Indirect Tumor Inhibitory Effects of MicroRNA-124 through Targeting EZH2 in The Multiple Myeloma Cell Line


Javid Sabour Takanlu, M.Sc, 1,2,#Arad Aghaie Fard, M.Sc, 1,#Saeed Mohammdi, Ph.D, 2,3Seyed Mohammad Ali Hosseini Rad, M.Sc, 4Saeid Abroun, Ph.D, 1,*Mohsen Nikbakht, Ph.D., 2,3,*
Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Hematology, Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences, Tehran, Iran
Hematologic Malignancies Research Center, Tehran University of Medical Sciences, Tehran, Iran
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Hematology, Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences, Tehran, Iran
Hematologic Malignancies Research Center, Tehran University of Medical Sciences, Tehran, Iran
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
*Corresponding Addresses: P.O.Box: 1411713131 Department of Hematology Faculty of Medical Sciences Tarbiat Modares University Tehran Iran P.O.Box: 14115111 Hematology Oncology and Stem Cell Transplantation Research Center Tehran University of Medical Sciences Tehran Iran Emails:abroun@modres.ac.ir,m-nikbakht@sina.tums.ac.ir

The first two authors equally contributed to this work.

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Abstract

Objective

Multiple myeloma (MM) is an incurable plasma cell malignancy. Several genetic and epigenetic changes affect numerous critical genes expression status in this disorder. CDKN2A gene is expressed at low level in almost all cases with MM disease. The mechanism of this gene down-regulation has remained controversial. In the present study, we targeted EZH2 by microRNA-124 (miR-124) in L-363 cells and assessed following possible impact on CDKN2A gene expression and phenotypic changes.

Materials and Methods

In this experimental study, growth inhibitory effects of miR-124 were measured by MTT assay in L-363 cell line. Likewise, cell cycle assay was measured by flowcytometery. The expression levels of EZH2 and CDKN2A were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR).

Results

qRT-PCR results showed induction of EZH2 gene expression after transduction of cells with lentivector expressing miR-124. The expression of CDKN2A was also upregulated as the result of EZH2 supression. Coincide with gene expression changes, cell cycle analysis by flow-cytometry indicated slightly increased G1-arrest in miR- transduced cells (P<0.05). MTT assay results also showed a significant decrease in viability and proliferation of miR- transduced cells (P<0.05).

Conclusion

It seems that assembling of H3K27me3 mark mediated by EZH2 is one of the key mechanisms of suppressing CDKN2A gene expression in MM disease. However, this suppressive function is applied by a multi-factor mechanism. In other words, targeting EZH2, as the core functional subunit of PRC2 complex, can increase expression of the downstream suppressive genes. Consequently, by increasing expression of tumor suppressor genes, myeloma cells are stopped from aberrant expansions and they become susceptible to regulated cellular death.