Expression of BCL 2 L 12 in acute leukemia patients : Potential association with clinical and prognostic factors

Introduction: Apoptosis is an important mechanism in both physiological and pathological conditions. The BCL2 family of proteins plays a critical role in regulation of apoptotic cell death. Up and down regulation of BCL2-like 12 (BCL2L12), a new member of the BCL2 family, has been reported in several malignancies. However, the expression level of BCL2L12 rarely has been studied in leukemia. This study was designed to investigate the mRNA expression of BCL2L12 in patients with acute leukemia. Materials and methods: 90 patients with acute leukemia as case group and 90 healthy persons as controls, were participated this study. RNA was extracted from peripheral blood samples. Expression level of BCL2L12 mRNA was evaluated by a quantitative real-time polymerase chain reaction (qRT-PCR) method and its association with clinical and laboratory findings was analyzed. Results: The expression of BCL2L12 mRNA was significantly lower in acute lymphoblastic leukemia (ALL) cases comparing the controls (P<0.001), while it was not significantly different in acute myeloid leukemia (AML) samples compared the control group. In addition, there were higher BCL2L12 level in females (than in males) and in patients with t(12;21) in ALL patients. There was no association between BCL2L12 expression level and other clinical and laboratory findings of AML patients. Conclusion: BCL2L12 seems play a role in the pathogenesis of ALL. Further studies with larger sample size is needed to clarify its probable impact on prognosis and therapeutic


Introduction
Apoptosis is an important mechanism in physiological conditions, such as embryonic development and tissue homeostasis as well as pathological conditions, such as cancer, neurodegenerative conditions and autoimmune diseases (1)(2)(3).Aberration in apoptotic pathways is one of the key components in the pathogenesis of lots of malignancies (1,4), including acute and chronic leukemias (2,5,6).In addition dysregulation of normal programmed cell death mechanisms plays an important role in cancer chemoresistance (7)(8).The BCL-2 family is one of the most important regulators of apoptosis, containing both anti-apoptotic and pro-apoptotic members (1,9).The pro-apoptotic members of the BCL2 family, including BAX, BAD, BID, and BCLXS, induce apoptosis, whereas the anti-apoptotic members, such as BCL2, BCLXL, and BCLW, suppress the apoptotic machinery (2)(3)(4).Over expression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-xL has been implicated in resistance to chemotherapy, whereas over expression of pro-apoptotic proteins such as Bax promotes apoptosis and sensitizes tumor cells to various anticancer drugs (10)(11)(12)(13).The BCL2-like 12 (BCL2L12) protein, a novel member of this family, structurally consists of a highly conserved BH2 domain, a BH3-like domain, and a prolin rich domain (5,12).BCL2L12 gene is located in 19q13.3 and composed of 7 coding exons and 6 intros which yield to a 334 amino acid long protein (12).To date, the precise function of BCL2L12 gene has not been determined and there are controversial data demonstrating that BCL2L12 involved in both pro-and antiapoptotic mechanisms (14).The BCL2L12 has been studied in several malignancies including chronic lymphocytic leukemia (CLL) (12) Moreover, a correlation between high Bcl-2 expression and poor response to chemotherapy has been reported in AML (25-27).In addition it has been shown that suppression of Bcl-2 expression promotes apoptosis and sensitizes AML cells to chemotherapy (26).In contrast, a relationship between high expression of BCL2 and increased disease-free survival in childhood ALL (29), as well as high BCL2 expression and higher complete remission rates in adults ALL have been reported (30).Moreover, elevated BCL2 expression is a good predictive factor for corticoresistance in T-ALL (31).As mentioned, data by different studies in various cancers, underlines the role of BCL2L12 in cancer genesis and behavior.However, the role of this protein in acute leukemias is not completely determined yet.There are only few reports about the association between BCL2L12 and AML; and also, to the best of our knowledge, studies about evaluation of BCL2L12 mRNA expression in patients with ALL have not been reported.Regarding these data, BCL2L12 mRNA expression by quantitative real-time polymerase chain reaction (qRT-PCR) method in acute Downloaded from jbrms.medilam.ac.ir at 3:52 IRST on Tuesday December 4th 2018 [ DOI: 10.18869/acadpub.jbrms.3.2.11 ] leukemia patients was evaluated in this study attempting to find its probable role in the pathogenesis of the disease.Furthermore, the association between BCL2-like12 expression level and other laboratory and clinical findings of patients was analyzed.

RNA extraction and cDNA synthesis:
The mononuclear blood cells of patients and controls were isolated from their peripheral blood samples using Ficoll gradient centrifugation.Total RNA was extracted by RNX plus™ kit (Cinnagen, Tehran, Iran) according to the manufacturer's instructions.The quality and quantity of the extracted RNA was evaluated by agarose gel electrophoresis.Total RNA was reversely transcribed using cDNA synthesis kit (thermo scientific, Lithuania).

BCL2L12 expression analysis:
The PCR primers for BCL2L12 or Beta-2microglobolin (B2M) were designed (23) as follows: BCL2L12 (gene of interest) forward, 3'-CCCTCGGCCTTGCTCTCT-5' and reverse, 3'-GGGCCACCAAAGCATAGAAG-5'.B2M (housekeeping or reference gene) forward, 3'-CAGCAAG GACTGGTCTTTCTAT-5' and reverse, 3'-GCGGCATCTTCAAACCTC-5 .'Real-time polymerase chain reactions were carried out using thermocycler (Applied Biosystems, Foster City, CA, USA) and SYBR green method by the means of TAKARA Master Mix (Japan).The reaction mixture was prepared by adding 10 µl Master Mix, 0.5 µl of each primer, 2 µl cDNA, 0.4 µl of Rox and 7.1 µl of nuclease free water in a total volume of 20 µl.The thermal conditions of the study consisted of an initial pre-denuturatun at 95 °C for 10 min followed by 40 cycles of denaturation at 95°C for 40 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 5 min.The relative BCL2L12 mRNA expression was calculated using the PCR threshold cycle number (CT) or comparative Ct (ΔΔCt) method.The relative fold change in gene expression = 2 -ΔΔCt, Where: ΔΔCt = ΔCt patients -ΔCt control group and ΔCt = Ct target gene (BCL2l12) -Ct reference gene (B2M) (42).In this method the amplification efficiencies of the BCL2L12 (Gene of interest) and reference genes must be approximately equal.In our study BCL2L12 and B2M PCR efficiencies were 94.13% and 98.99.

Statistical analysis
Data management and analysis was performed using SPSS (Statistical software for social analysis-version 11.5).The Kolmogorov-Smirnov test was applied to determine normality of data distribution and then, independent sample t-test and analysis of variance test (ANOVA test) were used for comparing continuous variables (such as levels of BCL2L12 mRNA) between the groups.Chi-Square was used to compare the non continuous variables and Pearson correlation test were used to determine relationship between variables.Differences were considered statistically significant at P<0.05.The t test showed no significant difference between the AML patients and control group (P = 0.15) in BCL2L12 mRNA expression but the mean level of BCL2L12 mRNA in ALL cases was significantly (P < 0.001) lower than the control group (6.15 vs 7.69).Tables 1 and 2 show mean level of BCL2L12 expression in different subgroups of patients (based on age, sex, …).There were not any statistically significant differences between BCL2L12 mRNA expression in different age and sex groups in AML cases and also in age groups in ALL patients.However the mean level of BCL2L12 mRNA in males were significantly (P = 0.01) lower than females (5.2 vs 6.6) in ALL cases.There were not any significant relationship between BCL2L12 expression and blood hemoglobin (P=0.82),RBC (P=0.85),WBC (P=0.78),PLT (P=0.20) in ALL cases and also between BCL2L12 and blood hemoglobin (P=0.82),RBC (P=0.42),WBC (P=0.31),PLT (P=0.89)age in ALL patients.Seven patients with t(12;21) positive in ALL group had significantly (P = 0.009) higher BCL2L12 expression compare to patients without this translocation (8.3 vs 5.7).In addition the BCL2L12 mRNA expression level was lower in AML group than the controls (7.11 vs. 7.62) but the differences were not statistically significant.In line to our study Thomadaki et al. examined the expression of this gene in 21 patients with de novo AML and did not find significant differences (p=0.14) between case and control groups (5.75 vs. 14.82)(35).In contrast to our results, Yu et al. reported a significantly (P < 0.01) lower level of BCL2L12 gene expression in 134 AML patients compared to control group (36).Although BCL2L12 expression in our AML patients was lower than control group (7.11 vs. 7.62) but the differences were not statistically significant.Lower BCL2L12 mRNA expression has been reported in other malignancies such as breast cancer (37) and higher BCL2L12 mRNA levels have been detected in CLL (15, 40), Nasopharyngeal carcinoma (16), bladder carcinoma (34).The dissimilarity between relative BCL2L12 expression in our study comparing the other studies (such as Thomadaki et al study) may be the result of using different reference genes; While we used B2M, they employed GAPDH as internal control (reference) gene.We did not find any article concerning relations between the levels of BCL2L12 mRNA with acute lymphoblastic leukemia.Similar study was done on relations between the levels of Bcl-2 with acute leukemia (29-31).In the ALL group, we found a higher BCL2L12 level in females (than in males) and in patients with t(12;21).In addition we did not find any association between BCL2L12 expression level and any of the clinical and laboratory findings of AML patients.In a similar study,

Table 1 .
The mean levels of BCL2L12 mRNA expression in different subgroups of the AML patients.

Table 2 .
The mean levels of BCL2L12 mRNA expression in different subgroups of the ALL patients.