To Investigate The MicroRNA Expression Profile In Diffuse Large B-cell Lymphoma

【Background】Non-Hodgkin ’s lymphoma(NHL) is a common malignant tumor of lymphoid tissue. The incidence rate of NHL ranks 12 th and the mortality rate ranks 10 th compared with other cancers. According to the cell oringination, the NHL can be divided into 3 groups: B-cell non-Hodgkin’s lymphoma, T-cell non-Hodgkin’s lymphoma, NK/T-cell non-Hodgkin’s lymphoma. Diffuse large B-cell lymphoma(DLBCL) is the most common type of NHL and accounts for 30% to 40% of NHL. DLCBL is a group of heterogeneous tumors with various clinical and pathologic features and shows rapid growth and highly invasive and metastatic features. Micro RNAs(mi RNAs) are a class of endogenous single-stranded and non-coding RNAs with a length of 21-25 nucleotides. Mi RNAs are highly conservative between different species. Mi RNAs can bind to one or more genes and negatively regulate gene expression at the post-transcriptional level by degradating target m RNA or repressing protein translation. Now, it is known that mi RNAs are involved in almost all biological processes, such as cell proliferation, differentiation, apoptosis, metabolism and tissue development. Studies have shown that mi RNAs function as oncogenes or tumor suppressors in the initiation and progression of tumors, so mi RNAs can be used as markers of early diagnosis and progonosis and potential targets for disease therapy. Abnormal expression of mi RNAs in lymphomas is frequently reported in recent years. Roehle has found that mi R-150, mi R-17-5p, mi R-145 and mi R-328 are specifically expressed in DLBCL. However, the role of mi RNAs in the pathogenesis of DLBCL has not been clearly eclucidated. Therefore, the analysis of mi RNA expression profile was performed in DLBCL tissues collected in Dali in this study.【Objective】In this study, the differential mi RNA expression profiles were analyzedin DLBCL compared with lymph node reactive hyperplasia. We predicted target genes of the differentially expressed mi RNAs and analyzed their functions. The purpose of this study was to investigate the molecular mechanism of carcinogenesis and development of DLBCL, and provide a theoretical basis for exploring markers of diagnosis and prognosis and targets for therapy.【Methods】We collected paraffin-embedded surgical or biopsy specimens from Affiliated Hospital of Dali University from March in 2012 to February in 2013. 10 cases of DLBCL tissues and 8 cases of lymph node reactive hyperplasia were selected as experimental group and control group, respectively. The differentially expressed mi RNAs were screened by mi RNA microarray and the target genes were predicted by bioinformatics methods. 1. mi RNA microarray: the seventh generation hybridization chip mi RCURYTMLNA(v.18.0) was used. 2. Total RNA was extracted using TRIzol(Invitrogen) and mi RNeasy mini kit(QIAGEN). The concentration and the purity of RNA were measured by Nano Drop spectrophotometer(ND-1000, Nano Drop Technologies), and the integrity of RNA was assessed by agarose gel electrophoresis. 3. mi RNA labelling: mi RNA labelling was performed using mi RCURY ? Hy3? / Hy5? Power labeling kit(Exiqon, Vedbaek, Denmark). 1μg of total RNA from each sample was labelled with Hy3 TM fluorescence at the 3’-terminal region. 4. mi RNA array hybridization: the labeled mi RNA was hybridizated with the mi RNA microarray chip. Subsequently, the chip was scanned by Axon Gene Pix 4000 B microarray scanner(Axon Instruments, Foster City, CA). 5. Data analysis: the images were analyzed by Gene Pix Pro6.0 software(Axon) to obtain data. Hierarchical clustering analysis was performed using MEV software(v4.6, TIGR). 6. Prediction of target genes: three databases(mirbase, miranda and targetscan) were used to predict the target genes of differentially expressed mi RNAs. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) databases were used to analyze the functions of target genes.【Results】1. Screening of differentially expressed mi RNAs: 59 mi RNAs were up-regulated and 67 mi RNAs were down-regulated in DLBCL compared with lymph node reactive hyperplasia. 2. Prediction of target genes: 67 target genes ofdown-regulated mi RNAs and 394 target genes of up-regulated mi RNAs were predicted by three kinds of softwares. 1042 target genes of down-regulated mi RNAs and 3500 target genes of up-regulated mi RNAs were predicted by two kinds of softwares. 3. Prediction of tumor-associated target genes: 3 micro RNAs and 4 target genes were identified to be associated with tumors by GO and pathway analysis and literatures summary. The micro RNAs were hsa-mi R-92a-3p, hsa-mi R-17-5p and hsa-mi R-155-3p. All of them were up-regulated in DLBCL. 4 target genes were ITGA5(hsa-mi R-92a-3p), CDKN1C(hsa-mi R-92a-3p), ITGB8(hsa-mi R-17-5p) and TP53INP1(hsa-mi R-155-3p).【 Conclusion 】 1. Hsa-mi R-92a-3p, hsa-mi R-17-5p and hsa-mi R-155-3p were up-regulated in DLBCL. We concluded that these mi RNAs may play important roles in the initiation, development, migration and invasion of DLBCL. 2. The predicted tumor-associated target genes were ITGA5(hsa-mi R-92a-3p), CDKN1C(hsa-mi R-92a-3p), ITGB8(hsa-mi R-17-5p) and TP53INP1(hsa-mi R-155-3p). 3. has-mi R-92a-3p and has-mi R-17-5p may regulate the expression of genes and promote the development of tumors in a collaborative manner. 4. There were no common target genes of has-mi R-92a-3p and hsa-mi R-155-3p, or has-mi R-17-5p and hsa-mi R-155-3p. 5. hsa-mi R-92a-3p, hsa-mi R-17-5p, hsa-mi R-155-3p probably function as oncogenes in DLBCL through binding to ITGA5(hsa-mi R-92a-3p), CDKN1C(hsa-mi R-92a-3p), ITGB8(hsa-mi R-17-5p) and TP53INP1(hsa-mi R-155-3p).