Limits...
Profiling and functional analyses of microRNAs and their target gene products in human uterine leiomyomas.

Zavadil J, Ye H, Liu Z, Wu J, Lee P, Hernando E, Soteropoulos P, Toruner GA, Wei JJ - PLoS ONE (2010)

Bottom Line: We found that a number of dysregulated microRNAs were inversely correlated with their targets at the protein level.Last, we functionally tested the repressor effects of selected cancer-related microRNAs on their predicted target genes in vitro.We found that some but not all of the predicted and inversely correlated target genes in ULMs can be directly regulated by microRNAs in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, New York University, New York, New York, United States of America.

ABSTRACT

Background: Human uterine leiomyomas (ULM) are characterized by dysregulation of a large number of genes and non-coding regulatory microRNAs. In order to identify microRNA::mRNA associations relevant to ULM pathogenesis, we examined global correlation patterns between the altered microRNA expression and the predicted target genes in ULMs and matched myometria.

Methodology/principal findings: Patterns of inverse association of microRNA with mRNA expression in ULMs revealed an involvement of multiple candidate pathways, including extensive transcriptional reprogramming, cell proliferation control, MAP kinase, TGF-beta, WNT, JAK/STAT signaling, remodeling of cell adhesion, and cell-cell and cell-matrix contacts. We further examined the correlation between the expression of the selected target gene protein products and microRNAs in thirty-six paired sets of leiomyomas and matched myometria. We found that a number of dysregulated microRNAs were inversely correlated with their targets at the protein level. The comparative genomic hybridization (CGH) in eight ULM patients revealed that partially shared deletions of two distinct chromosomal regions might be responsible for loss of cancer-associated microRNA expression and could thus contribute to the ULM pathogenesis via deregulation of target mRNAs. Last, we functionally tested the repressor effects of selected cancer-related microRNAs on their predicted target genes in vitro.

Conclusions/significance: We found that some but not all of the predicted and inversely correlated target genes in ULMs can be directly regulated by microRNAs in vitro. Our findings provide a broad overview of molecular events underlying the tumorigenesis of uterine ULMs and identify select genetic and regulatory events that alter microRNA expression and may play important roles in ULM pathobiology by positively regulating tumor growth while maintaining the non-invasive character of ULMs.

Show MeSH

Related in: MedlinePlus

Analysis of the miR-296 predicted target gene TSC2 and 11 let-7 predicted target genes in vitro.A Transient transfection analysis for luciferase reporter expression with TSC2 3′UTR in the presence and absence of miR-296. B Immunoblotting analysis of transient transfection analysis of miR-296 for TSC2 expression. TSC2 siRNA was used as a positive control antagonizing TSC2 expression. Block-iT = nonfucntional small RNA control. β-Actin was used as protein loading control. C Relative expression of let-7 predicted target genes (listed above) (y-axis) in transient transfection of nonfunctional small RNA (Block-iT (Controls), let-7c mimic and let-7 inhibitor (Anti-let-7). The relative expression levels were obtained in three cell lines of immortalized ULM cell line (ULM-3401), leiomyosarcoma cell lines (LMS-1, UT-1) (see Methods). T-bars indicate standard error of measurement. * = p value<0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2927438&req=5

pone-0012362-g004: Analysis of the miR-296 predicted target gene TSC2 and 11 let-7 predicted target genes in vitro.A Transient transfection analysis for luciferase reporter expression with TSC2 3′UTR in the presence and absence of miR-296. B Immunoblotting analysis of transient transfection analysis of miR-296 for TSC2 expression. TSC2 siRNA was used as a positive control antagonizing TSC2 expression. Block-iT = nonfucntional small RNA control. β-Actin was used as protein loading control. C Relative expression of let-7 predicted target genes (listed above) (y-axis) in transient transfection of nonfunctional small RNA (Block-iT (Controls), let-7c mimic and let-7 inhibitor (Anti-let-7). The relative expression levels were obtained in three cell lines of immortalized ULM cell line (ULM-3401), leiomyosarcoma cell lines (LMS-1, UT-1) (see Methods). T-bars indicate standard error of measurement. * = p value<0.05.

Mentions: We previously found that the product of TSC2 gene (tuberin) was significantly down regulated in ULMs [27], [34]. Downregulation of TSC2 was also found in this study (Figure S3). By correlation analysis, as illustrated in Figure 2, we found that predicted regulatory miR-296 was inversely correlated with TSC2 protein in 36 ULMs. TSC2 contains 41 exons with a very short 3′ untranslated region (3′UTR, <110 nt). The short TSC2 3′UTR may prevent microRNA regulation. However, in the TSC2 3′UTR immediately adjacent to the stop codon, there is a highly conserved sequence that harbors the complementary sites of miR-296 and a few other microRNAs. To study whether TSC2 is the target of miR-296, we prepared a TSC2 3′UTR reporter construct and examined the luciferase activity by treated cells with control, miR-296 mimic and inhibitor. There was no reduction of luciferase expression in cell treated with miR-296 (Figure 4A). We further examined whether miR-296 could inhibit TSC2 protein production. In comparison to TSC2 siRNA, no significant protein reduction was noted in cells treated with miR-296 (Figure 4B). The findings indicated that TSC2 was not a direct target of miR-296. The inverse correlation of TSC and miR-296 levels may thus be related to indirect or unrelated molecular mechanisms participating in ULM tumorigenesis.


Profiling and functional analyses of microRNAs and their target gene products in human uterine leiomyomas.

Zavadil J, Ye H, Liu Z, Wu J, Lee P, Hernando E, Soteropoulos P, Toruner GA, Wei JJ - PLoS ONE (2010)

Analysis of the miR-296 predicted target gene TSC2 and 11 let-7 predicted target genes in vitro.A Transient transfection analysis for luciferase reporter expression with TSC2 3′UTR in the presence and absence of miR-296. B Immunoblotting analysis of transient transfection analysis of miR-296 for TSC2 expression. TSC2 siRNA was used as a positive control antagonizing TSC2 expression. Block-iT = nonfucntional small RNA control. β-Actin was used as protein loading control. C Relative expression of let-7 predicted target genes (listed above) (y-axis) in transient transfection of nonfunctional small RNA (Block-iT (Controls), let-7c mimic and let-7 inhibitor (Anti-let-7). The relative expression levels were obtained in three cell lines of immortalized ULM cell line (ULM-3401), leiomyosarcoma cell lines (LMS-1, UT-1) (see Methods). T-bars indicate standard error of measurement. * = p value<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2927438&req=5

pone-0012362-g004: Analysis of the miR-296 predicted target gene TSC2 and 11 let-7 predicted target genes in vitro.A Transient transfection analysis for luciferase reporter expression with TSC2 3′UTR in the presence and absence of miR-296. B Immunoblotting analysis of transient transfection analysis of miR-296 for TSC2 expression. TSC2 siRNA was used as a positive control antagonizing TSC2 expression. Block-iT = nonfucntional small RNA control. β-Actin was used as protein loading control. C Relative expression of let-7 predicted target genes (listed above) (y-axis) in transient transfection of nonfunctional small RNA (Block-iT (Controls), let-7c mimic and let-7 inhibitor (Anti-let-7). The relative expression levels were obtained in three cell lines of immortalized ULM cell line (ULM-3401), leiomyosarcoma cell lines (LMS-1, UT-1) (see Methods). T-bars indicate standard error of measurement. * = p value<0.05.
Mentions: We previously found that the product of TSC2 gene (tuberin) was significantly down regulated in ULMs [27], [34]. Downregulation of TSC2 was also found in this study (Figure S3). By correlation analysis, as illustrated in Figure 2, we found that predicted regulatory miR-296 was inversely correlated with TSC2 protein in 36 ULMs. TSC2 contains 41 exons with a very short 3′ untranslated region (3′UTR, <110 nt). The short TSC2 3′UTR may prevent microRNA regulation. However, in the TSC2 3′UTR immediately adjacent to the stop codon, there is a highly conserved sequence that harbors the complementary sites of miR-296 and a few other microRNAs. To study whether TSC2 is the target of miR-296, we prepared a TSC2 3′UTR reporter construct and examined the luciferase activity by treated cells with control, miR-296 mimic and inhibitor. There was no reduction of luciferase expression in cell treated with miR-296 (Figure 4A). We further examined whether miR-296 could inhibit TSC2 protein production. In comparison to TSC2 siRNA, no significant protein reduction was noted in cells treated with miR-296 (Figure 4B). The findings indicated that TSC2 was not a direct target of miR-296. The inverse correlation of TSC and miR-296 levels may thus be related to indirect or unrelated molecular mechanisms participating in ULM tumorigenesis.

Bottom Line: We found that a number of dysregulated microRNAs were inversely correlated with their targets at the protein level.Last, we functionally tested the repressor effects of selected cancer-related microRNAs on their predicted target genes in vitro.We found that some but not all of the predicted and inversely correlated target genes in ULMs can be directly regulated by microRNAs in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, New York University, New York, New York, United States of America.

ABSTRACT

Background: Human uterine leiomyomas (ULM) are characterized by dysregulation of a large number of genes and non-coding regulatory microRNAs. In order to identify microRNA::mRNA associations relevant to ULM pathogenesis, we examined global correlation patterns between the altered microRNA expression and the predicted target genes in ULMs and matched myometria.

Methodology/principal findings: Patterns of inverse association of microRNA with mRNA expression in ULMs revealed an involvement of multiple candidate pathways, including extensive transcriptional reprogramming, cell proliferation control, MAP kinase, TGF-beta, WNT, JAK/STAT signaling, remodeling of cell adhesion, and cell-cell and cell-matrix contacts. We further examined the correlation between the expression of the selected target gene protein products and microRNAs in thirty-six paired sets of leiomyomas and matched myometria. We found that a number of dysregulated microRNAs were inversely correlated with their targets at the protein level. The comparative genomic hybridization (CGH) in eight ULM patients revealed that partially shared deletions of two distinct chromosomal regions might be responsible for loss of cancer-associated microRNA expression and could thus contribute to the ULM pathogenesis via deregulation of target mRNAs. Last, we functionally tested the repressor effects of selected cancer-related microRNAs on their predicted target genes in vitro.

Conclusions/significance: We found that some but not all of the predicted and inversely correlated target genes in ULMs can be directly regulated by microRNAs in vitro. Our findings provide a broad overview of molecular events underlying the tumorigenesis of uterine ULMs and identify select genetic and regulatory events that alter microRNA expression and may play important roles in ULM pathobiology by positively regulating tumor growth while maintaining the non-invasive character of ULMs.

Show MeSH
Related in: MedlinePlus