Differential Expressions Of MicroRNAs In The CD138~+Cells Of Multiple Myeloma Patients With Deletion Of Chromosome13

Objectives①To detect the chromosomal abnormalities in the CD138+cells from the bone marrow of multiple myeloma (MM) patients and analyse their relationship with the clinical implications.②To screen the differentially expressed miRNAs in the CD138+cells between chromosome13deletion positive and negative patients and search for the new molecular markers in MM patients with chromosome13deletion..③To screen the key target genes that may be involved in the regulation of miRNAs and further clarify the role of miRNAs in the pathogenesis of MM patients with chromosomal13deletion.Meterials and Methods①Collected specimens:29newly diagnosed multiple myeloma patients (M:F=20:9) were enrolled as experimental group,with a median age of60years old (range46to83).10healthy donors were chosen as controls. Fresh bone marrow was collected for test. Mononuclear cells were collected by density gradient centrifugation.CD138+cells were collected by immunomagnetic beads sorting.①FISH:In order to detect the chromosomal abnormalities and screen the samples for microRNA microarray experiments,5probes(P53, RBI, D13S319, IgH and1q21) were used for fluorescence in situ hybridization(FISH) experiments on29newly diagnosed MM patients.③Clinical analysis:Analyse the relationship between the cytogenetic abnormalities and the clinical features. ④microRNA microarray experiments:Extract the total RNAs and evaluate the quality of them. Separate and enrich the miRNAs using the mirVanaTM RNA isolation kit. The miRNAs were labeled by Cy3fluorophore and then be purfied by RNeasy Mini kit. Differentially expressed miRNAs were detected by the miRNA microarray. Stem-loop RT-PCR was used to validate the miRNA microarray experiments outcomes.⑤Bioinformatics analysis:The potential target genes of the detected differentially expressed miRNAs were predicted by online software. Then the regulatory network of miRNAs and target genes was established.Results1.FISH:①The positive rates of the RB1, D13S319, IgH,1q21and p53probes in the29MM patients were48.3%,44.8%,48.3%,24.1%,10.3%,respectively. All the5probes with positive signals were found in3.4%of the patients and all the5probes with negative signals were found in24.1%of the patients.51.7%of the patients were found to have both the RBI or D13S319positive signals.17.1%of the patients were found to have both the IgH translocation and13q.②The chemotherapy response analysis:Among the23cases of patients with complete follow-up,65.2%(15/23) achieved more than the MR efficacy. Response rate of patients using the chemotherapy based on dexamethasone was58.8%(10/17). The response rate of the patients using the bortezomib-based chemotherapy was83.3%(5/6).The comparison between the two was no significant difference. The presence response rate of patients with del(13q), IgH translocations57.1%(8/14),58.3%(7/12), respectively and there is no significant correlation between them. Among the6patients who take bortezomib, the response rate of13q-positive group was100%(3/3) and the group was66.7%(2/3). The response rate of IgH translocation positive group was100%(2/2) and the negative group e was75%(3/4).The13q-and IgH translocation positive groups have no significant differences with their negative groups.2. MicroRNA microarray experiments:For the subsequent miRNA microarray experiments,3cases with positive RBI, D13S319and negative IgH, p53,1q21probe signals were screened as experimental group and another3cases with all the5probes signals negatively were screened as control group. Five differentially expressed miRNAs were detected between the two groups. Of which one was up-regulated and four of them were down-regulated. Unsupervised cluster analysis showed that the experimental group and the control group can be separated by the five differentially expressed miRNAs. RT-PCR results are consistent with the microarray results.4. Target gene prediction and network analysis:In the5miRNAs, miR-424has the largest and miR-3679-5p has the least number of predicted target genes. By constructing the miRNA-target gene regulatory network, we obtained a series of target genes in the critical nodes of the network. We can guess that the gene of SMAD3may have a high biological significance as it has the maximum node degree in all of the target genes.Conclusions Immunomagnetic beads sorting for CD138+cells+FISH is a sensitive method for detecting the chromosomal abnormalities of MM patients. There is no statistically significant between the efficacy of different treatment options and FISH and therapeutic response although the incidence is different in the reaction. The multiple myeloma patients with the deletion of chromosome13have their specifically expressed miRNAs. We can guess that the miRNAs may be involved in the occurrence and development of this type of MM. The conclusion that the SMAD3and the TGF-β/Smad3-signaling pathway may be control the key factor and pathway which may be regulated by the needs to be further verified.