| 【Background】Lymphoma is the malignant neoplasm which is originated in lymph hematopoietic system, and is also a kind of solid tumor of the immune system[1].Lymphomas can be divided into two major categories: Hodgkin’s lymphoma(HL) and non-Hodgkin’s lymphoma(NHL). Almost 90% of lymphomas are NHL[1,2].According to the different origin of lymphocytes, NHL can be divided into three types:B-cell, T-cell and NK cell lymphoma. 70%~80% of NHL are B-cell types. Diffuse large B-cell lymphoma(DLBCL) is the most common, highly heterogeneous and invasive B-cell lymphoma with different pathologic types, showing rapid growth and various clinical features. DLBCL can be divided into three subtypes by microarray and Immunohistochemistry: germinal center B-cell-like(GCB), activated B-cell-like(ABC) and the third subtype. At present, 50% of the patients can be cured with R-CHOP Chemotherapy, but 40% of the patients are dead due to recurrence.Micro RNAs(mi RNAs) are a class of endogenous single-stranded and non-coding RNAs with a length of 20-24 nucleotides. 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. The main function of mi RNAs is to regulate the expression of genes related with the growth and development of organisms and diseases. mi RNAs are involved in the biological processes of cell proliferation, differentiation and apoptosis in human. In recent years, it has been reported that abnormal expression of mi RNAs plays a role of oncogene or tumor suppressor in many diseases, including cancers[7-9]. The expression of mi RNAs are critical effect factors of the occurrence and development of tumors, so the relationship between mi RNAs and tumors has become a focus in clinical and basic research.【Objective】In this study, in order to identify the differentially expressed mi RNAs in DLBCL, the mi RNA expression profiles were analyzed by mi RNA microarray. The expression of differentially expressed mi RNAs are verified by Real-time PCR and the significance of these mi RNAs are analyzed in combination with the clinical data. The tumor-related target genes of the differentially expressed mi RNAs are further predicted. This study is expected to provide a theoretical basis for exploring the roles of mi RNAs as potential target for diagnosis, treatment and prognosis of DLBCL.【Methods】The paraffin-embedded surgical or biopsy specimens are collected from several hospitals in Yunnan province(including the college of Clinical Medicine, Dali University; Infectious Disease Hospital of Yunnan Province; the first people’s Hospital of Yunnan, Kunming; Infectious Disease Hospital of Kunming) from December in 2012 to February in 2013, including 10 cases of DLBCL(tumor group)and 8 cases of RH(benign lesion group). 6 cases of HIV-DLBCL and 4 cases of non-HIV-DLBCL are included in the tumor group. 4 cases of HIV-RH and 4 cases of non-HIV-RH are included in the benign lesion group. The differentially expressed mi RNAs were screened by mi RNA microarray. Because of the small amounts of samples, there may exist individual differences. In order to select out mi RNAs of more reliable expression differences, the results of three groups(tumor group vs non-tumor group, HIV-DLBCL vs HIV-RH, non-HIV-DLBCL vs non-HIV-RH) were intersected to obtain the differentially expressed mi RNAs. The differential expression of mi RNAs which have been reported in literatures, are verified with Real-time PCR.The correlated between mi RNAs and clinic pathological features are further analyzed.Three databases(Targetscan, Miranda and Mirbase) were used to predict the target genes of differentially expressed mi RNAs. Combining with of literatures, Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) databases were used to analyze the functions of target genes.【Results】1. A total of 119 differentially expressed mi RNAs were identified in the tumor group compared with the non-tumor group, including 53 upregulated mi RNAs and 66 downregulated miRNAs.2. A total of 106 differentially expressed mi RNAs were identified in the HIV-DLBCL compared with the HIV-RH, including 58 upregulated mi RNAs and 48 downregulated miRNAs.3. A total of 58 differentially expressed mi RNAs were identified in the non-HIV-DLBCL and non-HIV-RH, including 18 upregulated mi RNAs and 40 downregulated mi RNAs.4.10 mi RNAs were obtained by intersection analysis, including 5 upregulated mi RNAs(mi R-3648, mir-17-5p, mi R-20a-5p, mi R-106a-5p, mi R-3116) and 5downregulated mi RNAs(mi R-3691-5p, mi R-4489, mi R-4423-3p, mi R-10b-5p,let-7c-5p).5. The miRNA expression detected by Real-time PCR was consistent with the results analyzed by mi RNA microarray. Compared with RH tissues, the expression of mi R-17-5p, mi R-106a-5p and mi R-20a-5p werw upregulated in DLBCL. In contrast,mi R-10b-5p and let-7c-5p were downregulated in DLBCL.6. mi R-17-5p significantly correlated with mi R-20a-5p(P <0.001).7. The expression levels of mi R-17-5p and mi R-20a-5p were associated with the clinical stages. With the increase of clinical stage, the relative expression of mi R-17-5p and mi R-20a-5p were also increased(P = 0.041, P = 0.043). mi R-20a-5p was also associated with the immune phenotypes of DLBCL. The expression of mi R-20a-5p was higher in GCB-DLBCL than that in non-GCB-DLBCL(P = 0.015).8. 1263 target genes of both mi R-17-5p and mi R-20a-5p were predicted by at least two kinds of softwares(Targetscan, Miranda and Mirbase). The target genes were identified to be associated with tumors by GO and pathway analysis and in combination with literatures. 3 target genes(CHD5, TRIM3, PTEN) were found to be associated with the pathogenesis and development of DLBCL.【Conclusion】1. Compared with the RH tissues, the expression of mi R-17-5p, mi R-106a-5p and mi R-20a-5p was upregulated in DLBCL, and the expression of mi R-10b-5p and let-7c-5p was downregulated in DLBCL. All of the five mi RNAs were involved in occurrence and development of DLBCL.2. miR-17-5p has shown a high correlation with miR-20a-5p, and the expression of both positively correlated with the clinical stages of DLBCL. It suggests mi R-17-5p and mi R-20a-5p function as oncogenes in DLBCL. The expression of mi R-20a-5p was significantly higher in GCB-DLBCL than that in non-GCB-DLBCL, suggesting that mi R-20a-5p may be associated with formation of germinal centre in DLBCL.3. These results indicated that mi R-17-5p and mi R-20a-5p may have synergistic effects through inhibiting the target genes(CHD5, TRIM3 and PTEN) in the progression of DLBCL, suggesting that they may become molecular markers for clinical stages.4. The differentially expressed mi RNAs in DLBCL and the target genes were worthy of further study, and they may be possible therapeutic targets for DLBCL. |
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