Methylation of NKILA in normal healthy controls and NHL cell lines
NKILA was reported to repress NF-κB signaling pathway [16–18] 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 (Fig. 1a). By MSP, methylation of NKILA was absent in all of normal peripheral blood buffy coats and normal tonsil tissues (Fig. 1b). Amongst NHL cell lines, NKILA was completely methylated (MM) in SU-DHL-6 and completely unmethylated (UU) in GRANTA-519, JEKO-1, MINO, REC-1, SP-53, KARPAS-299 and SU-DHL-1. However, neither U-MSP nor M-MSP signals were observed in SUP-T1 cells (Fig. 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 complete methylation of NKILA had a mean methylation percentage of 69.4%. In contrast, NHL cell lines with completely unmethylation of NKILA had a mean methylation percentage ranging from 5.0–6.5%, which confirmed the methylation status detected by MSP (Fig. 1d). These results indicated that NKILA was methylated in a tumor-specific manner in NHL cells.
Methylation and expression of NKILA in NHL cell lines
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 completed methylated for NKILA. Conversely, expression of NKILA was observed in other cell lines completely unmethylated for NKILA (Fig. 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 (Fig. 2b), with re-expression of NKILA (Fig. 2c). Hence, these data suggested that reversible silencing of NKILA was mediated by promoter DNA methylation in NHL cells.
Methylation of NKILA in primary NHL samples
To investigate the methylation of NKILA in NHL primary samples, MSP was performed with bisulfite-converted DNA in primary samples, including 26 mantle cell lymphoma (MCL), 56 DLBCL and 20 peripheral T-cell lymphoma (PTCL). MSP results showed that no methylation of NKILA was detected in primary MCL samples. However, NKILA was found to be methylated in 29 (51.79%) DLBCL and 4 (20%) PTCL cases (Fig. 3a-c), hence preferentially methylated in DLBCL than MCL (P < 0.0001) and PTCL (P = 0.007).
NKILA inhibiting IkBα phosphorylation and NF-κB activation
NKILA has been reported to suppress NF-κB signaling pathway 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 knock down NKILA and non-targeting siRNA was used as a control in SU-DHL-1. The qRT-PCR result confirmed NKILA was knocked down at 24-hours and 48-hours post-transfection (Fig. 4a). To further evaluate NKILA function in NF-κB signaling pathway, we examined IkBα phosphorylation and p65 nucleus translocation after NKILA knock-down, with augmentation of the NF-κB signaling by TNFα [19]. The result showed that NKILA knock-down led to increase of IkBα phosphorylation in SU-DHL-1 at both 24-hours and 48-hours post-transfection (Fig. 4b), which was associated with enhanced p65 translocated into nucleus compared to the control. Moreover, enhanced nuclear translocation of phosphorylated p65 ser536 was observed after NKILA knock-down (Fig. 4c). These results collectively indicated NKILA negatively regulated NF-κB signaling pathway by inhibiting IkBα phosphorylation, and reduced nuclear translocation of total and phosphorylated p65.
Effect of knock-down of NKILA on SU-DHL-1 cells
As NKILA plays as a negative regulator in NF-κB signaling pathway, 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. Knock-down of NKILA led to a significantly increased cell proliferation rate compared to the control (Fig. 5a). Furthermore, knock-down of NKILA resulted in reduced cell death in SU-DHL-1 (Fig. 5b). These results supportively indicated NKILA acted as a tumor suppressor lncRNA in SU-DHL-1.