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 . During routine pathology diagnosis, DLBCL are usually subclassified into GCB- and non-GCB DLBCL according to Hans’ algorithm . 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 . 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 . 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 . 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).