Introduction

Chronic lymphoid leukemia (CLL) is known as the most frequent type of leukemia in adults. CLL is characterized by mature-appearing monoclonal B cells with CD5, CD19, CD23 markers and reduction of membrane-bound IgM and IgD.1 CLL is the most common form of leukemia, accounting for ~30% of all cases. Similar to other cancers, the lack of early symptoms results in late detection of the disease and adverse sequelae.2 B-lymphocytes mature in bone marrow through rearrangement of immunoglobulin variable (V) gene segments which results in the formation of the code for an immunoglobulin molecule, used as the B-cell antigen receptor. This process includes rearrangement of heavy fragments of the VDJ gene that encodes the binding site of the receptor.2, 3 According to previous investigations, prognostic prediction in CLL is based on V-gene mutations, CD38, ZAP-70, deletion of chromosome. Expansion and somatic hypermutation of post-germinal center (memory) B cells are the most reported causes of IgVh mutation, and is the most crucial factor in this disease.3, 4, 5 In addition, various types of chromosomal deletions, such as 17q13, 6q21, 11q23, 13q14 and 17p13, have been observed in CLL patients.5, 6, 7 Dysfunction of TP53 due to regional deletion of chromosome 17 (17p13) has been reported to be associated with resistance to therapy with purine analogs and alkylating agents in CLL. Furthermore, ATM gene deletion on chromosome 11 (11q22) increases MDM2 activity which in turn decreases p53 pathway activity in CLL.8

MiRNAs are a class of small noncoding RNAs about 21–25 nucleotides long that regulate gene expression post-transcriptionally by binding to the 3′-untranslated region (UTR) of their mRNA targets, resulting in degradation or transcriptional repression of the targeted mRNA.9, 10, 11 MiRNAs control numerous cellular/molecular processes such as apoptosis, cell proliferation and differentiation.12, 13 Deregulated expression of miRNAs contributes to the initiation or development of numerous diseases such as cancer.10, 12 The role of miRNAs has been previously reported in cell cycle control, in a synergism with some transcription factors, namely c-myc, E2F or P53.14 miR-192 has been suggested to be a positive regulator of p53. This miRNA has a significant impact on cell differentiation, proliferation and apoptosis.2, 15, 16 Several studies have reported deregulated expression of miR-192 in biological samples from cancer patients.17, 18 However, only two studies have reported involvement of miR-192 in CLL: Georges et al. compared miR-192 expression in CLL versus CD5– cells and Di Lisio et al. assessed differentially expressed miRNAs in each type of lymphoma.19, 20

In the current study, the expression of miR-192 was analyzed in peripheral blood mononuclear cells (PBMCs) of CLL patients in comparison with healthy subjects in order to determine whether deregulated expression of this miRNA can be used as a diagnostic biomarker. Furthermore, pathway enrichment analysis was performed to unravel the possible role of miR-192 in the pathogenesis of CLL, and relevant signaling pathways which are affected by this miRNA in CLL.

Materials and methods

Diagnostic tests

Patients and samples

Blood samples were taken from patients with early-stage disease and random samples from healthy men and women. Forty samples, from 20 patients with CLL, diagnosed in the Omid Hospital (Isfahan, Iran), and 20 healthy controls were sampled during a period of 4 months, according to the Internal Review and the Ethics Boards of the Isfahan University of Medical Sciences. (Table 1). Exclusion criteria were: (i) CLL diagnosis more than 12 months before registration; (ii) clinical Binet stage B or stage C; (iii) need of therapy according to NCI guidelines; and (iv) age>70 years.21 After informed consent was obtained from the participants, 4 ml blood was collected in EDTA-containing tubes. Immediately after collection, the blood samples were transferred to a laboratory on ice. The Ethics Committee of the Omid Hospital approved the protocol of this study.

Table 1 Symptoms, clinical and biological features of patients included in this study
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Complete blood count

This test was performed using CA&XN-Series TM Automated Hematology Analyses. The Sysmex XN-Series uses fluorescence flow cytometry and three-dimensional highly sensitive flagging. This device determines nucleated red blood cells, white blood cell abnormalities, immature granulocytes, and, platelet immature cells and detects atypical lymphocytes. Stages of disease progression were seen in the complete blood count test in three CLL patients with this device as shown in Table 2 and Figure 1.

Table 2 Hematological parameters in three selected patients
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Figure 1
figure 1

Condition of CBC in three patients and progression of the disease (a–c). CBC, complete blood count; RBC, red blood cell; PLT, platelets; WBC, white blood cell; WDF, WBC differential; WNR, white cell nucleated.

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Peripheral blood mononuclear cells (PBMCs) isolation from blood

PBMCs were separated by density gradient lymphosep (Bio Sera, Kansas City, USA) according to the manufacturer’s protocol. Because mononuclear cells, monocytes and lymphocytes have lower density in comparison with erythrocytes and leukocytes (granulocytes), after centrifugation, they remain in an intermediate phase, while other cells are deposited. Four ml of blood was diluted at a ratio of 1:1 with physiological saline and gradually added to the 4 ml lymphoprep solution gradient in a falcon tube. Prepared falcons were centrifuged at 800 g for 30 min at room temperature, and then PBMCs were transferred from the middle phase into a 2 ml RNAase-free micro tube. After washing and cell counting, PBMCs were sedimented using centrifugation for 10 min at 250 g and were then frozen at −70 °C until the RNA extraction stage.

RNA extraction

Total RNA from PBMCs was extracted using miRNA Hybrid-R (Geneall, Seoul, Korea) based on the manufacturer's instructions. Quality of total RNA was determined at a 260/280 nm wavelength ratio measured by a NanoDrop spectrometer (Thermo Scientific, Waltham, MA, USA).

cDNA synthesis and real-time PCR

cDNA synthesis of miR-192 was performed using a commercial kit (Pars Genome, Tehran, Iran) and according to the manufacturer's leaflet. Real-time quantitative PCR reactions were accomplished in triplicate. Briefly, in a total volume of 10 μl, 20 ng μl−1 of cDNA product was added to a master mix, including 10 pmol μl−1 of miR-192 U6 (Housekeeping) primers (Pars genome) and 5 ml of SYBR premix ExTaq II (TaKaRa, Kusatsu, Shiga Prefecture, Japan). The run program was set at 95 °C for 5 mines followed by 40 cycles of 95 °C for 5 s, 60 °C for 20 s and 72 °C for 30 s.

Statistical analysis

Real-time PCR data analysis was performed using the ΔΔCT method and the final result was normalized by small nuclear RNA, U6, as an endogenous control. All statistical tests were performed using REST 2009 V2.0.13 and Graph Pad Prism statistical software, version 5.01 (Graph Pad, San Diego, CA, USA). For all tests a P-value of <0.05 was considered statistically significant.

Systematic pathway enrichment analysis

In molecular enrichment analysis on miR-192 targetome and signaling pathways, several databases were used, including the miRecords22 and miRTarBase23 databases, to obtain predicted and validated targets of miR-192. Then, to fulfill signaling pathway enrichment analysis, miR-192 targetome was imputed in the database for annotation, visualization and integrated discovery (DAVID) online database, version 6.7.24 This database provides the results from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify the signaling pathways and molecular networks with miR-192 targetome.25

Results

miR-192 expression in CLL patients versus healthy subjects

The expression of miR-192 was evaluated by quantitative real-time PCR method in two groups: CLL patients (n=20) and healthy subjects (n=20). Clinical and biological features of patients are shown in Supplementary Table 1. Data analysis using SPSS statistical (Chicago, IL, USA) and REST software (Newcastle, UK) indicated a significant reduction in the expression of miR-192 in patients with CLL compared with healthy individuals (P=0.002 ~2.5-fold; Figure 2).

Figure 2
figure 2

Comparison of RE CLL with control subjects. Downregulation of miR-192 in CLL patients. Quantitative PCR analysis of miR-192 expression level in PBMCs of CLL patients (n=20) and normal controls (n=20). Results are normalized to those of controls and are represented relative to expression of the small nuclear RNA U6. CLL, chronic lymphocytic leukemia; PBMCs, peripheral blood mononuclear cells.

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In this study, three variables associated with CLL (that is, WBC, lymphocyte and platelet counts) were checked using SPSS software. White blood cell counts were higher than the reference interval in 65% of patients, also lymphocyte and platelet counts in 90% and 45% of patients, respectively.

Molecular signaling pathway enrichment analysis

Using miRrecords and miRTarBase databases, 145 and 25 mRNAs were identified as validated and predicted targets of miR-192, respectively (Supplementary Table 1).

Predicted targets were approved by at least four prediction databases. All validated targets recovered from the miRTarBase database were supported by strong experimental evidence such as reporter assay, western blot and quantitative real-time PCR. Imputing gene symbols of selected miR-192 targetome into a useful annotation tool of DAVID determined statistically significant organization of imputed genes with several KEGG signaling pathways (Table 3). The set of imputed genes as miR-192 targetome was mostly enriched in several KEGG pathways involved in cell cycle and cancer signaling (Table 3). The KEGG pathways in cancer signaling are shown in Figure 3 and targets of miR-192 are indicated by red stars.

Table 3 The seven most statistically relevant KEGG signaling pathways with miR-192 targetome (DAIVD database)
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Figure 3
figure 3

Involvement ofmiR-192 targetome in cell cycle signaling pathway. For more information please see the KEGG database website: http://www.genome.jp/dbget-bin/www_bget?map04350.

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Discussion

In the present study, we showed a significant reduction in the expression of miR-192 in patients with CLL compared with healthy individuals. High expression of miR-192 inhibits metastatic colonization through increased apoptosis and decreased proliferation. CLL is defined as a disease of mature B cells. In CLL cancer cells originate from lymphoid cells, particularly B lymphocytes. These immature lymphocytes are observed in the blood and bone marrow. The monoclonal B cells in CLL express CD19, CD5, and CD23. CLL is observed in lymph nodes, spleen and other tissues.2 CLL leads to a decrease in platelet or erythrocyte count due to bleeding and bruising. Appropriate non-aggressive biomarkers with high sensitivity and specificity are required for the diagnosis of the disease. MiRNAs have emerged as biomarkers for the diagnosis and prognosis of different types of cancer.10, 12, 13, 26 Downregulation of miR-192 is correlated with the expression of Bcl-2, Zeb2 and VEGFA.17 In addition, miR-192 expression is correlated with gastric cancer in distinct metastasis.18 miR-192 can regulate p53, which enhances cyclin-dependent kinase inhibitor (CDKN1A/p21) and can adjust cell cycle arrest by inhibition of carcinogen. B-CLL cells are arrested in G0 (early G1) phase of the cell cycle that is controlled by cyclin d2 and d3 (cdk4). With stimulation of these cyclines, they could up-regulate.27 PTEN protein was not expressed in 28% of CLL patients with a normal PTEN genotype that pointed to LOH at 10q23.3. Inhibition of the PI3K/Akt signaling pathway in CLL may play a therapeutic role in the absence of PTEN protein.28 In addition, a decrease in PTEN increases PIP3 and Akt activation leading to the phosphorylation of Akt target proteins of miR-192. According to previous studies, B-cell lymphoma2 (BCL2) is a central player in promoting survival by inhibiting apoptosis and overexpression of Bcl2 in CLL.29 On the other hand, accumulating studies have demonstrated that miR-192 suppresses Bcl-2, resulting in enhanced apoptosis.17 Casitas B-lineage lymphoma (Cbl) is an E3 ubiquitin ligase of many tyrosine kinase receptors. The c-CBL and b-CBL genes are ubiquitously expressed with the highest levels in hematopoietic tissues.30 Many studies have indicated that Cbl plays an important role in CLL. miR-192 is able to active BCR-ABL and PI3K by phosphorylation. This process shows that miR-192 must be downregulated in this disease. Peroxisome proliferators–activate receptor (PPAR) α is a therapeutic target and a biological mediator of CLL. This pathway has been shown to activate proliferation in miR-192 by TCF.31 It can be concluded from Figure 3 that miR-192 can suppress BCR-ABL, PI3K-Akt and PPAR signaling pathways, by targeting the main elements of this pathway including CBL, Bcl2, and TCF. These results suggest the relevance of miR-192 as a valuable biomarker in CLL patients. However, more evidence from loss- and gain-of-function variants is required to confirm this theory. It can be assumed that due to its reduction, miR-192 may have a tumor suppressing role in this disease. This microRNA could be a marker for early diagnosis of CLL.

Conclusion

Despite achievements in monitoring of patients with CLL, an ideal biomarker for rapid and reliable diagnosis is still lacking. In this study, we investigated the transcript levels of miR-192 in CLL patients in comparison with normal subjects. We observed that the rate of miR-192 transcripts in CLL patients decreased. These observations proved that miR-192 can predict response to treatment. According to our results miR-192 could have an inducing role in CLL differentiation in possibly targeting some negative regulators of CLL differentiation. Our study of PBMCs in CLL patients showed downregulation in miR-192. This miRNA could be a potential marker for early diagnosis of CLL as well as a potential therapeutic target.