scholarly journals miR-1250-5p is a novel tumor suppressive intronic miRNA hypermethylated in non-Hodgkin’s lymphoma: novel targets with impact on ERK signaling and cell migration

2020 ◽  
Author(s):  
Min Yue Zhang ◽  
Lu Qian Wang ◽  
James Chim
Keyword(s):  
Dna Methylation ◽  
Cell Migration ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Host Gene ◽  
Erk Signaling ◽  
Primary Lymphoma ◽  
Promoter Dna Methylation ◽  
Dependent Cell ◽  
Promoter Dna

Abstract Background miR-1250 is localised to the second intron of AATK at chromosome 17q25. As a CpG island is present at the putative promoter region of its host gene, AATK, we postulated that the intronic miR-1250-5p is a tumor suppressor miRNA co-regulated with its host gene, AATK, by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). Methods AATK/miR-1250 methylation was studied in healthy controls, including ten normal peripheral blood buffy coat and eleven normal tonsils, ten lymphoma cell lines, and 120 primary lymphoma samples by methylation-specific PCR (MSP). The expression of miR-1250-5p and AATK was investigated by quantitative real-time PCR. Tumor suppressor properties of miR-1250-5p were demonstrated by over-expression of precursor miR-1250-5p in lymphoma cells. The target of miR-1250-5p was verified by luciferase reporter assay. Results AATK/miR-1250 methylation was absent in healthy peripheral blood and tonsils, but detected in five (50%) NHL cell lines. AATK/miR-1250 methylation correlated with repression of miR-1250-5p and AATK in NHL cell lines. In completely methylated SU-DHL-6 and SUP-T1 cells, treatment with 5-AzadC led to promoter demethylation and re-expression of both miR-1250-5p and AATK. In primary lymphoma samples, AATK/miR-1250 was frequently methylated in B-cell lymphoma (n = 41, 44.09%) and T-cell lymphoma (n = 9, 33.33%) with a comparable frequency (P = 0.318). In SU-DHL-1 cells, restoration of miR-1250-5p resulted in decreased cellular proliferation by MTS assay, increased cell death by trypan blue staining and enhanced apoptosis by annexin V-PI assay. Moreover, MAPK1 and WDR1 were verified as direct targets of miR-1250-5p by luciferase assay. In 39 primary NHLs, miR-1250-5p expression was shown to be inversely correlated with each of MAPK1 (P = 0.05) and WDR1 (P = 0.031) by qRT-PCR. Finally, in SU-DHL-1 cells, overexpression of miR-1250-5p led to repression of MAPK1 and WDR1 at both transcript and protein levels, with downregulation of phospho-ERK2 by Western-blotting and inhibition of SDF-1-dependent cell migration by transwell assay. Conclusions miR-1250-5p is a novel tumor suppressive intronic miRNA co-regulated and silenced by promoter DNA methylation of its host gene AATK in NHL. MAPK1 and WDR1 are novel miR-1250-5p direct targets rendering inhibition of MAPK/ERK signaling and SDF-1-dependent cell migration, hence implicated in survival and dissemination of lymphoma.

scholarly journals miR-1250-5p is a novel tumor suppressive intronic miRNA hypermethylated in non-Hodgkin’s lymphoma: novel targets with impact on ERK signaling and cell migration

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Min Yue Zhang ◽  
Lu Qian Wang ◽  
Chor Sang Chim
Keyword(s):  
Dna Methylation ◽  
Cell Migration ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Host Gene ◽  
Erk Signaling ◽  
Primary Lymphoma ◽  
Promoter Dna Methylation ◽  
Dependent Cell ◽  
Promoter Dna

Abstract Background miR-1250 is localised to the second intron of AATK at chromosome 17q25. As a CpG island is present at the putative promoter region of its host gene, AATK, we postulated that the intronic miR-1250-5p is a tumor suppressor miRNA co-regulated with its host gene, AATK, by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). Methods AATK/miR-1250 methylation was studied in healthy controls, including ten normal peripheral blood buffy coats and eleven normal tonsils, ten lymphoma cell lines, and 120 primary lymphoma samples by methylation-specific PCR (MSP). The expression of miR-1250-5p and AATK was investigated by quantitative real-time PCR. Tumor suppressor properties of miR-1250-5p were demonstrated by over-expression of precursor miR-1250-5p in lymphoma cells. The target of miR-1250-5p was verified by luciferase reporter assay. Results AATK/miR-1250 methylation was absent in healthy peripheral blood and tonsils, but detected in five (50%) NHL cell lines. AATK/miR-1250 methylation correlated with repression of miR-1250-5p and AATK in NHL cell lines. In completely methylated SU-DHL-6 and SUP-T1 cells, treatment with 5-AzadC led to promoter demethylation and re-expression of both miR-1250-5p and AATK. In primary lymphoma samples, AATK/miR-1250 was frequently methylated in B-cell lymphoma (n = 41, 44.09%) and T-cell lymphoma (n = 9, 33.33%) with a comparable frequency (P = 0.318). In SU-DHL-6 and SU-DHL-1 cells, restoration of miR-1250-5p resulted in decreased cellular proliferation by MTS assay, increased cell death by trypan blue staining and enhanced apoptosis by annexin V-PI assay. Moreover, MAPK1 and WDR1 were verified as direct targets of miR-1250-5p by luciferase assay. In 39 primary NHLs, miR-1250-5p expression was shown to be inversely correlated with each of MAPK1 (P = 0.05) and WDR1 (P = 0.031) by qRT-PCR. Finally, in SU-DHL-1 cells, overexpression of miR-1250-5p led to repression of MAPK1 and WDR1 at both transcript and protein levels, with downregulation of phospho-ERK2 by Western-blotting and inhibition of SDF-1-dependent cell migration by transwell assay. Conclusions miR-1250-5p is a novel tumor suppressive intronic miRNA co-regulated and silenced by promoter DNA methylation of its host gene AATK in NHL. MAPK1 and WDR1 are novel miR-1250-5p direct targets rendering inhibition of MAPK/ERK signaling and SDF-1-dependent cell migration, hence implicated in survival and dissemination of lymphoma.

scholarly journals Epigenetic silencing of miR-342-3p in B cell lymphoma and its impact on autophagy

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Min Yue Zhang ◽  
George A. Calin ◽  
Kit San Yuen ◽  
Dong Yan Jin ◽  
Chor Sang Chim
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Host Gene ◽  
Tumor Suppressor Function ◽  
Promoter Dna Methylation ◽  
Promoter Dna

Abstract Background miR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in carcinogenesis. Given the presence of a promotor-associated CpG island for its host gene, EVL, we hypothesized that intronic miR-342-3p is a tumor suppressor co-regulated with host gene by promoter DNA methylation in B cell lymphoma. Results By bisulfite pyrosequencing-verified methylation-specific PCR (MSP), EVL/MIR342 methylation was detected in five (50%) lymphoma cell lines but not normal peripheral blood and tonsils. EVL/MIR342 methylation correlated with repression of both miR-342-3p and EVL in cell lines. In completely methylated SU-DHL-16 cells, 5-AzadC treatment resulted in promoter demethylation and re-expression of miR-342-3p and EVL. In 132 primary lymphoma samples, EVL/MIR342 was preferentially methylated in B cell lymphomas (N = 68; 68.7%) than T cell lymphoma (N = 8; 24.2%) by MSP (P < 0.0001). Moreover, EVL/MIR342 methylation was associated with lower miR-342-3p expression in 79 primary NHL (P = 0.0443). In SU-DHL-16 cells, the tumor suppressor function of miR-342-3p was demonstrated by the inhibition of cellular proliferation and increase of cell death upon over-expression of miR-342-3p. Mechanistically, overexpression of miR-342-3p resulted in a decrease of LC3-II, a biomarker of autophagy, which was pro-survival for SU-DHL-16. Pre-treatment with 3-methyladenine, an autophagy inhibitor, abrogated tumor suppression associated with miR-342-3p overexpression. By luciferase assay, MAP1LC3B, a precursor of LC3-II, was confirmed as a direct target of miR-342-3p. Finally, in SU-DHL-16 cells, overexpression of miR-342-3p downregulated the known target DNMT1, with promoter demethylation and re-expression of tumor suppressor E-cadherin. Conclusions Intronic miR-342-3p is co-regulated with its host gene EVL by tumor-specific promoter DNA methylation in B cell lymphoma. The tumor suppressor function of miR-342-3p was mediated via inhibition of pro-survival autophagy by targeting MAP1LC3B and downregulation of DNMT1 with demethylation and re-expression of tumor suppressor genes.

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

2021 ◽  
Author(s):  
Min Yue Zhang ◽  
George Calin ◽  
Ming Dan Deng ◽  
Rex K.H. Au-Yeung ◽  
Lu Qian Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Death ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cellular Proliferation ◽  
Cell Lymphoma ◽  
Hodgkin's Lymphoma ◽  
Promoter Dna Methylation ◽  
Promoter Dna

Abstract 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.

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

Genes ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 128
Author(s):  
Min-Yue Zhang ◽  
George Calin ◽  
Ming-Dan Deng ◽  
Rex K. H. Au-Yeung ◽  
Lu-Qian Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Death ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Cell Lymphoma ◽  
Hodgkin's Lymphoma ◽  
Non Hodgkin's Lymphoma ◽  
Promoter Dna Methylation ◽  
Promoter Dna

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.

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

2020 ◽  
Author(s):  
Min Yue Zhang ◽  
Ming Dan Deng ◽  
Rex K.H. Au-Yeung ◽  
Lu Qian Wang ◽  
Chor Sang Chim
Keyword(s):  
Dna Methylation ◽  
Cell Death ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cellular Proliferation ◽  
Cell Lymphoma ◽  
Hodgkin's Lymphoma ◽  
Promoter Dna Methylation ◽  
Promoter Dna

Abstract 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.

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

2020 ◽  
Author(s):  
Min Yue Zhang ◽  
Ming Dan Deng ◽  
Lu Qian Wang ◽  
Rex K.H. Au-Yeung ◽  
Chor Sang Chim
Keyword(s):  
Dna Methylation ◽  
Cell Death ◽  
Tumor Suppressor ◽  
Hodgkin’S Lymphoma ◽  
Cellular Proliferation ◽  
Cell Lymphoma ◽  
Hodgkin's Lymphoma ◽  
Non Hodgkin's Lymphoma ◽  
Promoter Dna Methylation ◽  
Promoter Dna

Abstract 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.

scholarly journals Epigenetic silencing of LPP/miR-28 in multiple myeloma

2017 ◽  
Vol 71 (3) ◽  
pp. 253-258 ◽  
Author(s):  
Zhenhai Li ◽  
Kwan Yeung Wong ◽  
Godfrey Chi-fung Chan ◽  
Chor Sang Chim
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Cpg Island ◽  
Myeloma Cell ◽  
Tumour Suppressor ◽  
Host Gene ◽  
Healthy Controls ◽  
Promoter Dna Methylation ◽  
Promoter Dna ◽  
Partially Methylated

AimsmiR-28-5- is a tumour suppressor microRNA implicated in cancers. As a CpG island is absent in miR-28-5- but present in its host gene, LPP (LIM domain containing preferred translocation partner in lipoma), we hypothesized that miR-28-5p is epigenetically silenced by promoter DNA methylation of its host gene in multiple myeloma.MethodsMethylation-specific PCR, verified by quantitative bisulfite pyrosequencing, was employed to study methylation of LPP/miR-28 in healthy controls (n=10), human myeloma cell lines (HMCLs) (n=15), and primary myeloma marrow samples at diagnosis (n=49) and at relapse (n=18). Quantitative reverse transcription PCR was used to investigate expression of miR-28-5p, LPP and CCND1.ResultsLPP/miR-28 was completely unmethylated in all healthy controls and 12 (80%) HMCLs, but partially methylated in three (20%) HMCLs. Methylation of LPP/miR-28 correlated with low expression of miR-285p (p=0.012) and LPP (p=0.037) in HMCLs. In RPMI-8226R cells, in which LPP/miR-28 was partially methylated, 5-AzadC treatment led to demethylation of LPP/miR-28 and re-expression of both miR-28-5p (p=0.0007) and LPP (p=0.0007), whereas continuous culture without 5-AzadC restored LPP/miR-28 methylation and reduced expression of both miR-28-5p (p=0.0013) and LPP (p=0.0025). Moreover, a known miR-28-5p target, CCND1, was expressed at higher levels in HMCLs with LPP/miR-28 methylation than those without, consistent with a tumour suppressor role of miR-28-5p in myeloma. However, in primary samples, LPP/miR-28 was methylated in two (4.1%) at diagnosis, whereas none at relapse.ConclusionsThis is the first report of epigenetic regulation of the intronic miR-28-5p expression by promoter DNA methylation of its host gene, hence warrants further study in different cancers.

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