miR-17~92 Promotes Progression of ABC-DLBCL Lymphoma via Regulation of Canonical NF-kB Signaling

doi:10.21203/rs.3.rs-691597/v1

Download PDF

Research

miR-17~92 Promotes Progression of ABC-DLBCL Lymphoma via Regulation of Canonical NF-kB Signaling

https://doi.org/10.21203/rs.3.rs-691597/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoma characterized by constitutive NF-κB activation. Nevertheless, the role and mechanisms of miR-17~92 in contributing to the NF-κB activation in ABC-DLBCL are still elusive.

Methods: The expression of miR-17~92 primary transcript (MIR17HG) and NF-κB target genes was determined using RNA-sequencing. The expression of miR-17~92 was performed using microarray analysis. Plasmids carrying conditional over-expression and loss-of-function of miR-17~92 were respectively constructed and dual-luciferase reporter assay was used to validate the target gene of miR-17~92. Immunoprecipitation and polyubiquitination were further used to the study of potential mechanisms.

Results: Expression of MIR17HG was positively correlated with NF-κB activity, miR-17~92 activated the NF-κB signaling in ABC-DLBCL, and its over-expression promoted ABC-DLBCL cell growth, accelerated cell G1 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, miR-17~92 promoted NF-κB activation through directly targeting multiple ubiquitin-editing regulators to lead to increase the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex in ABC-DLBCL. We further found that miR-17~92 selectively activated IκB-α and NF-κB p65 but not NF-κB p52/p100, and high miR-17~92 expression was also associated with poorer outcome in ABC-DLBCL patients.

Conclusions: Taken together, miR-17~92 selectively activate the canonical NF-κB signaling via targeting ubiquitin-editing regulators to lead to constitutively NF-κB activation and poorer outcome, which is an innovative function of miR-17~92 and previously unappreciated regulatory mechanism of NF-κB activation in ABC-DLBCL. Targeting miR-17~92 may thus provide a novel bio-therapeutic strategy for ABC-DLBCL patients.

Cancer Biology

Diffuse large B-cell lymphoma

miR-17~92

NF-κB

ubiquitin-editing regulator

RIP1

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy, which is classified into germinal center B-cell-like (GCB), activated B-cell-like (ABC) and unclassified DLBCL based on gene expression profiling. They have markedly different clinical features, and ABC-DLBCL has distinctly worse prognosis compared to GCB-DLBCL [1]. It has been reported that constitutive NF-κB activation plays important role in lymphomagenesis of ABC-DLBCL and somatic mutations of genes, including CD79A/B, CARD11, A20, and MYD88, which are characteristically observed in ABC-DLBCL, are directly related to the constitutive NF-κB activation [2]. Nevertheless, close to 50% of ABC-DLBCL do not have these characteristic genetic mutations and mechanisms of NF-κB activation in these tumors are still elusive.

NF-κB activation is critical for B cell development and lymphoid organogenesis, and is mediated by homo- or hetero-dimers of NF-κB family members, including NF-κB1 (p50/p105), NF-κB2 (p52/p100), RelA (p65), RelB and Rel (c-Rel). Signaling pathways mediating NF-κB activation are primarily classified into canonical and non-canonical signaling, which involve the release of different NF-κB dimers from inactive cytoplasmic complexes into the nucleus [3]. Canonical NF-κB signaling relies on the phosphorylation of IκBs, particularly IκB-α, leading to nuclear translocation of various NF-κB dimers, predominantly the p50/p65, however, non-canonical NF-κB signaling leads to nuclear translocation of the p52/RelB dimer through a special mechanism which relies on the inducible procession of p100 instead of IκB-α. Until now, which signaling of them mediates the constitutive NF-κB activation still remains unclear in ABC-DLBCL. In addition, the interactions between microRNAs and NF-κB signaling have been also confirmed, suggesting that dysfunction of these interactions contributes to the development of NF-κB “driven” inflammation and tumors [4, 5]. MicroRNA-17 ~ 92 (miR-17 ~ 92) is an oncomiR cluster consisting of six distinct microRNAs, including miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92, which fall into four microRNA families (miR-17, miR-18, miR-19 and miR-92). MiR-17 ~ 92 is processed from the transcript of MIR17HG (also known as C13orf25), which is frequently amplified in lymphoma, leukemia and some solid tumors [6, 7]. MiR-17 ~ 92 is essential for B-cell development and its absence leads to inhibit B-cell development at pro-B to pre-B transition [8, 9], suggesting an important link between miR-17 ~ 92 and its functions during B-cell lymphopoiesis. Studies have also further shown that miR-17 ~ 92 targets distinct genes, including PHLPP2, PTEN, Bim, CDKN1A/p21, CD22, FCGR2B and Chek2, which contribute to the diverse pathways in different B-cell lymphoma subtypes [10–13]. But its exact function and mechanisms in DLBCL, especially in ABC-DLBCL in which NF-κB signaling is constitutively activated, are still unclear.

Here, we uncovered that miR-17 ~ 92 selectively activated the canonical but not non-canonical NF-κB signaling in ABC-DLBCL through directly targeting multiple ubiquitin-editing proteins, which leaded to regulate the K63- and K48-linked polyubiquitination of receptor-interacting protein 1 (RIP1) complex. Activation of the canonical NF-κB signaling further promoted tumor cell proliferation and chemoresistance as well as poorer survival. Our findings provided the rationale for targeting miR-17 ~ 92 as innovative therapeutics in ABC-DLBCL.

Patients

The diagnosis of two cohorts of 38 non-GCB and 26 ABC-DLBCL patients, who were from the Tianjin Medical University Cancer Institute and Hospital for discovery cohort and University of Nebraska Medical Center for validation cohort, respectively, was confirmed by an expert panel of hematopathologists based on WHO criteria and gene expression profiling. The study was approved by the ethics committee of Tianjin Medical University Cancer Institute and Hospital and University of Nebraska Medical Center. The informed consent was obtained from each participant.

Cell lines and primary normal B cells

The human ABC-DLBCL (U2932, Ly3, TMD8 and Ly10) and HEK293T cell lines were obtained and cultured in their respective medium supplemented with 10%~20% serum, and primary centroblasts (CD77⁺ CD38^hi), as normal B cells, were isolated and purified from fresh tonsils using magnetic microbeads (Miltenyi Biotec Inc., Auburn, CA, USA) as previously described [14]. Details were provided in the Supplementary material.

RNA extraction, RNA-sequencing and microarray analysis

Total RNA was respectively extracted from fresh frozen tissues of 38 non-GCB DLBCL patients using a RNeasy Mini Kit (Qiagen, Hilden, Germany) for RNA-sequencing and 26 ABC–DLBCL patients using mirVana™ miRNA isolation kit (Life Technologies, Grand Island, NY, USA) for microarray analysis of miRNA expression. And then all the total RNA were treated with RNase-free DNase I for 30 min at 37°C to remove residual DNA. Details were described in the Supplementary material.

Plasmids construction and NF-κB activity assay

The Tet-on recombinant plasmids including conditional over-expression of miR-17 ~ 92, which was named as pTIP-miR-17 ~ 92, and conditional loss-of-function of miR-17 ~ 92, which was named as pTRIPZ-miR-17 ~ 92 sponge, were respectively constructed as our previously report [10]. The diagram and map of these plasmids were shown in Supplementary Fig. S1. NF-κB activity was measured using the NF-κB Cignal reporter together with pTRIPZ-miR-17 ~ 92 sponge for transient cell transfection. Further details were provided in the Supplementary material.

Establishment of ABC-DLBCL cell lines with conditional over-expression or loss-of-function of miR-17 ~ 92

Virus was packaged into HEK293T cells and the virus pseudovirus particles were collected to infect the target cells to generate the ABC-DLBCL cells with conditional over-expression or loss-of-function of miR-17 ~ 92, which were further screened with puromycin and sorted by fluorescent expression using a FACSVantage (Bectom Dickinson, USA). Details were described in the Supplementary material.

Wild-type and mutant Luc-Target gene-3’UTR plasmids and luciferase reporter assays

Luc-Target gene-3’UTR plasmids (wild-type, WT) were constructed and the binding site’s seed sequence of miR-17 ~ 92 in Luc-Target genes-3’UTR plasmids were mutated using QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, USA) to generate corresponding mutant plasmids (mutant, Mut). All plasmids were sequenced to confirm their indentity. The primers were listed in Supplementary Table S1. Luciferase reporter assays were used to validate the direct target genes of miR-17 ~ 92. Details were provided in the Supplementary material.

Assays of cell growth, cycle and apoptosis, and real-time quantitative PCR

Cell counting and flow cytometry (Bectom Dickinson, CA, USA) were used to evaluate the growth, cycle and apoptosis. Three major miRNA numbers including miR-17-5p, miR-19b, miR-92a in miR-17 ~ 92 were determined using RT-qPCR, of which the gene-specific primers were purchased from Applied Biosystems (Foster City, USA). Six NF-κB downstream transcriptional target genes, including TNIP1, TNFα, NFKB2, CD83, BIRC3 and IRF1, were also determined using RT-qPCR, of which the primers were listed in Supplementary Table S2. Further details were described in the Supplementary material.

Immunoprecipitation, polyubiquitination of RIP1 complex and immunoblotting

RIP1 complex was immunoprecipitated from stably transduced ABC-DLBCL cell lines and the pull-down was immunoblotted to detect the K63- and K48-linked polyubiquitinations. Proteins were detected by immunoblotting. Details were described in the Supplementary material.

Survival and statistical analysis

Statistical analysis was performed using SPSS (v26.0). Survival analyses were performed using Kaplan-Meier curves and log-rank test. Two-sided P < 0.05 was considered as statistically significant.

MiR-17 ~ 92 enhances the NF-κB signaling in primary Non-GCB DLBCL tumors and ABC-DLBCL cells

DLBCL are subclassified into GCB- and ABC-DLBCL based on gene expression profiling [1]. During routine pathology diagnosis, DLBCL are usually subclassified into GCB- and non-GCB DLBCL according to Hans’ algorithm [15]. To characterize the relationship between miR-17 ~ 92 and NF-κB activity, we performed RNA-sequencing in 38 non-GCB DLBCL patients treated with R-CHOP, and found that miR-17 ~ 92 primary transcript (MIR17HG) was positively associated with the expression of a series of genes (Fig. 1, 0.011 ≤ P ≤ 0.037), which had been confirmed to be NF-κB downstream transcriptional target genes, including TNIP1, TNF-α, NFKB2, CD83, BIRC3, IRF-1, IRF-4, CCR7, NFKB1, CFLAR and TNFAIP2 [16–19]. These results suggested that miR-17 ~ 92 enhanced the NF-κB signaling in primary non-GCB DLBCL tumors.

To further evaluate whether miR-17 ~ 92 activated the NF-κB signaling in cells, we examined the effect of miR-17 ~ 92 on NF-κB activity. As shown in Fig. 2B, the luciferase activity of NF-κB Cignal reporter significantly increased after TNF-α treatment for 18h, and the increase in NF-κB luciferase activity was significantly attenuated (approximately 67.32%) with co-expression of miR-17 ~ 92 sponge, which suppressed miR-17 ~ 92 function (P < 0.01), suggesting miR-17 ~ 92 loss-of-function suppressed NF-κB activity in HEK293T cells. To investigate whether miR-17 ~ 92 regulated NF-κB activity in ABC-DLBCL cells, we examined the expression of miR-17 ~ 92 in them. Supplementary Fig. S2A showed that miR-17 ~ 92 cluster, including miR-17-5p, miR-19b and miR-92a, highly expressed in four ABC-DLBCL cell lines than primary normal B cells. U2932 and Ly3 had higher miR-17 ~ 92 expression, and they were selected to construct the conditional loss-of-function cells, whereas LY10 and TMD8 were selected to construct the conditional over-expression cells. These transduced cells were further confirmed by RT-qPCR (Supplementary Fig. S2B-C) and enhanced RFP or GFP assay (Supplementary Fig. S3). We then determined the expression of six NF-κB downstream transcriptional target genes in these stably transduced ABC-DLBCL cells, and found that the expression of these genes significantly decreased in U2932 and Ly3 cells upon miR-17 ~ 92 loss-of-function. Conversely, the levels of them significantly augmented in Ly10 and TMD8 cells upon miR-17 ~ 92 over-expression (Fig. 2C-D). These findings suggested that miR-17 ~ 92 indeed activated the NF-κB signaling in multiple types of cells, including ABC-DLBCL cells. Taken together, miR-17 ~ 92 enhanced the NF-κB signaling in primary non-GCB DLBCL tumors and ABC-DLBCL cells.

MiR-17 ~ 92 promotes tumor cell proliferation and enhances chemoresistance to NF-κB inhibitor in ABC-DLBCL cells

Next, we constructed the stably transduced cells to investigate the cell functions of ABC-DLBCL, which were induced by miR-17 ~ 92-mediating NF-κB activation. We found that miR-17 ~ 92 over-expression in Ly10 and TMD8 cells promoted cell growth, whereas miR-17 ~ 92 loss-of-function in U2932 and Ly3 cells inhibited cell growth (Fig. 3A). Meanwhile, miR-17 ~ 92 over-expression in LY10 and TMD8 cells promoted G1 to S phase transition; whereas miR-17 ~ 92 loss-of-function in U2932 and Ly3 cells blocked G1 to S phase transition, which resulted in G1 phase retention (Fig. 3B). BMS-345541 (abbreviated as BMS, Calbiochem, San Diego, USA) is a highly selective IKK inhibitor, which inhibits the NF-κB dependent transcription [20]. We also found that BMS treatment inhibited cell growth via blockading NF-κB activity in ABC-DLBCL cells (Fig. 3C, comparing control cells with or without BMS treatment). MiR-17 ~ 92 loss-of-function in U2932 and Ly3 cells enhanced the BMS-induced inhibition, whereas miR-17 ~ 92 over-expression in Ly10 and TMD8 cells partially attenuated the BMS-induced inhibition (Fig. 3C). Moreover, miR-17 ~ 92 loss-of-function sensitized the U2932 and Ly3 cells to apoptosis induced by BMS treatment; conversely, miR-17 ~ 92 over-expression rescued the Ly10 and TMD8 cells, at least partially, from BMS-induced apoptosis (Fig. 3D). Taken together, after augmenting the NF-κB activation, miR-17 ~ 92 promoted the cell proliferation and chemoresistance in ABC-DLBCL cells.

MiR-17 ~ 92 directly targets multiple ubiquitin-editing regulators

To expose the molecular mechanism for miR-17 ~ 92 enhancing NF-κB activity in ABC-DLBCL, we integrated literatures and searched for potential miR-17 ~ 92 target genes in NF-κB pathway through combinatorial prediction using TargetScan 7.0 and miRanda databases (Supplementary Table S3), and found that TNFAIP3 (also known as A20), CYLD, Rnf11 and KDM2A were the top four predicted miR-17 ~ 92 target genes according to the comprehensive scores. TNFAIP3, CYLD and Rnf11 were involved in editing ubiquitin as NF-κB negative regulators, whereas KDM2A was mainly involved in histone demethylation. Herein, the ubiquitin-editing regulators, including TNFAIP3, CYLD and Rnf11, were further validated and investigated in the process of NF-κB activation by miR-17 ~ 92. Integration of highly conserved sites of the 3’UTR of TNFAIP3, CYLD and Rnf11 binding to the seed sequence of miR-17 ~ 92 was listed in Supplementary Fig. S4. We constructed two TNFAIP3-3’UTR luciferase reporter plasmids containing TNFAIP3-3’UTR conserved miR-18a/-19a(b) binding sites, two CYLD-3’UTR luciferase reporter plasmids containing CYLD-3’UTR conserved miR-19a(b) binding sites, and three RNF11-3’UTR luciferase reporter plasmids containing Rnf11-3’UTR conserved miR-19a(b)/-92a binding sites. The corresponding point mutation plasmids of them were also constructed (Supplementary Fig. S5). Luciferase reporter assays revealed that Luc-TNFAIP3-3’ UTR plasmid 2 (P2), which harbored putative binding sites for miR-19a/b (Fig. 4A1), exhibited 38% lower luciferase activity compared to pGL3P vector, but Luc-TNFAIP3-3’ UTR P2 mutant returned to similar luciferase activity to pGL3P vector (Fig. 4A2). MiR-17 ~ 92 over-expression further decreased luciferase activity by 27% in wild-type but not mutant (Fig. 4A3), whereas miR-17 ~ 92 loss-of-function increased luciferase activity by 56% in wild-type but not mutant (Fig. 4A4). These results suggested that TNFAIP3-3’ UTR P2 but not P1 fragment (data not shown) was the direct target region of miR-17 ~ 92. Similarly, we verified that CYLD-3’UTR P1 but not P2 fragment, which harbored putative binding sites for miR-19a/b (Fig. 4B1-4), and RNF11-3’UTR P3 but not P1 and P2 fragments, which harbored putative binding sites for miR-92a (Fig. 4C1-4), were the direct target regions of miR-17 ~ 92. Therefore, our results demonstrated that multiple ubiquitin-editing regulators, including TNFAIP3, CYLD and Rnf11, were the direct targets of miR-17 ~ 92.

MiR-17 ~ 92 down-regulates multiple ubiquitin-editing proteins and activates the canonical NF-κB signaling in ABC-DLBCL cells

We further examined the role for miR-17 ~ 92-mediated ubiquitin-editing protein translations after targeting these genes. Figure 5A displayed that miR-17 ~ 92 loss-of-function up-regulated the levels of ubiquitin-editing proteins, including A20, CYLD and Rnf11, in U2932 and Ly3 cells; whereas miR-17 ~ 92 over-expression down-regulated these protein levels in Ly10 and TMD8 cells. Because these ubiquitin-editing proteins are the NF-κB negative regulators, the NF-κB signaling will be activated after down-regulating these proteins by miR-17 ~ 92. Next, we investigated that which one of canonical and non-canonical NF-κB pathways would be activated by miR-17 ~ 92 in ABC-DLBCL cells. Figure 5B revealed that miR-17 ~ 92 loss-of-function decreased the protein levels of phosphorylated-IκB-α and -p65 in U2932 and Ly3 cells, whereas the level of phosphorylated-p52/p100 was not changed. Conversely, miR-17 ~ 92 over-expression upregulated phosphorylated-IκB-α and -p65 levels in Ly10 and TMD8 cells, but not the level of phosphorylated-p52/p100. These results revealed that miR-17 ~ 92 selectively activated the canonical but not non-canonical NF-κB signaling in ABC-DLBCL.

MiR-17 ~ 92 is reported to also amplify the B-cell receptor (BCR) signaling via inhibiting the ITIM proteins [11]. BCR signaling is one of the NF-κB upstream pathways. We therefore selected the Ibrutinib, which is a highly selective and irreversible Bruton tyrosine kinase (BTK) inhibitor, to block the BCR signaling and further confirm the approach for miR-17 ~ 92 activating the canonical NF-κB signaling in ABC-DLBCL. Supplementary Fig. S6 revealed that Ibrutinib blocked the BCR signaling and inhibited the expression of phosphorylated-IκB-α and -p65, and miR-17 ~ 92 loss-of-function further amplified the inhibition effect in U2932 cells. Conversely, miR-17 ~ 92 over-expression partially reversed the inhibition effect in TMD8 cells. These results suggested that miR-17 ~ 92 selectively activated the canonical NF-κB signaling via targeting ubiquitin-editing proteins, including A20, CYLD and Rnf11, approach but not only BCR signaling in ABC-DLBCL.

MiR-17 ~ 92 regulates the K63-and K48-linked polyubiquitination in ABC-DLBCL cells

Rnf11 and A20 are involved in ubiquitination and interaction with RIP1 [21, 22] and RIP1 polyubiquitination is further involved in NF-κB activity [22–24]. CYLD is an ubiquitin-specific-processing protease according to the UniProt/SwissProt Protein Knowledgebase (http://www.uniprot.org/uniprot/Q9NQC7). We further exposed the ubiquitin-regulated mechanism of miR-17 ~ 92 in the process of the canonical NF-κB activation in ABC-DLBCL cells. Figure 6A showed that K63-linked polyubiquitination of RIP1 complex was up-regulated in a time-dependent manner upon miR-17 ~ 92 over-expression in TMD8 cells. Figure 6B revealed that miR-17 ~ 92 loss-of-function in U2932 cells significantly decreased the K63-linked polyubiquitination and enhanced the K48-linked polyubiquitination. Conversely, miR-17 ~ 92 over-expression in TMD8 cells significantly increased the K63-linked polyubiquitination and attenuated the K48-linked polyubiquitination. Previous report showed that ubiquitin-activating enzyme (E1) inhibitors should, in principle, block all functions of ubiquitination and are valuable tools for studying ubiquitination [25]. After blockading the polyubiquitination using PYR-41, which is the first E1 inhibitor, neither miR-17 ~ 92 loss-of-function nor miR-17 ~ 92 over-expression changed the level of phosphorylated-IκB-α and -p65 in U2932 and TMD8 cells (Supplementary Fig. S7), suggesting that PYR-41 inhibited the effect for miR-17 ~ 92 regulating the polyubiquitination of RIP1 complex in this process of the canonical NF-κB activation in ABC-DLBCL cells. Taken together, miR-17 ~ 92 promoted the formation of K63-linked polyubiquitination and decreased the K48-linked polyubiquitination of RIP1 complex via targeting multiple ubiquitin-editing proteins to activate the canonical NF-κB signaling in ABC-DLBCL cells.

MiR-17 ~ 92 is correlated with poorer outcome in ABC-DLBCL patients

To validate the correlations between miR-17 ~ 92 expression and overall survival, we performed the microarray analysis of miRNA expression in 26 ABC-DLBCL patients treated with R-CHOP and found that patients with high miR-17 ~ 92 expression tended to have poorer overall survival; however, there were too few patients for a statistical significance (Fig. 6C, P > 0.05). Moreover, among 38 non-GCB DLBCL patients studied by RNA-sequencing, 34 completed clinical follow-up (range, 0.5–89.1 months). We found that patients with high miR-17 ~ 92 primary transcript expression had significantly poorer overall survival (Fig. 6D, P = 0.043).

Studies have demonstrated that characteristic “driver” genes, including CD79A/B, CARD11, A20 and MYD88, frequently mutate in approximate 50% ABC-DLBCL patients to further result in the constitutive NF-κB activation [2, 26–28]. Nevertheless, for those ABC-DLBCL patients without characteristic genetic “driver” mutations, mechanisms of NF-κB activation are still elusive. Although a report showed that miR-17 ~ 92 could induce DLBCL through regulating several pathways including BCR signaling, which further activated the NF-κB pathway [29]. However, the BCR signaling is only one of the NF-κB upstream pathways. It remains poorly understood whether miR-17 ~ 92 can activate the NF-κB signaling via other upstream approaches in DLBCL, especially in ABC-DLBCL. Our results demonstrated that miR-17 ~ 92 could activate the NF-κB signaling to lead to constitutively NF-κB activation through directly targeting multiple ubiquitin-editing proteins in ABC-DLBCL, which was different from the BCR approach. Hence, our findings are significant, since it implies that miR-17 ~ 92 is an important and previously unappreciated regulatory mechanism during NF-κB activation for ABC-DLBCL.

In this study, three ubiquitin-editing proteins, including TNFAIP3, CYLD and Rnf11, were preferentially predicted and validated to be the direct target genes of miR-17 ~ 92 in the process of NF-κB activation in ABC-DLBCL. However, whether other regulators, including Tax1bp1, Itch and Traf3 which are not the ubiquitin-editing proteins, are also the direct target genes of miR-17 ~ 92 to contribute to the NF-κB activation in ABC-DLBCL is unclear. It needs to be further validated in future. Besides, the binding of TNFα to TNF receptor can induce the trimerization and then recruits several proteins, including RIPs, TRADD, TRAF2/5, cIAP1/2, NEMO and others, to form complex (herein named RIPs complex). RIPs are the important and true adaptors in NF-κB signaling by interacting with upstream signaling cassettes through well-characterized protein-binding domains, and recruiting IKKα/β through NEMO binding [30]. RIPs consist of seven RIP family members (RIP1 ~ 7), but RIP1 is a key regulator of NF-κB activation [30, 31]. After formation of RIP1 complex, cIAP1/2 and Ubc5 catalyze the RIP1 polyubiquitination. Alternatively, TRAF2 and TRAF5 can also be polyubiquitinated due to their RING domains [32, 33]. In this study, RIP1 antibody was applied to pull down the entire RIP1 complex and then it was confirmed to be polyubiquitinated in this process of NF-κB activation by miR-17 ~ 92 in ABC-DLBCL. It means that any members of RIP1 complex are possible to be polyubiquitinated. It is unclear that which member of RIP1 complex is indeed polyubiquitinated. Further studies using high throughput analysis of post-translational modifications of proteins may help to answer this key question.

In addition, our previous studies using miRNA array analysis demonstrated that miR-17 ~ 92 overexpressed in various B-cell lymphomas, including ABC-DLBCL[14]. In this study, we also found that four ABC-DLBCL cell lines showed high miR-17 ~ 92 level using RT-qPCR method. Another report displayed that amplification of C13orf25 locus on chromosome 13 was frequently detected in GCB-DLBCL but not ABC-DLBCL [34]. Actually expect for gene amplification, miR-17 ~ 92 over-expression is also collaboratively regulated by a number of oncogenic transcription factors and histone modification, including MYC, E2F, Spi-1, Fli-1 and H3K4 trimethylation [7, 35]. Our previous study also revealed that miR-17 ~ 92 over-expression might not correlate well with the C13orf25 gene amplification, in which miR-17 ~ 92 expression was the highest in DHL16 cells, but without C13orf25 amplification based on Chip-chip assay [14]. Therefore, miR-17 ~ 92 over-expression in ABC-DLBCL may be secondary to the activation of c-MYC and/or other oncogenic signaling pathways, since miR-17 ~ 92 is one of the c-MYC downstream targets [36], which is often upregulated in ABC-DLBCL. Otherwise, miR-17 ~ 92 sponge was used as miR-17 ~ 92 competitive inhibitor to investigate the miR-17 ~ 92 loss-of-function in ABC-DLBCL cells in this study. Actually, miRNA sponges have been increasingly applied in miRNA loss-of-function studies due to its high specificity and strong inhibition of target miRNAs, which suppress miRNA targets at least as strongly as chemically modified antisense oligonucleotides [37, 38].

In summary, this study demonstrated that miR-17 ~ 92 selectively activated the canonical but not non-canonical NF-κB signaling via directly targeting multiple ubiquitin-editing proteins, including TNFAIP3, CYLD and Rnf11, leading to increase the K63-linked polyubiquitination and attenuate the K48-linked polyubiquitination of RIP1 complex, and activation of the canonical NF-κB signaling further promoted cell proliferation and enhanced chemoresistance in ABC-DLBCL (Fig. 6E). Our findings imply that targeting miR-17 ~ 92 may provide a novel bio-therapeutic strategy for ABC-DLBCL patients.

DLBCL: Diffuse large B-cell lymphoma; ABC-DLBCL: Activated B-cell like diffuse large B-cell lymphoma; Non-GCB DLBCL: Non-germinal center B-cell-like diffuse large B-cell lymphoma; GCB-DLBCL: Germinal center B-cell-like diffuse large B-cell lymphoma; miRNA or miRs: microRNAs; NF-κB: nuclear factor kappa-B; RIP1: receptor-interacting protein 1; WHO: World Health Organization; RT-qPCR: reverse transcription quantitative polymerase chain reaction;

Acknowledgements

We greatly appreciate Dr. Charles Wood, working in University of Nebraska, for international training help, and Tianjin Marvel Biology Technology Co., Ltd for assistance with sequencing and data handling.

Authors' contributions

Kai Fu, Huilai Zhang and Huaqing Wang conceived and designed research. Xianhuo Wang, Xiaoyan Zhang and Chengfeng Bi performed experiments and interpreted results of experiments. Xianhuo Wang and Xiaoyan Zhang analyzed data and prepared figures. Xianhuo Wang and Xin Huang performed statistical and bioinformatics analysis. Wing C. Chan and Julie M. Vose provided material support. Timothy W. McKeithan and Bin Meng provided suggestions and reviewed pathology. Xianhuo Wang and Kai Fu drafted paper, edited and revised manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by the National Institutes of Health [grant numbers D43 TW001429].

Availability of data and materials

The datasets generated and/or analyzed during the present study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The study was approved by the ethics committee of Tianjin Medical University Cancer Institute and Hospital and University of Nebraska Medical Center. The informed consent was obtained from each participant.

Competing interests

The author declares no competing interest exists.

Consent for publication

Not applicable.

Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000; 403(6769): 503-11.
Compagno M, Lim WK, Grunn A, Nandula SV, Brahmachary M, Shen Q, et al. Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma. Nature. 2009; 459(7247):717-21.
Taniguchi K, Karin M. NF-κB, inflammation, immunity and cancer: coming of age. Nat Rev Immunol. 2018; 18(5): 309-24.
Tong L, Yuan Y, Wu S. Therapeutic microRNAs targeting the NF-kappa B signaling circuits of cancers. Adv Drug Deliv Rev. 2015; 81:1-15.
Wu J, Ding J, Yang J, Guo X, Zheng Y. MicroRNA roles in the nuclear factor kappa B signaling pathway in cancer. Front Immunol. 2018; 9:546.
Ota A, Tagawa H, Karnan S, Tsuzuki S, Karpas A, Kira S, et al. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res. 2004; 64(9): 3087-95.
Lambo S, Gröbner SN, Rausch T, Waszak SM, Schmidt C, Gorthi A, et al. The molecular landscape of ETMR at diagnosis and relapse. Nature. 2019; 576(7786): 274-80.
Ventura A, Young AG, Winslow MM, Lintault L, Meissner A, Erkeland SJ, et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell. 2008; 132(5):875-86.
Wu Y, Schutt S, Paz K, Zhang M, Flynn RP, Bastian D, et al. MicroRNA-17-92 is required for T-cell and B-cell pathogenicity in chronic graft-versus-host disease in mice. Blood. 2018; 131(17): 1974-86.
Rao E, Jiang C, Ji M, Huang X, Iqbal J, Lenz G, et al. The miRNA-17∼92 cluster mediates chemoresistance and enhances tumor growth in mantle cell lymphoma via PI3K/AKT pathway activation. Leukemia. 2012; 26(5):1064-72.
Inomata M, Tagawa H, Guo YM, Kameoka Y, Takahashi N, Sawada K. MicroRNA-17-92 down-regulates expression of distinct targets in different B-cell lymphoma subtypes. Blood. 2009; 113(2): 396-402.
Psathas JN, Doonan PJ, Raman P, Freedman BD, Minn AJ, Thomas-Tikhonenko A. The Myc-miR-17-92 axis amplifies B-cell receptor signaling via inhibition of ITIM proteins: a novel lymphomagenic feed-forward loop. Blood. 2013; 122(26): 4220-9.
Mihailovich M, Bremang M, Spadotto V, Musiani D, Vitale E, Varano G, et al. miR-17-92 fine-tunes MYC expression and function to ensure optimal B cell lymphoma growth. Nat Commun. 2015; 6: 8725.
Ji M, Rao E, Ramachandrareddy H, Shen Y, Jiang C, Chen J, et al. The miR-17-92 microRNA cluster is regulated by multiple mechanisms in B-cell malignancies. Am J Pathol. 2011; 179:1645-56.
Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004; 103: 275-82.
Kim SW, Ramasamy K, Bouamar H, Lin AP, Jiang D, Aguiar RC. MicroRNAs miR-125a and miR-125b constitutively activate the NF-κB pathway by targeting the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20). Proc Natl Acad Sci U S A. 2012; 109:7865-70.
Boddicker RL, Kip NS, Xing X, Zeng Y, Yang ZZ, Lee JH, et al. The oncogenic transcription factor IRF4 is regulated by a novel CD30/NF-κB positive feedback loop in peripheral T-cell lymphoma. Blood. 2015; 125:3118-27.
Mburu YK, Egloff AM, Walker WH, Wang L, Seethala RR, van Waes C, et al. Chemokine receptor 7 (CCR7) gene expression is regulated by NF-κB and activator protein 1 (AP1) in metastatic squamous cell carcinoma of head and neck (SCCHN). J Biol Chem. 2012; 287: 3581-90.
Chen CC, Liu HP, Chao M, Liang Y, Tsang NM, Huang HY, et al. NF-κB-mediated transcriptional upregulation of TNFAIP2 by the Epstein-Barr virus oncoprotein, LMP1, promotes cell motility in nasopharyngeal carcinoma. Oncogene. 2014; 33:3648-59.
Burke JR, Pattoli MA, Gregor KR, Brassil PJ, MacMaster JF, McIntyre KW, et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J Biol Chem. 2003; 278: 1450-6.
Connor MK, Seth A. A central role for the ring finger protein RNF11 in ubiquitin-mediated proteolysis via interactions with E2s and E3s. Oncogene. 2004; 23: 2089-95.
Shembade N, Parvatiyar K, Harhaj NS, Harhaj EW. The ubiquitin-editing enzyme A20 requires RNF11 to down-regulate NF-kappaB signalling. EMBO J. 2009; 28:513-22.
Ea CK, Deng L, Xia ZP, Pineda G, Chen ZJ. Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol Cell. 2006; 22:245-57.
Wertz IE, O'Rourke KM, Zhou H, Eby M, Aravind L, Seshagiri S, et al. De-ubiquitination and ubiquitin ligase domains of A20 down-regulate NF-kappaB signalling. Nature. 2004; 430: 694-9.
Yang Y, Kitagaki J, Dai RM, Tsai YC, Lorick KL, Ludwig RL, et al. Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res. 2007; 67: 9472-81.
Davis RE, Ngo VN, Lenz G, Tolar P, Young RM, Romesser PB, et al. Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature. 2010; 463: 88-92.
Ngo VN, Young RM, Schmitz R, Jhavar S, Xiao W, Lim KH, et al. Oncogenically active MYD88 mutations in human lymphoma. Nature. 2011; 470: 115-9.
Lim KH, Yang Y, Staudt LM. Pathogenetic importance and therapeutic implications of NF-κB in lymphoid malignancies. Immunol Rev. 2012; 246: 359-78.
Sandhu SK, Fassan M, Volinia S, Lovat F, Balatti V, Pekarsky Y, et al. B-cell malignancies in microRNA Eμ-miR-17~92 transgenic mice. Proc Natl Acad Sci U S A. 2013; 110: 18208-13.
Hayden MS, Ghosh S. Shared principles in NF-kB signaling. Cell. 2008; 132: 344-62.
Berger SB, Kasparcova V, Hoffman S, Swift B, Dare L, Schaeffer M, et al. Cutting Edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice. J Immunol. 2014; 192: 5476-80.
Park ES, Choi S, Shin B, Yu J, Yu J, Hwang JM, et al. Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) negatively regulates the TRAF2 ubiquitin-dependent pathway by suppressing the TRAF2-sphingosine 1-phosphate (S1P) interaction. J Biol Chem. 2015; 290: 9660-73.
Tao T, Cheng C, Ji Y, Xu G, Zhang J, Zhang L, et al. Numbl inhibits glioma cell migration and invasion by suppressing TRAF5-mediated NF-κB activation. Mol Biol Cell. 2012; 23: 2635-44.
Lenz G, Wright GW, Emre NC, Kohlhammer H, Dave SS, Davis RE, et al. Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways. Proc Natl Acad Sci U S A. 2008; 105:13520-5.
Mendell JT. miRiad roles for the miR-17-92 cluster in development and disease. Cell. 2008; 133: 217-22.
O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature. 2005; 435: 839-43.
Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007; 4:721-6.
Barta T, Peskova L, Hampl A. MiRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico. Sci Rep. 2016; 6:36625.

Download PDF

Version 1

posted

You are reading this latest preprint version

miR-17~92 Promotes Progression of ABC-DLBCL Lymphoma via Regulation of Canonical NF-kB Signaling

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Patients

Cell lines and primary normal B cells

RNA extraction, RNA-sequencing and microarray analysis

Plasmids construction and NF-κB activity assay

Establishment of ABC-DLBCL cell lines with conditional over-expression or loss-of-function of miR-17 ~ 92

Wild-type and mutant Luc-Target gene-3’UTR plasmids and luciferase reporter assays

Assays of cell growth, cycle and apoptosis, and real-time quantitative PCR

Immunoprecipitation, polyubiquitination of RIP1 complex and immunoblotting

Survival and statistical analysis

Results

MiR-17 ~ 92 enhances the NF-κB signaling in primary Non-GCB DLBCL tumors and ABC-DLBCL cells

MiR-17 ~ 92 promotes tumor cell proliferation and enhances chemoresistance to NF-κB inhibitor in ABC-DLBCL cells

MiR-17 ~ 92 directly targets multiple ubiquitin-editing regulators

MiR-17 ~ 92 down-regulates multiple ubiquitin-editing proteins and activates the canonical NF-κB signaling in ABC-DLBCL cells

MiR-17 ~ 92 regulates the K63-and K48-linked polyubiquitination in ABC-DLBCL cells

MiR-17 ~ 92 is correlated with poorer outcome in ABC-DLBCL patients

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1