1. Introduction
Chronic myeloid leukemia is a malignant myeloproliferative disease, mainly caused by a reciprocal translocation between chromosomes 9 and 22 (9; 22) (q34; q11), resulting in the Philadelphia chromosome (Ph), and the corresponding
BCR::ABL1 fusion gene, which is the main biomarker of this pathology [
1,
2].
The resulting transcriptional fusion encodes the oncoprotein
BCR::ABL1, which is a constitutively active tyrosine kinase, whose activity stimulates leukemogenesis [
3]. Therefore, treatment is carried out with tyrosine kinase inhibitors (TKIs), and despite this being a highly efficient treatment, approximately 13% of patients develop resistance to this class of compounds [
4].
Patient monitoring through molecular quantification of the
BCR::ABL1 transcript is highly relevant for evaluating the prognosis of patients with CML. Treatment failure is related to mechanisms of resistance to TKIs, the origin of which may be a mutation in the coding region of the kinase domain of the
BCR::ABL1 tyrosine kinase. Several mutations in this domain have already been described, such as T315I, V299L, G250E, F317L, Y253H, E255K/V, F359V/C/I, and L248V [
5] among others. Although CML has
BCR::ABL1 as both a diagnostic and monitoring marker, the search for other plasma biomarkers are important for the prognosis of the disease. Among the biomolecules that can be explored with this potential are non-coding RNAs (ncRNAs), which can help in the identification of altered or modulated targets in different phases of the disease; this can be extremely relevant in clinical use, as they can be related to resistance to the chemotherapy drugs used.
MicroRNAs make up a broad and well-studied class of ncRNAs, being correlated with many signaling pathways [
6] playing a role in the regulation of gene expression, RNA maturation, protein synthesis, and can also have their activity regulated at a post-transcriptional level [
7,
8,
9].
Considering the presented information, this study aimed to evaluate the dynamics of microRNA expression in samples from patients with CML, correlating the results with the stage of the disease and BCR::ABL1 transcript quantification to help in the disease follow-up and prognosis.
3. Discussion
Several studies have shown that MicroRNAs are associated with different biological pathways and have a great influence on the transduction of cell signaling [
30]. The involvement of microRNAs in the development, progression, and resistance to chemotherapy in CML [
31] shows that they can be important targets for studies related to chronic myeloid leukemia and other types of cancer. The present study addressed different clinical cohorts, forming different points of analysis aiming to evaluate possible biomarkers.
To achieve this, a screening of 35 microRNAs was first carried out in plasma samples from a cohort of patients with CML. These microRNAs were selected according to previous data in the literature, with some being correlated to leukemias in different contexts, such as diagnosis, prognostic assessment, and Treatment-Free Remission protocol, while some were selected based on their scientific relevance in other types of cancer. The results obtained showed that miR-7-5p had a significant differential expression among groups of patients separated according to their percentage of BCR::ABL1 expression.
The expression of miR-7-5p was significantly increased in samples from patients with a high percentage of BCR::ABL1 transcript compared to healthy controls. Surprisingly, as observed in the results, this microRNA had the highest expression in some CML patients treated with Imatinib and with a high percentage of BCR::ABL1, while other patients in this same group presented values similar to the average of controls. This result suggested that microRNA 7-5p may be related to the progression of the disease, considering that the increase in its expression could be related to the increase in the percentage of BCR::ABL1 transcript. These results led us to perform two additional analyses: single point analysis with RNA samples obtained from leukocytes (buffy coat) from the same patients, and analysis at various follow-up points of patients with CML.
To identify the origin of miR-7-5p in plasma, we evaluated its expression in leukocytes from the same patients. Unlike what was observed in plasma, miR-7-5p did not have a differential expression in the patients’ leukocytes. The increase of miR-7-5p in plasma could be related to the release of extracellular vesicles containing microRNA-7-5p or other factors, such as the hypothesis that this microRNA is being secreted by cells of the bone marrow and not directly from leukocytes in the bloodstream.
As another approach to investigate the role of miR-7-5p in CML, we evaluated a follow-up cohort of samples from the same patients collected at consecutive points throughout their progression with Imatinib treatment. Similar to what was found in the previous stage, miR-7-5p showed different behaviors in relation to the course of the disease in each patient, indicating a heterogeneous behavior of this target, suggesting that it can be modulated and altered by several factors.
These results indicate that microRNA-7-5p may be related to the expression of the BCR::ABL1 transcript. Some points of the analysis showed sudden increases in the expression of miR-7-5p, indicating that it can be a monitoring biomarker. Furthermore, it was also observed that microRNA-7 had a coincident progression relationship with the BCR::ABL1 transcript, where both increased or decreased at the same time, which may be related only to the BCR::ABL1 transcript and not with the prediction of prognosis. However, monitoring more patients for longer periods of time is necessary to confirm this hypothesis.
Another approach of the present study evaluated patients who started treatment with Imatinib, and, as they had excellent progression, were enrolled for treatment-free remission. All of these patients had undetectable BCR::ABL1 t at all follow-up points. In this context, the molecular monitoring of BCR::ABL1 expression in TFR patients together with the plasma quantification of miR-7-5p aimed to observe whether miR-7-5p could serve as an anticipatory marker of increased expression of BCR::ABL1, indicating the need to resume discontinuation for these patients.
Of the 4 TFR patients, 3 maintained stable miR-7-5p expression at all points of their follow-up, indicating that there may be some type of relationship with the worsening prognosis and the increase in the percentage of leukemic cells. Two patients had unstable results, such as patient 1 who had an instability where they had an increase in the expression of miR-7-5p in the third and fourth points. Considering the instability in patient 5, it can be hypothesized that there are other factors interfering in miR-7-5p expression. More follow-up points would be necessary so that we can draw any conclusions.
MicroRNA-7 has been studied in several types of cancer [
32], such as lung, hepatocellular, breast, gliomas, colorectal, hematological neoplasms, among others [
33]. This microRNA has been characterized as a tumor suppressor in several types of cancer [
34] and has also been related to the modulation of signaling pathways [
35,
36]. MicroRNA-7 may have a functional performance related to the inhibition of DNA repair mechanisms, carried out by PARP-1 and BRCA [
37]. A study indicated that there is a relationship between ANRIL/miR-7 in which microRNA-7 may function as a tumor suppressor in T-cell acute lymphoblastic leukemia [
32]. A possible miRNA-TET2 pathway was also identified, where microRNAs, including miR-29b, miR-101, miR-125b, miR-29c, and miR-7 are overexpressed and thus may be involved in the pathogenesis of AML [
38].
In the context of chronic myeloid leukemia, the roles for miR-7 are not yet well understood. Jiang et al. (2017) carried out studies demonstrating that miR-7 inhibited cell proliferation and promoted apoptosis in K562 cell lines focusing on the
BCR::ABL1/PI3K/AKT signaling pathway [
39]. The same study demonstrated that microRNA-7 may also be related to the sensitization of the K562 cell to Imatinib [
39]. Considering the studies already carried out and the results obtained in this work, we can hypothesize that these pathways may be related to changes in the expression of microRNA-7/
BCR::ABL1.
There is little evidence about the relationship between microRNA-7-5p and chronic myeloid leukemia patients. However, the present work found evidence that miR-7-5p may be related to chronic myeloid leukemia in a non-specific way, mainly considering its relationship with the BCR::ABL1 transcript. It is also important to highlight that the modern study of oncology, mainly focused on diagnosis, prognosis, and therapeutic evolution, is linked to individual differences and patient profiles. Therefore, the identification of a single sensitive and specific biomarker for a type of neoplasm is unlikely, requiring studies with a panel of biomarkers and the stratification of patients with a given neoplasm to elucidate more sensitive and specific biomarkers. This reiterates the importance of microRNAs as excellent accessories and mainly individualized biomarkers, being important in precision medicine, which is the main objective of clinical oncology, as it directs efforts, resources, time, and patient survival, which increases their quality of life.
4. Materials and Methods
4.1. Casuistry
Peripheral blood samples from patients with CML were collected at Erasto Gaertner Hospital (Curitiba, PR, Brazil), for research projects approved according to CAAE 08809419.0.0000.0098 and 53207021.5.0000.0098. The microRNAs addressed in this project were previously selected based on studies related to microRNAs in CML, consisting of 35 miRNAs: miR-7-5p, miR-17-5p, miR-19b-3p, miR-20a-5p, miR-21-5p, miR-23a-3p, miR-25-3p, miR-27b-3p, miR-29a-3p, miR-29b-3p, miR-92a-1-5p, miR-93-5p, miR-103a-3p, miR-106b-5p, miR-122-5p, miR-125b-5p, miR-130a-3p, miR-130a-5p, miR-142-5p, miR-148b-3p, miR-150-5p, miR-155-5p, miR-186-5p, miR-192-5p, miR-193b-3p, miR-199a-3p, miR-205-5p, miR-214-5p, miR-221-3p, miR-221-5p, miR-331-5p, miR-361-5p, miR-451, miR-486-5p, and miR-494-3p. The miRNAs were evaluated in cohorts of CML patients, who were divided into groups and selected considering the following variables: age, sex, type of leukemia, time of diagnosis, and type and duration of treatment. Thus, the cohorts analyzed at single-point samples were: (1) High, samples from Imatinib-treated patients, with BCR::ABL1 > 0.1% (n = 30); (2) ND, samples from Imatinib-treated patients, with BCR::ABL1 < 0.1% or undetectable (n = 20); (3) Healthy Control (HC) samples from healthy blood donors (n = 20); and a fourth group of serial samples; and (4) Follow-up cohort: samples from 11 patients that contained several collection points, at different points in the treatment, to evaluate the evolution of the disease through the quantification of BCR::ABL1 by RT-qPCR.
4.2. Buffy Coat RNA Extraction
After plasma separation by centrifugation, the buffy coat was subjected to total RNA extraction using the QIAamp® RNA Blood Mini Kit (Qiagen, Hilden, Germany). The extracted RNA was quantified in NanoDrop™ One (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 °C until use.
4.3. Quantification of BCR::ABL1
Total RNA extracted from the buffy coat was analyzed by RT-qPCR according to the protocol described by Marin et al., 2023 [
40].
4.4. Synthesis of Endogenous cDNA from Buffy Coat Samples
After extracting the RNA from the buffy coat, this RNA was used to quantify the BCR::ABL1 transcript, as described in Marin et al., 2023, and for the quantification of microRNAs. The endogenous miUSB-U6 was used to quantify microRNAs, and cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), following the manufacturer’s instructions.
4.5. Extraction of Plasma RNA
Before starting the extraction, 1 µL of cel-miR-39-3p template at 1 nM (Spike-in-control) was added to each 100 µL of plasma. Then the total RNA was extracted using the MagmaxTM mirVanaTM Total RNA Isolation Kit (Thermo Fisher Scientific), following the instructions for use for RNA isolation from serum and plasma samples.
4.6. cDNA Synthesis of Free MicroRNAs in Plasma
For reverse transcription, the TaqManTM Advanced miRNA cDNA Synthesis kit (Thermo Fisher Scientific) was used, following the manufacturer’s instructions.
4.7. Real-Time PCR of microRNAs
For the relative quantification of miRNAs, assays were used with TaqMan probes specific for each microRNA (Thermo Fisher Scientific) and the TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific). The PCR reaction was carried out on a QuantStudio 5TM real-time PCR platform (Thermo Fisher Scientific) using the exogenous microRNA cel-miR-39-3p as a normalizer. The qPCRs were performed following the manufacturer’s protocol, in duplicate for each sample and always using a negative control (the no template control, NTC).
4.8. Statistical Analyses
The data were calculated using the 2
−ΔΔCT methodology [
41] where the results obtained for single-point analyses were performed using the Graphpad Prism 7 software. Expression data was normalized with the median of the Ct values of endogenous control miUSB-U6 and the exogenous control cell- miR-39-3p.
5. Conclusions
35 microRNAs were evaluated, namely: miR-7-5p, miR-17-5p, miR-19b-3p, miR-20a-5p, miR-21-5p, miR-23a-3p, miR-25-3p, miR-27b-3p, miR-29a-3p, miR-29b-3p, miR-92a-1-5p, miR-93-5p, miR-103a-3p, miR-106b-5p, miR-122-5p, miR-125b-5p, miR-130a-3p, miR-130a-5p, miR-142-5p, miR-148b-3p, miR-150-5p, miR-155-5p, miR-186-5p, miR-192-5p, miR-193b-3p, miR-199a-3p, miR-205-5p, miR-214-5p, miR-221-3p, miR-221-5p, miR-331-5p, miR-361-5p, miR-451, miR-486-5p, and miR-494-3p. This work showed that the majority of microRNAS analyzed do not have a relationship with chronic myeloid leukemia. However, it was also observed that microRNA 7-5p may have a relationship with chronic myeloid leukemia and the BCR::ABL1 transcript in different ways.