Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin
Notice bibliographique
Résumé
Abstract INTRODUCTION It is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known. METHODS Human myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia). RESULTS BMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05). CONCLUSION Our results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».