miR-181d/RBP2/NF-κB p65 Feedback Loop Promotes Chronic Myeloid Leukemia Blast Crisis

Minran Zhou Qilu Hospital of Shandong University Xiaolin Yin Qilu Hospital of Shandong University Lixin Zheng Shandong University Yue Fu Qilu Hospital of Shandong University Yue Wang Qilu Hospital of Shandong University Zelong Cui Qilu Hospital of Shandong University Zhenxing Gao Qilu Hospital of Shandong University Xiaoming Wang Qilu Hospital of Shandong University Tao Huang Qilu Hospital of Shandong University Jihui Jia Shandong University Chunyan Chen (  chency@sdu.edu.cn ) Qilu Hospital of Shandong University https://orcid.org/0000-0002-5919-7624


Background
Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease that is originated from hematopoietic stem cells and characterized by the BCR-ABL fusion gene [1]. The natural course of the disease includes a chronic phase (CP), an accelerated phase (AP) and a blast phase (BP). In the chronic phase of CML (CML-CP), the leukemia cells still retain a certain ability to differentiate into mature cells; while in blast phase of CML (CML-BP), a large number of invasive primitive and immature cells accumulate [2]. At present, CML-CP patients have a good response to tyrosine kinase inhibitors (TKIs), and most of them survive for a long time. However, some patients are still insensitive to TKIs or develop drug resistance, leading to an accelerated CML phase and/or blast phase. Once they progress into CML-BP, the curative effect is poor, and the fatality rate is extremely high. Besides, the etiology of CML malignant transformation is complex and highly heterogeneous [3]. Therefore, it is becoming urgent to explore the molecular mechanisms of blast crisis transition and develop new therapeutic targets.
Epigenetics mean that the gene expression undergoes heritable changes without changes in the gene DNA sequence [4]. Epigenetic modi cations include DNA methylation, histone modi cation and noncoding RNA regulation, which affect cell proliferation, differentiation and apoptosis [5,6]. MicroRNAs (miRNAs) are a class of small non-coding RNAs, which negatively or positively regulate genes expression, by directly binding to the 3'untranslated regions (3'UTR) of the target genes mRNAs [7]. They are also vital players in the development of leukemia. A recent study showed that miR-150 expression, was downregulated in the transition period between the CML chronic phase and the blast crisis [8]. Moreover, the expression of miR-328 was lost in blast crisis of CML and its overexpression could promote cell differentiation and inhibit cell proliferation, by inducing the survival factor PIM1 and suppressing the binding between the translational regulator poly(rC)-binding protein hnRNP E2 and CEBPA [9]. Besides, miR-223 which forms a feedback loop with the cell cycle regulator gene E2F1 was found to be underexpressed in acute myeloid leukemia (AML) [10]. MiR-29 was downregulated in KIT-related AML.
The mutual regulation of miR-29 and SP1/ NF-κB/HDAC forms a feedback regulation network, which mediates the pathogenesis of KIT-related AML [11]. MiR-181 family was overexpressed in AML, which blocked cell differentiation by inhibiting the expression of PRKCD, CTDSPL and CAMKK1 [12]. However, whether miR-181d misregulation is involved in CML progression, is unknown.
The misregulation of histone-modifying enzymes, including methyltransferases and demethylases, mediates tumorigenesis. Recent studies have found that histone methyltransferase is involved in the pathogenesis of CML blast crisis. For instance, the retinoblastoma-interacting zinc-nger protein 1 (RIZ1) and H3K9 histone methyltransferase, are downregulated in CML-BP. The overexpression of RIZ1 can inhibit cell proliferation, induce apoptosis and promote differentiation by inhibiting the IGF-1 signaling pathway [13,14]. The expression of BMI1, that belongs to the Polycomb-group of proteins, gradually increased in CML progression, which makes this protein an effective marker of CML-BP [15]. We have found that the histone demethylase RBP2 mediates the blast crisis of CML through a negative regulation of miR-21 [16]. However, the mechanism of RBP2 underexpression in CML-BP, has not yet been studied.
The Nuclear Factor-kappa B (NF-κB) family plays an important role in CML progression by regulating cell proliferation and apoptosis and there is an NF-κB/TNF-α feedback loop, in leukemic primary cells, which promotes leukemia progression [17]. The inhibitors of NF-κB signaling pathway PS-1145 and AS602868, signi cantly induced the apoptosis of CML primary cells [18,19]. Parthenolide, that inhibits NF-κB transcription, greatly promoted the apoptosis of leukemia cells in the blast crisis phase [20]. Furthermore, recent studies showed that there is an interaction between NF-κB and histone-modifying enzymes, which are involved in the development of in ammation and tumors, such as JMJD3, FBXL11 and JMJD2B [21][22][23]. However, whether RBP2 could regulate NF-κB expression, remains largely unknown.
In this study, we aimed to de ne whether miR-181d could mediate CML progression through a miR-181d/RBP2/NF-κB p65 feedback loop. In CML, miR-181d overexpression repressed RBP2 expression, which resulted in p65 overexpression. Moreover, high p65 expression reversely upregulated the level of mature miR-181d, which formed a feedback loop and promoted CML blast crisis transition.

Patients and bone marrow samples
Patients' bone marrow samples were collected between July 2010 and June 2018, from the Department of Hematology, Qilu Hospital of Shandong University, Jinan, China. The patients were newly diagnosed CML-CP (n=42) and CML-BP (n=15). Mononuclear cells were isolated from the samples by Ficoll-Hypaque density-gradient centrifugation and stored at -80°C. The study was approved by the Ethics Committee of Qilu Hospital of Shandong University, and also accorded with the Helsinki Declaration of 1975, as revised in 1983.

Cell lines and cell culture
The human cell line K562, HL60 and HEK-293 were cultured at 37 °C, 95% air and 5% CO2 in RPMI 1640, containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and without antibiotics. The cells were cultured on 6,12 and24-well plates for 18 to 24 h before the start of the experiments.

RNA extraction and qRT-PCR
Total RNA was extracted from the human bone-marrow samples and the cells using different treatments of Trizol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, the extracted RNA was reverse transcribed using PrimeScript RT reagent Kit using the gDNA Eraser (Takara, Japan). The cDNAs were then subjected to SYBR Green-based real-time PCR analysis. RBP2 and p65 mRNA levels were normalized to that of the human β-actin. The mRNA level of the mature miR-181d was normalized to that of U6. The probes for RBP2 used were Hs00231908_m1 (Applied Biosystems). The primers for miR-181d and U6 were MQP-0101 and MQP-0201 (Ribobio). The other primers used in qRT-PCR assays are listed in Table 2 and the expression was calculated by the 2 -ΔΔCt method.
Subsequently, immunoblots were probed with ECL detection reagent (Millipore) and according to standard protocols.

Immunostaining
The mononuclear cells, that were isolated from patient bone-marrow samples, were used to prepare cytospins with glass slides xed by a polyformaldehyde xation solution. The samples were stained with anti-RBP2 antibody (1:150, Abcam) and anti-p65 antibody (1:150, Abcam) overnight at 4°C, followed by an incubation with a horseradish peroxidase-conjugated secondary antibody for 30 min.

Immunohistochemistry
Paraffin embedded slides were deparaffinized, rehydrated and subjected to antigen-retrieval using citric acid buffer. The endogenous peroxidase was deactivated by H2O2. The slides were blocked using a 10% goat serum solution and incubated with the corresponding primary antibodies overnight at 4°C. The used antibodies were: anti-RBP2 antibody (1:150, Abcam), anti-p65 antibody (1:150, Abcam) and anti-Ki67 antibody (1:100, Abcam). Next, the slides were incubated with a secondary antibody, followed by a colorimetric detection using a DAB staining kit (Vector Laboratories, USA).
The treated cells were incubated with EDU for two hours before uorescence detection. Then, the cells were smeared on glass slides, xed with 4% paraformaldehyde for 30 minutes and then stained using a Cell-Light™ EDU Apollo®488 In Vitro Imaging Kit (RioBio, China), and following the manufacturer's instructions. The slides were examined by confocal laser scanning microscopy.

Chromatin immunoprecipitation (ChIP) assay
For the ChIP assay, the Cell Signaling ChIP assay protocol was used. The precipitated DNA samples were detected by PCR. The PCR primers for p65 and miR-181d promoters are listed in Table 2. 2.10 Luciferase reporter assay MiR-181d mimics/inhibitor (Ribobio, Guangzhou, China), RBP2 wild-type/mutant 3'UTR and the internal control vector TK plasmids were transfected into HL60 and HEK-293 cells. The plasmids containing RBP2 wild-type, RBP2-mutant, defective in demethylase activity (RBP2 H483A), p65 promoter wild or binding site mutant plasmids, and the internal control vector TK plasmid, were transfected into HL60 and HEK-293 cells. The p65 expression plasmid/siRNA, miR-181d promoter wild, or binding site mutant plasmids, and the internal control vector TK plasmid were transfected into HL60 and HEK-293 cells. After 24 or 48 h of incubation, luciferase activity was measured using a Luciferase Assay System (Promega, Madison, WI, USA) and according to the manufacturer's protocol. were treated with 2Gy dose of radiation. After 24 hours, 1× 10 6 K562 cells were subcutaneously injected into the right or left ank of the mice. Tumor growth was monitored every 3 days. From the seventh day, miR-181d antagomir (miR30002821-4-5) or control, were injected into the tumor every 3 days. The total period lasted 16 days. All animal procedures were approved by Qilu Hospital of Shandong University

Tumor xenograft model
Research Ethics Committee. The animal study also accorded with the ARRIVE guidelines [24].

Statistical analysis
All experiments were repeated at least three times. The data were expressed as mean ± standard deviation. Student's t-test was used to compare the means between the two groups using the GraphPad Prism for Windows, version 5.00 (GraphPad Software, La Jolla, CA, USA). The p-values of < 0.05 were considered statistically signi cant.

miR-181d is overexpressed in CML-BP and promotes leukemia cell proliferation
To examine whether miR-181d is critical in CML progression, we measured the expression level of mature miR-181d in bone-marrow samples of newly diagnosed CML-CP patients (n = 42) and CML-BP patients (n = 15). The relative clinical characteristics of these patients are shown in Table 1. The mature miR-181d level was higher in the CML-BP samples than in the CML-CP samples (Fig. 1A). Therefore, miR-181d is overexpressed during CML progression. Furthermore, we explored the potential role of miR-181d in leukemia cell proliferation by transfecting miR-181d mimics or inhibitor into K562 and HL60 cells. The cells transfected with miR-181d mimics proliferated at a higher rate compared to the NC mimics transfection, and the cells transfected with miR-181d inhibitor proliferated at a slower rate (Figs. 1B, C).

miR-181d directly targets the 3'UTR of histone demethylase RBP2
To investigate the mechanisms by which miR-181d promotes cell proliferation, we investigated the potential role of miR-181d in the regulation of RBP2 expression by modulating miR-181d expression levels via transfecting miR-181d mimics or inhibitor into K562 and HL60 cells. RBP2 mRNA and protein levels were signi cantly decreased in K562 and HL60 cells overexpressing miR-181d when compared to control cells ( Fig. 2A and B). On the other hand, miR-181d inhibition in K562 and HL60 cells, resulted in RBP2 increased mRNA and protein levels ( Fig. 2A and B). Using the target prediction programs miRanda and TargetScan, we found that the 3'UTR of RBP2 mRNA contains a conserved miR-181d binding site (Fig. 2C). To verify this nding, the RBP2 3'UTR, containing the putative miR-181d binding site, and its mutant 3'UTR (with mutated miR-181d binding site) were cloned downstream of the luciferase open reading frame. These luciferase reporter constructs were co-transfected into HL60 and HEK-293 cells with miR-181d mimics or inhibitor. The luciferase activity of RBP2 3'UTR was repressed by the miR-181d mimics transfection and this repression was abrogated when miR-181d binding site was. Oppositely, the miR-181d inhibitor upregulated the luciferase activity of RBP2 3'UTR and this upregulation was also abrogated when miR-181d binding site was mutated (Fig. 2D-G). These data demonstrated that RBP2 was a direct target of miR-181d.

RBP2 directly targets p65 promoter and inhibits its expression in histone demethylase dependent manner
Several studies have shown that p65 is pivotal in promoting cell proliferation and our previous study con rmed that RBP2 mediates CML blast crisis. However, it is not clear whether p65 is involved in this pathogenesis. By transfecting RBP2 wild-type or RBP2-mutant (defective in demethylase activity, RBP2 H483A) plasmids or the control plasmid into K562 and HL60 cells, we compared p65 expression in differently treated cells. We found that RBP2 overexpression, signi cantly downregulated p65 mRNA and protein levels (Fig. 3A, B and C). However, RBP2 H483A plasmid, which also overexpresses RBP2 but without demethylase activity, could not inhibit p65 expression ( Fig. 3B and C). These results suggest that RBP2 negatively regulates p65 expression and this was dependent on its enzyme activity. Furthermore, to determine whether p65 is a direct target of RBP2, we identi ed two potential RBP2 binding sites in the promoter of p65 (Fig. 3D). We constructed two p65 promoter plasmids, that included the rst RBP2 binding site (p65 pro1) and the second RBP2 binding site (p65 pro2), and which were cotransfected into the HL60 and HEK-293 cells with the RBP2 plasmid. The promoter activity of p65 pro1 was signi cantly decreased after RBP2 overexpression in HL60 and HEK-293 cells, while no signi cant change was observed in p65 pro2 promoter activity ( Fig. 3E and F). Moreover, accompanied by the mutation of the binding site in p65 pro1, the promoter activity of p65 pro1-mut was unchanged after RBP2 overexpression in HL60 and HEK-293 cells (Fig. 3G, H). To determine the association between RBP2 and p65 promoter, a chromatin immunoprecipitation assay was performed, and which showed that RBP2 bound to the promoter region of the p65 promoter in K562 and HL60 cells, (Fig. 3I and J). Furthermore, in K562 and HL60 cells transfected with the RBP2 plasmid, RBP2 overexpression increased its association with p65 promoter sequences ( Fig. 3K and L). Consistent with its demethylase activity, which is speci c for tri-and dimethylated lysine 4 on histone 3, RBP2 overexpression also remarkably reduced H3K4 tri/dimethylation at the promoter region of p65 ( Fig. 3K and L). Therefore, the results demonstrate that RBP2 reduces H3K4 tri/dimethylation by its binding to the p65 promoter region, which negatively regulates its expression.

Ectopic expression of p65 in CML-BP enhances leukemia cell proliferation
To determine whether p65 is critical player in CML progression, we measured p65 expression in bonemarrow samples from patients with CML-CP and CML-BP. Compared with CML-CP samples, p65 mRNA and protein levels were signi cantly higher in the CML-BP samples (Fig. 4A, B).
Moreover, to explore the potential mechanism by which miR-181d promotes CML progression, the p65 plasmids or siRNA were transfected into K562 and HL60 cells. The cells transfected with p65 plasmid proliferated at a higher rate compared to the control plasmid; while the cells transfected with p65 siRNA proliferated at a lower rate (Fig. 4C, D). These data indicate that ectopic p65 expression in CML-BP cells enhances leukemia cell proliferation.

p65 directly modulates miR-181d expression, which forms a positive feedback loop
It is worth mentioning that we found miR-181d to be positively regulated by p65 (Fig. 5A-C). To explore this mechanism, we found that there were two classical binding sites of p65 in the miR-181d promoter (Fig. 5D). To investigate whether miR-181d is a direct target of p65, luciferase reporters' plasmids were constructed and which included the two potential binding sites (miR-181d pro1 and pro2), respectively.
The overexpression of p65 signi cantly increased the luciferase activity of miR-181d pro1; while p65 siRNA greatly inhibited miR-181d pro1 luciferase activity (Fig. 5E-H). However, the p65 plasmid and siRNA did not affect the luciferase activity of miR-181d pro2 (Fig. 5E-H). Furthermore, we constructed a mutant luciferase reporter for miR-181d pro1 (miR-181d pro1-mut) and observed no changes in the promoter activity of miR-181d pro1-mut when p65 was overexpressed or inhibited ( Fig. 5I-L). Besides, to explore the association between p65 and the miR-181d promoter, we applied a series of ChIP assays and we found that p65 bound to the promoter region of the miR-181d promoter in K562 and HL60 cells (Fig. 5M and N). Taken together, the above results show that p65 directly targets miR-181d and forms a positive feedback loop to promote the transition of CML blast crisis.

miR-181d inhibition suppresses leukemia cell proliferation in vivo
We further examined the oncogenic role of miR-181d in CML blast crisis in vivo. When the tumor diameters reached 8-9 mm, miR-181d antagomir was injected into the tumor every 3 days, and then the mice were sacri ced on the sixteenth day (Fig. 6A). The tumors, that were treated with miR-181d antagomir, were smaller compared to the control tumors treated, both in size and weight (Fig. 6B-D). It was proved that miR-181d antagomir signi cantly inhibited miR-181d expression in vivo (Fig. 6E). Besides, IHC con rmed that RBP2 levels were increased and that p65 levels were decreased in tumors treated with miR-181d antagomir (Fig. 6F, G, H). Ki67 staining con rmed the in vivo tumor growth results (Fig. 6F, I). Overall, these data indicate, that the inhibition of miR-181d, suppresses leukemia cell proliferation in vivo, and that miR-181d might be an effective target for inhibiting CML blast transformation.

Discussion
Recent studies have demonstrated that epigenetic misregulation plays vital roles in leukemia. The ectopic expression of miR-181 family blocked cell differentiation by inhibiting the expression of PRKCD, CTDSPL and CAMKK1, which promoted AML pathogenesis [12]. MiR-181a-1/b-1 deletion in mice, inhibited the development of T-cell acute lymphoblastic leukemia, which was induced by Notch1 [25]. Nonetheless, whether miR-181d plays important roles in the CML blast transformation have not been clearly identi ed.
In this study, we demonstrated that miR-181d mediates CML malignant transformation and that it is overexpressed in bone-marrow samples of CML-BP. Following this, we determined that miR-181d overexpression signi cantly promoted leukemia cell proliferation, and its greatly suppressed cell proliferation. The possible mechanism by which miR-181d regulates cell proliferation was reported to be associated with the regulation of KNAIN2, CDKN3 and CYLD [26][27][28]. In this study, we revealed a new mechanism and showed that miR-181d promotes leukemia cell proliferation by directly targeting and negatively regulating the histone demethylase RBP2.
The retinoblastoma binding protein 2 (RBP2) is a member of the JARID family of proteins, which has a histone demethylase activity by speci cally demethylating tri-and di-methylated lysine 4 of histone 3 (H3K4) [29][30][31]. Our previous study showed that a low RBP2 expression could not repress miR-21 expression, which promoted the transition of CML from CP to BP [16]. In this study, the results characterized the mechanism by which RBP2 was underexpressed in CML blast crisis, which further revealed the importance of epigenetic misregulation in CML blast transformation.
The AT-rich interaction domain of RBP2 can recognize a speci c DNA sequence CCGCCC [32] that is contained in the promoter region of p65. We found that RBP2 directly and negatively regulates p65 expression by binding to its promoter, which depended on its enzyme activity. RBP2 overexpression signi cantly downregulated p65 mRNA and protein levels. However, RBP2 H483A plasmid, which is also overexpress RBP2, but without demethylase activity, could not inhibit p65 expression. Furthermore, Luciferase reporter assay and ChIP assay showed that RBP2 directly binds to p65 promoter.
Recent studies have shown that miRNAs play important roles in in ammation and cancer by regulating the NF-κB pathway [33][34][35][36]. Nevertheless, whether miRNAs could be reversely regulated by NF-κB is largely unknown. Here, we found that NF-κB/p65 upregulates the expression of miR-181d and forms a feedback loop. Mechanistically, miR-181d proximal promoter contains the classical binding sites of NF-κB/p65 (GGGRNNYYCC R-purine, Y-pyrimidine, N-arbitrary base) [37]. The overexpression of p65 signi cantly upregulated miR-181d expression and activated its promoter activity; while p65 inhibition greatly downregulated miR-181d expression and suppressed miR-181d promoter activity. However, when the binding site was mutated, the corresponding activation, or inhibition disappeared. Furthermore, the ChIP assay showed that p65 directly binds to the promoter sequence of miR-181d.
In summary, we provide evidence for the existence of a new epigenetic mechanism that is involved in CML blast transformation (Fig. 6J).

Conclusions
Taken together, the non-coding RNA miR-181d is overexpressed in CML-BP, which promotes leukemia cell proliferation. Mechanismally, miR-181d downregulated the level of histone demethylase RBP2, which inhibited p65 expression in leukemia cells by its binding to the p65 promoter and demethylating the tri/dimethylated H3K4 region in the p65 promoter locus. Conversely, p65 upregulated the level of mature miR-181d by directly binding to its promoter. These ndings might point to a way to build new diagnostic markers for CML blast crisis.

Declarations
Ethics approval and consent to participate Informed consent was obtained from all participants that were included in the study. The animal study was approved by Qilu Hospital of Shandong University Research Ethics Committee.

Consent for publication
Each person provided signed informed consent for publication of the results of the study.

Availability of data and materials
All data generated or analyzed during this study were included in this published article and its additional le.       wild-type promoter by p65, including the rst p65 binding site (miR-181d pro1) and the second p65