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Introduction

Acute myeloid leukemia (AML) is a cancer of the white blood cells characterized by the clonal proliferation of myeloid progenitor cells in the bone marrow and peripheral blood (1). AML accounts for ~80% of all acute leukemia cases in adults, and the incidence of this disease has been revealed to increase with age (2,3). At present, AML is curable in 35–40% of patients <60 years old; however, among patients >60 years of age, cases of full recovery are less common (5–15%) (1,4). Due to the fact that survival rates remain relatively low (overall 5-year survival is <5% in older patients), novel therapeutics and treatment strategies are required (3).

The etiology of AML is not yet fully known, but there are a number of genetic factors that may predispose patients to this disease, including chromosomal translocations (Breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1), and mutations in FMs-Related Tyrosine Kinase 3 (FLT3), Tumor Protein 53 and Additional Sex Combs Like 1, Transcriptional Regulator genes (5). Among the genetic factors that are involved in the development of AML, are the runt-related transcription factor 1 (RUNX1) and runt-related transcription factor 3 (RUNX3) genes. These genes belong to the runt domain transcription factor family, which is responsible for encoding the DNA-binding α-subunits of the RUNT domain transcription factor and serve an important role in the regulation of transcription (6,7). However, they may be dysregulated in human cancer cells (as a result of mutations, translocations or inactivation), and therefore potentially serve a role in the pathogenesis of cancer (6,8). The RUNX1 gene, which is located on chromosome 21 (locus q21.22), serves an important role in hematopoiesis during embryonic development (9). Furthermore, it is responsible for the formation of hematopoietic stem cells and progenitor cells due to its expression in all hematopoietic sites (10). Previous studies have demonstrated that the chromosomal translocations and mutations in the RUNX1 gene may be associated with several types of leukemia, including AML (11,12).

The RUNX3 gene also encodes transcription factors and is responsible for the regulation of a number of other genes, including Transforming growth factor-β and Notch 1 pathways, Core-Binding Factor β subunit, ETS proto-oncogene 1 and ETS proto-oncogene 2 transcription factor genes (11). The RUNX3 gene is located on chromosome 1 (locus p36) (11,13) and is highly expressed in all hematopoietic stem cells (14). Furthermore, the RUNX3 gene is hypothesized to act as a tumor suppressor. There is evidence to suggest that the inactivation of this gene is associated with the development of various types of cancer, including breast cancer (15). Additionally, deletion of this gene is associated with hyperplasia of the gastric mucosa and gastric cancer development (16,17). Continuous research regarding the potential functions of RUNX family genes in tumor development and the influence of these genes on the expression of other genes may assist in the early detection of cancer, and the development of more effective treatment modalities and novel therapeutics for patients with cancer (8). The role of the RUNX1 and RUNX3 genes in AML have, thus far, not been completely elucidated. Therefore, the aim of the present study was to evaluate the mRNA expression level of the RUNX1 and RUNX3 genes in patients with AML.

Materials and methods

Sample collection and ethics statement

The investigated group comprised of 43 (22 female and 21 male) patients who had been diagnosed with AML at the Hematology Clinic, Medical University of Lodz (Lodz, Poland). The mean age at the time of diagnosis was 57.9 years (58.6 for females and 57.4 for males; age range 17–80 years). Patients were divided into subgroups according to the French-American-British (FAB) classification of AML (18). Peripheral blood (March 2016-May 2017) was used for research and the samples obtained were residual material remaining following routine blood tests. The Ethics Committee of the Medical University of Lodz approved the present study (RNN/88/16/KE). Written informed consent was obtained from the patients for participation in the study.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the blood cells of participants using the Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland), according to the manufacturer's protocol. The purity of obtained RNA was determined by the A260/280 ratio (DNA/RNA absorbance to protein absorbance). Absorbance at 260 nm was used to determine the amount of RNA required for reverse transcription. Isolated RNA samples were stored at −76°C until further analysis. RNA samples were reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. The thermocycling parameters were as follows: 25°C for 10 min, then 37°C for 120 min and 85°C for 5 min. The final concentration of RNA in the reaction mixture in all samples was 0.02 µg/µl. The presence of cDNA was verified through PCR amplification normalized to GAPDH (19). PCR was performed for qualitative analysis of mRNA expression RUNX1 and RUNX3. Amplification was performed according to the manufacturer's protocol for the AccuTaq™LA DNA Polymerase kit (Sigma Aldrich; Merck KGaA, Darmstadt, Germany). The reaction mixture consisted of 1 µl cDNA template, 0.7 µl of 10 µM each primer, 3.5 µl of 1.5 mM 10× PCR buffer without MgCl2 (Sigma Aldrich; Merck KGaA), 0.7 µl of 25 mM MgCl2 reagent, 0.4 µl of 0.2 mM dNTP (deoxynucleotides) mix, 0.2 µl of 0.5 U AccuTaq LA DNA Polymerase and distilled water to the final volume of 21 µl. Primers used in the present study are listed in Table I. A negative control, without cDNA template, was included in every experiment. Amplification was performed using an MJ Mini Personal Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min, denaturation at 92°C for 1 min, primer annealing at 58°C for RUNX1 and 56°C for RUNX3 for 30 sec, elongation at 72°C for 45 sec and final elongation at 72°C for 7 min. Electrophoresis on a 2% agarose gel was used to assess the products of PCR amplification. The sizes of the reaction products were as follows: RUNX1, 96 bp and RUNX3, 120 bp. qPCR was used for quantitative assessment of RUNX1, RUNX3 and GAPDH mRNA expression, and reactions were performed in a Rotor-Gene™ 6000 thermocycler (Corbett Life Science; Qiagen GmbH, Hilden, Germany). GAPDH is a housekeeping gene, the expression of which is often used to normalize mRNA levels between samples (19). The reaction mixture consisted of 5 µl RT HS-PCR Mix Sybr® B (A&A Biotechnology), 0.7 µl of 10 µM each primer, 1 µl cDNA template and nuclease-free water to a final volume of 10 µl. Experiments for investigated and reference genes were performed in triplicate and reactions were performed in separate tubes. A negative control, without cDNA template, in triplicate was also included in every experiment. The reaction parameters were as follows: Initial denaturation at 95°C for 10 min, denaturation at 95°C for 10 sec, primer annealing at 55°C for RUNX1 and 58°C for RUNX3 for 15 sec, elongation at 72°C for 20 sec. In order to assess the specification of products, analysis of melting curves was performed following amplification. The 2−ΔΔCq method was used to estimate relative changes in gene expression determined by RT-qPCR analysis (20). The mean Cq values of GAPDH, RUNX1 and RUNX3 genes were used in subsequent calculations.

Table I. Primers used for reverse transcription-quantitative polymerase chain reaction analysis.

Table I.

Primers used for reverse transcription-quantitative polymerase chain reaction analysis.

Gene	Forward primer	Reverse primer
GAPDH	5′-TGGTATCGTGGAAGGACTCATGAC-3′	5′-ATGCCAGTGAGCTTCCCGTTCAGC-3′
RUNX1	5′-AGTGGAAGAGGGAAAAGC-3′	5′-ATCCACTGTGATTTTGATGG-3′
RUNX3	5′-ATGACGAGAACTACTCCG-3′	5′-TCAGGGTGAAACTCTTCC-3′

[i] RUNX1, runt-related transcription factor 1; RUNX3, runt-related transcription factor 3.

Statistical analysis

Statistical analyses were performed using STATISTICA 12.5 (StatSoft Inc., Tulsa, OK, USA). A comparative statistical analysis was performed using the non-parametric U Mann-Whitney test in the absence of normality of relative levels of RUNX1 and RUNX3 gene expression. P<0.05 was considered to indicate a statistically significant difference.

Results

Relative RUNX1 and RUNX3 gene expression level with sex and age of diagnosis

All 43 samples exhibited GAPDH expression. The presence of RUNX1 and RUNX3 gene expression was also identified in all selected samples. Quantitative analyses revealed that the transcript level of RUNX1 and RUNX3 genes varied among selected cases. It ranged between 0.13 and 18.37, with a median value 0.73 for the RUNX1 gene and between 0.04 and 8.54 with a median value of 1.28 for the RUNX3 gene. The investigated group comprised 22 females and 21 males. Statistically significant differences between patient sex and relative RUNX1 expression were identified (P=0.044). Levels were higher and varied more among females (Fig. 1). However, no significant differences were identified between sex and relative RUNX3 gene expression (P=0.130; data not shown). Another compared parameter was age at the time of AML diagnosis. The mean age was 57.9, 58.6 years for females and 57.4 years for males; however, no statistically significant associations were identified between age at the time of diagnosis and mRNA expression of RUNX1 (P=0.970) or RUNX3 (P=0.469).

Figure 1. Associations between relative RUNX1 gene expression level and patient sex. Statistically significant differences were observed between female and male patients (P=0.044). RUNX1, runt-related transcription factor 1; F, female; M, male; SE, standard error; SD, standard deviation.

Relative RUNX1 and RUNX3 gene expression level with FAB classification and mortality

Patients were also divided into subgroups according to the FAB classification of AML (18). Full details are presented in Table II. Statistical analysis revealed no significant associations between FAB classification subgroups and relative RUNX1 (P=0.746) and RUNX3 (P=0.771) expression. Relative expression was also compared with mortality among the enrolled patients. The results indicated that there is a statistically significant association between the relative expression of RUNX3 and mortality among patients (P=0.036). Mortality was more frequent among patients with higher RUNX3 expression levels (Fig. 2); however, no such association was observed between mortality and RUNX1 expression (P=0.445, data not shown).

Figure 2. Associations between relative RUNX3 gene expression level and mortality. Statistically significant differences were observed among subgroups (P=0.036). RUNX3, runt-related transcription factor 3; SE, standard error; SD, standard deviation.

Table II. French-American-British classification of investigated patients.

Table II.

French-American-British classification of investigated patients.

Diagnosis	Number of cases	RUNX1 P-value	RUNX3 P-value
AML undefined	21 (10F, 11M)
AML 0	1F
AML1	3 (2F, 1M)
AML2	8 (5F, 3M)	0.746	0.771
AML3	2 (1F, 1M)
AML4	4 (2F, 2M)
AML5	3 (1F, 2M)
AML6	1M

[i] RUNX1, runt-related transcription factor 1; RUNX3, runt-related transcription factor 3; AML, acute myeloid leukemia; F, female; M, male.

Discussion

Due to the presence of various mutations in the RUNX1 and RUNX3 genes in patients with AML, we hypothesized that these genes may influence mRNA formation and may contribute to the different levels of expression among the investigated cases. To the best of our knowledge, the present study is the first to present the RUNX1 and RUNX3 gene expression levels in patients with AML determined by RT-qPCR analysis in a Polish population, as previous studies have only been conducted in Chinese populations thus far.

The RUNX1 gene serves an important role in hematopoiesis (9). Its abnormal expression is present in various malignancies, including ovarian cancer, cytogenetically normal AML (CN-AML) and breast cancer (21–24). However, the significance of the RUNX1 gene in cancer development is not fully known. Previous studies have suggested that the RUNX1 gene functions as a tumor suppressor in AML (25), and that loss of the RUNX1 gene may lead to weak differentiation and leukemia development (26). One previous study has reported that a normal expression level of RUNX1 gene inhibits cell proliferation and promotes differentiation of hematopoietic progenitor cells (21). By contrast, deactivating the RUNX1 gene may cause amplification of myeloid progenitors and the subsequent development of AML. A previous study undertaken by Silva et al (25) suggested that the RUNX1 gene acts as a classical tumor suppressor gene; however, other studies have suggested that RUNX1 functions as an oncogene and that it may cause AML development due to its pro-survival role in leukemia cell proliferation (27–30). The results of these studies also suggested that the prognostic impact in CN-AML depends on the RUNX1 expression level. A study undertaken by Goyama et al (28) reported that overexpression of the RUNX1 gene inhibited the growth of regular cord blood cells by inducing myeloid differentiation. It was suggested that the RUNX1 gene may be a valuable novel marker for risk stratification in patients with AML and that it is an excellent candidate for anticancer-targeted therapy due to the modulation of its post-translational modifications (29).

It is now hypothesized that, due to its expression level, the RUNX1 gene may serve a role as a tumor promoter or tumor suppressor in different types of cancer and hematological malignancies including AML (21). A study undertaken by Fu et al (21) estimated RUNX1 expression using microarrays and revealed that a high level of RUNX1 mRNA expression in CN-AML was associated with a poorer overall survival (OS) and event-free survival (EFS) than low RUNX1 mRNA expression. The median OS and EFS times in patients with a higher RUNX1 expression level were poorer than that of the low RUNX1 expression group (P=0.009 and P=0.011, respectively). Among 157 patients with CN-AML with a higher RUNX1 gene expression, significantly more patients exhibited the FAB M2 subtype than in the group with lower RUNX1 gene expression. Furthermore, the RUNX1 high expression group included significantly more patients with the FAB M1 subtype than the RUNX1 low expression group (P=0.0014), suggesting that the leukemia cells from patients with a high expression of RUNX1 are derive from relatively less mature cells. According to this aforementioned study, RUNX1 gene expression may have prognostic significance in AML and it may be a biomarker of an unfavorable outcome in CN-AML, where overexpression of the RUNX1 gene is widespread among patients (high expression of RUNX1 is associated with poorer disease outcomes) (21).

These results differed from those obtained in the present study, where there were no associations among mortality, FAB classification of AML and the expression level of RUNX1. Furthermore, the present study revealed statistically significant differences in RUNX1 gene expression levels between females and males; as females tended to exhibit a higher and more variable expression level. This suggested that sex may affect RUNX1 expression, thereby influencing the process of leukemia development.

The RUNX3 gene is involved in neurogenesis and thymopoiesis, and serves a role as a tumor suppressor in gastric cancer (7,31–33). A study undertaken by Jiang et al reported that the RUNX3 expression level is associated with breast cancer development and that it is decreased in this type of cancer. The principal cause for this inactivation mechanism may be hypermethylation in the promoter region (34). A study undertaken by Cheng et al (7) demonstrated that RUNX3 gene expression was an independent prognostic factor in childhood AML, and that a higher RUNX3 gene expression level was associated with a shorter EFS and OS time (7). The results of a study undertaken by Lacayo et al (35) also demonstrated that a higher level of RUNX3 gene expression was associated with a shortened EFS rate in childhood AML. However, this study was conducted on patients belonging to an FLT3 mutant group, which may have also affected EFS (35). Based on these aforementioned studies, it is possible that the RUNX3 gene expression level is associated with a shorter survival time in childhood AML (7,35). Furthermore, according to the study undertaken by Cheng et al (7), the RUNX3 gene expression level was not associated with age or sex. However, in a group of patients with a lower RUNX3 gene expression level, this was significantly associated with the presence of t(8;21) or inv(16) translocations (7). Lower RUNX3 gene expression levels were frequently identified in patients with FAB M2 and M4 AML subtypes. Furthermore, RUNX3 was significantly underexpressed in the prognostically favorable subgroup of AML with the t(8;21) and inv(16) translocations (7).

The RUNX3 expression level differed among the patients enrolled in the present study, and the study undertaken by Cheng et al (7) obtained similar levels of RUNX3 expression in patients with childhood AML, although the results of the present study were more varied and slightly higher. This may be due to differences in age between the investigated groups. Cheng et al (7) identified no statistically significant associations between clinicopathological features (sex, age or FAB classification) and relative RUNX3 expression level. The results obtained in the present study are comparable, as no associations between sex or age at the time of diagnosis or FAB classification and RUNX3 expression were identified. Statistically significant differences were identified between the expression level and the incidence of mortality among patients, as mortality occurred more frequently in the group with a higher RUNX3 expression level. These observations are also similar to those reported by Cheng et al (7) which leads to the conclusion that RUNX3 may serve as a potential prognostic factor in AML.

The lack of an association between the selected clinicopathological features and relative RUNX1 and RUNX3 expression may be a limitation of the present study, particularly due to the relatively small group of investigated patients. Future studies would benefit from an increased number of patients and the collection of more detailed clinical information, including the results of peripheral blood morphology, previously applied treatment, percent of blasts in bone marrow.

The results of the present study suggested that sex may be associated with the expression level of the RUNX1 gene and may influence the difference in the process of AML development between females and males. Based on the results of earlier studies (7,35) and those of the present study, RUNX3 may serve as a potential novel prognostic factor. Patients with a higher RUNX3 expression level generally have poorer outcomes. However, the obtained results must be confirmed in a larger cohort.

Acknowledgements

Not applicable.

Funding

The present study was supported by statutory funds of the Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Medical University of Lodz (grant no. 503/3-015-02/503-31-001) and funds of the Faculty of Pharmacy, Medical University of Lodz (grant nos. 502-03/3-015-02/502-34-089 and 502-03/3-015-02/502-34-088).

Availability of data and materials

All data generated or analyzed during the present study are included in this published article.

Author's contributions

AK and DS planned and conducted experiments, and assisted in the preparation of the manuscript for publication. MŻ and AJ conducted experiments. EB planned and supervised experiments, and assisted in the preparation of the manuscript for publication.

Ethics approval and consent to participate

The Ethics Committee of the Medical University of Lodz approved the present study (RNN/88/16/KE). Written informed consent was obtained from the patients for participation in the study.

Consent for publication

Written informed consent was obtained from the patients for the publication of their data.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References