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MicroRNA-223 is a novel negative regulator of HSP90B1 in CLL.

Rodríguez-Vicente AE, Quwaider D, Benito R, Misiewicz-Krzeminska I, Hernández-Sánchez M, de Coca AG, Fisac R, Alonso JM, Zato C, de Paz JF, García JL, Sarasquete ME, Hernández JÁ, Corchado JM, González M, Gutiérrez NC, Hernández-Rivas JM - BMC Cancer (2015)

Bottom Line: These results were confirmed at the protein level by western blot.Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients.By contrast, the presence of rs2307842 was not related to the outcome.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Hematología, IBSAL, IBMCC, CIC, Universidad de Salamanca, CSIC, Hospital Universitario, Salamanca, Spain. anaerv@hotmail.com.

ABSTRACT

Background: MicroRNAs are known to inhibit gene expression by binding to the 3'UTR of the target transcript. Downregulation of miR-223 has been recently reported to have prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients.

Methods: By applying next-generation sequencing techniques we have detected a common polymorphism (rs2307842), in 24% of CLL patients, which disrupts the binding site for miR-223 in HSP90B1 3'UTR. We investigated whether miR-223 directly targets HSP90B1 through luciferase assays and ectopic expression of miR-223. Quantitative real-time polymerase chain reaction and western blot were used to determine HSP90B1 expression in CLL patients. The relationship between rs2307842 status, HSP90B1 expression and clinico-biological data were assessed.

Results: HSP90B1 is a direct target for miR-223 by interaction with the putative miR-223 binding site. The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients. These results were confirmed at the protein level by western blot. Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients. By contrast, the presence of rs2307842 was not related to the outcome.

Conclusions: HSP90B1 is a direct target gene of miR-223. Our results provide a plausible explanation of why CLL patients harboring miR-223 downregulation are associated with a poor outcome, pointing out HSP90B1 as a new pathogenic mechanism in CLL and a promising therapeutic target.

No MeSH data available.


Related in: MedlinePlus

HSP90B1is a direct target of miR-223. (A) 3′untranslated region (3′UTR) of HSP90B1 (263 nt length) with a predicted binding site for miR-223 at 204–210 nt (grey box). The figure shows the mature miR-223 sequence (hsa-miR-223) aligned with HSP90B1 3′UTR wild type (WT, up), and with the polymorphism (VAR, below). The seed region is shown in bold. The rs2307842 polymorphism (in grey) disrupts the putative binding site for miR-223 by deleting the last three nucleotides of the seed region. (B) Luciferase reporter assays to confirm targeting of HSP90B1 3′UTR by miR-223. Ectopic miR-223 expression inhibits the wild-type but not the variant HSP90B1 3′UTR reporter activity in HEK293 cells. Cells were co-transfected with miR-223 precursor/negative control (NC) miRNA and with either wild-type (WT) or variant (VAR) HSP90B1 3′UTR reporter construct. Luciferase activity assay was performed 24 h after transfection. The columns represent normalized relative luciferase activity by means with 95% confidence intervals from 4 independent experiments (Mann–Whitney test, *P < 0.05). (C) and (D) Ectopic miR-223 expression reduced both HSP90B1 mRNA (C) and protein (D) expression in H929 cell line (WT) but not in MM1S (VAR). Cells were transfected with miR-223 precursors and negative controls. After 24 h, cells were analyzed for HSP90B1 expression by qRT-PCR (C) and western blot (D). The data shown are representative of 3 independent experiments (Mann–Whitney test, *P < 0.05).
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Fig1: HSP90B1is a direct target of miR-223. (A) 3′untranslated region (3′UTR) of HSP90B1 (263 nt length) with a predicted binding site for miR-223 at 204–210 nt (grey box). The figure shows the mature miR-223 sequence (hsa-miR-223) aligned with HSP90B1 3′UTR wild type (WT, up), and with the polymorphism (VAR, below). The seed region is shown in bold. The rs2307842 polymorphism (in grey) disrupts the putative binding site for miR-223 by deleting the last three nucleotides of the seed region. (B) Luciferase reporter assays to confirm targeting of HSP90B1 3′UTR by miR-223. Ectopic miR-223 expression inhibits the wild-type but not the variant HSP90B1 3′UTR reporter activity in HEK293 cells. Cells were co-transfected with miR-223 precursor/negative control (NC) miRNA and with either wild-type (WT) or variant (VAR) HSP90B1 3′UTR reporter construct. Luciferase activity assay was performed 24 h after transfection. The columns represent normalized relative luciferase activity by means with 95% confidence intervals from 4 independent experiments (Mann–Whitney test, *P < 0.05). (C) and (D) Ectopic miR-223 expression reduced both HSP90B1 mRNA (C) and protein (D) expression in H929 cell line (WT) but not in MM1S (VAR). Cells were transfected with miR-223 precursors and negative controls. After 24 h, cells were analyzed for HSP90B1 expression by qRT-PCR (C) and western blot (D). The data shown are representative of 3 independent experiments (Mann–Whitney test, *P < 0.05).

Mentions: By applying a custom-made data analysis pipeline, we have annotated the detected variants, including reported single-nucleotide polymorphisms (SNPs), genomic location, predicted miRNA binding sites, consequences of the variant in transcripts (i.e. synonymous, missense) and protein function prediction for those variants that are predicted to result in an aminoacid sustitution. In one out of four CLL patients (25%) we identified a 4-bp insertion/deletion polymorphism (−/GACT) in 3′UTR of HSP90B1, filled as rs2307842 (102865778-102865781b) in the NCBI SNP database. Rs2307842 results in the deletion of four nucleotides in 3′UTR sequence, three of them being part of the predicted binding site for miR-223 (Figure 1A). According to the databases, UCSC Genome Browser, NCBI and Ensembl, the reference genome contains the ′GACT′ sequence. The major allele in the European population, according to the NCBI SNP database, is ′GACT′ (allele frequency: 0.79 ± 0.06), whereas the 4-bp deletion has a minor allele frequency of 0.21 ± 0.06. Thus, we considered the individuals carrying the ′GACT′ sequence as wild-type (WT) and the individuals with the 4 bp-deletion as variants (VAR). We hypothesized that this deletion disrupts the binding site for miR-223, thereby increasing the translation of HSP90B1.Figure 1


MicroRNA-223 is a novel negative regulator of HSP90B1 in CLL.

Rodríguez-Vicente AE, Quwaider D, Benito R, Misiewicz-Krzeminska I, Hernández-Sánchez M, de Coca AG, Fisac R, Alonso JM, Zato C, de Paz JF, García JL, Sarasquete ME, Hernández JÁ, Corchado JM, González M, Gutiérrez NC, Hernández-Rivas JM - BMC Cancer (2015)

HSP90B1is a direct target of miR-223. (A) 3′untranslated region (3′UTR) of HSP90B1 (263 nt length) with a predicted binding site for miR-223 at 204–210 nt (grey box). The figure shows the mature miR-223 sequence (hsa-miR-223) aligned with HSP90B1 3′UTR wild type (WT, up), and with the polymorphism (VAR, below). The seed region is shown in bold. The rs2307842 polymorphism (in grey) disrupts the putative binding site for miR-223 by deleting the last three nucleotides of the seed region. (B) Luciferase reporter assays to confirm targeting of HSP90B1 3′UTR by miR-223. Ectopic miR-223 expression inhibits the wild-type but not the variant HSP90B1 3′UTR reporter activity in HEK293 cells. Cells were co-transfected with miR-223 precursor/negative control (NC) miRNA and with either wild-type (WT) or variant (VAR) HSP90B1 3′UTR reporter construct. Luciferase activity assay was performed 24 h after transfection. The columns represent normalized relative luciferase activity by means with 95% confidence intervals from 4 independent experiments (Mann–Whitney test, *P < 0.05). (C) and (D) Ectopic miR-223 expression reduced both HSP90B1 mRNA (C) and protein (D) expression in H929 cell line (WT) but not in MM1S (VAR). Cells were transfected with miR-223 precursors and negative controls. After 24 h, cells were analyzed for HSP90B1 expression by qRT-PCR (C) and western blot (D). The data shown are representative of 3 independent experiments (Mann–Whitney test, *P < 0.05).
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Related In: Results  -  Collection

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Fig1: HSP90B1is a direct target of miR-223. (A) 3′untranslated region (3′UTR) of HSP90B1 (263 nt length) with a predicted binding site for miR-223 at 204–210 nt (grey box). The figure shows the mature miR-223 sequence (hsa-miR-223) aligned with HSP90B1 3′UTR wild type (WT, up), and with the polymorphism (VAR, below). The seed region is shown in bold. The rs2307842 polymorphism (in grey) disrupts the putative binding site for miR-223 by deleting the last three nucleotides of the seed region. (B) Luciferase reporter assays to confirm targeting of HSP90B1 3′UTR by miR-223. Ectopic miR-223 expression inhibits the wild-type but not the variant HSP90B1 3′UTR reporter activity in HEK293 cells. Cells were co-transfected with miR-223 precursor/negative control (NC) miRNA and with either wild-type (WT) or variant (VAR) HSP90B1 3′UTR reporter construct. Luciferase activity assay was performed 24 h after transfection. The columns represent normalized relative luciferase activity by means with 95% confidence intervals from 4 independent experiments (Mann–Whitney test, *P < 0.05). (C) and (D) Ectopic miR-223 expression reduced both HSP90B1 mRNA (C) and protein (D) expression in H929 cell line (WT) but not in MM1S (VAR). Cells were transfected with miR-223 precursors and negative controls. After 24 h, cells were analyzed for HSP90B1 expression by qRT-PCR (C) and western blot (D). The data shown are representative of 3 independent experiments (Mann–Whitney test, *P < 0.05).
Mentions: By applying a custom-made data analysis pipeline, we have annotated the detected variants, including reported single-nucleotide polymorphisms (SNPs), genomic location, predicted miRNA binding sites, consequences of the variant in transcripts (i.e. synonymous, missense) and protein function prediction for those variants that are predicted to result in an aminoacid sustitution. In one out of four CLL patients (25%) we identified a 4-bp insertion/deletion polymorphism (−/GACT) in 3′UTR of HSP90B1, filled as rs2307842 (102865778-102865781b) in the NCBI SNP database. Rs2307842 results in the deletion of four nucleotides in 3′UTR sequence, three of them being part of the predicted binding site for miR-223 (Figure 1A). According to the databases, UCSC Genome Browser, NCBI and Ensembl, the reference genome contains the ′GACT′ sequence. The major allele in the European population, according to the NCBI SNP database, is ′GACT′ (allele frequency: 0.79 ± 0.06), whereas the 4-bp deletion has a minor allele frequency of 0.21 ± 0.06. Thus, we considered the individuals carrying the ′GACT′ sequence as wild-type (WT) and the individuals with the 4 bp-deletion as variants (VAR). We hypothesized that this deletion disrupts the binding site for miR-223, thereby increasing the translation of HSP90B1.Figure 1

Bottom Line: These results were confirmed at the protein level by western blot.Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients.By contrast, the presence of rs2307842 was not related to the outcome.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Hematología, IBSAL, IBMCC, CIC, Universidad de Salamanca, CSIC, Hospital Universitario, Salamanca, Spain. anaerv@hotmail.com.

ABSTRACT

Background: MicroRNAs are known to inhibit gene expression by binding to the 3'UTR of the target transcript. Downregulation of miR-223 has been recently reported to have prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients.

Methods: By applying next-generation sequencing techniques we have detected a common polymorphism (rs2307842), in 24% of CLL patients, which disrupts the binding site for miR-223 in HSP90B1 3'UTR. We investigated whether miR-223 directly targets HSP90B1 through luciferase assays and ectopic expression of miR-223. Quantitative real-time polymerase chain reaction and western blot were used to determine HSP90B1 expression in CLL patients. The relationship between rs2307842 status, HSP90B1 expression and clinico-biological data were assessed.

Results: HSP90B1 is a direct target for miR-223 by interaction with the putative miR-223 binding site. The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients. These results were confirmed at the protein level by western blot. Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients. By contrast, the presence of rs2307842 was not related to the outcome.

Conclusions: HSP90B1 is a direct target gene of miR-223. Our results provide a plausible explanation of why CLL patients harboring miR-223 downregulation are associated with a poor outcome, pointing out HSP90B1 as a new pathogenic mechanism in CLL and a promising therapeutic target.

No MeSH data available.


Related in: MedlinePlus