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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
James Chim
University of Hong Kong
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2427-915X
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
miR-1250-5p, non-Hodgkin’s lymphoma, DNA methylation, tumor suppressor miRNA, MAPK1, WDR1
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This preprint is under consideration at Cell Communication and Signaling. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
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
James Chim
University of Hong Kong
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2427-915X
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 21 Sep, 2020
No community comments so far
Editorial decision: Major Revision
On 18 Oct, 2020
Review #1 received
Received 06 Oct, 2020
Reviewer #1 agreed
On 01 Oct, 2020
Reviewers invited
Invitations sent on 20 Sep, 2020
First submitted
On 18 Sep, 2020
Editor assigned
On 18 Sep, 2020
Submission checks complete
On 17 Sep, 2020
Editor invited
On 17 Sep, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5