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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 versican 3′UTR reduces cell proliferation and tumor growth.(a) Mouse breast carcinoma cells 4T1 was transfected with a fragment of versican 3′UTR (700 bp) or a control vector. Pooled cells were obtained. RNA was isolated from the pooled cells and the expression of 3′UTR was confirmed by RT-PCR. (b) Cell proliferation assays were performed in cells transfected with the control vector or the 3′UTR in low serum conditions (1.5% FBS) by cell counting after trypan blue staining. *, P<0.05. Error bars indicate SD (n = 5). (c) Cell cycle analysis by FACS confirmed that the vector-transfected cells had greater population of G2/M cells than the VerUTR-transfected cells. Representative data is shown. (n = 3). (d) The cells were injected subcutaneously into BALB/c mice. Tumor size was recorded weekly and tumor growth curve was obtained for a period of four weeks. Asterisks indicate significance. *, P<0.05. Error bars indicate SEM (n = 3).
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pone-0013599-g001: Expression of versican 3′UTR reduces cell proliferation and tumor growth.(a) Mouse breast carcinoma cells 4T1 was transfected with a fragment of versican 3′UTR (700 bp) or a control vector. Pooled cells were obtained. RNA was isolated from the pooled cells and the expression of 3′UTR was confirmed by RT-PCR. (b) Cell proliferation assays were performed in cells transfected with the control vector or the 3′UTR in low serum conditions (1.5% FBS) by cell counting after trypan blue staining. *, P<0.05. Error bars indicate SD (n = 5). (c) Cell cycle analysis by FACS confirmed that the vector-transfected cells had greater population of G2/M cells than the VerUTR-transfected cells. Representative data is shown. (n = 3). (d) The cells were injected subcutaneously into BALB/c mice. Tumor size was recorded weekly and tumor growth curve was obtained for a period of four weeks. Asterisks indicate significance. *, P<0.05. Error bars indicate SEM (n = 3).

Mentions: An expression construct was generated to study the function of 3′UTR. The conserved region of versican 3′UTR (2285–3000 bp, Genebank access number, NM_001126336.1) was cloned and inserted in front of a CMV promoter producing the construct VerUTR (Figure 1a). The construct was stably expressed in a mouse breast carcinoma cell line, 4T1, and its expression was confirmed by RT-PCR. This cell line was chosen because of its compatibility with BALB/c mice without rejection of transplanted cells by the host′s immune system. Injecting these cells into the mice represents an isogenic relationship between the host and the tumor cells, and allows the studies of molecularly modified tumor cells. Tumor growth and metastatic invasion induced by the 4T1 cells closely mimic human breast cancer progression, and is an established animal model for stage IV human breast carcinoma.


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 versican 3′UTR reduces cell proliferation and tumor growth.(a) Mouse breast carcinoma cells 4T1 was transfected with a fragment of versican 3′UTR (700 bp) or a control vector. Pooled cells were obtained. RNA was isolated from the pooled cells and the expression of 3′UTR was confirmed by RT-PCR. (b) Cell proliferation assays were performed in cells transfected with the control vector or the 3′UTR in low serum conditions (1.5% FBS) by cell counting after trypan blue staining. *, P<0.05. Error bars indicate SD (n = 5). (c) Cell cycle analysis by FACS confirmed that the vector-transfected cells had greater population of G2/M cells than the VerUTR-transfected cells. Representative data is shown. (n = 3). (d) The cells were injected subcutaneously into BALB/c mice. Tumor size was recorded weekly and tumor growth curve was obtained for a period of four weeks. Asterisks indicate significance. *, P<0.05. Error bars indicate SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2963607&req=5

pone-0013599-g001: Expression of versican 3′UTR reduces cell proliferation and tumor growth.(a) Mouse breast carcinoma cells 4T1 was transfected with a fragment of versican 3′UTR (700 bp) or a control vector. Pooled cells were obtained. RNA was isolated from the pooled cells and the expression of 3′UTR was confirmed by RT-PCR. (b) Cell proliferation assays were performed in cells transfected with the control vector or the 3′UTR in low serum conditions (1.5% FBS) by cell counting after trypan blue staining. *, P<0.05. Error bars indicate SD (n = 5). (c) Cell cycle analysis by FACS confirmed that the vector-transfected cells had greater population of G2/M cells than the VerUTR-transfected cells. Representative data is shown. (n = 3). (d) The cells were injected subcutaneously into BALB/c mice. Tumor size was recorded weekly and tumor growth curve was obtained for a period of four weeks. Asterisks indicate significance. *, P<0.05. Error bars indicate SEM (n = 3).
Mentions: An expression construct was generated to study the function of 3′UTR. The conserved region of versican 3′UTR (2285–3000 bp, Genebank access number, NM_001126336.1) was cloned and inserted in front of a CMV promoter producing the construct VerUTR (Figure 1a). The construct was stably expressed in a mouse breast carcinoma cell line, 4T1, and its expression was confirmed by RT-PCR. This cell line was chosen because of its compatibility with BALB/c mice without rejection of transplanted cells by the host′s immune system. Injecting these cells into the mice represents an isogenic relationship between the host and the tumor cells, and allows the studies of molecularly modified tumor cells. Tumor growth and metastatic invasion induced by the 4T1 cells closely mimic human breast cancer progression, and is an established animal model for stage IV human breast carcinoma.

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