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MicroRNA-140-5p inhibits hepatocellular carcinoma by directly targeting the unique isomerase Pin1 to block multiple cancer-driving pathways

View Article: PubMed Central - PubMed

ABSTRACT

Hepatocellular carcinoma (HCC) is the second leading cause of cancer related-death. As a major common regulator of numerous cancer-driving pathways and a unique therapeutic target, the prolyl isomerase Pin1 is overexpressed in a majority of HCCs, whereas the mechanism underlying Pin1 overexpression remains elusive. Here we find that miR-140-5p inhibits HCC by directly targeting Pin1 to block multiple cancer-driving pathways. Bioinformatics analysis, miRNA binding and functional assays identify that miR-140-5p directly interacts with the 3′UTR of Pin1 and inhibits Pin1 translation. Furthermore, like stable Pin1 knockdown, moderate overexpression of miR-140-5p not only eliminates Pin1, but also inhibits cells growth and metastasis. Importantly, these effects of miR-140-5p are largely rescued by reconstitution of Pin1. Moreover, miR-140-5p inhibits multiple Pin1-dependent cancer pathways and suppresses tumor growth in mice. The clinical significance of these findings has been substantiated by the demonstrations that miR-140-5p is frequently down-regulated and inversely correlated with Pin1 overexpression in HCC tissues and cell lines. Given prevalent miR-140-5p downregulation in other cancers and major impact of Pin1 overexpression on activating numerous cancer-driving pathways including global miRNA downregulation, the miR-140-5p/Pin1 axis may play a major role in tumorigenesis and offer promising therapeutic targets for HCC and other cancers.

No MeSH data available.


Pin1 is a direct target of miR-140-5p in HCC.(a) Bioinformatic prediction of candidate miRNAs targeting the Pin1 3′UTR. (b) Schematic diagram showing the predicted binding region of miRNAs in Pin1 3′UTR. (c) The predicted binding sites for miR-140-5p in Pin1 3′UTR. The red nucleotides are the seed-pairing target sites of miRNAs. (d) Bioinformatic analyses show Pin1 as a promising target of miR-140-5p (top panel) and miR-140-5p reduces Pin1 expression (bottom panel), as assayed by a luciferase reporter. The seed sequences of miR-140-5p within the Pin1 3′UTR are evolutionarily highly conserved across mammals as marked red. Capitalized letters are the conserved binding sites that directly interact with miR-140-5p. Dual-luciferase assay showed that miR-140-5p and miR-200s reduce luciferase activity by about 50%. (e) MiR-140-5p targets wild-type Pin1 3′UTR, but not its mutant. Luciferase constructs bearing a Pin1 3′UTR (WT) or Pin1 3′UTR containing mutated binding sequences of miR-140-5p (Mut) were cotransfected with miR-140-5p. Results showed that miR-140-5p reduces luciferase activity by 50%, but that was abolished when miR-140-5p binding sequences on Pin1 3′UTR was mutated. (f) MiR-140-5p downregulates Pin1 and cyclin D1 expression, as detected by Western blot analysis. β-actin served as loading control. (g) MiR-140-5p downregulates Pin1 at the translational level. Huh7 cells were infected with lentiviruses expressing miR-140-5p followed by selection with indicated concentration of puromycin. The expression of miR-140-5p and Pin1 protein were significantly increased and decreased, respectively, while Pin1 mRNA level had no significant change. In all panels, bar graphs represent mean ± SEM of three independent experiments. The statistical significance of all tests was accepted for P < 0.05.
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f1: Pin1 is a direct target of miR-140-5p in HCC.(a) Bioinformatic prediction of candidate miRNAs targeting the Pin1 3′UTR. (b) Schematic diagram showing the predicted binding region of miRNAs in Pin1 3′UTR. (c) The predicted binding sites for miR-140-5p in Pin1 3′UTR. The red nucleotides are the seed-pairing target sites of miRNAs. (d) Bioinformatic analyses show Pin1 as a promising target of miR-140-5p (top panel) and miR-140-5p reduces Pin1 expression (bottom panel), as assayed by a luciferase reporter. The seed sequences of miR-140-5p within the Pin1 3′UTR are evolutionarily highly conserved across mammals as marked red. Capitalized letters are the conserved binding sites that directly interact with miR-140-5p. Dual-luciferase assay showed that miR-140-5p and miR-200s reduce luciferase activity by about 50%. (e) MiR-140-5p targets wild-type Pin1 3′UTR, but not its mutant. Luciferase constructs bearing a Pin1 3′UTR (WT) or Pin1 3′UTR containing mutated binding sequences of miR-140-5p (Mut) were cotransfected with miR-140-5p. Results showed that miR-140-5p reduces luciferase activity by 50%, but that was abolished when miR-140-5p binding sequences on Pin1 3′UTR was mutated. (f) MiR-140-5p downregulates Pin1 and cyclin D1 expression, as detected by Western blot analysis. β-actin served as loading control. (g) MiR-140-5p downregulates Pin1 at the translational level. Huh7 cells were infected with lentiviruses expressing miR-140-5p followed by selection with indicated concentration of puromycin. The expression of miR-140-5p and Pin1 protein were significantly increased and decreased, respectively, while Pin1 mRNA level had no significant change. In all panels, bar graphs represent mean ± SEM of three independent experiments. The statistical significance of all tests was accepted for P < 0.05.

Mentions: Pin1 overexpression has been observed in ~70% human HCC patients29, but little is known about molecular mechanisms leading to Pin1 overexpression in HCC. To identify whether Pin1 is regulated by miRNAs, we used miRNA target prediction programs miRnada43, TargetScan21 and PITA44 to search for candidate miRNAs for Pin1 (Supplementary Table S1). We identified eleven candidate miRNAs with the potential targeting the 3′UTR of Pin1, which were predicted by at least two of the three bioinformatics databases. Among these eleven candidates, miR-140-5p and miR-200s (miR-200b/200c/429) were predicted by all of the three databases (Fig. 1a) and found to be frequently downregulated in HCC145. Interestingly, in addition to a miR-200s target site, as reported2122, Pin1 3′UTR has a predicted miR-140-5p target site (Fig. 1b and c).


MicroRNA-140-5p inhibits hepatocellular carcinoma by directly targeting the unique isomerase Pin1 to block multiple cancer-driving pathways
Pin1 is a direct target of miR-140-5p in HCC.(a) Bioinformatic prediction of candidate miRNAs targeting the Pin1 3′UTR. (b) Schematic diagram showing the predicted binding region of miRNAs in Pin1 3′UTR. (c) The predicted binding sites for miR-140-5p in Pin1 3′UTR. The red nucleotides are the seed-pairing target sites of miRNAs. (d) Bioinformatic analyses show Pin1 as a promising target of miR-140-5p (top panel) and miR-140-5p reduces Pin1 expression (bottom panel), as assayed by a luciferase reporter. The seed sequences of miR-140-5p within the Pin1 3′UTR are evolutionarily highly conserved across mammals as marked red. Capitalized letters are the conserved binding sites that directly interact with miR-140-5p. Dual-luciferase assay showed that miR-140-5p and miR-200s reduce luciferase activity by about 50%. (e) MiR-140-5p targets wild-type Pin1 3′UTR, but not its mutant. Luciferase constructs bearing a Pin1 3′UTR (WT) or Pin1 3′UTR containing mutated binding sequences of miR-140-5p (Mut) were cotransfected with miR-140-5p. Results showed that miR-140-5p reduces luciferase activity by 50%, but that was abolished when miR-140-5p binding sequences on Pin1 3′UTR was mutated. (f) MiR-140-5p downregulates Pin1 and cyclin D1 expression, as detected by Western blot analysis. β-actin served as loading control. (g) MiR-140-5p downregulates Pin1 at the translational level. Huh7 cells were infected with lentiviruses expressing miR-140-5p followed by selection with indicated concentration of puromycin. The expression of miR-140-5p and Pin1 protein were significantly increased and decreased, respectively, while Pin1 mRNA level had no significant change. In all panels, bar graphs represent mean ± SEM of three independent experiments. The statistical significance of all tests was accepted for P < 0.05.
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Related In: Results  -  Collection

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f1: Pin1 is a direct target of miR-140-5p in HCC.(a) Bioinformatic prediction of candidate miRNAs targeting the Pin1 3′UTR. (b) Schematic diagram showing the predicted binding region of miRNAs in Pin1 3′UTR. (c) The predicted binding sites for miR-140-5p in Pin1 3′UTR. The red nucleotides are the seed-pairing target sites of miRNAs. (d) Bioinformatic analyses show Pin1 as a promising target of miR-140-5p (top panel) and miR-140-5p reduces Pin1 expression (bottom panel), as assayed by a luciferase reporter. The seed sequences of miR-140-5p within the Pin1 3′UTR are evolutionarily highly conserved across mammals as marked red. Capitalized letters are the conserved binding sites that directly interact with miR-140-5p. Dual-luciferase assay showed that miR-140-5p and miR-200s reduce luciferase activity by about 50%. (e) MiR-140-5p targets wild-type Pin1 3′UTR, but not its mutant. Luciferase constructs bearing a Pin1 3′UTR (WT) or Pin1 3′UTR containing mutated binding sequences of miR-140-5p (Mut) were cotransfected with miR-140-5p. Results showed that miR-140-5p reduces luciferase activity by 50%, but that was abolished when miR-140-5p binding sequences on Pin1 3′UTR was mutated. (f) MiR-140-5p downregulates Pin1 and cyclin D1 expression, as detected by Western blot analysis. β-actin served as loading control. (g) MiR-140-5p downregulates Pin1 at the translational level. Huh7 cells were infected with lentiviruses expressing miR-140-5p followed by selection with indicated concentration of puromycin. The expression of miR-140-5p and Pin1 protein were significantly increased and decreased, respectively, while Pin1 mRNA level had no significant change. In all panels, bar graphs represent mean ± SEM of three independent experiments. The statistical significance of all tests was accepted for P < 0.05.
Mentions: Pin1 overexpression has been observed in ~70% human HCC patients29, but little is known about molecular mechanisms leading to Pin1 overexpression in HCC. To identify whether Pin1 is regulated by miRNAs, we used miRNA target prediction programs miRnada43, TargetScan21 and PITA44 to search for candidate miRNAs for Pin1 (Supplementary Table S1). We identified eleven candidate miRNAs with the potential targeting the 3′UTR of Pin1, which were predicted by at least two of the three bioinformatics databases. Among these eleven candidates, miR-140-5p and miR-200s (miR-200b/200c/429) were predicted by all of the three databases (Fig. 1a) and found to be frequently downregulated in HCC145. Interestingly, in addition to a miR-200s target site, as reported2122, Pin1 3′UTR has a predicted miR-140-5p target site (Fig. 1b and c).

View Article: PubMed Central - PubMed

ABSTRACT

Hepatocellular carcinoma (HCC) is the second leading cause of cancer related-death. As a major common regulator of numerous cancer-driving pathways and a unique therapeutic target, the prolyl isomerase Pin1 is overexpressed in a majority of HCCs, whereas the mechanism underlying Pin1 overexpression remains elusive. Here we find that miR-140-5p inhibits HCC by directly targeting Pin1 to block multiple cancer-driving pathways. Bioinformatics analysis, miRNA binding and functional assays identify that miR-140-5p directly interacts with the 3&prime;UTR of Pin1 and inhibits Pin1 translation. Furthermore, like stable Pin1 knockdown, moderate overexpression of miR-140-5p not only eliminates Pin1, but also inhibits cells growth and metastasis. Importantly, these effects of miR-140-5p are largely rescued by reconstitution of Pin1. Moreover, miR-140-5p inhibits multiple Pin1-dependent cancer pathways and suppresses tumor growth in mice. The clinical significance of these findings has been substantiated by the demonstrations that miR-140-5p is frequently down-regulated and inversely correlated with Pin1 overexpression in HCC tissues and cell lines. Given prevalent miR-140-5p downregulation in other cancers and major impact of Pin1 overexpression on activating numerous cancer-driving pathways including global miRNA downregulation, the miR-140-5p/Pin1 axis may play a major role in tumorigenesis and offer promising therapeutic targets for HCC and other cancers.

No MeSH data available.