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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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Effects of overexpression of miR-433 on TYMS mRNA levels in HeLa cells. Hela cells were transfected with pre-miR-negative control (NC), pre-miR-433 (miR-433) or TYMS siRNA (0.75, 1.5 and 3.0 nM). The relative TYMS mRNA level was normalized with β-actin. Results represent the mean ± S.D. for four independent experiments. Relative TYMS mRNA levels were expressed as ratios of the relative TYMS mRNA of the precursor for the negative control. *, P < 0.05, compared with the negative control determined by Dunnett’s multiple comparison tests.
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Figure 3: Effects of overexpression of miR-433 on TYMS mRNA levels in HeLa cells. Hela cells were transfected with pre-miR-negative control (NC), pre-miR-433 (miR-433) or TYMS siRNA (0.75, 1.5 and 3.0 nM). The relative TYMS mRNA level was normalized with β-actin. Results represent the mean ± S.D. for four independent experiments. Relative TYMS mRNA levels were expressed as ratios of the relative TYMS mRNA of the precursor for the negative control. *, P < 0.05, compared with the negative control determined by Dunnett’s multiple comparison tests.

Mentions: To verify the effect of the overexpression of miR-433 on TYMS mRNA levels, HeLa cells were temporarily transfected with the pre-miR-negative control, pre-miR-433 or TYMS siRNA (positive control) and TYMS mRNA levels were quantitated by real-time RT-PCR analysis using ACTB for normalization. The overexpression of miR-433 significantly decreased TYMS mRNA levels in a precursor concentration-dependent manner; reduced by approximately 50% at 3.0 nM of pre-miR-433 as compared with the negative control (Figure 3).


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)

Effects of overexpression of miR-433 on TYMS mRNA levels in HeLa cells. Hela cells were transfected with pre-miR-negative control (NC), pre-miR-433 (miR-433) or TYMS siRNA (0.75, 1.5 and 3.0 nM). The relative TYMS mRNA level was normalized with β-actin. Results represent the mean ± S.D. for four independent experiments. Relative TYMS mRNA levels were expressed as ratios of the relative TYMS mRNA of the precursor for the negative control. *, P < 0.05, compared with the negative control determined by Dunnett’s multiple comparison tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effects of overexpression of miR-433 on TYMS mRNA levels in HeLa cells. Hela cells were transfected with pre-miR-negative control (NC), pre-miR-433 (miR-433) or TYMS siRNA (0.75, 1.5 and 3.0 nM). The relative TYMS mRNA level was normalized with β-actin. Results represent the mean ± S.D. for four independent experiments. Relative TYMS mRNA levels were expressed as ratios of the relative TYMS mRNA of the precursor for the negative control. *, P < 0.05, compared with the negative control determined by Dunnett’s multiple comparison tests.
Mentions: To verify the effect of the overexpression of miR-433 on TYMS mRNA levels, HeLa cells were temporarily transfected with the pre-miR-negative control, pre-miR-433 or TYMS siRNA (positive control) and TYMS mRNA levels were quantitated by real-time RT-PCR analysis using ACTB for normalization. The overexpression of miR-433 significantly decreased TYMS mRNA levels in a precursor concentration-dependent manner; reduced by approximately 50% at 3.0 nM of pre-miR-433 as compared with the negative control (Figure 3).

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