DNA methylation‐mediated silencing of microRNA‐204 enhances T cell acute lymphoblastic leukemia by up‐regulating MMP‐2 and MMP‐9 via NF‐κB

Abstract T cell acute lymphoblastic leukaemia (T‐ALL) is a highly aggressive haematological cancer of the bone marrow. The abnormal expression of microRNAs (miRNAs) is reportedly involved in T‐ALL development and progression. Thus, we aimed to decipher the involvement of miR‐204 silencing mediated by DNA methylation in the occurrence of T cell acute lymphoblastic leukaemia (T‐ALL). miR‐204 expression was determined in bone marrow and peripheral blood samples from T‐ALL patients by real‐time quantitative PCR (RT‐qPCR) with its effect on cell proliferation evaluated by functional assays. In addition, bisulphite sequencing PCR was employed to detect the DNA methylation level of the miR‐204 promoter region, and the binding site between miR‐204 and IRAK1 was detected by luciferase assay. We found that miR‐204 was down‐regulated in T cells of T‐ALL patients, which was caused by the increased DNA methylation in the promoter region of miR‐204. Moreover, overexpression of miR‐204 inhibited T‐ALL cell proliferation while enhancing their apoptosis through interleukin receptor‐associated kinase 1 (IRAK1), which enhanced the expression of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 through activation of p‐p65. Thus, miR‐204 modulated MMP‐2 and MMP‐9 through IRAK1/NF‐κB signalling pathway, which was confirmed by in vivo assay. Taken together, DNA methylation‐mediated miR‐204 silencing increased the transcription of IRAK1, thus activating the NF‐κB signalling pathway and up‐regulating the downstream targets MMP‐2/MMP‐9.


| INTRODUC TI ON
T cell acute lymphoblastic leukaemia (T-ALL) is a highly aggressive haematological malignancy caused by the overproduction of T cells. T-ALL contributes to 15% of ALL cases in children and 25% of those in adults, and its overall survival rate is quite low, being only 60%-70% in children and 30%-40% in adults. [1][2][3] Although recent papers have reported the potential molecular mechanisms leading to T-ALL, 4,5 sufficient understanding of the metastatic mechanisms of T-ALL is still lacking and in need for further investigation.
Increasing evidence suggests that methylation of microRNAs (miRNAs) and activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathway are involved in T-ALL development and progression. 6 Indeed, NF-ĸB signalling is an important pathway in T-ALL cells, whereby sirtuin1 can physically interact with and deacetylate the NF-ĸB subunit of p65/ RELA at lysine 310 residue, thus resulting in transcriptional inhibition. 7 Moreover, NF-κB which is recognized as an important factor in normal inflammatory, immune and carcinogenesis, is found to be activated in T-ALL. 8 Importantly, activation of NF-κB in cancer-associated leucocytes drives cytokine secretion that facilitates metastasis via activation of the C-X-C chemokine receptor type 7 (CXCR7). 9 Yang et al revealed that miR-101 reduced T-ALL cell proliferation and invasion by targeting CXCR7/signal transducer and the activator of transcription 3 (STAT3) signalling pathway. 10 Previous research has indicated that miR-204 expression is down-regulated in T-ALL, 11 and that the cancer development and progression is promoted by miR-204 silencing due to DNA methylation of the miR-204 promoter. 12 Interleukin-1 receptor-associated kinase (IRAK) contributes significantly in the pathogenesis of inflammatory autoimmune disorders, 13 and T-ALL cells express elevated levels of IRAK1 mRNA, as well as increased proportions of activated IRAK1. 14 Bioinformatics tools predicted that IRAK1 was a possible target of miR-204. It has previously been reported that IRAK1 was an upstream signalling component of the NF-κB signalling pathway. 15 Of note, previous research has shown that NF-κB was overexpressed in T-ALL cells, indicating that NF-κB signalling pathway may be potentially involved in the molecular mechanism of T-ALL. 16 However, the specific mechanisms underlying DNA methylation of miR-204 promoter in T-ALL remain to be identified.
In this study, we measured the expression of miR-204 and the methylation level of its promoter region in specimens from T-ALL patients and in T-ALL cell lines, and studied the effects of the downstream regulatory gene IRAK1 in changing the expression of NF-κB and ultimately promoting proliferation and metastasis of T-ALL cells.
In so doing, we test the hypothesis that miR-204 silencing mediated by DNA methylation regulates the IRAK1/NF-κB signalling in the development of T-ALL.

| Ethics statement
This study has been reviewed and approved by the Medical Ethics

| Clinical tissue samples
Peripheral blood and bone marrow samples were collected from 16 healthy volunteers (the normal group) and 32 patients who were diagnosed as T-ALL (the T-ALL group) at Zhangzhou Affiliated Hospital of Fujian Medical University from 2012 to 2016. The patients had received no clinical treatment prior to sampling. The CD3 + kit (Invitrogen, Carlsbad, CA, USA) and CD2 + kit (STEMCELL Tech., Vancouver, BC, Canada) were used to sort the T cells from blood of healthy volunteers and T-ALL patients.

| RNA extraction and real-time quantitative PCR (RT-qPCR)
Total RNA was extracted using the Trizol kit (15596026, Invitrogen, Carlsbad, CA, USA). Synthesis of cDNA from mRNA was generated using the PrimeScript RT reagent kit (RR047A, Takara, Japan),  Table 1. Results were calculated using the 2 −△△CT method.

| Protein extraction and quantification
Proteins were extracted using protease inhibitor-contained RIPA buffer (Boster, Wuhan, China). The concentration of total proteins

| Dual-luciferase reporter assay
The binding site between miR-204 and IRAK1 was predicted by the Primer Premier 5.0 software, and the binding sequences were obtained to construct luciferase-tagged plasmids. The fragments of

| Statistical analysis
All statistical analyses were completed with SPSS 21.0 software (IBM, Armonk, NY, USA). Data were shown as the mean ± standard deviation from at least three independent experiments performed in triplicate. First, tests for normality and homogeneity of variance were performed to confirm that the data conformed to normality and homogeneity of variance. The data between two different groups were analysed by unpaired t test, the data between multiple groups were analysed by one-way analysis of variance (ANOVA) and Tukey's post hoc test, and the data between multiple groups at different time points were analysed by two-way ANOVA. A value of P < 0.05 was indicative of significant statistical difference.

| DNA methylation induces decreased miR-204 in T-ALL
Previous studies suggested that miR-204 under-expressed in the T cells of T-ALL patients. 11 To determine the role of miR-204 in T-ALL, here we first analysed the expression of miR-204 in T cells collected from 32T-ALL patients and 16 healthy volunteers using RT-qPCR.
The results showed that miR-204 expression was down-regulated in T cells of T-ALL patients compared to those of healthy volunteers ( Figure 1A), which is consistent with the previous report that

| Overexpression of miR-204 inhibits the proliferation and enhances apoptosis of T-ALL cells
To unveil the effect of miR-204 on the proliferation of T-ALL cells, we transfected miR-204 into the Jurkat cells. RT-qPCR confirmed that miR-204 expression was significantly increased in miR-204 mimictransfected Jurkat cells (Figure 2A). By using CCK-8 assay as well as Hoechst and PI double staining assay, we found that the proliferation of T-ALL cells was inhibited ( Figure 2B) and cell death was increased ( Figure 2C)

| miR-204 directly targets the 3'UTR of IRAK1 in Jurkat cells
To study the effect of miR-204 on T-ALL, we first predicted IRAK1 as a target of miR-204 through scrutiny of the relevant website ( Figure 3A).
To further confirm the role of IRAK1 in the occurrence of T-ALL, we

| Inhibition of IRAK1 modulated by miR-204 inhibits the proliferation of T-ALL cells and enhances apoptosis
To further unveil whether miR-204 affects T-ALL through modulation of IRAK1, we transfected Jurkat cells with mimic-NC + oe-NC, mimic-miR-204 + oe-NC and mimic-miR-204 + oe-IRAK1.
RT-qPCR results showed that miR-204 expression in the mimic-miR-204 + oe-NC and mimic-miR-204 + oe-IRAK1 groups were significantly increased compared to the mimic-NC + oe-NC group

| MiR-204 inhibits IRAK1 to enhance the expression of MMP-2/MMP-9 through activation of p-p65
To confirm the role of MMP-2 and MMP-9 in the occurrence of T-ALL, we investigated the effect of related proteins on T-ALL cells using Western blot analysis. It has been known for some time that in NK cells IRAK1 is the upstream effector of the NF-κB signalling pathway, 15 which is highlyexpressed in T-ALL patients in proportion to disease progression. 16 MMP-2 and MMP-9 are important downstream proteins of the NF-κB signalling pathway, 17 and play vital roles in T-ALL. 18 Our results proved that the expression levels of p-p65, MMP-2 and MMP-9 in T cells from T-ALL patients were significantly increased compared with the levels in healthy volunteers ( Figure 5A), suggesting that MMP-2 and MMP-9 contributed to the occurrence of T-ALL.
Subsequently, oe-NC + si-NC, oe-IRAK1 + si-NC and oe-IRAK1 + si-p65 were transfected into Jurkat cells to determine whether IRAK1 affects the expression of MMP-2 and MMP-9 through the NF-κB signalling pathway. As shown in Figure 5B,

| D ISCUSS I ON
Recent papers have reported that methylation of miRNAs and activation of NF-κB signalling pathway were involved in T-ALL development and progression. 8,19 In our investigation, we explored the In our present study, we found that miR-204 targeted inhibition of IRAK1 expression, thereby inhibiting T-ALL cell proliferation and promoting T-ALL cell apoptosis. IRAK1 is known as a widely expressed serine/threonine kinase, which has a regulatory effect on signalling downstream to Toll-like and Interleukin-1 Receptors, and furthermore, IRAK1 is reported to be highly expressed in all types of T-ALL and to exert regulatory functions in T-ALL cell lines. 24 Partially consistent with our present findings, the expression of IRAK1 mRNA and the levels of IRAK1 have been found elevated in T-ALL cells, and IRAK4 signalling was implicated in as having a critical role in T-ALL proliferation and chemo-resistance. 14 Recent findings showed that that miR-146a directly targets IRAK1 and TRAF6 to alleviate cardiac ischaemia and reperfusion injury. 25 Hence, a wide body of evidence support our model that miR-204 targets IRAK1 to suppress the proliferation and promote the apoptosis of T-ALL cells. are assumed to be particularly important for cell transmigration, and the association of cell surface between MMP-2, MMP-9 and integrins has been implicated in the progression and growth of chronic myeloid leukaemia and acute myeloid leukaemia cells. 26 In addition, the activity of MMP-2 and MMP-9 may be related to an aggravated course of acute myeloid leukaemia, 27 implying an interplay between MMP-2/MMP-9 and T-ALL. Importantly, NF-κB is an essential regulator of immune function, 28 and inhibition of the NF-κB pathway can induce cell growth inhibition and apoptosis. 29 Wu et al demonstrated that NF-κB stimulated by Asb2α induced degradation and dissociation of IκBα, contributing to T-ALL. 30 Inhibition of NF-κB signalling was shown to impair leukaemic T cell growth, which has also been observed in acute T cell leukaemia mouse models. 31 Of note, others have documented that the activities of MMP-2 and MMP-9 were suppressed by kisspeptin through inhibition of the nuclear translocation of the NF-κB. 32 Similarly, Tu et al found that loss of miR-146b-5p up-regulated MMP-9 expression via the NF-κB signalling pathway, to promote T-ALL cell migration and invasion. 6 Very importantly, previous evidence also highlighted that IRAK-1 could lead to the activation of NF-κB. 33 In conclusion, our study elucidated the possible pathway that DNA methylation at the promotor region causes miR-204 silencing, whereas overexpression of miR-204 targets IRAK1, resulting in increased expression of the downstream NF-κB signalling pathway and MMP-2/MMP-9, thereby alleviating T-ALL ( Figure 7). These findings suggest that miR-204 impedes T-ALL growth and metastasis by targeting IRAK1 and highlight the potential role of miR-204/ NF-κB signal pathway in targeted therapy of T-ALL patients.

ACK N OWLED G M ENTS
We would like to acknowledge the reviewers for their helpful comments on this paper.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.