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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

Hsp90b1 is upregulated in CLL patients with the rs2307842 polymorphism andIGHV-unmutated status, as assessed by qRT-PCR and western blot analysis. Box plots show the relative upregulation of HSP90B1 mRNA in CLL patients with (A) rs2307842 (VAR) and (B) IGHV unmutated genes (UM) compared with wild-type CLL patients (WT) and the mutated cases (MUT), respectively. The thick line inside the box plot indicates the median expression levels and the box shows the 25th and 75th percentiles, while the whiskers show the maximum and minimum values. Outliers are represented by open circles. Statistical significance was determined by the Mann–Whitney U test (P < 0.05). (C) Representative lysates of purified B lymphocytes from CLL patients were prepared and Hsp90b1 protein levels were analyzed by western blot. B-actin served as loading control. Representative blots from three CLL patients are shown: #1 patient with IGHV unmutated genes (UM CLL), #2 wild-type for rs2307842 and with IGHV mutated genes (WT&MUT CLL) and #3 patient with rs2307842 (VAR CLL).
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Fig2: Hsp90b1 is upregulated in CLL patients with the rs2307842 polymorphism andIGHV-unmutated status, as assessed by qRT-PCR and western blot analysis. Box plots show the relative upregulation of HSP90B1 mRNA in CLL patients with (A) rs2307842 (VAR) and (B) IGHV unmutated genes (UM) compared with wild-type CLL patients (WT) and the mutated cases (MUT), respectively. The thick line inside the box plot indicates the median expression levels and the box shows the 25th and 75th percentiles, while the whiskers show the maximum and minimum values. Outliers are represented by open circles. Statistical significance was determined by the Mann–Whitney U test (P < 0.05). (C) Representative lysates of purified B lymphocytes from CLL patients were prepared and Hsp90b1 protein levels were analyzed by western blot. B-actin served as loading control. Representative blots from three CLL patients are shown: #1 patient with IGHV unmutated genes (UM CLL), #2 wild-type for rs2307842 and with IGHV mutated genes (WT&MUT CLL) and #3 patient with rs2307842 (VAR CLL).

Mentions: We have performed qRT-PCR in a total of 97 CLL samples: 25 out of them were CLL patients with rs2307842 (VAR-CLLs) and 72 were wild-type (WT-CLLs). qRT-PCR results showed that HSP90B1 was overexpressed in VAR-CLLs (P = 0.001) (Figure 2A). To gain insight into its influence on gene expression, we have measured HSP90B1 mRNA levels in the paired normal fraction of 50 cases (13 VAR-CLLs and 37 WT-CLLs). As expected, the results showed that B lymphocytes (tumor fraction) from VAR-CLLs showed a higher level of HSP90B1 expression than B lymphocytes from WT-CLLs (P = 0.001), and also from the normal cells from the same patients (VAR-CLLs) (P < 0.001) (Additional file 3: Figure S1). However, no changes in HSP90B1 mRNA expression were observed between tumor and normal fractions in CLLs without the SNP (P = 0.201). Thus, rs2307842 influenced HSP90B1 overexpression only in the tumor fraction of the CLL patients with the polymorphism. Of note, we also observed overexpression of HSP90B1 in patients with Figure 2B). The overexpression was also confirmed in the tumor fraction of the purified paired samples (data not shown).IGHV unmutated genes (UM-CLLs, n = 52) in comparison with mutated cases (MUT-CLLs, n = 45) (P = 0.003) (Figure 2B.Figure 2


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)

Hsp90b1 is upregulated in CLL patients with the rs2307842 polymorphism andIGHV-unmutated status, as assessed by qRT-PCR and western blot analysis. Box plots show the relative upregulation of HSP90B1 mRNA in CLL patients with (A) rs2307842 (VAR) and (B) IGHV unmutated genes (UM) compared with wild-type CLL patients (WT) and the mutated cases (MUT), respectively. The thick line inside the box plot indicates the median expression levels and the box shows the 25th and 75th percentiles, while the whiskers show the maximum and minimum values. Outliers are represented by open circles. Statistical significance was determined by the Mann–Whitney U test (P < 0.05). (C) Representative lysates of purified B lymphocytes from CLL patients were prepared and Hsp90b1 protein levels were analyzed by western blot. B-actin served as loading control. Representative blots from three CLL patients are shown: #1 patient with IGHV unmutated genes (UM CLL), #2 wild-type for rs2307842 and with IGHV mutated genes (WT&MUT CLL) and #3 patient with rs2307842 (VAR CLL).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Hsp90b1 is upregulated in CLL patients with the rs2307842 polymorphism andIGHV-unmutated status, as assessed by qRT-PCR and western blot analysis. Box plots show the relative upregulation of HSP90B1 mRNA in CLL patients with (A) rs2307842 (VAR) and (B) IGHV unmutated genes (UM) compared with wild-type CLL patients (WT) and the mutated cases (MUT), respectively. The thick line inside the box plot indicates the median expression levels and the box shows the 25th and 75th percentiles, while the whiskers show the maximum and minimum values. Outliers are represented by open circles. Statistical significance was determined by the Mann–Whitney U test (P < 0.05). (C) Representative lysates of purified B lymphocytes from CLL patients were prepared and Hsp90b1 protein levels were analyzed by western blot. B-actin served as loading control. Representative blots from three CLL patients are shown: #1 patient with IGHV unmutated genes (UM CLL), #2 wild-type for rs2307842 and with IGHV mutated genes (WT&MUT CLL) and #3 patient with rs2307842 (VAR CLL).
Mentions: We have performed qRT-PCR in a total of 97 CLL samples: 25 out of them were CLL patients with rs2307842 (VAR-CLLs) and 72 were wild-type (WT-CLLs). qRT-PCR results showed that HSP90B1 was overexpressed in VAR-CLLs (P = 0.001) (Figure 2A). To gain insight into its influence on gene expression, we have measured HSP90B1 mRNA levels in the paired normal fraction of 50 cases (13 VAR-CLLs and 37 WT-CLLs). As expected, the results showed that B lymphocytes (tumor fraction) from VAR-CLLs showed a higher level of HSP90B1 expression than B lymphocytes from WT-CLLs (P = 0.001), and also from the normal cells from the same patients (VAR-CLLs) (P < 0.001) (Additional file 3: Figure S1). However, no changes in HSP90B1 mRNA expression were observed between tumor and normal fractions in CLLs without the SNP (P = 0.201). Thus, rs2307842 influenced HSP90B1 overexpression only in the tumor fraction of the CLL patients with the polymorphism. Of note, we also observed overexpression of HSP90B1 in patients with Figure 2B). The overexpression was also confirmed in the tumor fraction of the purified paired samples (data not shown).IGHV unmutated genes (UM-CLLs, n = 52) in comparison with mutated cases (MUT-CLLs, n = 45) (P = 0.003) (Figure 2B.Figure 2

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