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Expression of versican 3'-untranslated region modulates endogenous microRNA functions.

Lee DY, Jeyapalan Z, Fang L, Yang J, Zhang Y, Yee AY, Li M, Du WW, Shatseva T, Yang BB - PLoS ONE (2010)

Bottom Line: We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels.In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN.Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions.

View Article: PubMed Central - PubMed

Affiliation: Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada.

ABSTRACT

Background: Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined.

Methods and findings: Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector.

Conclusion: Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities.

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Related in: MedlinePlus

Expression of VerUTR affects miRNA levels.RNAs from pooled cell lines and primary tissues of the VerUTR transgenic and wild type mice were isolated. By Real-time PCR, relative quantities of miRNAs were determined and compared with the use of miRNA-specific primers. Primary organs from twelve mice at the age between four to six months were analyzed in these experiments. Quantitative values of miRNA levels were normalized with U6 RNA levels for comparison. Representative data is shown. (a) In the lung tissues, the levels of miR-199a-3p were about 39% lower in the VerUTR transgenic mice compared with that in the wildtype. In contrast, there was no significant difference between transgenic and wildtype kidney tissues. **, p<0.01. Error bars, SD (n = 3). (b) The levels of miR-199a-3p were compared between primary tissues and cancer cell lines. There is a 29% reduction in miR-199a-3p levels in the liver tissues of transgenic mice compared with that of wildtype. In the 4T1 cell line, cells transfected with VerUTR showed 73% reduction of miR-199a-3p compared with cells transfected with the control vector. **, p<0.01. Error bars, SD (n = 4). (c) Levels of miR-136 were significantly lowered in the lung tissues of transgenic mice compared with that of wildtype by 40%. There was no significant difference in miR-136 levels between the cells transfected with either VerUTR or the control vector. *, p<0.05. Error bars, SD (n = 3). (d) Expression of miR-136 in lung and kidney tissues of the same mice was inspected. *, p<0.05. Error bars, SD (n = 3). (e) Expression of unrelated microRNAs miR-17-5p and miR-328 were analyzed to ensure that the subjects are comparable and the levels of other miRNAs were not affected.
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pone-0013599-g005: Expression of VerUTR affects miRNA levels.RNAs from pooled cell lines and primary tissues of the VerUTR transgenic and wild type mice were isolated. By Real-time PCR, relative quantities of miRNAs were determined and compared with the use of miRNA-specific primers. Primary organs from twelve mice at the age between four to six months were analyzed in these experiments. Quantitative values of miRNA levels were normalized with U6 RNA levels for comparison. Representative data is shown. (a) In the lung tissues, the levels of miR-199a-3p were about 39% lower in the VerUTR transgenic mice compared with that in the wildtype. In contrast, there was no significant difference between transgenic and wildtype kidney tissues. **, p<0.01. Error bars, SD (n = 3). (b) The levels of miR-199a-3p were compared between primary tissues and cancer cell lines. There is a 29% reduction in miR-199a-3p levels in the liver tissues of transgenic mice compared with that of wildtype. In the 4T1 cell line, cells transfected with VerUTR showed 73% reduction of miR-199a-3p compared with cells transfected with the control vector. **, p<0.01. Error bars, SD (n = 4). (c) Levels of miR-136 were significantly lowered in the lung tissues of transgenic mice compared with that of wildtype by 40%. There was no significant difference in miR-136 levels between the cells transfected with either VerUTR or the control vector. *, p<0.05. Error bars, SD (n = 3). (d) Expression of miR-136 in lung and kidney tissues of the same mice was inspected. *, p<0.05. Error bars, SD (n = 3). (e) Expression of unrelated microRNAs miR-17-5p and miR-328 were analyzed to ensure that the subjects are comparable and the levels of other miRNAs were not affected.

Mentions: In our previous studies, miR-199a-3p was found to regulate two extracellular matrix proteins, versican and fibronectin [24], and it has also been shown to be important during development [34]. Expression of miR-199a-3p was analyzed in the primary lung and kidney of VerUTR transgenic and wildtype mice. Reduction in miRNA expression was observed in these primary tissues. There was a 39% reduction in miR-199a-3p expression in lung tissues but no significant changes were found between the transgenic and wildtype kidney tissues (Figure 5a). We reasoned that different tissues might exhibit different levels of miR-199a-3p expression, but more importantly, different tissues might respond differently to the presence of 3′UTR. Using primary liver tissues as another example, we detected a 29% reduction in miR-199a-3p expression in the presence of 3′UTR (Figure 5b). In addition, cells transfected with 3′UTR lost 79% of its miR-199a-3p compared with the control cells.


Expression of versican 3'-untranslated region modulates endogenous microRNA functions.

Lee DY, Jeyapalan Z, Fang L, Yang J, Zhang Y, Yee AY, Li M, Du WW, Shatseva T, Yang BB - PLoS ONE (2010)

Expression of VerUTR affects miRNA levels.RNAs from pooled cell lines and primary tissues of the VerUTR transgenic and wild type mice were isolated. By Real-time PCR, relative quantities of miRNAs were determined and compared with the use of miRNA-specific primers. Primary organs from twelve mice at the age between four to six months were analyzed in these experiments. Quantitative values of miRNA levels were normalized with U6 RNA levels for comparison. Representative data is shown. (a) In the lung tissues, the levels of miR-199a-3p were about 39% lower in the VerUTR transgenic mice compared with that in the wildtype. In contrast, there was no significant difference between transgenic and wildtype kidney tissues. **, p<0.01. Error bars, SD (n = 3). (b) The levels of miR-199a-3p were compared between primary tissues and cancer cell lines. There is a 29% reduction in miR-199a-3p levels in the liver tissues of transgenic mice compared with that of wildtype. In the 4T1 cell line, cells transfected with VerUTR showed 73% reduction of miR-199a-3p compared with cells transfected with the control vector. **, p<0.01. Error bars, SD (n = 4). (c) Levels of miR-136 were significantly lowered in the lung tissues of transgenic mice compared with that of wildtype by 40%. There was no significant difference in miR-136 levels between the cells transfected with either VerUTR or the control vector. *, p<0.05. Error bars, SD (n = 3). (d) Expression of miR-136 in lung and kidney tissues of the same mice was inspected. *, p<0.05. Error bars, SD (n = 3). (e) Expression of unrelated microRNAs miR-17-5p and miR-328 were analyzed to ensure that the subjects are comparable and the levels of other miRNAs were not affected.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2963607&req=5

pone-0013599-g005: Expression of VerUTR affects miRNA levels.RNAs from pooled cell lines and primary tissues of the VerUTR transgenic and wild type mice were isolated. By Real-time PCR, relative quantities of miRNAs were determined and compared with the use of miRNA-specific primers. Primary organs from twelve mice at the age between four to six months were analyzed in these experiments. Quantitative values of miRNA levels were normalized with U6 RNA levels for comparison. Representative data is shown. (a) In the lung tissues, the levels of miR-199a-3p were about 39% lower in the VerUTR transgenic mice compared with that in the wildtype. In contrast, there was no significant difference between transgenic and wildtype kidney tissues. **, p<0.01. Error bars, SD (n = 3). (b) The levels of miR-199a-3p were compared between primary tissues and cancer cell lines. There is a 29% reduction in miR-199a-3p levels in the liver tissues of transgenic mice compared with that of wildtype. In the 4T1 cell line, cells transfected with VerUTR showed 73% reduction of miR-199a-3p compared with cells transfected with the control vector. **, p<0.01. Error bars, SD (n = 4). (c) Levels of miR-136 were significantly lowered in the lung tissues of transgenic mice compared with that of wildtype by 40%. There was no significant difference in miR-136 levels between the cells transfected with either VerUTR or the control vector. *, p<0.05. Error bars, SD (n = 3). (d) Expression of miR-136 in lung and kidney tissues of the same mice was inspected. *, p<0.05. Error bars, SD (n = 3). (e) Expression of unrelated microRNAs miR-17-5p and miR-328 were analyzed to ensure that the subjects are comparable and the levels of other miRNAs were not affected.
Mentions: In our previous studies, miR-199a-3p was found to regulate two extracellular matrix proteins, versican and fibronectin [24], and it has also been shown to be important during development [34]. Expression of miR-199a-3p was analyzed in the primary lung and kidney of VerUTR transgenic and wildtype mice. Reduction in miRNA expression was observed in these primary tissues. There was a 39% reduction in miR-199a-3p expression in lung tissues but no significant changes were found between the transgenic and wildtype kidney tissues (Figure 5a). We reasoned that different tissues might exhibit different levels of miR-199a-3p expression, but more importantly, different tissues might respond differently to the presence of 3′UTR. Using primary liver tissues as another example, we detected a 29% reduction in miR-199a-3p expression in the presence of 3′UTR (Figure 5b). In addition, cells transfected with 3′UTR lost 79% of its miR-199a-3p compared with the control cells.

Bottom Line: We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels.In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN.Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions.

View Article: PubMed Central - PubMed

Affiliation: Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada.

ABSTRACT

Background: Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined.

Methods and findings: Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector.

Conclusion: Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities.

Show MeSH
Related in: MedlinePlus