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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

Targeting of PTEN by miR-144 and miR-136.(a) Cell lysates prepared from the VerUTR and control cells were analyzed on western blot probed with anti-PTEN antibody. Increased PTEN expression was detected in the VerUTR cells, while protein loading is equal as exhibited by actin staining. (b) Lysates prepared from lung and kidney of VerUTR transgenic and wildtype mice were probed with anti-PTEN antibody by western blot analysis. Increased PTEN expression was observed in the organs of the VerUTR transgenic mice. (c) 4T1 cells were transiently transfected with VerUTR and siRNAs against 3′UTR or control sequence. As a negative control, 4T1 cells were also transfected with control vector and control sequence. Lysates were prepared from transfected cells and analyzed by western blot probed with anti-PTEN and anti-actin antibodies. Decreased PTEN expression was observed when the VerUTR was co-transfected with siRNAs against VerUTR. (d) Hypothetical targeting site by miR-144 in the 3′UTR of PTEN was cloned and mutated (in red), generating two constructs luc-PTEN-144 and luc-PTEN-144-mut. In luciferase activity assays, U343 cells were co-transfected with luc-PTEN-144 or luc-PTEN-144-mut with miR-144. Luciferase activity of mutated construct was higher than that of the construct expressing original 3′UTR. **, p<0.001. Error bars, SD (n = 3). (e) miR-136's potential targeting sequence in the 3′UTR of PTEN was cloned and mutated (in red), generating two constructs luc-PTEN-136 and luc-PTEN-136-mut. In luciferase activity assays, U343 cells were co-transfected with luc-PTEN-136 or luc-PTEN-136-mut with miR-136. Repression on luciferase activity was removed when miR-136 targeting site was mutated. **, p<0.001. Error bars, SD (n = 3). (f) Luciferase reporter vector harboring the PTEN 3′UTR was co-transfected with VerUTR construct in increasing amounts of plasmid DNA complemented with the control vector in U343 cells. Increased rations of VerUTR bound more endogenous miRNAs and thus freeing the translation of luciferase protein, resulting in higher levels of luciferase activities.
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pone-0013599-g004: Targeting of PTEN by miR-144 and miR-136.(a) Cell lysates prepared from the VerUTR and control cells were analyzed on western blot probed with anti-PTEN antibody. Increased PTEN expression was detected in the VerUTR cells, while protein loading is equal as exhibited by actin staining. (b) Lysates prepared from lung and kidney of VerUTR transgenic and wildtype mice were probed with anti-PTEN antibody by western blot analysis. Increased PTEN expression was observed in the organs of the VerUTR transgenic mice. (c) 4T1 cells were transiently transfected with VerUTR and siRNAs against 3′UTR or control sequence. As a negative control, 4T1 cells were also transfected with control vector and control sequence. Lysates were prepared from transfected cells and analyzed by western blot probed with anti-PTEN and anti-actin antibodies. Decreased PTEN expression was observed when the VerUTR was co-transfected with siRNAs against VerUTR. (d) Hypothetical targeting site by miR-144 in the 3′UTR of PTEN was cloned and mutated (in red), generating two constructs luc-PTEN-144 and luc-PTEN-144-mut. In luciferase activity assays, U343 cells were co-transfected with luc-PTEN-144 or luc-PTEN-144-mut with miR-144. Luciferase activity of mutated construct was higher than that of the construct expressing original 3′UTR. **, p<0.001. Error bars, SD (n = 3). (e) miR-136's potential targeting sequence in the 3′UTR of PTEN was cloned and mutated (in red), generating two constructs luc-PTEN-136 and luc-PTEN-136-mut. In luciferase activity assays, U343 cells were co-transfected with luc-PTEN-136 or luc-PTEN-136-mut with miR-136. Repression on luciferase activity was removed when miR-136 targeting site was mutated. **, p<0.001. Error bars, SD (n = 3). (f) Luciferase reporter vector harboring the PTEN 3′UTR was co-transfected with VerUTR construct in increasing amounts of plasmid DNA complemented with the control vector in U343 cells. Increased rations of VerUTR bound more endogenous miRNAs and thus freeing the translation of luciferase protein, resulting in higher levels of luciferase activities.

Mentions: Another gene of interest was Phosphotase and Tensin Homolog, or PTEN. PTEN has been shown to function as a tumor suppressor, where loss or mutation of this gene leads to cancer predisposition [31]. Similar to Rb1, PTEN is also known as a negative cell cycle regulator by arresting cells in G1 phase [32]. Increased expression of PTEN resulted in decreased cell proliferation. To confirm these results, pooled cells transfected with VerUTR or the control vector were lysed and probed with anti-PTEN antibody for western blot analysis. We observed an elevation of PTEN expression in the VerUTR-transfected cells (Figure 4a). When primary tissues were examined for PTEN expression, similar results were obtained but were less significant in kidney tissues (Figure 4b). Staining of PTEN in the primary lung tissues also yield a similar result, while expression of other proteins were not influenced (not shown). To confirm that VerUTR could antagonize PTEN for miRNA binding, we performed knockdown experiments using siRNA against versican 3′UTR and detected an elevation of PTEN expression in the VerUTR-transfected cells (Figure 4c). Knockdown of VerUTR by siRNA abolished the increased PTEN expression to the same level seen in cells transfected with the control vector.


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)

Targeting of PTEN by miR-144 and miR-136.(a) Cell lysates prepared from the VerUTR and control cells were analyzed on western blot probed with anti-PTEN antibody. Increased PTEN expression was detected in the VerUTR cells, while protein loading is equal as exhibited by actin staining. (b) Lysates prepared from lung and kidney of VerUTR transgenic and wildtype mice were probed with anti-PTEN antibody by western blot analysis. Increased PTEN expression was observed in the organs of the VerUTR transgenic mice. (c) 4T1 cells were transiently transfected with VerUTR and siRNAs against 3′UTR or control sequence. As a negative control, 4T1 cells were also transfected with control vector and control sequence. Lysates were prepared from transfected cells and analyzed by western blot probed with anti-PTEN and anti-actin antibodies. Decreased PTEN expression was observed when the VerUTR was co-transfected with siRNAs against VerUTR. (d) Hypothetical targeting site by miR-144 in the 3′UTR of PTEN was cloned and mutated (in red), generating two constructs luc-PTEN-144 and luc-PTEN-144-mut. In luciferase activity assays, U343 cells were co-transfected with luc-PTEN-144 or luc-PTEN-144-mut with miR-144. Luciferase activity of mutated construct was higher than that of the construct expressing original 3′UTR. **, p<0.001. Error bars, SD (n = 3). (e) miR-136's potential targeting sequence in the 3′UTR of PTEN was cloned and mutated (in red), generating two constructs luc-PTEN-136 and luc-PTEN-136-mut. In luciferase activity assays, U343 cells were co-transfected with luc-PTEN-136 or luc-PTEN-136-mut with miR-136. Repression on luciferase activity was removed when miR-136 targeting site was mutated. **, p<0.001. Error bars, SD (n = 3). (f) Luciferase reporter vector harboring the PTEN 3′UTR was co-transfected with VerUTR construct in increasing amounts of plasmid DNA complemented with the control vector in U343 cells. Increased rations of VerUTR bound more endogenous miRNAs and thus freeing the translation of luciferase protein, resulting in higher levels of luciferase activities.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2963607&req=5

pone-0013599-g004: Targeting of PTEN by miR-144 and miR-136.(a) Cell lysates prepared from the VerUTR and control cells were analyzed on western blot probed with anti-PTEN antibody. Increased PTEN expression was detected in the VerUTR cells, while protein loading is equal as exhibited by actin staining. (b) Lysates prepared from lung and kidney of VerUTR transgenic and wildtype mice were probed with anti-PTEN antibody by western blot analysis. Increased PTEN expression was observed in the organs of the VerUTR transgenic mice. (c) 4T1 cells were transiently transfected with VerUTR and siRNAs against 3′UTR or control sequence. As a negative control, 4T1 cells were also transfected with control vector and control sequence. Lysates were prepared from transfected cells and analyzed by western blot probed with anti-PTEN and anti-actin antibodies. Decreased PTEN expression was observed when the VerUTR was co-transfected with siRNAs against VerUTR. (d) Hypothetical targeting site by miR-144 in the 3′UTR of PTEN was cloned and mutated (in red), generating two constructs luc-PTEN-144 and luc-PTEN-144-mut. In luciferase activity assays, U343 cells were co-transfected with luc-PTEN-144 or luc-PTEN-144-mut with miR-144. Luciferase activity of mutated construct was higher than that of the construct expressing original 3′UTR. **, p<0.001. Error bars, SD (n = 3). (e) miR-136's potential targeting sequence in the 3′UTR of PTEN was cloned and mutated (in red), generating two constructs luc-PTEN-136 and luc-PTEN-136-mut. In luciferase activity assays, U343 cells were co-transfected with luc-PTEN-136 or luc-PTEN-136-mut with miR-136. Repression on luciferase activity was removed when miR-136 targeting site was mutated. **, p<0.001. Error bars, SD (n = 3). (f) Luciferase reporter vector harboring the PTEN 3′UTR was co-transfected with VerUTR construct in increasing amounts of plasmid DNA complemented with the control vector in U343 cells. Increased rations of VerUTR bound more endogenous miRNAs and thus freeing the translation of luciferase protein, resulting in higher levels of luciferase activities.
Mentions: Another gene of interest was Phosphotase and Tensin Homolog, or PTEN. PTEN has been shown to function as a tumor suppressor, where loss or mutation of this gene leads to cancer predisposition [31]. Similar to Rb1, PTEN is also known as a negative cell cycle regulator by arresting cells in G1 phase [32]. Increased expression of PTEN resulted in decreased cell proliferation. To confirm these results, pooled cells transfected with VerUTR or the control vector were lysed and probed with anti-PTEN antibody for western blot analysis. We observed an elevation of PTEN expression in the VerUTR-transfected cells (Figure 4a). When primary tissues were examined for PTEN expression, similar results were obtained but were less significant in kidney tissues (Figure 4b). Staining of PTEN in the primary lung tissues also yield a similar result, while expression of other proteins were not influenced (not shown). To confirm that VerUTR could antagonize PTEN for miRNA binding, we performed knockdown experiments using siRNA against versican 3′UTR and detected an elevation of PTEN expression in the VerUTR-transfected cells (Figure 4c). Knockdown of VerUTR by siRNA abolished the increased PTEN expression to the same level seen in cells transfected with the control vector.

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