1Department of Hematology, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran.
2Hematology-Oncology and Stem Cell Transplantation Research Center, Tehran, Iran.
3Cell Therapy and Hematopoietic Stem Cell Transplantation Research Center, Tehran, Iran.
4Hematologic Malignancies Research Center, Tehran University of Medical Sciences, Tehran, Iran.
5Comprehensive Cancer Research Center, Mazandaran University of Medical Science, Sari, Iran.
Recent studies have devoted much attention to non-protein-coding transcripts in relation to a wide range of malignancies.
Altered expression of
Chronic lymphocytic leukemia (CLL), a common blood cancer in western world, is characterized by proliferation and accumulation of neoplastic CD5high B-cells in bone marrow, peripheral blood, and secondary lymphoid organs . It mainly affects the elderly, and the mean age at diagnosis is about 70 years. CLL is more common in male subjects. About 25–30% of all new leukemia cases can be attributed to CLL in the western population. However, CLL is rare in Asia and accounts for only about 5% of all leukemia cases . Several factors have been reported to increase the risk of CLL including occupational exposure to specific substances (ethylene oxide, benzene, 1,3-butadiene, and pesticides), ethnicity, positive family history of lymphoproliferative disorders, and genetic predisposition . CLL has a heterogeneous nature and involves numerous genetic and epigenetic alterations. Some patients need interventions due to progressive disease course, whereas subjects with indolent course require no therapy for years .
Studies have revealed that only 2% of the human genome sequences are protein-coding genes, whereas the majority of transcriptome does not encode proteins. Non-coding RNAs (ncRNAs) are divided into regulatory and housekeeping classes. Regulatory ncRNAs are subcategorized, depending on the nucleotide length, into long (>200 nt) and short (<200 nt) ncRNAs. Long ncRNAs (lncRNAs) play important roles in multiple biological processes such as DNA repair, regulation of gene expression, transcription, and translation . Some lncRNAs, including
Although the precise molecular mechanisms by which MALAT1 may contribute to CLL pathogenesis have not been fully elucidated yet, several observations support the involvement of this lncRNA in CLL. These include its role in cell cycle regulation and p53-mediated response to DNA damage, as well as in mRNA processing. The emerging role of MALAT1 in hematologic malignancies prompted us to quantify its expression in patients with CLL to establish whether its deregulation may be associated with disease progression.
In this study, we examined 60 subjects including 30 CLL patients and 30 healthy individuals in the period between 2016 and 2018. The CLL group consisted of 18 male and 12 female patients (mean age, 56.4 yr) and the control group included 15 male and 15 female subjects (mean age, 69.16 yr). Diagnosis of patients was based on standard guidelines including complete blood count, peripheral blood smear, and immunologic tests. The Ethics Committee of Tarbiat Modarres and Tehran University of Medical Science approved the study content and patients were given written informed consent. Notably, in our two years follow-up all patients were alive and only six of them received chemotherapy, as follows: one patient received chlorambucil and prednisolone, one patient received bendamustine and rituximab, and the rest four patients received R-CVP chemotherapy. Due to the limited number of patients in the different treatment groups, we did not evaluate MALAT1 expression in relation to treatment.
About 10 mL of fasting venous blood were taken from all study participants. Blood mononuclear cells (MNCs) were isolated by ficoll lymphodex medium (Inno-Train Diagnostik, Kronberg, Germany). In brief, 5 mL of EDTA-containing blood were diluted by sterile phosphate buffer saline (PBS) in a 1:1 ratio and layered over 7 mL of lymphocyte separating medium in a sterile Falcon tube. The tube was centrifuged at 400 g for 30 min and the MNC-containing ring was transferred into a new tube and washed with PBS. Total RNA extraction was performed using RiboEx reagent (GeneAll, Seoul, Korea) After determination of RNA purity using a spectrophotometer, cDNA synthesis was performed by Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). In each reaction tube, 1–5 µg of total RNA, 1 µL of random hexamer primer, 4 µL of 10× buffer, 2 µL of dNTP mix, 1 µL of M-MuLV Reverse Transcriptase and nuclease-free water (up to 20 µL) were added. In order to evaluate
Patient's bone marrow cells were cultured in complete medium containing RPMI 1640, fetal calf serum, and antibiotics for 24 h at 37℃. For the harvesting step, Colsemide and KCl were added to cultures. Next, the cells were fixed by methanol and acetic acid (3:1 ratio) solution. FISH was performed on prepared slides using a panel of probes to detect chromosomal anomalies including deletion of 13q14.3, 17p13, 11q22.3, 6q21, trisomy 12, and c-MYC translocation.
SPSS software (version 18) was applied for data analysis. One-way ANOVA, independent t-test or Mann-Whitney U test was used to compare quantitative data between CLL patients and healthy controls. A
Demographic and clinical information on study participants is listed in Table 1. White blood cells and lymphocyte count were higher in CLL patients. Platelet count and hemoglobin levels were significantly higher in healthy controls. There were no gender-related differences in the groups. According to FISH findings, 20% of patients showed a normal chromosomal pattern. Del13q14 was the most common cytogenetic abnormality as it was found in 53.3% of patients, followed by del 11q22 (16.6%), del 17p13.1 (10%), trisomy 12 (10%), and c-MYC amplification (3.3%).
The expression levels of
Malignancies are a global health issue and are the 3rd cause of mortality in Iran. In this context, the increase in the recorded incidence of blood cancers in Iran can be attributed to several causes including improvement in cancer diagnosis setting and registry and increase in cancer-related risk factors or unknown etiologies .
With the advancement of biotechnological tools, studies have devoted much attention to genetic alterations as an inseparable part of cancer. In this respect, non-protein-coding transcripts such as lncRNAs play a critical role in regulating cellular functions and may be related to both tumor suppressive and oncogenic characteristics. According to recent evidences, lncRNAs are involved in many principal cellular mechanisms such as transcriptional and post-transcriptional regulation, chromatin rearrangement, and nuclear-cytoplasm trafficking . Deregulation of lncRNA expression has been reported in a variety of human diseases, from neurologic disorders (multiple sclerosis and Alzheimer's disease) to several solid (breast, lung, bladder) and hematologic cancers (lymphoma and leukemia) .
Initially described as a metastasis biomarker of lung cancer,
Previously, the overexpression of
There are several factors related to CLL prognosis including VH mutation status, zeta-associated protein (ZAP-70) and CD38 expression, and chromosomal aberrations . We determined chromosomal abnormalities in patients using the FISH method and 80% of them presented cytogenetic abnormalities. On the contrary, findings from the northeast of Iran (Mashhad) revealed a lower frequency of chromosomal abnormalities in CLL patients (45.5%); however, in line with our data, del13q14 was the most frequent alteration. In Korean patients, a lower overall frequency of abnormalities was observed, compared to western populations and the present report, and also a different frequency distribution, as trisomy 12 represented the most common chromosomal alteration found . Despite the high incidence of chromosomal anomalies in this study, no significant relationships were found between the level of
In conclusion, a high expression of oncogenic
Relative expression of MALAT1 (2−Δct) in CLL and control group using β2m as internal control gene. Increased expression of