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Downregulation of miR-217 correlates with resistance of Ph(+) leukemia cells to ABL tyrosine kinase inhibitors.

Nishioka C, Ikezoe T, Yang J, Nobumoto A, Tsuda M, Yokoyama A - Cancer Sci. (2014)

Bottom Line: Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3'-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI.In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune-deficient mice that were treated with the combination of 5-AzadC and dasatinib.Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKI.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Japan.

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Forced expression of miR-217 sensitizes dasatinib-resistant K562 (K562DR) cells to dasatinib. (a) The sequence of miR-217 and its potential matching site in the DNMT3A 3′-UTR. DNMT3A 3′-UTR mutant vector with deletion of 4 bp (CAUG) in the binding site of miR-217 was generated by using the PrimeSTAR Mutagenesis Basal Kit. (b) Reporter gene assay. miR-217 overexpressed K562DR cells were transfected with either DNMT3A 3′-UTR WT or mutant luciferase reporter vector. The pRL-SV40-Luciferase (Renilla luciferase) vector was co-transfected for normalization. After 48 h, cells were harvested and subjected to the reporter assay. (c) Real-time RT-PCR. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (d) Western blot analysis. K562DR cells were transfected with control or miR-217 expression vector. These cells were subjected to western blot analysis to monitor the levels of the indicated proteins. Each lane was loaded with 30 μg of whole protein lysate. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). (e) MTT assay. K562DR cells were transfected with control or miR-217 expression vector. These cells were plated in 96-well plates and cultured with dasatinib (1, 5 or 10 nM). After 96, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Downregulation of DNMT3A in K562DR cells sensitizes these cells to dasatinib. (f) Real-time RT-PCR. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. RNA was extracted from these cells. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (g) MTT assay. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. These cells were plated in 96-well plates and cultured with dasatinib (10 nM). At the indicated time point, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Mut, mutation; WT, wild type.
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fig02: Forced expression of miR-217 sensitizes dasatinib-resistant K562 (K562DR) cells to dasatinib. (a) The sequence of miR-217 and its potential matching site in the DNMT3A 3′-UTR. DNMT3A 3′-UTR mutant vector with deletion of 4 bp (CAUG) in the binding site of miR-217 was generated by using the PrimeSTAR Mutagenesis Basal Kit. (b) Reporter gene assay. miR-217 overexpressed K562DR cells were transfected with either DNMT3A 3′-UTR WT or mutant luciferase reporter vector. The pRL-SV40-Luciferase (Renilla luciferase) vector was co-transfected for normalization. After 48 h, cells were harvested and subjected to the reporter assay. (c) Real-time RT-PCR. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (d) Western blot analysis. K562DR cells were transfected with control or miR-217 expression vector. These cells were subjected to western blot analysis to monitor the levels of the indicated proteins. Each lane was loaded with 30 μg of whole protein lysate. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). (e) MTT assay. K562DR cells were transfected with control or miR-217 expression vector. These cells were plated in 96-well plates and cultured with dasatinib (1, 5 or 10 nM). After 96, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Downregulation of DNMT3A in K562DR cells sensitizes these cells to dasatinib. (f) Real-time RT-PCR. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. RNA was extracted from these cells. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (g) MTT assay. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. These cells were plated in 96-well plates and cultured with dasatinib (10 nM). At the indicated time point, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Mut, mutation; WT, wild type.

Mentions: Recent studies show that miR-29b and miR-140 regulate expression of DNMT, including DNMT1 and DNMT3A.15–18 However, the role of miR-217 in the regulation of DNMT remains unknown. Computational miRNA target analysis from miRNA databases identified homology between miR-217 and the 3′-UTR of the human DNMT3A gene, but not DNMT1 genes, prompting us to elucidate the link between miR-217 and DNMT3A (Fig.2a). We explored the effect of miR-217 on transcriptional activity of DNMT3A by using DNMT3A 3′-UTR luciferase reporter vector (Fig.2b). The luciferase activity in miR-217 stably expressing K562DR cells were less than that in control miRNA transfected cells (Fig.2b, Fig. S2). We further deleted four nucleotides (CAUG) from the miR-217 binding site of DNMT3A 3′-UTR and transfected this mutant construct in miR-217 stably expressing K562DR cells (Fig.2b). This mutant abrogated the miR-217/DNMT3A interaction, as evidenced by unchanged luciferase activity (Fig.2b). These results suggest that miR-217 negatively regulates expression of DNMT3A in leukemia cells. In fact, downregulated levels of DNMT3A mRNA and protein were noted in miR-217 stably expressing K562DR cells (Fig.2c,d). Intriguingly, forced expression of miR-217 in K562DR cells sensitized these cells to dasatinib (5 or 10 nM, 96 h), as measured by the MTT assay (Fig.2e). To explore the involvement of the elevated levels of DNMT3A in the acquisition of drug resistance of K562DR cells, we suppressed DNMT3A by siRNA and tested the sensitivity of these cells to dasatinib. Both DNMT3A siRNA1 and DNMT3A siRNA2 effectively downregulated the levels of DNMT3A in K562DR cells (Fig.2f). As expected, DNMT3A-depleted K562DR cells regained sensitivity to dasatinib (Fig.2g).


Downregulation of miR-217 correlates with resistance of Ph(+) leukemia cells to ABL tyrosine kinase inhibitors.

Nishioka C, Ikezoe T, Yang J, Nobumoto A, Tsuda M, Yokoyama A - Cancer Sci. (2014)

Forced expression of miR-217 sensitizes dasatinib-resistant K562 (K562DR) cells to dasatinib. (a) The sequence of miR-217 and its potential matching site in the DNMT3A 3′-UTR. DNMT3A 3′-UTR mutant vector with deletion of 4 bp (CAUG) in the binding site of miR-217 was generated by using the PrimeSTAR Mutagenesis Basal Kit. (b) Reporter gene assay. miR-217 overexpressed K562DR cells were transfected with either DNMT3A 3′-UTR WT or mutant luciferase reporter vector. The pRL-SV40-Luciferase (Renilla luciferase) vector was co-transfected for normalization. After 48 h, cells were harvested and subjected to the reporter assay. (c) Real-time RT-PCR. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (d) Western blot analysis. K562DR cells were transfected with control or miR-217 expression vector. These cells were subjected to western blot analysis to monitor the levels of the indicated proteins. Each lane was loaded with 30 μg of whole protein lysate. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). (e) MTT assay. K562DR cells were transfected with control or miR-217 expression vector. These cells were plated in 96-well plates and cultured with dasatinib (1, 5 or 10 nM). After 96, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Downregulation of DNMT3A in K562DR cells sensitizes these cells to dasatinib. (f) Real-time RT-PCR. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. RNA was extracted from these cells. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (g) MTT assay. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. These cells were plated in 96-well plates and cultured with dasatinib (10 nM). At the indicated time point, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Mut, mutation; WT, wild type.
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fig02: Forced expression of miR-217 sensitizes dasatinib-resistant K562 (K562DR) cells to dasatinib. (a) The sequence of miR-217 and its potential matching site in the DNMT3A 3′-UTR. DNMT3A 3′-UTR mutant vector with deletion of 4 bp (CAUG) in the binding site of miR-217 was generated by using the PrimeSTAR Mutagenesis Basal Kit. (b) Reporter gene assay. miR-217 overexpressed K562DR cells were transfected with either DNMT3A 3′-UTR WT or mutant luciferase reporter vector. The pRL-SV40-Luciferase (Renilla luciferase) vector was co-transfected for normalization. After 48 h, cells were harvested and subjected to the reporter assay. (c) Real-time RT-PCR. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (d) Western blot analysis. K562DR cells were transfected with control or miR-217 expression vector. These cells were subjected to western blot analysis to monitor the levels of the indicated proteins. Each lane was loaded with 30 μg of whole protein lysate. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). (e) MTT assay. K562DR cells were transfected with control or miR-217 expression vector. These cells were plated in 96-well plates and cultured with dasatinib (1, 5 or 10 nM). After 96, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Downregulation of DNMT3A in K562DR cells sensitizes these cells to dasatinib. (f) Real-time RT-PCR. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. RNA was extracted from these cells. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (g) MTT assay. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. These cells were plated in 96-well plates and cultured with dasatinib (10 nM). At the indicated time point, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Mut, mutation; WT, wild type.
Mentions: Recent studies show that miR-29b and miR-140 regulate expression of DNMT, including DNMT1 and DNMT3A.15–18 However, the role of miR-217 in the regulation of DNMT remains unknown. Computational miRNA target analysis from miRNA databases identified homology between miR-217 and the 3′-UTR of the human DNMT3A gene, but not DNMT1 genes, prompting us to elucidate the link between miR-217 and DNMT3A (Fig.2a). We explored the effect of miR-217 on transcriptional activity of DNMT3A by using DNMT3A 3′-UTR luciferase reporter vector (Fig.2b). The luciferase activity in miR-217 stably expressing K562DR cells were less than that in control miRNA transfected cells (Fig.2b, Fig. S2). We further deleted four nucleotides (CAUG) from the miR-217 binding site of DNMT3A 3′-UTR and transfected this mutant construct in miR-217 stably expressing K562DR cells (Fig.2b). This mutant abrogated the miR-217/DNMT3A interaction, as evidenced by unchanged luciferase activity (Fig.2b). These results suggest that miR-217 negatively regulates expression of DNMT3A in leukemia cells. In fact, downregulated levels of DNMT3A mRNA and protein were noted in miR-217 stably expressing K562DR cells (Fig.2c,d). Intriguingly, forced expression of miR-217 in K562DR cells sensitized these cells to dasatinib (5 or 10 nM, 96 h), as measured by the MTT assay (Fig.2e). To explore the involvement of the elevated levels of DNMT3A in the acquisition of drug resistance of K562DR cells, we suppressed DNMT3A by siRNA and tested the sensitivity of these cells to dasatinib. Both DNMT3A siRNA1 and DNMT3A siRNA2 effectively downregulated the levels of DNMT3A in K562DR cells (Fig.2f). As expected, DNMT3A-depleted K562DR cells regained sensitivity to dasatinib (Fig.2g).

Bottom Line: Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3'-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI.In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune-deficient mice that were treated with the combination of 5-AzadC and dasatinib.Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKI.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Japan.

Show MeSH
Related in: MedlinePlus