Background The long non-coding RNA (lncRNA) NKILA, localized to 20q13.31, is a negative regulator of NF-κB signaling. As a CpG island is embedded in the promoter region of NKILA, NKILA is hypothesized as a tumor suppressor lncRNA reversibly silenced by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). Methods Methylation-specific PCR (MSP) and quantitative bisulfite pyrosequencing were performed to detect the methylation of NKILA in normal peripheral blood buffy coats, normal tonsils tissue, NHL cell lines and NHL primary samples. SU-DHL-6 cells were treated with 5-Aza-2'-deoxycytidine for reversal of methylation-associated NKILA silencing. Tumor suppressor properties and biological function of NKILA were demonstrated by knockdown of NKILA in SU-DHL-1 cells. Results By pyrosequencing-verified MSP, NKILA was unmethylated in normal healthy controls, including 10 peripheral blood buffy coats and 11 tonsils tissue, but completely methylated in one (10%) NHL cell line SU-DHL-6. Among the lymphoma cell lines, by semi-quantitative RT-PCR, methylation of NKILA was inversely correlated with its expression. In the completely methylated SU-DHL-6 cells, hypomethylation treatment with 5-Aza-2'-deoxycytidine resulted in promoter demethylation and re-expression of NKILA transcript. In 102 NHL primary samples, NKILA methylation was observed in none of mantle cell lymphoma (MCL) cases, but in 29 (51.79%) diffuse large-B cell lymphoma (DLBCL) and 4 (20%) peripheral T-cell lymphoma (PTCL) cases, hence preferentially methylated in DLBCL than MCL (P < 0.0001) and PTCL (P = 0.007). Mechanistically, 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, knock-down of NKILA in SU-DHL-1 cells led to decreased cell death and increased cellular proliferation, indicating a tumor suppressor role of NKILA in NHL cells. Conclusion NKILA is a tumour suppressor lncRNA frequently hypermethylated in DLBCL. Promoter DNA methylation-mediated NKILA silencing led to increase of cellular proliferation and decrease of cell death via repression of NF-κB signaling in NHL cells.

Background: 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, we hypothesized that NKILA is a tumor suppressor lncRNA reversibly silenced by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). Results: By pyrosequencing-verified methylation-specific PCR (MSP), NKILA was unmethylated in normal healthy controls, including 10 peripheral blood buffy coats and 11 normal tonsils tissue, but completely methylated in one (10%) NHL cell line SU-DHL-6. Among the lymphoma cell lines, by semi-quantitative RT-PCR, methylation of NKILA was inversely correlated with its expression. In the completely methylated SU-DHL-6 cells, hypomethylation treatment with 5-Aza-2'-deoxycytidine resulted in promoter demethylation and re-expression of NKILA transcript. In NHL primary samples (n=102), NKILA methylation was observed none of mantle cell lymphoma (MCL) cases, but in 29 (51.79%) diffuse large-B cell lymphoma (DLBCL) and 4 (20%) peripheral T-cell lymphoma (PTCL) cases, hence preferentially methylated in DLBCL than MCL (P < 0.0001) and PTCL (P = 0.007). Mechanistically, knockdown of NKILA resulted in promoting IkBα phosphorylation, which was associated with nucleus translocation of total p65 and phosphorylated p65 in SU-DHL-1 cells, hence constitutive NF-κB activation. Functionally, knock-down of NKILA in SU-DHL-1 cells led to decreased cell death and increased cellular proliferation, indicating a tumor suppressor role of NKILA in NHL cells. Conclusions: NKILA was a tumour suppressor lncRNA frequently hypermethylated in DLBCL. Promoter DNA methylation-mediated NKILA silencing led to increase of cellular proliferation and decrease of cell death via repression of NF-κB signaling in NHL cells.

Background: 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, we hypothesized that NKILA is a tumor suppressor lncRNA reversibly silenced by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). Methods: Methylation-specific PCR (MSP) and quantitative bisulfite pyrosequencing were performed to detect the methylation of NKILA in normal peripheral blood buffy coats, normal tonsils tissue, NHL cell lines and NHL primary samples. SU-DHL-6 cells were treated with 5-Aza-2'-deoxycytidine for reversal of methylation-associated NKILA silencing. Tumor suppressor properties of NKILA were demonstrated by knockdown of NKILA in SU-DHL-1 cells.Results: By pyrosequencing-verified MSP, NKILA was unmethylated in normal healthy controls, including 10 peripheral blood buffy coats and 11 normal tonsils tissue, but completely methylated in one (10%) NHL cell line SU-DHL-6. Among the lymphoma cell lines, by semi-quantitative RT-PCR, methylation of NKILA was inversely correlated with its expression. In the completely methylated SU-DHL-6 cells, hypomethylation treatment with 5-Aza-2'-deoxycytidine resulted in promoter demethylation and re-expression of NKILA transcript. In NHL primary samples (n=102), NKILA methylation was observed none of mantle cell lymphoma (MCL) cases, but in 29 (51.79%) diffuse large-B cell lymphoma (DLBCL) and 4 (20%) peripheral T-cell lymphoma (PTCL) cases, hence preferentially methylated in DLBCL than MCL (P < 0.0001) and PTCL (P = 0.007). Mechanistically, knockdown of NKILA resulted in promoting IkBα phosphorylation, which was associated with nucleus translocation of total p65 and phosphorylated p65 in SU-DHL-1 cells, hence constitutive NF-κB activation. Functionally, knock-down of NKILA in SU-DHL-1 cells led to decreased cell death and increased cellular proliferation, indicating a tumor suppressor role of NKILA in NHL cells. Conclusions: NKILA was a tumour suppressor lncRNA frequently hypermethylated in DLBCL. Promoter DNA methylation-mediated NKILA silencing led to increase of cellular proliferation and decrease of cell death via repression of NF-κB signaling in NHL cells.