| Objective: Chronic myeloproliferative neoplasma(c MPN)is a class of Clonal diseases Originated in hematopoietic stem cells, is a kind of malignant hematopoietic diseases.In 2008, a detailed list of Chronic myeloproliferative neoplasma was discussed by WHO including primary myelofibrosis(PMF), polycythemia vera(PV), essential thrombocythemia(ET), chronic neutrophilic leukemia(CNL), chronic eosinophilic leukemia/high eosinophil syndrome(CEL/HES), mast cell disease(MCD)and chronic myeloproliferative neoplasms unclassified(c MPN-U). And V617 F mutations in the JAK gene exists in patients with PMF, PV and ET, this kind of disease causes the increase of granulocytes, erythrocytes and platelets in peripheral blood as a result of bone marrow cell proliferation. The common clinical manifestations of this kind of disease are thrombosis, hemorrhage, milt intumescent, into leukemia, PV and ET have a tendency to transform to MF. Immunomodulatory drugs(IMi Ds) are important part of the treatment scheme of a variety of malignant diseases[1]. Thalidomide is a representative drug of IMi Ds, Early studies suggested that generate and anti vascular its induced oxidative stress may be the mechanism of teratogenic effects[2,3]. In recent years, in a landmark documents shows that cereblon(CRBN) is the first target for thalidomide teratogenicity[4].E3 ubiquitin ligase compounds is made of CRBN、DDB1、ROC1、CUL4 atc,it can regulate DNA’s replication repair and transcription[5、6、7]. CRBN is a related factor of E3 ubiquitin ligase compounds, it can happen ubiquitination in vitro and in vivo[4]. Studies have shown that thalidomide’s teratogenicity achieved by blocking CRBN’s ubiquitination[8]. When thalidomide or lenalidomide play a role, CRBN can’t form to E3 ubiquitin ligase compounds. Thalidomide and some similar drugs,for example lenalidomide and pomalidomide are widely used in the treatment of chronic myeloproliferative neoplasms, multiple myeloma and myelodysplastic syndrome and other malignant diseases[9].Studise about multiple myeloma(MM) show that CRBN is necessary to immunomodulatory drugs for treating.It’s also observed that CRBN expression level decreasing in MM patients with primary resistance to the drug lenalidomide[8、10]. The reaction of thalidomide maintenance therapy and CRBN expression levels in patients showed a positive correlation[11、12]. However, there is still little research concerned expression of CRBN for therapy for Chronic myeloproliferative neoplasma with immunomodulator.This experiment by detecting the expression of CRBN m RNA and protein in bone marrow and blood mononuclear cells in patients with cMPN,analysis of cMPN patients with bone marrow mononuclear cells CRBN gene expression and blood mononuclear cells CRBN protein level,to study the effect of CRBN expression level and clinical curative of thalidomide treatment.Methods:This study collected 24 cases(MF 5 cases,PV 7 cases,ET 12 cases) c MPN pateints’ bone marrow and 7 cases pateints’ blood,the patients were first diagnosed in Second Hospital of Hebei Medical University,set to these patients initial treatment group.Set the 11 patients after oral thalidomide therapy 50mg/d to treatment group and collect their blood.In the treatment group,3 cases treated with thalidomide less than 6 months,4 cases treated with thalidomide 6-12 months,4 cases treated with thalidomide more than 12 months. Collect 10 cases bone marrow and blood of patients with non-malignant hematological disease and set to control group. Application of PT-PCR detection the expressoin level of CRBN m RNA in bone marrow mononuclear cells,Western-blot method is applied to detect the expression level of cereblon protein in blood mononuclear cells.Results:1 cereblon m RNA expressionRT-PCR results showed that the bone marrow BM-MNCs cereblon m RNA relative expression quantity in initial treatment group is(1.0333±0.2723). The CRBN relative expression quantity in MF, PV, ET group respectively is:(1.1814±0.2257),(0.9943±0.3377),(0.9943±0.2484). The CRBN relative expression quantity in control group is(0.5699±0.9474)(see Fig.1, Tabel 1). The relative expression levels of all c MPN patients cereblon m RNA were elevated,comparing with control group,the differences were statistically significant(P=0.000); The relative expression levels of cereblon m RNA in untreated MF patients and untreated PV patients compared with no statistically significant differences(P=0.183), The relative expression levels of cereblon m RNA in untreated MF patients and untreated ET patients compared with no statistically significant differences(P=0.144), and the relative expression levels of cereblon m RNA in untreated ET patients and untreated PV patients compared with no statistically significant differences(P=0.100)(see Tabel 1). There were 13 cases with JAK2V617 F mutation positive and 11 cases with JAK2V617 F mutation negative in the initial treatment group,the relative expression levels of cereblon m RNA in the two groups are(0.9743±0.2912),(1.1031±0.2427), the two groups showed no significant difference(P=0.259), and the two groups were statistically significant when compared with the control group(P=0.000)(see Fig.2, Tabel 2).2 Expression level of cereblon proteinWestern Blot results showed that the relative expression quantity of MNCs cereblon protein in initial treatment group、treated group、control group respectively is:(1.550±0.223)pg/ml,(1.028±0.182)pg/ml,(0.911±0.161)pg/ml,(see Fig.3, Tabel 3). In the treated group, the relative expression level of 3 cases treated with thalidomide less than 6 months, 4 cases treated with thalidomide 6-12 months, 4 cases treated with thalidomide more than 12 months respectively is(1.181±0.133)pg/ml,(0.996±0.571)pg/ml,(0.945±0.248)pg/ml. With the continued growth of oral thalidomide time, CRBN protein expression level was decreased(see Fig.4, Tabel 4). The expression levels of cereblon protein in all c MPN patients increased. Differences in initial treatment group compared with the treatment group was statistically significant(P=0.000). Initial treatment group compared with the control group difference was statistically significant(P=0.000). The treatment group CRBN protein levels tended to increase compared with the control group, but the difference was not statistically significant(P=0.160)(see Tabel 3). Set the 7 cases without thalidomide treated to the first group, the 3 cases treated with thalidomide less than 6 months to the second group, the 4 cases treated with thalidomide 6-12 months to the third group, 4 cases treated with thalidomide more than 12 months to the fourth group, 10 cases in control group to the fifth group.and the cereblon protein expression level of the five groups are(1.550±0.223)pg/ml,(1.181±0.133)pg/ml,(0.996±0.571)pg/ml,(0.945±0.248)pg/ml,(0.911±0.161)pg/ml, the CRBN protein expression levels in each group on a downward trend. The first group has a statistically significant difference compared to the second group(P=0.007), has statistically significant differences compared to the third, fourth, fifth group too(P=0.000). The second group and the third group, the fourth group compared the differences were not statistically significant(P=0.197, P=0.104). The second group and the fifth group have a statistically significant difference compared(P=0.034). The third group and the fourth group, the fifth group compared the differences were not statistically significant(P=0.697, P=0.435). The fourth group and the fifth group showed no significant difference(P =0.749)(see Tabel 4).3 Correlation between expressions of CRBN m RNA and CRBN proteinCereblon m RNA relative expressions of 7 patients in initial treatment group were 1.0000, 1.1617, 0.5428, 1.1327, 1.1759, 1.4321, 0.8564. cereblon protein expressions were 1.566 pg/ml, 1.431 pg/ml, 1.164 pg/ml, 1.632 pg/ml, 1.732 pg/ml, 1.849 pg/ml, 1.479 pg/ml. It shows that positive correlation between the two data sets, the correlation coefficient is 0.714, This is statistically significant(P=0.024)(see Fig.5).Conclusions:1 The expression levels of cereblon m RNA and protein in the newly diagnosed patients of c MPN are higher than that in control group and after thalidomide therapy patients’.2 In the newly diagnosed patients,the expression level of cases with JAK2V617 F mutation positive was no significant difference compared with expression level of ones with JAK2V617 F mutation negative. This proves that no correlation between the expression of CRBN and JAKV617 F mutation.3 With the extension of oral thalidomide treatment time, the expression levels of CRBN protein decreased gradually.4 The CRBN gene and protein’s expression level may have some relevance. |
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