Aberrant Expression Of miRNA and mRNA Of Cell Cycle and Adhesion-Related Genes In Bone Marrow Stroma Cells Derived From Patients With Multiple Myeloma

Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of malignant plasma cells (PC) within the bone marrow. The bone marrow microenvironment such as bone marrow stroma cells (BMSC) supports MM disease progression, resistance to chemotherapy, protects the tumor cells against apoptosis and causes osteolytic bone disease and angiogenesis.

The aim of this study was to identify constitutive genetic alterations in BMSC derived from patients with MM (MM-BMSC) in comparison to BMSC from healthy donors. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates and were further expanded in cell culture. We studied 25 MM patients and 5 healthy donors. Senescence status was determined in passage 1 of cell cultures. MM-BSMC displayed a considerably higher percentage of senescence cells (p<0,05). We investigated the expression of cell cycle and adhesion-associated genes (CCNE1, CCND1, CDKN1B, VCAM, ICAM, IKK-alpha) in BMSC (passage 4) using SYBR-Green Real-Time PCR and relative quantification by linear regression.

A downregulation of CCNE1 (p=0,05), CDKN1B (p=0,29), and an upregulation of CCND1 (p=0,05), VCAM-1 (p=0,33), ICAM-1 (p=0,33), and IKK-alpha (p=0,05) was demonstrated. Furthermore, the expression profile of miRNAs, targeting the analyzed mRNA genes or correlating with senescence, was studied (miR-16, miR-221, miR-126, miR-223, miR-485-5p and miR-519d). For miRNA detection treatment with Poly(A)-Polymerase and cDNA-Synthesis with a Poly(T)VN-Adaptor primer were carried out following an amplification with an universal reverse primer corresponding to the adaptor sequence and a miRNA-specific primer. miR-16, miR-223, miR-485-5p and miR-519d were significantly upregulated, (p=0,02; p=0,004; p=0,02; and p=0,002, respectively), whereas miR-221 and miR-126 showed no considerable differences to BMSC obtained from healthy donors. Next we investigated incubation of immunmodulatory drug Lenalidomid in BMSC cultures. Cells were treated with 10 µM Lenalidomid over 72 hours and expression was normalized to a 0,01 % DMSO control. Significant difference in gene expression were visible for ICAM-1 (p=0,01). For CDKN1B (p=0,16) and VCAM-1 (p=0,12) we demonstrated changes in gene expression.

Our results indicate aberrant expression of cell cycle and adhesion-related genes, such as CCNE1, CCND1 and CDKN1B VCAM-1, ICAM-1 and IKK-alpha in MM-BMSC as compared with healthy donors. Furthermore, we found significant upregulation of miR-16, miR-223, miR-485-5p and miR-519d. Further investigation are needed to determine molecular mechanisms in MM-BMSC and PC interaction that lead to constitutive changes in BMSC characteristics and gene expression patterns.