Epigenetic Silencing of Tumor Suppressor lncRNA NKILA: Implication on NF-κB Signaling in Non-Hodgkin’s Lymphoma

The long non-coding RNA (lncRNA) NKILA, localized to 20q13.31, is a negative regulator of NF-κB signaling implicated in carcinogenesis. As a CpG island is embedded in the promoter region of NKILA, it is hypothesized as a tumor suppressor lncRNA silenced by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). By pyrosequencing-verified methylation-specific PCR, NKILA methylation was detected in 1/10 (10%) NHL cell lines, but not in normal peripheral blood buffy coats or tonsils. NKILA methylation correlated with the repression of NKILA in cell lines. Hypomethylation treatment with 5-Aza-2′-deoxycytidine resulted in promoter demethylation and the re-expression of NKILA. In 102 NHL primary samples, NKILA was methylated in 29 (51.79%) diffuse large B-cell lymphoma (DLBCL) and 4 (20%) peripheral T-cell lymphoma cases, but unmethylated in all 26 mantle cell lymphoma cases. Mechanistically, the knockdown of NKILA resulted in promoting IkBα phosphorylation, associated with nucleus translocation of total p65 and phosphorylated p65 in SU-DHL-1 cells, hence constitutive NF-κB activation. Functionally, the knockdown of NKILA in SU-DHL-1 cells led to decreased cell death and increased cellular proliferation. Collectively, NKILA was a tumor suppressor lncRNA frequently hypermethylated in DLBCL. Promoter DNA methylation-mediated NKILA silencing resulted in increased cellular proliferation and decreased cell death via the repression of NF-κB signaling in NHL.


Introduction
Non-Hodgkin's lymphoma (NHL) encompasses a heterogeneous group of diseases, including B-cell, T-cell and natural killer (NK)-cell lymphoma based on the origin and lineage of tumor cells [1]. B-cell lymphoma comprises more than 70% of all lymphomas, while T-cell lymphoma accounts for 10-15% of all lymphomas [2]. Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma are the two most common subtypes of NHL [3], whereas NK-cell lymphoma is an aggressive subtype rare in Western countries [4]. The incidence of NHL was 6.7/100,000 among males and 4.7/100,000 among females worldwide, which ranked as the 8th and 10th of all types of cancers, respectively [5]. The clinical features of NHL patients include painless lymphadenopathy, organomegaly and the presence or absence of B symptoms (night sweats, weight loss > 10% and fever with temperature > 38 • C) [6].
Long non-coding RNAs (lncRNAs) are broadly defined as a class of non-coding RNAs measuring >200 nucleotides [14,15]. lncRNAs play essential roles in regulating multiple biological processes, including cellular metabolism, organogenesis and carcinogenesis [16][17][18]. LncRNA, NKILA (NF-KappaB Interacting LncRNA), localized to 20q13.31, was firstly found to be downregulated in breast cancer. Overexpression of NKILA resulted in the inhibition of metastasis and the increase in apoptosis by repression of NF-κB signaling activity in breast cancer cells, indicating the tumor suppressor property of NKILA [19]. Moreover, the NF-κB signaling pathway is constitutively activated and, hence, implicated in the pathogenesis of NHL. However, the function of NKILA in lymphoma remains unknown.
As a CpG island is present at the promoter region of NKILA, we postulated that NKILA is a tumor suppressor lncRNA reversibly silenced by promoter DNA methylation in NHL. Herein, the methylation of NKILA will be studied in NHL, and the role of NKILA in lymphomagenesis will also be investigated.

Patient Samples
A total of 102 formalin fixed, paraffin-embedded (FFPE) or fresh frozen diagnostic lymph node biopsy tissues, including 56 DLBCL, and 26 mantle cell lymphoma (MCL) and 20 peripheral T-cell lymphoma (PTCL) cases, were acquired from 5 hospitals in Hong Kong, China (Queen Mary Hospital, Kwong Wah Hospital, Princess Margaret Hospital, United Christian Hospital and Pamela Youde Nethersole Eastern Hospital). The diagnosis of lymphoma was based on the WHO (World Health Organization, Geneva, Switzerland) classification [20]. Eleven FFPE tonsil tissues were also obtained from healthy individuals undergoing tonsillectomy. Our study was approved by the Institutional Review Board of Queen Mary Hospital.

DNA Extraction
DNA from NHL cell lines and healthy peripheral blood was extracted with a DNA Blood Mini kit (Qiagen). DNA extraction from frozen patient biopsies was conducted with an automated DNA extraction system (DNA Tissue Kit from Qiagen). DNA extraction from FFPE tissues were performed by using QIAamp DNA FFPE Tissue Kit (Qiagen).

Methylation-Specific Polymerase Chain Reaction (MSP)
Sodium bisulfite conversion was conducted with an EpiTect Bisulfite Kit (Qiagen). Afterwards, MSP was performed in bisulfite-treated DNA with two sets of primers, which were specific to unmethylated (U-MSP) or methylated (M-MSP) DNA sequences. MSP primers were designed at the CpG island upstream to the NKILA gene by the online tool Methprimer (http://www.urogene.org/methprimer/ (accessed on 31 May 2019). Details of the primer sequence and PCR condition for MSP are listed in Table 1. For each MSP reaction, the enzymatically methylated control DNA (CpGenome Universal Methylated DNA; Chemicon/Millipore, Billerica, MA, USA) was used as the positive control for M-MSP and the negative control for U-MSP.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated with a Direct-zol™ RNA MiniPrep kit (Zymo Research), followed by reverse transcription with SuperScript ® III (Invitrogen, Carlsbad, CA, USA). The qRT-PCR was performed with a SYBR ® Select Master Mix (ABI, Bedford, MA, USA), and the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. The relative quantity of NKILA expression was calculated by the method of 2 −∆∆Ct and normalized against the endogenous control. The primer sequences for NKILA and GAPDH are listed in Table 1 [19,21].

Quantitative Bisulfite Pyrosequencing
The promoter region of NKILA overlapped with the amplicon of MSP was amplified in the bisulfite converted DNA with methylation-unbiased primers. Primer sequences and condition for PCR were listed as follows: forward primer, 5 -GTT GGG GAG AGG GTA TAG-3 ; reverse primer, 5 -Biotin-CTC CTC CTC CTC ATT CAA ATC-3 and condition, 56 • C/45 x/1.5 mM MgCl 2 . A Qiagen PyroMark PCR Kit was employed to perform PCR amplification. Afterwards, a stretch of DNA containing 10 consecutive CpG dinucleotides were pyrosequenced with a sequencing primer, 5 -GTT AGG GGA GGG GGT G-3 , on a PSQ 96MA system and analyzed using PyroQ-CpG 1.0.9. software.

Knockdown of NKILA
RNA interference by small interfering RNA (siRNA) was used to knockdown the expression of NKILA. Briefly, SU-DHL-1 cells were seeded at a density of 0.5 × 10 6 /mL in a six-well plate and transfected with NKILA siRNA (n541256, Ambion, Austin, TX, USA) or Silencer Negative Control (Ambion) at a final concentration of 150 nM with an RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA). The transfected cells were cultured for 24 h or 48 h.

Cell Proliferation and Cell Death
After 24 h and 48 h transfection, viable cells were analyzed by trypan blue exclusion and the cell number was measured by the Countess II (Invitrogen). Cell proliferation was calculated by normalization with the negative control at 48 h post-transfection. Cell death was analyzed by trypan blue exclusion at 24 h post-transfection. All experiments were repeated at least three times.
Nuclear and cytosol fractionations of SU-DHL-1 cells were prepared by the Nuclear/Cytosol Fractionation Kit (BioVision, Milpitas, CA, USA), according to the manufacturers' instructions. Western blot was then performed in these samples.

Statistical Analysis
The difference of cell proliferation and cell death between SU-DHL-1 cells transfected with NKILA siRNA and Silencer Negative Control were compared by Student's t-test. The difference of the NKILA methylation frequency in different subtypes of NHL primary samples was analyzed by the χ 2 test. All p-values were 2-sided; p < 0.05 was considered as the significant difference.

NKILA Was Methylated in a Tumor-Specific Manner in NHL Cells
NKILA was reported to repress the NF-κB signaling pathway [19,22,23], which is constitutively activated and implicated in lymphomagenesis. There is a CpG island at the promoter region of NKILA. Hence, the methylation status of NKILA was investigated by MSP in the bisulfite-converted DNA of normal healthy controls, including 10 peripheral blood buffy coats and 11 normal tonsil tissues, in addition to 10 NHL cell lines. Direct sequencing of M-MSP products from methylated positive control DNA demonstrated that all unmethylated cytosines were converted into thymidines after PCR, whereas all methylated cytosines remained unchanged, indicating the complete bisulfite conversion and specificity of MSP ( Figure 1A). By MSP, the methylation of NKILA was absent in all of normal peripheral blood buffy coats and normal tonsil tissues ( Figure 1B Figure 1C). Furthermore, the methylation status of NKILA in NHL cell lines detected by MSP was verified by quantitative bisulfite pyrosequencing. SU-DHL-6 cells with a complete methylation of NKILA had a mean methylation percentage of 69.4%. In contrast, NHL cell lines with complete unmethylation of NKILA had a mean methylation percentage ranging from 5.0% to 6.5%, which confirmed the methylation status detected by MSP ( Figure 1D). These results indicated that NKILA was methylated in a tumor-specific manner in NHL cells.

The Expression of NKILA Was Inversely Correlated with Promoter DNA Methylation
To explore the relationship between promoter DNA methylation and the expression of NKILA, semi-quantitative RT-PCR of NKILA was performed in NHL cell lines.
As demonstrated by the DNA gel, no expression of NKILA was detected in SU-DHL-6 cells that was completely methylated for NKILA. Conversely, the expression of NKILA was observed in other cell lines completely unmethylated for NKILA (Figure 2A). Furthermore, to study whether promoter DNA methylation was associated with reversible silencing of NKILA, SU-DHL-6 cells, which were completely methylated for NKILA, were treated with a demethylating agent, 5-AzadC, for 7 days. Upon treatment with 5-AzadC, the promoter of NKILA was demethylated, as illustrated by the emergence of U-MSP signal ( Figure 2B), with the re-expression of NKILA ( Figure 2C). Hence, these data suggested that the reversible silencing of NKILA was mediated by promoter DNA methylation in NHL cells.

NKILA Was Differentially Methylated in NHL Primary Samples
To investigate the methylation of NKILA in NHL primary samples, MSP was performed with bisulfite-converted DNA in primary samples, including 26 MCL, 56 DLBCL and 20 PTCL cases. The MSP results showed that no methylation of NKILA was detected in primary MCL samples ( Figure 3A). However, NKILA was found to be methylated in 29 (51.79%) DLBCL and 4 (20%) PTCL cases ( Figure 3B,C), hence preferentially methylated in DLBCL than MCL (p < 0.0001) and PTCL (p = 0.007).

NKILA Inhibited IkBα Phosphorylation and NF-κB Activation
NKILA has been reported to suppress NF-κB signaling pathways by blocking IkBα phosphorylation in breast cancer, non-small cell lung cancer and nasopharyngeal carcinoma [19,22,23]. To elucidate NKILA function in lymphoma, NKILA-targeted siRNA was used to knockdown NKILA and non-targeting siRNA was used as a control in SU-DHL-1 cells. The qRT-PCR result confirmed that NKILA was knocked down at 24 h and 48 h posttransfection ( Figure 4A). To further evaluate NKILA function in NF-κB signaling pathways, we examined IkBα phosphorylation and p65 nucleus translocation after NKILA knockdown, with an augmentation of the NF-κB signaling by TNFα [19]. The result showed that NKILA knockdown led to an increase in IkBα phosphorylation in SU-DHL-1 cells at both 24 h and 48 h post-transfection ( Figure 4B), which was associated with enhanced p65 being translocated into the nucleus, compared with the control. Moreover, the enhanced nuclear translocation of phosphorylated p65 ser536 was observed after NKILA knockdown ( Figure 4C). These results collectively indicated that NKILA negatively regulated NF-κB signaling pathways, by inhibiting IkBα phosphorylation and reducing nuclear translocation of total and phosphorylated p65.

NKILA Was a Tumor Suppressor lncRNA in SU-DHL-1 Cells
As NKILA was a negative regulator in NF-κB signaling pathways, its tumor suppressor function in lymphoma was further explored by examining cell proliferation and cell death in SU-DHL-1 cells that were completely unmethylated for NKILA. The knockdown of NKILA led to a significantly increased cell proliferation rate compared with the control ( Figure 5A). Furthermore, the knockdown of NKILA resulted in reduced cell death in SU-DHL-1 cells ( Figure 5B). These results supportively indicated NKILA acted as a tumor suppressor lncRNA in SU-DHL-1 cells.

Discussion
Several observations were made in this study. Firstly, we showed that NKILA was methylated in NHL cell lines and NHL primary samples, but unmethylated in normal controls, hence methylated in a tumor-specific manner in NHL. This was consistent with a tumor-specific pattern of the methylation of tumor suppressor protein-coding genes, such as p16 and p15 [24], and non-coding tumor suppressor microRNAs, such as miR-342-3p [25] and miR-1250-5p [26], in NHL. However, this contrasted with the tissue-but not tumorspecific pattern of methylation, such as miR-373 [27] and miR-127 [28], which were shown to be methylated in both normal counterparts and tumor cells, hence likely unimportant in carcinogenesis.
Secondly, in primary NHL samples, NKILA was frequently methylated in DLBCL, but not in MCL or PTCL samples. Indeed, NHL is highly heterogenous with different genetic and epigenetic features [1,20]. For instance, a microarray-based DNA methylation study in 367 hematological neoplasms demonstrated that promoter DNA hypermethylation was more frequent in precursor B and T lymphoid neoplasias and mature B-cell lymphomas of a germinal center origin (such as DLBCL, FL and Burkitt's lymphoma), than in mature T-cell lymphomas, such as PTCL [29]. Therefore, differential methylation of NKILA can be accounted for the difference in cell origin and their inherent pathogenetic mechanisms.
Thirdly, to our knowledge, this is the first study that reported that promoter DNA methylation mediated the reversible silencing of NKILA in NHL, which is evidenced by an inverse correlation between the NKILA methylation and its expression in NHL cell lines, and the re-expression of NKILA upon demethylation treatment in cell lines with a complete methylation of NKILA. Apart from promoter DNA methylation, NKILA have been reported to be regulated by other mechanisms. For instance, Huang et al. [30] showed that, in cytotoxic T lymphocytes, NKILA transcription induced by antigen stimulation was mediated by an increase in the acetylation of histones (H4ac, H3K27ac and H3K9ac) at the promoter region of NKILA, suggesting the expression of NKILA regulated by histone modification. In addition, in breast cancer cells, some oncogenic microRNAs, such as miR-103 or miR-107, could directly target and downregulate the expression of NKILA, which was confirmed by the luciferase reporter assay [19].
Fourthly, NKILA was first shown to suppress NF-κB signaling in breast cancer. It can directly bind to the NF-κB/IκB complex, and potentially mask the phosphorylation site of IκB, thereby suppressing IKK-induced IκB phosphorylation and, hence, NF-κB activity [19]. As the constitutive activation of the NF-κB signaling pathway is the hallmark of many lymphoid malignancies, including Hodgkin's lymphoma and DLBCL [31], and hence implicated in lymphomagenesis, it is essential to comprehend the precise function of NKILA and its interaction with NF-κB in lymphomas. To the best of our knowledge, this is the first study showing the role of NKILA in lymphomas. Previous studies reported that phosphorylation p65 at ser536 led to the enhanced transcriptional activity of NF-κB [32][33][34]. We observed that the knockdown of NKILA led to the upregulation of NF-κB signaling pathways by promoting IκBα phosphorylation, and the consequent nuclear translocation of total p65 and phosphorylated p65 in lymphoma cells. Furthermore, as a transcription factor, NF-κB regulates the expression of multiple downstream effectors, enhancing cell proliferation, inhibiting cell apoptosis or promoting cell migration and invasion [35]. Herein, in NHL cells, the downregulation of NKILA resulted in an increase in cellular proliferation and decrease in cell death, consistent with the tumor suppressor role of NKILA in multiple tumors, including melanoma, lung cancer, rectal cancer, laryngeal cancer and breast cancer [19,22,[36][37][38][39][40]. In addition, NKILA has also been shown to inhibit tumor invasion and migration in epithelial cancers, such as breast cancer, hepatocellular carcinoma and tongue squamous cell carcinoma [19,39,40]. Collectively, these results suggested that the NKILA can inhibit cellular proliferation and induce cell death by suppressing the NF-κB signaling pathway in NHL cells.

Conclusions
Taken together, the epigenetic silencing of lncRNA NKILA was mediated by promoter DNA methylation in a tumor-specific manner in NHL. Frequent hypermethylation of NKILA was preferentially detected in DLBCL patients. NKILA exerted its tumor suppressive property by the inhibition of cellular proliferation and increase of cell death, in association with suppression of the NF-κB signaling pathway via inducing IκBα phosphorylation, and the consequent nucleus translocation of total p65 and phosphorylated p65 in NHL cells. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: All data generated or analyzed during this study are included in this published article.