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MicroRNA-433 negatively regulates the expression of thymidylate synthase (TYMS) responsible for 5-fluorouracil sensitivity in HeLa cells.

Gotanda K, Hirota T, Matsumoto N, Ieiri I - BMC Cancer (2013)

Bottom Line: The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01).The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05).The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Thymidylate synthase (TYMS) is an important folate-dependent enzyme in DNA synthesis and an important target for cancer chemotherapy. High TYMS expression levels in tumors are generally associated with resistance to 5-fluorouracil (5-FU). The cause of the variability in TYMS expression is still not fully understood, however, only a small proportion of the TYMS expression can be explained by TYMS genetic polymorphisms. The purpose of this study is to identify novel microRNAs (miRNAs) which regulate the expression of TYMS and to determine whether miRNAs binding to the 3'-untranslated region (UTR) of TYMS mRNA affect the proliferation of HeLa cells treated with 5-FU.

Methods: An in silico search was performed to find potential binding sites of miRNAs in TYMS mRNA. The efficacy of predicted miRNAs at the 3'-UTR of TYMS mRNA was evaluated using a dual-luciferase reporter assay. TYMS mRNA and protein expression in HeLa cells was quantified with real-time RT-PCR and Western blotting, respectively. The effects of miR-433 on cell proliferative activity were determined by WST-8 assay.

Results: The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01). The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05). Furthermore, miR-433 increased inhibition of cell proliferation in HeLa cells treated with 5-FU at over 2.0 μM.

Conclusion: The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells. This is the first report showing that a miRNA regulating TYMS expression has a significant impact on sensitivity to 5-FU treatment.

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Repressive regulation of luciferase activity including the human 3′-UTR of TYMS by miR-433. Reporter constructs containing the full-length 3′-UTR of TYMS were transiently introduced with 3.0 nM of pre-miR-negative control (NC) or pre-miR-433 (miR-433) into HeLa cells. Luciferase activity was normalized to Renilla luciferase activity. Relative luciferase activity is expressed as a ratio of the Renilla luciferase activity of each precursor. Each column represents the mean ± S.E. for three independent experiments. *, P < 0.01, compared with the precursor for the negative control determined by an unpaired two-sample t-test. †, P < 0.05, compared with pGL3 + 6-bp deletion for the negative control determined by an unpaired two-sample t-test. N.S.: not significant.
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Figure 2: Repressive regulation of luciferase activity including the human 3′-UTR of TYMS by miR-433. Reporter constructs containing the full-length 3′-UTR of TYMS were transiently introduced with 3.0 nM of pre-miR-negative control (NC) or pre-miR-433 (miR-433) into HeLa cells. Luciferase activity was normalized to Renilla luciferase activity. Relative luciferase activity is expressed as a ratio of the Renilla luciferase activity of each precursor. Each column represents the mean ± S.E. for three independent experiments. *, P < 0.01, compared with the precursor for the negative control determined by an unpaired two-sample t-test. †, P < 0.05, compared with pGL3 + 6-bp deletion for the negative control determined by an unpaired two-sample t-test. N.S.: not significant.

Mentions: The sequence complementary to the miR-433-binding site and two full-length human TYMS 3′-UTR sequences (the wild type or 6-bp deletion) were inserted downstream of the luciferase reporter gene. We measured luciferase reporter activity as the effects of repressive post-transcriptional regulation by miR-433. The luciferase vector with the 6-bp deletion allele showed significantly lower activity than the wild-type allele in the negative control miRNA-transfected cells (P < 0.05, Figure 2). On the other hand, over expression of miR-433 significantly decreased the reporter activity in the cells with the 3′-UTR-inserted vectors compared to the negative control.


MicroRNA-433 negatively regulates the expression of thymidylate synthase (TYMS) responsible for 5-fluorouracil sensitivity in HeLa cells.

Gotanda K, Hirota T, Matsumoto N, Ieiri I - BMC Cancer (2013)

Repressive regulation of luciferase activity including the human 3′-UTR of TYMS by miR-433. Reporter constructs containing the full-length 3′-UTR of TYMS were transiently introduced with 3.0 nM of pre-miR-negative control (NC) or pre-miR-433 (miR-433) into HeLa cells. Luciferase activity was normalized to Renilla luciferase activity. Relative luciferase activity is expressed as a ratio of the Renilla luciferase activity of each precursor. Each column represents the mean ± S.E. for three independent experiments. *, P < 0.01, compared with the precursor for the negative control determined by an unpaired two-sample t-test. †, P < 0.05, compared with pGL3 + 6-bp deletion for the negative control determined by an unpaired two-sample t-test. N.S.: not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750578&req=5

Figure 2: Repressive regulation of luciferase activity including the human 3′-UTR of TYMS by miR-433. Reporter constructs containing the full-length 3′-UTR of TYMS were transiently introduced with 3.0 nM of pre-miR-negative control (NC) or pre-miR-433 (miR-433) into HeLa cells. Luciferase activity was normalized to Renilla luciferase activity. Relative luciferase activity is expressed as a ratio of the Renilla luciferase activity of each precursor. Each column represents the mean ± S.E. for three independent experiments. *, P < 0.01, compared with the precursor for the negative control determined by an unpaired two-sample t-test. †, P < 0.05, compared with pGL3 + 6-bp deletion for the negative control determined by an unpaired two-sample t-test. N.S.: not significant.
Mentions: The sequence complementary to the miR-433-binding site and two full-length human TYMS 3′-UTR sequences (the wild type or 6-bp deletion) were inserted downstream of the luciferase reporter gene. We measured luciferase reporter activity as the effects of repressive post-transcriptional regulation by miR-433. The luciferase vector with the 6-bp deletion allele showed significantly lower activity than the wild-type allele in the negative control miRNA-transfected cells (P < 0.05, Figure 2). On the other hand, over expression of miR-433 significantly decreased the reporter activity in the cells with the 3′-UTR-inserted vectors compared to the negative control.

Bottom Line: The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01).The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05).The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Thymidylate synthase (TYMS) is an important folate-dependent enzyme in DNA synthesis and an important target for cancer chemotherapy. High TYMS expression levels in tumors are generally associated with resistance to 5-fluorouracil (5-FU). The cause of the variability in TYMS expression is still not fully understood, however, only a small proportion of the TYMS expression can be explained by TYMS genetic polymorphisms. The purpose of this study is to identify novel microRNAs (miRNAs) which regulate the expression of TYMS and to determine whether miRNAs binding to the 3'-untranslated region (UTR) of TYMS mRNA affect the proliferation of HeLa cells treated with 5-FU.

Methods: An in silico search was performed to find potential binding sites of miRNAs in TYMS mRNA. The efficacy of predicted miRNAs at the 3'-UTR of TYMS mRNA was evaluated using a dual-luciferase reporter assay. TYMS mRNA and protein expression in HeLa cells was quantified with real-time RT-PCR and Western blotting, respectively. The effects of miR-433 on cell proliferative activity were determined by WST-8 assay.

Results: The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01). The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05). Furthermore, miR-433 increased inhibition of cell proliferation in HeLa cells treated with 5-FU at over 2.0 μM.

Conclusion: The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells. This is the first report showing that a miRNA regulating TYMS expression has a significant impact on sensitivity to 5-FU treatment.

Show MeSH
Related in: MedlinePlus