| Objective: Multiple myeloma (multiple myeloma, MM) is a bloodsystem tumor caused by the malignant proliferation of plasma cells, withanemia, renal impairment, repeated infection, soluble osseous bone destructionfor the clinical manifestations, the incidence rates of multiple myeloma arehigh in elderly persons., the sex ratio has no difference. Immunomodulatorydrugs is the foundation of many malignancies treatment[1], thalidomide, wasoriginally used for pregnant women sedative, notorious for itsteratogenicity.Earlier studies think thalidomide induced oxidative stress andanti-angiogenesis, may be the mechanism of action of its teratogenicity[2,3].Recently in a landmark documents show that cereblon (CRBN) is the firsttarget for thalidomide teratogenicity[4].Thalidomide combining CRBN inhibition of E3ubiquitin ligasecompounds (CRBN, DDB1and CUL4). The function of this kind ofcompounds regulate DNA repair, replication and transcription, the inhibitionof compounds by thalidomide preventing a very important protein (such as cutfibroblast growth factor8) in embryonic body development degradation causeteratogenic effect[4]. But whether the teratogenic effect of thalidomide and itsrelated immunomodulatory drugs IMIDs was related to their anti-myelomaactive-ty, recently,it is showed that the expression of CRBN is also necessaryto the anti-myeloma activity for thalidomide and its derivatives--lenalidomideand pomalidomide[5].It’s also observed that CRB-N expression leveldecreasing in MM patients with primary resistance to the druglenalidomide[5,6]. Instead, reducing CRBN expression does not affect patientswith MM to other reagent, such as bortezomib, dexamethasone and melphalan [4]. A recent study found that there is a positive correlation between CRBNwith the reaction of maintenance treatment on the thalidomide andlenalidomide with dexamethasone therapy in MM Patients[7,8]. In addition, weand others have observed that patients with relapse and refractory CRBNgenetic mutations, this support CRBN play a key role in all sorts of IMiDstreatment[9]. However, many patients with low level of CRBN, have noobvious mutation, thus transcription or transcription factors (such as theregulation of microRNA) may influence CRBN expression and response forthe IMID therapy[10].This experiment by detecting the expression of CRBN mRNA andprotein in bone marrow mononuclear cells in patients with MM, analysis ofMM patients with bone marrow mononuclear cells CRBN gene expressionand protein level, to study the effect of CRBN expression level and clinicalcurative of thalidomide treatment.Methods: RT-PCR and Western Blot method were used to theexpressi-ng level of cereblon mRNA and proptein in patients with multiplemye-loma,which were collected from the second hospital of hebei medicaluniversity，among them20cases of new diagnosis patients with MM, set toinitial group，30cases for bone marrow get more than PR after treatment andgroup set to reaction. Of which20cases use thalidomide50mg QD fortreatment,8cases with thalidomide75mg QD for treatment,1case withthalidomide100mg QD for treatment,1case with thalidomide200mg QDfor treatment; Select20cases of normal control group.Application of PT-PCR detection the expression level of cereblon mRNA between groups ofmononuclear cell (BM-MNCs), Western method is applied to detect theexpression level of cereblon protein.Results:1cereblon mRNA expressionRT-PCR results showed that the relative expression quantity of BM- MNCs cereblon mRNA in Pre-treatment group, the well reaction group andnormal control group respectively is:(2.47±1.55),(1.27±0.60） and(0.65±0.21).The relative expression levels of all MM patients cereblon mRNAwere elevated, comparing with normal control group the differences werestatistically significant (p=0.000); The expression of cereblon mRNA leveldecline in well reaction group then pre-treatment group but still higher thanthe normal control group, the difference was statistically significant (p=0.000). Without thalidomide treated was the first group, treated withthalidomide1-3months was the second group,4to6months was the thirdgroup,7-9month was the fourth group,10-12months was the fifth groupand more then12months was the sixth group，and the relative expressions ofthe CRBN mRNA of six groups are (2.47±1.55),(1.83±0.52）,(1.59±0.45),(1.58±0.48),(0.83±0.37), and (0.68±0.19) respectively, the relative expressionof CRBN mRNA in each group declined gradually, there is no statisticaldifference between each groups among1group,2group,3group,4and5groups (p=0.053), there is no statistical difference between each groupsamong2,3,4,5,6group (p=0.299), there is statistically significant between1group and6group (p=0.004).2The expression level of cereblon proteinWestern blot results showed that the relative expression quantity of BM-MNCs cereblon protien in Pre-treatment group, the well reaction group andnormal control group respectively is:(1.52±0.08) pg/ml,(1.09±0.06) pg/mland(0.87±0.09) pg/ml.The relative expression levels of all MM patientscereblon protein were elevated, comparing with normal control group thedifferences were statistically significant (p=0.000); The expression ofcereblon protein level declined in well reaction group then pre-treatmentgroup but still higher than the normal control group, the difference wasstatistically significant (p=0.000). Without thalidomide treated was the firstgroup, treated with thalidomide1-3months was the second group,4to6 months was the third group,7-9month was the fourth group,10-12monthswas the fifth group and more then12months was the sixth group，and therelative CRBN protein in each group is(1.52±0.08) pg/ml,(1.17±0.08）pg/ml,(1.09±0.04) pg/ml,(1.09±0.02) pg/ml,(1.04±0.03) pg/ml,(1.03±0.03)pg/ml, the relative expression of CRBN protein in each group declinedgradually, each groups have statistical difference among1group,2group,3group (p=0.000), there is no statistical difference between each groupsamong2,3,4,5,6group (p=0.300).3relationship between the curative effect of Pre-treatment group andexpression level of CRBNRT-PCR results showed that before and after the treated whith1course ortreated whith more than1course to get PR in pre-treatment group, the relativeexpression level of CRBN mRNA respective is (3.50±0.71）,(1.65±0.48)，patients do not get PR before and after the treated in pre-treatment group therelative expression level of CRBN mRNA respective is (0.56±0.11),(0.50±0.14)，normal control group, the relative expression level of CRBNmRNA is (0.64±0.20). Patients achieve PR CRBN mRNA expression levelrelatively significantly higher than the group that do not get the PR patients,the difference is statistically significant (p=0.000), and significantly higherthan the control group, after treatment the genetic level declined, but stillhigher than that of normal control group, the difference is statisticallysignificant (p=0.000). Patients who were not reach PR relative expressionlevel of CRBN mRNA has no obvious change between before and aftertreatment, there was no statistically significant difference (p=0.733), andthere was no statistically significant difference compared with normal controlgroup (p=0.733).Western blot showed that before and after the treated whith1course ortreated whith more than1course to get PR in pre-treatment group, the relativeexpression level of CRBN protein respective is (1.51±0.07) pg/ml,(1.10±0.07) pg/ml，patients do not get PR before and after the treated in pre-treatmentgroup the relative expression level of CRBN protein respective is (0.94±0.24）pg/ml,(0.88±0.22) pg/ml，and the relative expression level of CRBN proteinin normal control group is (0.86±0.08) pg/ml. Patients achieve PR CRBNprotein expression level relatively significantly higher than the group that donot get the PR patients, the difference is statistically significant (p=0.000),and significantly higher than the control group, after treatment the proteinlevel declined, but still higher than that of normal control group, the differenceis statistically significant (p=0.000). Patients who were not reach PR relativeexpression level of CRBN protein has no obvious change between before andafter treatment, there was no statistically significant difference (p=0.733),and there was no statistically significant difference compared with normalcontrol group (p=0.733).Conclusion:1Pre-treatment MM patients CRBN expression level is higher than MMpatients after thalidomide treatment confirming CRBN is targets forthalidomide; For pre-treatment MM patients, patients reach PR the expressionlevel of CRBN showed higher than that patients who w-ere not reach PR，confirming that CRBN may be associated with the treatment efficacy of MM.2As different time of thalidomide maintenance treatment, CRBNexpression level decreased gradually, the genetic level decreased slower thanthe decreased of protein levels.3CRBN mRNA and protein expression may exist a certain correlation. |
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