Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults, despite better therapeutic options and improved survival of the patients, and treatment resistance is a major clinical challenge of DLBCL where approximately 40% of the patients have refractory disease or relapse. Therefore, it is urgent to find new drug targets and effective therapeutic drugs to improve the survival of DLBCL patients.
Nowadays, microarray analysis has become a widely used tool for generating gene expression data on a genomic scale and emerged as a promising and efficient tool for screening significant genetic or epigenetic alterations in carcinogenesis. MiRNAs are known as a large family of short ncRNAs. Its structurally consist of 19-22 nucleotides in length and functionally as one of the main regulators of gene expression in important biological and physiological environment like cell growth, apoptosis, proliferation, differentiation, cell motility, angiogenesis as well as disease formation, progression importantly in cancer cell invasion, migration, and metastasis. In the present study, we analyzed the differentially expressed miRNAs of DLBCL and LRH two groups via Human miRNA Array. A total of 2550 microRNAs were detected, FC≥2 and P ≤0.05 were used as the screening criteria, 204 differential miRNAs were screened, among which 54 was up-regulated and 150 were down-regulated. Studies have shown that miRNAs play an important role in the development of tumor. MiRNAs are also involved in the B cell development, such as B cell receptor, B cell migration/adhesion, the production of follicles, plasma cells, and memory B cells and so on. With the extensive development of miRNAs in various fields, miRNAs are expected to receive more and more attention, such as molecular targets for diagnosis, prognosis prediction and treatment of DLBCL. Beheshti et al discovered circulating miRNA signature in a Smurf2-deficient mouse model that spontaneously develops DLBCL. They investigated this 10-miRNA signature (miR-15a, let-7c, let-7b, miR-27a, miR-10b, miR-18a, miR-497, miR-130a, miR24, and miR-155), and derived let-7b, let-7c, miR-18a, miR-24, and miR-15a with a classification rate of 91% for serum from patients with DLBCL versus normal controls. These circulating miRNAs seemed to distinguish between DLBCL subtypes and disease characteristics for clinicopathological diagnosis. Ting et al found that miR-155, miR-17/92, miR-21, miR-224, and mir-146b-5p have value in predicting treatment response to chemotherapy in DLBCL. They suggested that miRNAs can be employed as an indicator to predict relapse or refractoriness after treatment with DLBCL. So our study screen 204 differential miRNAs will provide a basis for researchers in identifying the pathogenesis of DLBCL, could serve as reliable biomarkers for precise diagnosis, and as therapeutic targets for improvement of therapeutic efficacy in DLBCL in the future.
MiRNAs are an important regulator of gene expression, because they will eventually lead to a decrease in the observed mRNA expression of target genes. Therefore, we predicted target genes of the 204 differentially expressed miRNAs with 3 databases（Targetscan，microRNAorg，PITA）. We found 7522 target genes of 204 differential miRNAs. Our results will provide a theoretical basis for other researchers to study the occurrence and development of DLBCL.
MiRNAs regulated target genes or itself to activate or inhibit signaling pathways, have also become a research hotspot for tumor development and therapeutic targets in DLBCL. Furthermore, KEGG analysis was implemented to determine the roles of these target genes. We found that target genes were enriched in Pathway in cancers, MAPK signal pathway, Regulation of actin cytoskeleton, Focal adhesion, Endocytosis, Wnt signal pathway, Axon guidance, Calcium signal pathway, and PI3K/AKT signal pathway. Shim H et al pointed out that miR-124 is decreased in DLBCL, and that miR-124 is a tumor suppressor by targeting NF-κB p65 in B-cell lymphoma. Zhao CC et al indicated that SMAD5-AS1 inhibits DLBCL proliferation by sponging miR-135b-5p to up-regulate adenomatous polyposis coli expression and inactivate classic Wnt/β-catenin pathway. Yoon S et al found that the PI3K/Akt signaling pathway is strongly enriched with targets of a few miRNAs in DLBCL. In our study, some of the signaling pathways, such as pathway in cancers, MAPK signal pathway, Wnt signal pathway, PI3K/AKT signal pathway, which we found are consistent with relevant studies in DLBCL. But we also found a number of novel signaling pathways, such as Regulation of actin cytoskeleton, Focal adhesion, Endocytosis, Axon guidance, Calcium signal pathway, which are speculated to be related to the occurrence and development of DLBCL. The role of these newly discovered signaling pathways in DLBCL needs to be further studied.
According to our previous studies and literature review, the PI3K/AKT signaling pathway plays an important regulatory role in the occurrence and progress of DLBCL. So in this study we verified 8 differentially expressed miRNAs, which potential target gene maybe regulates the PI3K/AKT signaling pathway by qRT-PCR in DLBCL and LRH. We found that miR-19b-3p, miR-193a-3p, miR-370-3p and miR-490-5p are low expression in DLBCL (Figure 7A), and showed statistically differences (P < 0.05). The expression of miR-630 in DLBCL was high and the difference was statistically significant (P < 0.05). Compared with three groups of GCB, non-GCB and LRH, GCB and non-GCB showed statistically differences in the lower expression of miR-19b-3p, miR-193a-3p, miR-370-3p, miR-490-5p, and in the high expression of miR-630 in DLBCL (P < 0.05). There was no significant difference in miRNA expression between GCB and non-GCB DLBCL (Figure 7B).These differentially expressed miRNAs play a significant regulatory role in a variety of tumors, but there are few or no relevant studies in DLBCL.
MiR-19b-3p has been declared to be associated with favorable or unfavorable events in several cancers, its role is controversial depending on the tumor, and could be good non-invasive biomarkers for cancer detection. Tang Y et al indicated that miRNA plays an important role in the occurrence and development of intrahepatic cholangiocarcinoma (ICC). They collected 94 pairs of specimens of ICC tissues and adjacent tissues, and 5 ml of peripheral blood of 342 ICC patients who underwent ICC resection before and one week after surgery. Luciferase activity assay was confirmed the regulation of miR-19b-3p on coiled-coil domain containing 6 (CCDC6). The results showed that miR-19b-3p levels were significantly higher in ICC tissues compared with adjacent tissues. Serum miR-19b-3p levels of ICC patients tended to decline after surgery, and were related to lymph node metastasis and histological grading of ICC. They confirmed that miR-19b-3p promoted the ICC cell proliferation, epithelial-mesenchymal transition (EMT), inhibited apoptosis, and knockdown of CCDC6 reversed these effects. These results suggested that serum mir-19b-3p level is an important biomarker for ICC diagnosis, and targeting the miR-19b-3p-CCDC6 axis may be a promising strategy for ICC treatment. Song M et al confirmed that the roles of miR-19b-3p in pancreatic cancer. In this study human pancreatic cancer cell line was transfected with miR-19b-3p mimic and inhibitor. They found that miR-19b-3p overexpression promoted pancreatic cancer proliferation while miR-19b-3p inhibition decreased that. Flow cytometry analysis of cell cycle indicated that miR-19b-3p overexpression increased the percentage of pancreatic cancer in S phase while miR-19b-3p inhibition decreased that. The study demonstrates that miR-19b-3p promotes pancreatic cancer cells proliferation. Park EJ et al indicated that the transfection of the miR-19b-3p impeded breast cancer cell migration. They also found Aquaporin-5 (AQP5) plays a role in breast cancer cell migration, and using bioinformatic analyses identified miR-19b-3p as putative regulator of AQP5 mRNA. Finally, it was confirmed that miR-19b-3p can inhibit the migration of breast cancer cells through exosome-mediated delivery by targeting AQP5. Marcuello M et al showed that a plasma 6-miRNA signature (miR-15b-5p, miR-18a-5p, miR-29a-3p, miR-335-5p, miR-19a-3p and miR-19b-3p) could distinguish between colorectal cancer (CRC) or advanced adenomas (AA) and healthy individuals (controls). The study was included 213 individuals (CRC, 59, AA, 74, controls, 80). MiRNA expression was quantified by real-time RT-qPCR and data analysis was performed by logistic regression. They described in plasma, serum from patients with AA or CRC presented significant differences in the 6-miRNA signature compared to controls. The serum 6-miRNA signature could be a useful strategy to improve diagnostic performances of current CRC screening programmes. We can see that miR-19b-3p has been implicated in some cancers, but its role is controversial, and no relevant studies were noted in DLBCL.
Many studies recently presented the crucial role of the miR-193 family, which comprise miR-193a-3p, miR-193a-5p, miR-193b-3p, and miR-193b-5p in health and disease biological processes by interaction with special target gene and signal pathway, which mainly act as a tumor suppressor. Wang SS et al validated that miR-193a-3p is an anti-oncogene that plays an important role in health and disease biology by interacting with specific targets and signals, and it also inhibited the propagation and facilitated the apoptosis of hepatocellular carcinoma (HCC) cells. HCC patients with a higher level of miR-193a-3p expression had a favorable overall survival. They suggested that miR-193a-3p can be used as a promising biomarker for the diagnosis and therapeutic target of HCC in the future. Lin M et al aimed to explore the role and mechanism of miR-193a-3p in CRC. They found that the expression levels of miR-193a-3p in human CRC cell lines were significantly decreased compared with that in normal colonic epithelium cell line. Then, plasminogen activator urokinase (PLAU) was verified as a direct target gene of miR-193a-3p. Over-expression of miR-193a-3p inhibited proliferation, migration and angiogenesis of CRC cell, however, forced expression of PLAU could rescue the inhibitory effects. Chen ZM et al found that miR-193a-3p expression in pancreatic ductal adenocarcinoma (PDAC) tissue was significantly lower than in non-cancerous tissue. When overexpressing miR-193a-3p in PDAC cells, their multiplication ability was significantly inhibited, apoptosis was accelerated, and cell cycle was blocked in the G1 and G2/M phases. MiR-193a-3p may function as a tumor inhibitor in PDAC advance. In our study, we found that miR-193a-3p is low expression in DLBCL, and showed statistically differences compared with LHR (P < 0.05). We speculated the functional role of this miRNA in DLBCL considered as a tumor suppressor, however, according to our knowledge. There is no relevant report in DLBCL.
MiR-370-3p plays an important regulatory role in a variety of tumors, and growing evidence has suggested that it is down-regulated and acts as a suppressor in numerous cancers. Many studies indicated that it plays a regulatory role on tumors through the regulation of target genes, can increase the sensitivity to chemotherapy drugs, and can be also used as a biomarker or a therapeutic tool in tumors. Li LM et al investigated that the role of miR-370-3p in chronic myeloid leukemia (CML) inhibits cell proliferation and induces CML cell apoptosis by suppressing PDLIM1/Wnt/β-catenin signaling. They concluded that the expression of miR-370-3p has markedly decreased in the peripheral blood mononuclear cells of patients with CML and in cell lines. The miR-370-3p in CML cells up-regulated proliferation, and down-regulated apoptosis. Nadaradjane A et al investigated whether miR-370-3p can be used in vivo to increase the anti-glioblastoma multiforme (GBM) effect of temozolomide (TMZ). They used the model of LN18-induced GBMs in mice. The data indicated that the miRNA-370-3p/TMZ treatment was two times more efficient than the TMZ treatment for decreased the tumor volume. They supported that miR-370-3p could be used as a therapeutic tool for anti-GBM therapy. Leivonen SK et al profiled miRNAs of matched primary and relapsed DLBCL by next-generation sequencing. Thirteen miRNAs showed significant differential expression between primary and relapse specimen. MiR-370-3p was markedly down-regulated in most relapsed DLBCL samples, and over-expression of miR-370-3p regulated target genes MAP3K8, PIK3R1, PIK3CG, PI3KCD, SYK, and resulted in down-regulated mRNA levels. They validated that miR-370-3p down-regulate genes on the PI, MAPK, and BCR signaling pathways, and enhanced chemosensitivity of DLBCL cells in vitro. The results demonstrated that differentially expressed miRNAs promote disease progression by regulating key cell survival pathways and mediating chemical sensitivity, thus represented potential novel therapeutic targets. Our current experiment found that miR-370-3p was under-expressed in DLBCL, suggesting that it was may be inhibited the occurrence and development of DLBCL. Research of miR-370-3p in patient with DLBCL was rare, which calls for further study.
Recent studies have found that miR-490-5p is related to the occurrence and development of tumors, and play an important role in a variety of tumors. Wang J et al found that the expression of miR-490-5p was dramatically down-regulated in neuroblastoma (NB) tissues and cell lines using qRT- PCR. They found that significantly decreased miR-490-5p levels were correlated with lymph-node metastasis, and poor survival prognosis in NB patients, by used the Pearson Chi-square test and Kaplan-Meier analysis. The significantly over-expression of the miR-490-5p suppressed cell proliferation migration, invasion, induced cell cycle G0/G1 arrest, and cell apoptosis in NB cell lines. Myeloma overexpressed gene (MYEOV) was confirmed as a target gene of miR-490-5p by luciferase reporter assay. The results were demonstrated for the first time that miR-490-5p functions as a tumor suppressor in NB by targeting MYEOV. Nevertheless Xiang M et al shown that miR-490-5p promoted proliferation of bladder cancer cells, inhibited apoptosis of the cells. They suggested that miR-490-5p has potential to become a new target for the future treatment of bladder cancer. Yu Y et al explored targeting relationship between miR-490-5p and epithelial cell transforming sequence 2 (ECT2) in HCC, and influences of miR-490-5p and ECT2 on the stemness of HCC cells. They found that miR-490-5p was remarkably downregulated, and ECT2 was upregulated in HCC tissues compared with adjacent tissues. The expression of miR-490-5p was positively correlated with OS and DFS in patients with HCC. Over-expression of miR-490-5p restrained the proliferation, sphere formation ability, and stemness of HCC cells. MiR-490-5p inhibited the stemness of HCC cells through repressing the expression of ECT2. In our study, differential expression of miRNAs between DLBCL and LRH was screened, and low expression of miR-490-5p was found in DLBCL with a statistical difference (p=0.001). We speculated that miR-490-5p might be involved in the occurrence and development of DLBCL, but no relevant studies were conducted on its regulatory mechanism in DLBCL.
Evidence has demonstrated that miR-630 is involved in multiple processes in cancer development and progression. Valera VA et al performed miRNA profiling in young prostate cancer (EO-PCa) patients and compared with PCa in older men (LO-PCa). They found that compared with EO-PCa and its normal counterpart, miR -630 was significantly up-regulated. Differentially expressed miRNAs provided insights into the molecular mechanisms involve in this PCa subtype. Chen L et al observed that up-regulation of miR-630 inhibited oxaliplatin uptake in renal cell carcinoma by targeting organic cation transporter. Pan X-M et al firstly provided the evidence that miR-630 inhibited papillary thyroid carcinoma(PTC) cell growth, metastasis, epithelial-mesenchymal transition by suppressing JAK2/STAT3 signal pathway, and that a potential therapeutic strategy through enhancing miR-630 expression might benefit PTC patients. Li GW et al found that the expression of miR-630 was decreased in osteosarcoma (OS)tissues and cell lines. A low level of miR-630 was associated with late clinical stage and distant metastasis. Clinical assay indicated that down-regulation of miR-630 is closely linked to poor prognosis, and was an independent prognostic indicator for overall survival of OS patients. Functional studies showed that over-expression of miR-630 inhibited cell growth, colony formation, migration, invasion, EMT pathway, and promoted apoptosis in OS. In this experiment we screened of DLBCL and LRH differentially expressed miRNAs, found that miR-630 for differentially expressed miRNAs. Used fresh frozen tissue by qRT - PCR test, verification miR-630 with high expression in DLBCL (P = 0.007). We hypothesized that miR-630 may be involved in the occurrence or development of DLBCL, but no relevant studies have been conducted on DLBCL. Since it plays an important role in other tumors, it is worth further study in DLBCL.