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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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Prediction of miRNA-binding sites in the 3′-UTR of TYMS mRNA. (A) The locations of the 6-bp deletion (indicated as arrow) and predicted miR-433-binding site in the 3′-UTR of TYMS mRNA. (B) Homology between the 3′-UTR of TYMS mRNA and the predicted target sequence of miR-433. Numbers indicate mRNA positions.
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Figure 1: Prediction of miRNA-binding sites in the 3′-UTR of TYMS mRNA. (A) The locations of the 6-bp deletion (indicated as arrow) and predicted miR-433-binding site in the 3′-UTR of TYMS mRNA. (B) Homology between the 3′-UTR of TYMS mRNA and the predicted target sequence of miR-433. Numbers indicate mRNA positions.

Mentions: To identify miRNAs targeting the 3′-UTR of TYMS mRNA, we used the programs TargetScan (http://www.targetscan.org) and MiRanda (http://www.microrna.org). MiR-433 was selected as a candidate miRNA binding to the 3′-UTR of TYMS mRNA based on matching sites (Figure 1). The 6-bp deletion (rs16430) was located 22 bp downstream from the predicted miR-433-binding site.


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)

Prediction of miRNA-binding sites in the 3′-UTR of TYMS mRNA. (A) The locations of the 6-bp deletion (indicated as arrow) and predicted miR-433-binding site in the 3′-UTR of TYMS mRNA. (B) Homology between the 3′-UTR of TYMS mRNA and the predicted target sequence of miR-433. Numbers indicate mRNA positions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Prediction of miRNA-binding sites in the 3′-UTR of TYMS mRNA. (A) The locations of the 6-bp deletion (indicated as arrow) and predicted miR-433-binding site in the 3′-UTR of TYMS mRNA. (B) Homology between the 3′-UTR of TYMS mRNA and the predicted target sequence of miR-433. Numbers indicate mRNA positions.
Mentions: To identify miRNAs targeting the 3′-UTR of TYMS mRNA, we used the programs TargetScan (http://www.targetscan.org) and MiRanda (http://www.microrna.org). MiR-433 was selected as a candidate miRNA binding to the 3′-UTR of TYMS mRNA based on matching sites (Figure 1). The 6-bp deletion (rs16430) was located 22 bp downstream from the predicted miR-433-binding site.

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