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NR4A1 Inhibition Synergizes with Ibrutinib in Killing Mantle Cell Lymphoma Cells

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to identify NR4A1 targets by RNA-seq and high-throughput data analysis and verify these genes by quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods. Methods: Jeko/Rec-1 Cas9 control and NR4A1 sgRNA stable cell lines were generated with tet-on system vector, sg RNAs were induced for 48 hours after doxycycline addition, mRNA was extracted and used for RNA sequencing. After TopHat analysis followed by Cufflinks, down/up regulated genes list was generated. qRT–PCR validation was then performed using SYBR Green assays. Results: Using an optimized data analysis workflow and with a fold change ≥1.3 and p value <0.05, thousands of genes were changed. Altered expression of 6 genes was then confirmed with qRT–PCR, demonstrating the high qulity of the RNA-seq method.

ORGANISM(S): Homo sapiens

PROVIDER: GSE106092 | GEO | 2017/10/25

SECONDARY ACCESSION(S): PRJNA415591

REPOSITORIES: GEO

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Project description:Mantle cell lymphoma (MCL) is an aggressive B-cell non-HodgkinM-bM-^@M-^Ys lymphoma (NHL). In cancers, tumour suppressive microRNAs may be silenced by DNA hypermethylation. By microRNA profiling, miR-155-3p was significantly upregulated upon demethylation treatment of MCL cell lines with 5-aza-2M-bM-^@M-^Y-deoxycytidine (5-azadC). Methylation-specific PCR, verified by pyrosequencing, showed complete methylation of miR-155-3p in one MCL cell line (REC-1). 5-azadC treatment of REC-1 led to demethylation and re-expression of miR-155-3p. Over-expression of miR-155-3p led to increased sub-G1 apoptotic cells and reduced cellular viability, demonstrating its tumour suppressive properties. By luciferase assay, lymphotoxin-beta (LT-M-NM-2) was validated as a miR-155-3p target. In 31 primary MCL, miR-155-3p was found hypermethylated in 6(19%) cases. To test if methylation of miR-155-3p was MCL-specific, miR-155-3p methylation was tested in an additional 191 B-cell, T-cell and NK-cell NHLs, yielding miR-155-3p methylation in 66(34.6%) including 36(27%) non-MCL B-cell, 24(53%) T-cell and 6(46%) of NK-cell lymphoma. Moreover, in 72 primary NHL samples with RNA, miR-155-3p methylation correlated with miR-155-3p downregulation (p=0.030), and LT-M-NM-2 upregulation (p=0.004). Collectively, miR-155-3p is tumour suppressive microRNA hypermethylated in MCL and other NHL subtypes. As miR-155-3p targets LT-M-NM-2, which is an upstream activator of the non-canonical NF-kB signalling, miR-155-3p methylation is potentially important in lymphomagenesis Total RNA isolated from MINO and JEKO-1 before and after 5-azadC treatment were converted into cDNA by MegaplexTM RT Primers and TaqManM-BM-. MicroRNA Reverse Transcription Kit. cDNA was pre-amplified using MegaplexTM PreAmp Primer and loaded onto 384-well format TaqmanM-BM-. human microRNA array A V2.0 & B V3.0. Real-time PCR was performed on 7900HT Real-Time PCR system and raw data were analyzed normalizing to mean of three endogenous controls (U6snRNA, RNU44 and RNU48). Relative microRNA levels were determined by M-NM-^TM-NM-^TCt using endogenous controls and untreated controls using SDS 2.4 and RQ manager 1.2. All experimental procedures and analyses were performed according to manufacturerM-bM-^@M-^Ys instruction, using reagents, system and softwares acquired from Applied Biosystems (Foster City, USA).

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NR4A1 Inhibition Synergizes with Ibrutinib in Killing Mantle Cell Lymphoma Cells

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