Study On The Role Of DLK1Gene In Hematopoiesis Regulation In Patients With Bone Marrow Failure Diseases The Prognostic Value Of Thrombocytopenia In Patients With Primary Mvelodysplastic Syndromes

BackgroundDLK1is a member of the epidermal growth factor-like homeotic protein family whose expression is known to modulate the differentiation signals of several kinds of cells, and thought to participate in hematopoiesis, whereas abnormally expressed in a variety of blood diseases. DLK1mRNA levels were high in CD34+cells from patients with Myelodysplastic syndromes (MDS) which suggests a potential role of DLK1in the pathogenesis of the disease. Aplastic anemia (AA), also known as bone marrow failure disease, has an increased amount of fat cells in bone marrow as its typical characteristic, meanwhile the gene play an important role in inhibition of preadipocytes differentiation, suggesting that it may also participate in the pathogenesis of AA.ObjectiveThe present study was aimed to investigate the role of DLK1mRNA and soluble protein expression levels in MDS and AA and its clinical significance, explore the possible mechanism of DLK1overexpression in the function of bone marrow hematopoetic stem cell (HSC) and also the role of DLK1in the pathogenesis of MDS by hematopoietic-specific promoter HS21/45vav to high expression DLK1in vivo.MethodsWe examined the DLK1mRNA level in bone marrow mononuclear cells (BMMNC) and mesenchymal stem cells (MSC) from MDS and AA respectively by real time PCR; tested the soluble DLK1protein level in the serum and bone marrow plasma of MDS and AA by ELISA. And then we successfully constructed hM HS21/45vav-DLKl vector which was integrated to the C57BL/6J genome DNA. Real time PCR and western blot comfirmed high expression of DLK1in the hematopoietic system of transgenic animal models. To analyze the regulating effect of DLK1on the hematopoetic system by apoptosis, HSC colony-forming cell (CFC) assays, CFU-F assays and co-culture experiments. Results1. We detected DLK1mRNA level in BMMNC from59cases of patients with MDS,50cases of patients with A A by real time PCR. Our results showed that Dlkl mRNA levels were higher in samples from MDS patients than those from healthy control (p=0.049), and were much higher in MDS patients with myelofibrosis than that without myelofibrosis (P=0.034).Whereas Dlkl mRNA levels were lower in samples from AA patients than those from healthy control (p=0.001), and positively correlated with absolute reticulocyte count and CD3+CD8+T lymphocyte ratio(p=0.006,0.018respectively). In the50AA patients,14cases were severe AA and the remaining were non-severe AA, DLK1mRNA levels were much lower in the former than that in the latter (p=0.022).2. DLK1soluble protein levels in the serum of AA patients were distinctly lower than that in MDS and control (both P value<0.001), and negatively correlated with ferritin (P=0.005). However the protein levels in MDS patients were higher than that of healthy control, but there’s no statistical significance. Data of19MDS patients with expression of DLK1beyond upper limited was analyzed, showing DLK1correlated positively with CFU-E, BFU-E and IL-2as well(p=0.007,0.036,0.005respectively), while negatively with ferritin(p=0.041).DLK1protein levels in the bone marrow plasma of MDS patients were also much higher than that in A A (p=0.038).3. In the BMMSC of MDS patients, DLK1mRNA level was higher than the normal controls, but without statistical significance. Whereas the expression level in AA patients was significantly lower than that in MDS and controls (p=0.031,0.025respectively).4. DLK1expression in BMMNC RNA levels and serum protein levels were both obviously higher in hypo-MDS patients than those from AA patients (p=0.030,0.012), suggesting a clinical significance in these two bone marrow failure diseases differentiation.5. We successfully constructed DLK1over-expression mouse model. There was no significant change in mouse phenotypes. Compared with normal control, in vitro colony forming ability of bone marrow hematopoietic stem/progenitor cells and mesenchymal stem cells of transgenic mice was almost the same. However the result of co-culture shows:MSCs of transgenic mice which over-expressed DLK1inhibited the proliferation capacity of BFU-E, CFU-G, CFU-M, besides the size of the colones was much smaller and the morphology seemed more loose compared to the controls, especially the BFU-E. IL-6mRNA level in MSCs of transgenic mouse was evidently higher than that of wild type (p=0.021), but no difference between the BM cells of transgenic mouse and that of controls. Also no significant difference in the expression levels of TNF-a and SDF-1was between the two groups of mouse in either the whole BM cells or MSCs.ConclusionIn Bone marrow failure diseases, DLK1level was high in patients with MDS, and the increased expression may inhibit erythroid progenitor cell proliferation,and was associated with the formation of bone marrow fibrosis. Moreover, MSCs with over-expressed DLK1negatively regulated hematopoiesis in transgenic mouse.In AA patients, DLK1level was low and the expression level indicated the severity of AA disease to a certain extent. DLK1May be involved in the pathogenesis of AA by affecting the differentiation of bone marrow mesenchymal stem cells.In summary, dysregulation of DLK1expression in Bone marrow, whether; up or down, may cause an imbalance in the BM microenvironment resulting in the inhibition of hematopoiesis in the Bone marrow failure diseases. Furthermore, detection the expression of DLK1showed clinical significance in the differential diagnosis of the two diseases. Objective To investigate the prognostic value of thrombocytopenia in patients with primary myelodysplastic syndromes (MDS)Methods Four hundred and nineteen primary MDS patients were retrospectively analyzed. Kaplan-Meier method, Log-rank test and COX regression model were used to evaluate factors that influence the prognosis.Results Two hundred and fifty-six cases(61.1%) had thrombocytopenia (PLT<100×109/L), one hundred and three cases(24.6%) had severe thrombocytopenia (PLT<30×109/L). Overall survival (OS) tended to shorten along with the decreasing of platelet count. Univariate analysis indicated PLT<30x109/L, MCV<95fl, LDH>300U/L, lymphocyte-like micromegakaryocyte, nucleated RBC PAS positive, IPSS cytogenetic intermediate-and poor-risk were all related with poor prognosis. Moreover, The prognosis of patients with RCMD, RAEB-Ⅰ or RAEB-Ⅱ was poorer than the other subgroups. Among these parameters, PLT<30×109/L, MCV<95fl, IPSS cytogenetic intermediate-and poor-risk group and RCMD, RAEB-Ⅰ, RAEB-Ⅱ had independent prognostic significance in multivariate analysis. Modified WPSS prognostic model was proposed by adopting PLT, MCV, chromosomal karyotype and WHO classification. The OS of patients with low risk (0to1), intermediate-1risk (2to3), intermediate-2risk (4to5)and high risk (6to7) were59m,28m,14m and4m, respectively, and there was a statistically significant difference between the groups (p<0.05).Conclusion Severe thrombocytopenia indicated unfavorable prognosis, in combination with MCV, chromosomal karyotype and WHO classification, a modified WPSS prognostic model was proposed and worked well for prognostic indication in patients with MDS.