The Expressions Of Mitoferrin-1/-2and ABCB10and Relationship With Mitochondrial Iron-overload In MDS

ObjectiveThe aim is to study the expressions of Mitoferrin-1,Mitoferrin-2and ABCB10at the gene and protein level respectively in patients with myelodysplastic syndromes(MDS),in order to reveal the reasons for mitochondrial iron overload in MDS patients,to explore the role of mitochondrial iron metabolism abnormalities in the pathogenesis of MDS,and to provide the basis for iron chelation therapy.Object and Methods1. To select newly diagnosed MDS patients (23cases of adults and8cases of children) for the experimental roup,immune thrombocytopenia (ITP) of newly diagnosed patients (12adults and8children) as the similar healthy control group of this experiment. The inclusion criteria of experimental group refered to the2008World Health Organization (WHO) reclassification of myelodysplastic syndromes (MDS). To be enrolled in this study,patients had to be previously untreated with red blood cell transfusions and iron chelating agents.The experimental and control group were excluded from other blood diseases,severe infection,thyroid hyperfunction and others with abnormal iron metabolism.2. To extract bone marrow fluid from the experimental group and the control group with sterile syringe,smear a few pieces quickly,extract each2-4ml of bone marrow fluid again and put into EDTA anticoagulant tube fastly.3. Each3ml of fasting venous blood was extracted from the experimental and control group in the early morning,anticoagulant with heparin and sent to the biochemistry department of our hospital to detect serum iron metabolism indicators.4. Bone marrow smears of the experimental and control group were done with iron staining respectively.To observe the distributions of extracellular iron and intracellular iron,record and capture images to save.5. To separate bone marrow mononuclear cells by density gradient centrifugation, distribute into2tubes for the extraction of total RNA and protein,and store them at-80℃.6. To extract the total RNA from bone marrow mononuclear cells with reference to the Trizol reagent manual,detect the purity and integrity of the extracted RNA.To synthesize the cDNA by applying Fermentas first strand reverse transcription kit. To detect the expressions of Mitoferrin-1,Mitoferrin-2and ABCB10mRNA by real-time PCR.7. To extract the total protein from bone marrow mononuclear cells using RIPA lysis buffer. To detect the expressions of Mitoferrin-1,Mitoferrin-2and ABCB10protein by Western Blot.Results1. The expressions of Mitoferrin-1mRNA,Mitoferrin-2mRNA and ABCB10mRNA in adult MDS patients compared with the control group showed no statistically significant difference (P>0.05).2.(1)The expression of Mitoferrin-1protein in adult low-risk MDS(RA and RARS) was higher than the control group,and the difference was statistically significant (P<0.001);(2) the expression of Mitoferrin-1protein in high-risk MDS(RAEB) compared with the control group showed no statistically significant difference (P>0.05);(3) the expression of Mitoferrin-1protein in RARS was higher than RA,and the difference was statistically significant (P<0.001);(4) the expression of Mitoferrin-2protein in MDS patients compared with the control group showed no statistically significant difference (P>0.05);(5) the expression of ABCB10protein in low-risk MDS was higher than the control group,and the difference was statistically significant (P<0.001);(6) the expression of ABCB10protein in high-risk MDS compared with the control group showed no statistically significant difference (P>0.05).(7) the expression of ABCB10protein in RARS was higher than RA,and the difference was statistically significant (P<0.001).3. The expressions of Mitoferrin-1mRNA,Mitoferrin-2mRNA and ABCB10mRNA in MDS children compared with the control group showed no statistically significant difference (P>0.05).4.(1)The expression of Mitoferrin-1protein in2cases of RA children was higher than the control group;(2) the expression of Mitoferrin-1protein in RAEB children compared with the control group showed no statistically significant difference (P>0.05);(3) the expression of Mitoferrin-2protein in MDS children compared with the control group showed no statistically significant difference (P>0.05);(4) the expression of ABCB10protein in2cases of RA children was higher than the control group;(5) the expression of ABCB10protein in RAEB children compared with the control group showed no statistically significant difference (P>0.05).Conclusion1. The expressions of Mitoferrin-1and ABCB10in MDS were not significantly higher than the control at the gene level.The expressions of Mitoferrin-1and ABCB10in MDS showed heterogeneity at the protein level,higher than the control in low-risk group (RA, RARS), no significantly higher than the control in high-risk group(RAEB).2. The expression of Mitoferrin-2in MDS was not significantly higher than the control at both gene and protein level.3. The expression of Mitoferrin-1in MDS may be regulated by post-transcriptional mechanism.The inappropriate increased stabilization of Mitoferrin-1mediated by ABCB10may be involved in the occurrence of mitochondrial iron overload in some patients of MDS(especially RARS).4. Mitoferrin-2may not be responsible for the occurrence of mitochondrial iron overload in MDS.