNA-155 overexpression and As2O3 combined with miRNA-155 inhibition expression group were respectively:(1.5±0.236)%, (4.7±3.101)%, (11.7± 3.027)%, (11.2±3.1)% and (61.2±4.561)%. The As2O3 combined with miRNA-155 inhibition expression group is more obvious and more significant pathological changes than the other groups, the difference between groups was statistically significant (P< 0.05). As2O3 group, negative control group, and As2O3 combined with miRNA-155 overexpression were no significant difference in apoptosis rate (P> 0.05).9. By Targetscan online miRNA prediction software, predicted FOXO3a as miRNA-155 target genes. The protein expression of FOXO3a was detected by Western Blot. It was found that the expression level of FOXO3a was lower in the miRNA-155 overexpression group than the negative control group, while the expression of FOXO3a protein was higher in the miRNA-155 inhibition expre-ssion group. Through the gray analysis software analyzed the expression level of FOXO3a protein had significant difference between miRNA-155 inhibiiton group and the other two groups (P< 0.05), and there was no significant difference (P> 0.05) between miRNA overexpression group and negative control group.Conclusion:1. Small RNA of NK cells, SNK-6 cells and YTS cells were done high-throughput sequencing by Solexa sequencing technology. The results was analyses by advanced bioinformatics assay, and we found the expression level of miRNA-155 in SNK-6 cells was significantly higher than normal NK cells, also have significant differences than YTS cell line. This suggested that miRNA-155 may play an important role in SNK-6 cells.2. MiRNA-155 expression level in NK/T cell lymphoma in patients was high, suggesting that miRNA-155 not only maybe associated with the development of NK/T cell lymphoma, but also may be a molecular marker of the potential efficacy.3. Based on the biological functions research of miRNA-155 in NK/T cell lymphoma cell lines SNK-6 cells, it is found that miRNA-155 may be correlated with SNK-6 cell proliferation, apoptosis, cell cycle and drug sensitivity. Meanwhile, miRNA-155 may affect NK/T lymphoma biological function through the FOXO3a role pathway. |
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