The aims of the study were to: (i) establish predictors of long-term and profound response at the molecular level to lenalidomide (LEN) treatment of patients with multiple myeloma (MM); (ii) identify alterations in gene expression and in the activity of various molecular pathways elicited by long-lasting exposure to LEN; (iii) recognize potentially targetable pathways induced by long-term exposure to LEN to select the best treatment regimen after relapse or even to provide research rationale for new treatment regimens in the future.
To achieve these goals, bone marrow was collected from 8 patients with newly diagnosed multiple myeloma (NDMM) and 8 patients with multiple myeloma treated with lenalidomide and dexamethasone (RD), who achieved at least a very good partial response (VGPR) after treatment.
In order to obtain a population of CD138+ cells from the bone marrow, an immunomagnetic isolation method was used. For this purpose, the CD138 MicroBeads kit, human (Miltenyi Biotec, Auburn, AL, USA) was used.
RNA was isolated from CD138+ cells from each patient using the RNAqueous-Micro Kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s recommendations. RNA concentration and quality were measured using TapeStation 4510 (Agilent Technologies, Santa Clara, CA, USA).
The Illumina Stranded mRNA Prep kit (Illumina, San Diego, CA, USA) and IDT for Illumina RNA UD Indexes Set A, Ligation (Illumina, San Diego, CA, USA) were used for global gene transcription analysis. 500 ng of RNA from each sample was used to prepare the library. The final loading library concentration was 1.2 pM. Sequencing parameters were in accordance with the manufacturer’s recommendations (read length: 2 × 75 bp; read type: paired end; dual index reads; the number of cycles per index read was 10). Sequencing was carried out on a NextSeq 550 instrument (Illumina, San Diego, CA, USA) using NextSeq 500/550 High Output Kit v2.5 (150 cycles) reagents.
In order to validate the NGS method, the expression level of selected genes was measured using qRT-PCR. The genes were selected based on NGS results that appear to be important in the development of the body’s response to lenalidomide. Primers for each gene were designed using the BLAST program. 0.1 µg of total RNA was used for reverse transcription. Reactions were performed using a First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). qPCR reactions were performed using the SYBR Green PCR Master Mix kit (Bio-Rad, Hercules, CA, USA). The GAPDH gene was used as the endogenous control gene. CFX Maestro Software (Bio-Rad, Hercules, CA, USA) was used to analyze gene expression using the comparative Ct quantification (2ΔCt) method.
Bioinformatics analysis was performed in the R environment. Genes with a fold change greater than 2 or less than − 2 were considered differentially expressed genes (DEGs) (p < 0.05).
A detailed description of the procedures can be found in article: Łuczkowska, K., Kulig, P., Baumert, B. & Machaliński, B. The evidence that 25(OH)D3 and VK2 MK-7 vitamins influence the proliferative potential and gene expression profiles of multiple myeloma cells and the development of resistance to bortezomib. Nutrients 14(23), 5190 (2022).
A global increase in gene expression was found in the RD group compared to NDMM, suggesting the involvement of epigenetic mechanisms. Moreover, upregulation of genes controlling the interaction within MM niche was detected. Next, genes controlling immune response were upregulated. In particular, the gene encoding the IL-17 receptor was overexpressed in the RD group which is a novel finding.
Please consult the Readme.txt file for additional information.
