Competing Endogenous RNA Network in Newly Diagnosed Multiple Myeloma by Genetic Microarray

Competing Endogenous RNA Network in Newly Diagnosed Multiple Myeloma by Genetic Microarray

Multiple myeloma (MM) is a malignant blood disorder characterized by the clonal proliferation of plasma cells, leading to an increase in monoclonal immunoglobulins in the blood and urine, and causing damage to target organs. The disease is known for its high genetic heterogeneity, and recent studies have highlighted the role of long non-coding RNAs (lncRNAs) in the formation, progression, and metastasis of MM. lncRNAs, along with circular RNAs (circRNAs), function as microRNA (miRNA) sponges, indirectly binding miRNAs and influencing the post-transcriptional regulation of target gene expression. This interaction is known as the competing endogenous RNA (ceRNA) relationship, which has emerged as a crucial mechanism in cancer biology. However, the understanding of ceRNA relationships in MM, particularly in newly diagnosed patients, remains limited.

To explore these relationships, we employed genetic microarray technology, which has become a reliable method for analyzing nucleic acid sequences and is widely used in precision medicine. In this study, we utilized the Affymetrix microarray platform to detect and analyze the expression of lncRNAs, circRNAs, miRNAs, and mRNAs in bone marrow cells from newly diagnosed MM patients. Our goal was to identify differentially expressed RNAs and construct a ceRNA network to better understand the pathogenesis of MM.

We recruited ten newly diagnosed, untreated MM patients from Beijing Chaoyang Hospital between July 2018 and April 2019. All patients met the 2014 International Myeloma Working Group (IMWG) updated criteria for MM diagnosis. Exclusion criteria included recurrent or refractory MM, critical disease, patients preparing for autologous stem cell transplantation, and those with plasmacytoma. Bone marrow specimens were also obtained from ten healthy volunteers as a control group. The study was approved by the ethics committee of Beijing Chaoyang Hospital, and all participants provided informed consent. The research complied with the Declaration of Helsinki and its amendments.

Mononuclear cells were isolated from 8 mL of fresh bone marrow using lymphocyte separation medium (Ficoll PLUS; GE Healthcare) and red blood cell lysis buffer (Solarbio) within two hours of harvesting. Total RNA was purified using the miRNeasy Serum/Plasma kit (Qiagen), and its purity and integrity were assessed by agarose gel electrophoresis. The human ClariomTM D array (Affymetrix) was used to detect and analyze the expression of lncRNAs, circRNAs, miRNAs, and mRNAs based on hybridization signal intensity. GeneChip Command Console Software (AGCC version 4.0; Affymetrix) was employed to generate a ceRNA network using the target prediction database.

Statistical analysis was performed using the t-test to compare the means of two samples, with a significance threshold of P < 0.05. Differentially expressed genes were identified when the fold-change (FC) was ≥2.0. Pearson correlation coefficients between matched molecules (e.g., lncRNA and miRNA) were calculated based on expression data, with a cutoff of 0.99 and a significance threshold of P < 0.05. The quantity of miRNA response elements and combining scores were used to evaluate the competency of ceRNA molecules.

Using the Affymetrix human ClariomTM D chip, we screened a total of 135,731 genes and identified differentially expressed genes as follows: 234 lncRNAs (1.7‰), 557 circRNAs (4.1‰), 122 miRNAs (0.9‰), and 709 mRNAs (5.2‰). Only a small number of RNAs were involved in the ceRNA network: nine lncRNAs, 42 circRNAs, eight miRNAs, and 51 mRNAs. The degree value (DV) indicated the total quantity of a gene related to other genes, and we identified molecules with the highest DVs: hsa_miR-4772-3p (DV:24), three upstream lncRNAs (RPL4P4, RPSAP19, and BMS1P5), and 13 circRNAs (hsa_circ_0004646, hsa_circ_0069826, hsa_circ_0012001, hsa_circ_0070485, hsa_circ_0064902, hsa_circ_0089337, hsa_circ_0035979, hsa_circ_0067793, hsa_circ_0004409, hsa_circ_0002929, hsa_circ_0004086, hsa_circ_0075924, and hsa_circ_0003069). All upstream molecules shared the downstream mRNA RPL37A, which codes for ribosomal protein L37.

Our analysis revealed that the ceRNA network involves a series of complex relationships between lncRNAs, circRNAs, miRNAs, and mRNAs. The ceRNA relationship has been previously shown to be a compelling mechanism in MM pathogenesis, with activation or inhibition of the miRNA/mRNA axis controlling protein production. In our study, one of the most activated RNAs in the ceRNA network was hsa_miR-4772-3p, which plays a role in ribosome synthesis. Three lncRNAs and 13 circRNAs sponging hsa_miR-4772-3p form the miR-4772-3p/RPL37A axis, while mRNAs RPL10A, RPL23, RPL37A, and RPL7L1 code for structural proteins of the ribosome skeleton, such as ribosomal protein 37. This suggests that the activities of the ceRNA network may increase protein production in newly diagnosed MM patients, consistent with the disease characteristics.

Additionally, our findings indicate that hsa_miR-618 and hsa_miR-1284 may be involved in the MM mechanism as other centers of the ceRNA network. miR-618 has been shown to play a role in tumor inhibition and to suppress metastasis in gastric cancer by negatively regulating the transcription of transforming growth factor-b2, a cytokine that promotes proliferation and suppresses apoptosis in endothelial cells. Similarly, miR-1284 has been reported to suppress the proliferation, migration, and invasion of breast cancer cells by targeting transcription factor zinc finger protein 2 to decrease tumor growth. These findings suggest that miRNAs can act as proto-oncogenes or tumor suppressors in the mechanism of RNA action, potentially causing protein expression disorders. We speculate that ceRNA relationships cause an imbalance in gene expression, affecting the proliferation process of MM cells.

In conclusion, our study provides preliminary evidence of RNAs in a ceRNA relationship using genetic microarray analysis of a large number of aberrantly expressed RNAs from patients with newly diagnosed MM. This offers new insights into the pathogenesis of MM and identifies RNA molecules that could be used as novel targets for new countermeasures against the disease. The ceRNA network, particularly the miR-4772-3p/RPL37A axis, may play a significant role in increasing protein production in MM patients, highlighting the potential for therapeutic interventions targeting these RNA interactions.

doi.org/10.1097/CM9.0000000000001108

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