CORRELATION STUDY BETWEEN THREE DIFFERENT GENES EXPRESSION AND CHRONIC MYELOID LEUKEMIA IN IRAQ

The following study was conducted to investigate the correlation between the expression of three different genes (NOB1, DDX47, CD101 ( with the occurrence and development of chronic myeloid leukemia (CML) in Iraq. The difference in the expression of these genes between patients and healthy controls was studied. Moreover the correlation of age and gender with CML occurrence and comparing with control was also examined. Results showed significant increases in mean of gene expression level (ΔCt) of patient groups for all genes compared to the corresponding ΔCt means in control group, also the gene expression folding (2 -∆∆Ct ) reflect significant differences in the expression of these genes and CD101, mRNA showed the highest level in CML patients which reached to (3.44), while NOB1 and DDX47 recorded (2.90 and 1.08) respectively. On the other hand no significant differences were recorded according to age and gender between CML patients and control, CML disease could affect any age and both male and female. Key wards: NOB1, CD101, DDX47, pateints, age, gender. ةيقارعلا ةيعارزلا مولعلا ةلجم 2021 : 52 ) 3 :) 611 619 يئاطلاو يروبجلا تانيج ثلاثل ينيجلا ريبعتلا نيب طابترلاا هسا رد هفلتخم قا رعلا يف نمزملا يوقنلا مدلا ضاضيباو يئاطلا يرخف اهم يروبجلا ىنثم ريده ثحاب دعاسم ذاتسا صلختسملا هقلاعلا نع يرحتلل هسا ردلا هذه ءا رجا مت نيب هفلتخم تانيج ثلاثل ينيجلا ريبعتلا ( NOB1, DDX47,CD101 ) و ثودح روطتو لا مدلا ضاضيبا ضرم يوقن بعتلا يف قرفلا هسا رد مت اضيا .قا رعلا يف نمزملا اقباس هروكذملا تانيجلل ينيجلا ري نيب .ءاحصلاا ةرطيسلا هعومجمو ىضرملا نمزملا يوقنلا مدلا ضاضيبا ثودحب سنجلاو رمعلا هقلاع صحف مت كلذ ىلا هفاضا ا .ةرطيسلا عم هتنراقمو ظ يبعتلا طسوتم يف يونعم دايدزا دوجو جئاتنلا تره عم هنراقملاب تانيجلا لكلو ىضرملل ينيجلا ر طسوتم يف هيونعم تاقورف دوجو جئاتننلا تلجس اضياو ةرطيسلا ا نيح يف تانيجلل ينبجلا فاعضلاا ريبعت نيج رهظ CD101 ىلعا طسوتم ( غلب ثيح نمزملا يوقنلا مدلا ضاضيبا ىضرم يفريبعتلا يف 3.44 نم لك لجس امنيب ) NOB1 و DDX47 ( 2.90 و 1.08 ىلا هبسن ءاحصلااو ىضرملا نيب هيونعم تاقورف دوجو جئاتنلا رهضت مل ىرخلاا هيحانلا نم .بقاعتلا ىلع ) ناو سنجلاو رمعلا نمزملا يوقنلا مدلا ضاضيبا ضرم .ثانلااو روكذلا نم لكو رمع يا بيصي يا نكمي , رمع ,ىضرم , سنج . NOB1, CD101, DDX47 هيحاتفملا تاملكلا : Received:22/6/2020, Accepted:6/9/2020 Iraqi Journal of Agricultural Sciences –2021:52(3):611-619 Aljoubory & Altaee 612 INTRODUCTION As a myeloproliferative disorder, chronic myeloid leukemia (CML) characterized by enhanced myeloidic cell proliferation. The disease progresses through different phases: chronic, accelerated and undifferentiated blast crises. A cytogenetic abnormality is the charestristics of CML consisting of a reciprocal translocation between the q arms of chromosomes 9 and 22, t(9;22): the chromosome in Philadelphia (7). The oncogene abl being transferred from the q arm of chromosome 9 into the q arm of chromosome 22 in the bcr region as a result of this translocation and a chimeric protein with heavy activation of the tyrosine kinase was encoded by the fusion gene bcr/abl (1). This work laid the foundation for the discovery of a tyrosine kinase inhibitor (TKI) as CML therapy (3). Hydroxyurea and eventually interferons and allogeneic transplantation of stem cells were used as effective treatment. However the development of imatinib and other drugs has changed the concept of disease treatment (10). CML patients commonly have few symptoms, common symptoms include abdominal fullness due to splenomegaly, anemia-related fatigue, bone pain associated with a packed bone marrow, bleeding, weight loss, malaise or night sweats. An enlarged spleen is often visible during physical examination whereas an enlarged liver or purpura is less normal (13). NOB1: Nin one binding 1 human gene is composed of nine exons and eight introns, and is located on chromosome 16q22.1 and it give RNA-binding protein NOB1 (19). Ribosome assembly required over 210 protein and RNA. The modification and cleavage of the initial rRNA, folding of the rRNA, and binding of ribosomal proteins and 5S RNA was mediated by these factors (17). However, pre-rRNA processing required the involving of NOB1. NOB1 cleaves 20S rRNA intermediate at cleavage site D to produce the mature 18S rRNA in a late cytoplasmic processing step. Abnormal expression of NOB1 in leukemia has been detected (6). DDX47: DDX47 (DEAD box polypeptide 47) is a member of the DEAD box family of proteins. Many conserved motifs which include the highly conserved DEAD (AspGlu-Ala-Asp) amino acid sequence motif were conserved by this family. The function as ATP-dependent RNA helicases is the major activity of DEAD box proteins (20). All aspects of RNA metabolism and function which include pre-mRNA splicing, RNA synthesis, RNA degradation, RNA export, RNA translation, RNA secondary structure formation, ribosome biogenesis, and the assembly of RNP complexes were mediated by DEAD proteins as helicases. DDX47 can transfer between the cytoplasm and the nucleus, with an RNA-independent ATPase activity. DEAD box RNA helicases are located on chromosome 12p13.1 and it is the key components of life (9). CD101: (Cluster of Differentiation 101) is a protein coding of the gene located on chromosome 1p13.1 (18). However, inhibition of T-cells proliferation induced by CD3, inhibition of expression of IL2 on activated Tcells and secretion of it, inhibition of tyrosine kinases that are required for IL2 formation and cellular proliferation are one of the roles of CD101. It may also be a marker of CD4 + and CD56 + leukemic tumor cells (12). The following study was aimed to investigate the correlation between the expression of three different genes (NOB1, DDX47, CD101 ( with the occurrence and development of chronic myeloid leukemia (CML). MATERIALS AND METHODS Patients and Controls : In this study fortyfour CML patients with an age ranging between (19-70) years old that were divided a according to gender into (19) male and (25) female were obtained from medical city (Baghdad Teaching Hospital/ Baghdad/Iraq), in addition to forty-six healthy controls with an age ranging between (19-62) years old and that were divided a according to gender into (25) male and (21) female. The characteristic peripheral blood smear analysis and complete blood profiling along with bone marrow examination data of the patients is the basis for diagnosis of CML. Blood collection: one ml blood sample was taken from each patient and healthy control and collected in TRIzol TM Reagent containing tube for RT-qPCR analysis. Total RNA Extraction with TRIzol: RNA was isolated from sample according to the Iraqi Journal of Agricultural Sciences –2021:52(3):611-619 Aljoubory & Altaee 613 protocol of TRIzolTM Reagent as the following steps: Sample lysis: For each tube, 0.5 mL of blood was added to 0.5 mL of TRIzol TM Reagent, the lysate was homogenized by pipetting up and down several times. For three phase's separation: 0.2 mL of chloroform was added to the lysate for each tube, then the tube cap secured. All mixes were Incubate for 2–3 minutes then centrifuge for 10 minutes at 12,000 rpm. Then, the mixture was separated into a lower organic phase, interphase, and a colorless upper aqueous phase. The RNA containing aqueous phase was transferred into a another clean tube. For RNA precipitation: to the aqueous phase, isopropanol (0.5 ml) was added and incubated for 10 minutes then centrifuge for 10 minutes at 12,000 rpm. Total RNA was precipitate formed a white gel-like pellet at the bottom of the tube. Supernatant was then discarded. For RNA washing: For each tube, 0.5mL of 70% ethanol was added and vortex briefly then centrifuge for 5 minutes at 10000 rpm. Ethanol then aspirated and air-dried the pellet. For RNA solubility: pellets were incubated in a water bath or heat block set at 55–60°C for 10–15 minutes after rehydration in 100μl of Nuclease Free Water. RNA purity and concentration measurements: the purity and concentration of the extracted lysates were determined by spectrophotometer nano drop (Q5000 (UVVIS), the concentration is measured in ng /μl unit, and the purity is measured by optical density (OD) ratio 260 / 280nm (DNA and protein absorption wave length ). The accepted purity of RNA is 1.72. According to the lowest concentration of samples the concentration of RNA samples was normalized by using the following equation: Vn = Co*V0 / Cn Where, Co is the lowest concentration, VO is the normalized volume (equal to 100 μL, Cn current sample concentration and Vn is the volume of current sample that will diluted by T.D.W to generate totally 100 μl. Primer design: The cDNA sequences of (NOB1, DDX47, CD101 and TEGT) genes were obtained from the NCBI GenBank database. RT-qPCR primers were designed using Primer Premier 3 software with melting temperature between 60 to 65 C, primer length between 18 to 23 nucleotides, and PCR amplicon length within 75 to 150 base pair as shown in Table 1. Table 1. Primers sequences Primer Name Sequence Annealing


‫العراقية‬ ‫الزراعية‬ ‫العلوم‬ ‫مجلة‬
. The oncogene abl being transferred from the q arm of chromosome 9 into the q arm of chromosome 22 in the bcr region as a result of this translocation and a chimeric protein with heavy activation of the tyrosine kinase was encoded by the fusion gene bcr/abl (1). This work laid the foundation for the discovery of a tyrosine kinase inhibitor (TKI) as CML therapy (3). Hydroxyurea and eventually interferons and allogeneic transplantation of stem cells were used as effective treatment. However the development of imatinib and other drugs has changed the concept of disease treatment (10). CML patients commonly have few symptoms, common symptoms include abdominal fullness due to splenomegaly, anemia-related fatigue, bone pain associated with a packed bone marrow, bleeding, weight loss, malaise or night sweats. An enlarged spleen is often visible during physical examination whereas an enlarged liver or purpura is less normal (13). NOB1: Nin one binding 1 human gene is composed of nine exons and eight introns, and is located on chromosome 16q22.1 and it give RNA-binding protein NOB1 (19). Ribosome assembly required over 210 protein and RNA. The modification and cleavage of the initial rRNA, folding of the rRNA, and binding of ribosomal proteins and 5S RNA was mediated by these factors (17). However, pre-rRNA processing required the involving of NOB1. NOB1 cleaves 20S rRNA intermediate at cleavage site D to produce the mature 18S rRNA in a late cytoplasmic processing step. Abnormal expression of NOB1 in leukemia has been detected (6). DDX47: DDX47 (DEAD box polypeptide 47) is a member of the DEAD box family of proteins. Many conserved motifs which include the highly conserved DEAD (Asp-Glu-Ala-Asp) amino acid sequence motif were conserved by this family. The function as ATP-dependent RNA helicases is the major activity of DEAD box proteins (20). All aspects of RNA metabolism and function which include pre-mRNA splicing, RNA synthesis, RNA degradation, RNA export, RNA translation, RNA secondary structure formation, ribosome biogenesis, and the assembly of RNP complexes were mediated by DEAD proteins as helicases. DDX47 can transfer between the cytoplasm and the nucleus, with an RNA-independent ATPase activity. DEAD box RNA helicases are located on chromosome 12p13.1 and it is the key components of life (9). CD101: (Cluster of Differentiation 101) is a protein coding of the gene located on chromosome 1p13.1 (18). However, inhibition of T-cells proliferation induced by CD3, inhibition of expression of IL2 on activated Tcells and secretion of it, inhibition of tyrosine kinases that are required for IL2 formation and cellular proliferation are one of the roles of CD101. It may also be a marker of CD4 + and CD56 + leukemic tumor cells (12). The following study was aimed to investigate the correlation between the expression of three different genes (NOB1, DDX47, CD101 ( with the occurrence and development of chronic myeloid leukemia (CML).

MATERIALS AND METHODS Patients and Controls
: In this study fortyfour CML patients with an age ranging between (19-70) years old that were divided a according to gender into (19) male and (25) female were obtained from medical city (Baghdad Teaching Hospital/ Baghdad/Iraq), in addition to forty-six healthy controls with an age ranging between (19-62) years old and that were divided a according to gender into (25) male and (21) female. The characteristic peripheral blood smear analysis and complete blood profiling along with bone marrow examination data of the patients is the basis for diagnosis of CML. Blood collection: one ml blood sample was taken from each patient and healthy control and collected in TRIzol TM Reagent containing tube for RT-qPCR analysis. Total RNA Extraction with TRIzol: RNA was isolated from sample according to the protocol of TRIzolTM Reagent as the following steps: Sample lysis: For each tube, 0.5 mL of blood was added to 0.5 mL of TRIzol TM Reagent, the lysate was homogenized by pipetting up and down several times. For three phase's separation: 0.2 mL of chloroform was added to the lysate for each tube, then the tube cap secured. All mixes were Incubate for 2-3 minutes then centrifuge for 10 minutes at 12,000 rpm. Then, the mixture was separated into a lower organic phase, interphase, and a colorless upper aqueous phase. The RNA containing aqueous phase was transferred into a another clean tube. For RNA precipitation: to the aqueous phase, isopropanol (0.5 ml) was added and incubated for 10 minutes then centrifuge for 10 minutes at 12,000 rpm. Total RNA was precipitate formed a white gel-like pellet at the bottom of the tube. Supernatant was then discarded. For RNA washing: For each tube, 0.5mL of 70% ethanol was added and vortex briefly then centrifuge for 5 minutes at 10000 rpm. Ethanol then aspirated and air-dried the pellet. For RNA solubility: pellets were incubated in a water bath or heat block set at 55-60°C for 10-15 minutes after rehydration in 100μl of Nuclease Free Water. RNA purity and concentration measurements: the purity and concentration of the extracted lysates were determined by spectrophotometer nano drop (Q5000 (UV-VIS), the concentration is measured in ng /µl unit, and the purity is measured by optical density (OD) ratio 260 / 280nm (DNA and protein absorption wave length ). The accepted purity of RNA is 1.7-2. According to the lowest concentration of samples the concentration of RNA samples was normalized by using the following equation: Vn = Co*V0 / Cn Where, Co is the lowest concentration, VO is the normalized volume (equal to 100 µL, Cn current sample concentration and Vn is the volume of current sample that will diluted by T.D.W to generate totally 100 µl. Primer design: The cDNA sequences of (NOB1, DDX47, CD101 and TEGT) genes were obtained from the NCBI GenBank database. RT-qPCR primers were designed using Primer Premier 3 software with melting temperature between 60 to 65 C, primer length between 18 to 23 nucleotides, and PCR amplicon length within 75 to 150 base pair as shown in Table 1. Table 1 Gene Expression cDNA synthesis from mRNA: The kit that was used to assess the expression of (NOB1, DDX47, and IGSF2) genes was GoTaq ® 1-Step RT-qPCR System (Promega, USA). It is a reagent system for quantitative analysis of RNA using a one -step RT-qPCR protocol.
According to the manufacturer instructions, the procedure was performed with a 20 μl reaction volume. To be reversely transcribed the total RNA volume was (4μl).
Protocol: Reverse transcription reactions should be assembled in RNase-free environment. The RNA templates and all reagents were thawed, and each solution was mixed gently. The RT FDmix tubes were placed on PCR tube rack. The reaction component was added to the RT FDmix tube as in Table 2. up to 20 μl Tubes were placed in a thermal cycler program as in Table 3   Table 3. Thermal cycler steps of conditions cDNA Reverse Transcription Synthesized cDNA was immediately used as template for PCR or for long-term storage at -20°C.

Quantitative Real Time PCR (qRT-PCR):
The expression levels of (NOB1, DDX47, and CD101) genes were estimated by the reverse transcription -quantitative polymerase chain reaction (qRT-PCR) method, which is a sensitive technique for the quantification of steady-state mRNA levels. To confirm the expression of target gene, quantitative real time qRT-PCR SYBR Green assay was used. Primers sequences for NOB1, DDX47, and CD101 genes were designed in the current study and synthesized by Alpha DNA Ltd (Canada) and stored lyophilized at (-23°C). Primers sequences are shown in Table (1). The mRNA levels of endogenous control gene TEGT were amplified and used to normalize the mRNA levels of the tested genes. TEGT primers sequences are also shown in Table (1).

Primer preparation
A primer for (NOB1, DDX47, CD101 and TEGT) working solution were prepared from the lyophilized primers after dissolving in nuclease free water according to the manufacture, to make a stock solution with a concentration of 100 µM for each primers and stored at (-23°C). A working solution with a concentration of 10 µM was prepared by diluting 10µL of primers stock solution in 90 µL of nuclease free water and stored at (-23°C) until use.

Quantitative Real Time PCR (qRT-PCR)
Run: Quantitative Real Time PCR (qRT-PCR) was performed using the MIC-4 Realtime PCR System (AUSTRALIA). The gene expression levels and fold change were quantified by measuring the threshold cycle (Ct) employing the 2xqPCR Master Mix Kits components. Every reaction was done in a duplicate. The required volume was calculated according to Table 4.

Table 4. Components of quantitative real-time PCR used in gene expression experiment
The cycling protocol was programed for the following optimized cycles and according to the thermal profile as shown in Table 5   Table 5

. Thermal profile of genes expression
Using the real-time cycler software, the threshold cycle (CT) was calculated for each sample. All samples were run in duplicate and mean values were calculated (Appendix 3).
Expression data of selected genes were normalized against housekeeping.The ΔΔCt method by (5) was used as was recommended for data analysis and results were expressed as Step 4 Step 3 Step 2 Step 1

. Amplification plots of TEGT gene by RT-PCR
A representative melt curve NOB1 gene for samples analyzed by RT-PCR is given in Figure 5, in which, a single peak was observed for the amplicons. Such findings are interpreted that the melt curve represented a pure, single amplicon for each sample, and the specificity of amplification was considered to be great with intercalating dye assay.

Figure 5. Melt curve of NOB1 gene amplicons after RT-qPCR analysis showing single peaks
Expression level of NOB1, CD101, and DDX47 genes in the studied groups: The ΔCt mean of NOB1 gene in blood samples of CML patients was (21.90), CD101 ΔCt mean was (23.44) and for DDX47 ΔCt mean was (18.08). There was a significant p<0.05 increases in ΔCt mean of patient groups for all genes compared to the corresponding ΔCt means in control group as shown in Table 6. The expression of genes showed significant variation between the CML patients and control. Such findings are consistent with previous study (10,16), which generally reported the correlation of the high percentage of expressing these genes in CML patients. The current study highlighted the importance of these genes in occurrence and development of chronic myeloid leukemia and results clearly indicate an involvement of all the studied genes in the CML invasion and metastasis. Since the reason for high expression of previous genes in CML patients is presently unclear but may be related to disturbances in this type of cancer leading to anomalies in the production of these genes. The correlation of these genes with other types of cancer was also recorded by some researches (14 , 17). In conclusion, NOB1, CD101 and DDX47 mRNA expression were significantly upregulated in CML tissue, in comparison with controls. These results suggest that increased genes expression might be a useful diagnostic marker and might also become a potential target in the treatment of chronic myeloid leukemia in Iraq. Correlation of age and gender with CML and control groups: result revealed in Table 9 and 10 relation of age and gender with CML in comparison with control. Results showed that no significant differences p> 0.05 were recorded for both age and gender between CML patients and controls, similar results were also obtained by (4,8). The percentage of patient male was 21.11% while control male record 27.78%. Moreover patients female showed 27.78% percentage while control female showed 23.33% percentage as shown in Table 8. Also the mean patient's age was 40.36 while the mean control age was 41.00 as shown in Table 9. These findings suggest that CML can occur in both male and female and can affect all ages.