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Secreted uPAR isoform 2 (uPAR7b) is a novel direct target of miR-221.

Falkenberg N, Anastasov N, Schaub A, Radulovic V, Schmitt M, Magdolen V, Aubele M - Oncotarget (2015)

Bottom Line: For the first time, the uPAR isoform 2 was demonstrated as direct target for miR-221/-222.Inhibition of miR-221 reduced uPAR protein expression and expression of the tumor cell invasion markers vimentin and RHOC.These results demonstrate a direct and positive regulation of the secreted uPAR isoform 2 by miR-221, increasing its protein expression, a prerequisite for malignancy, while the other uPAR isoforms (1, 3 and 4) are indirectly regulated through miR-10b and miR-221/-222.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, German Research Center for Environmental Health, Neuherberg, Germany.

ABSTRACT
miR-221/-222 and components of the urokinase-type plasminogen activator system (uPAS) are associated with metastasis and poor prognosis in breast cancer, including the triple-negative subtype (TNBC). Modification of components of uPAS and involved miRNAs may contribute to targeted therapy for breast cancer patients. miR-221-/-222-overexpressing or miR-221-depleted cells were employed for qRT-PCR and Western blots to show associations of uPAR with miR-221/-222. To substantiate direct targeting of miR-221/-222 within 3' UTR of the uPAR isoform 2, in silico analysesand in vitro assays were conducted. Significant associations between miR-221 and uPAR isoform 2 expressions were observed at the mRNA and protein levels in breast cancer cells representing TNBC. For the first time, the uPAR isoform 2 was demonstrated as direct target for miR-221/-222. Inhibition of miR-221 reduced uPAR protein expression and expression of the tumor cell invasion markers vimentin and RHOC. These results demonstrate a direct and positive regulation of the secreted uPAR isoform 2 by miR-221, increasing its protein expression, a prerequisite for malignancy, while the other uPAR isoforms (1, 3 and 4) are indirectly regulated through miR-10b and miR-221/-222. By targeting uPAR isoforms and/or miRNA-221/-222, the diagnosis and therapy of breast cancer, in particular in TNBC, could be significantly improved.

No MeSH data available.


Related in: MedlinePlus

miR-221 directly regulates uPAR isoform 2 (uPAR7b) and indirectly uPAR isoforms 1, 3, 4 and miR-10b(A) qRT-PCR for expression analysis of miR-221 and miR-10b as well as of uPAR isoforms 1, 3, 4 and isoform 2 (uPAR7b). Data represent the means and ± SD (n = 3). The Student's t-test was used for statistical analysis: * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative Western blot analyses of protein expression of uPAR (in the lysate and supernatant) as well as of vimentin, RHOC and actin in the cell lysates are shown. (C) Schematic illustration showing possible molecular mechanisms regulating expression of membrane anchored uPAR isoforms 1, 3 and 4 or the secreted uPAR isoform 2.
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Figure 4: miR-221 directly regulates uPAR isoform 2 (uPAR7b) and indirectly uPAR isoforms 1, 3, 4 and miR-10b(A) qRT-PCR for expression analysis of miR-221 and miR-10b as well as of uPAR isoforms 1, 3, 4 and isoform 2 (uPAR7b). Data represent the means and ± SD (n = 3). The Student's t-test was used for statistical analysis: * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative Western blot analyses of protein expression of uPAR (in the lysate and supernatant) as well as of vimentin, RHOC and actin in the cell lysates are shown. (C) Schematic illustration showing possible molecular mechanisms regulating expression of membrane anchored uPAR isoforms 1, 3 and 4 or the secreted uPAR isoform 2.

Mentions: In ectopically miR-221- or miR-222-overexpressed HEK293T cells, the miR-221 overexpression stronger and more significantly affected luciferase activity than miR-222 (Figure 3C). Therefore, to investigate the association of overexpressed miR-221 with uPAR in more detail, Western blot and qRT-PCR analyses were performed in those cancer cells, which endogenously overexpress miR-221 or in miR-221-depleted MDA-MB-231 cells. When miR-221 was inhibited by anti-miR-221 in MDA-MB-231 cells (p < 0.001, Figure 4A), the mRNA expression of all four uPAR isoforms was almost unchanged compared with non-infected MDA-MB-231 cells (Figure 4A). Regarding protein expressions, a reduction of uPAR in the cell lysate and almost complete reduction of uPAR in the supernatant (indicating secreted uPAR7b) along with reduced levels of the tumor cell invasion marker vimentin following miR-221 inhibition were observed (Figure 4B) while uPAR mRNA levels were not affected. In addition, positive regulation and elevated uPAR protein expression was shown previously using Western blot analysis of ectopically miR-221-overexpressing SKBR3 cells [12] and elevated uPAR mRNA levels of all four isoforms were detected by qRT-PCR in miR-221-overexpressing SKBR3 cells (data not shown).


Secreted uPAR isoform 2 (uPAR7b) is a novel direct target of miR-221.

Falkenberg N, Anastasov N, Schaub A, Radulovic V, Schmitt M, Magdolen V, Aubele M - Oncotarget (2015)

miR-221 directly regulates uPAR isoform 2 (uPAR7b) and indirectly uPAR isoforms 1, 3, 4 and miR-10b(A) qRT-PCR for expression analysis of miR-221 and miR-10b as well as of uPAR isoforms 1, 3, 4 and isoform 2 (uPAR7b). Data represent the means and ± SD (n = 3). The Student's t-test was used for statistical analysis: * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative Western blot analyses of protein expression of uPAR (in the lysate and supernatant) as well as of vimentin, RHOC and actin in the cell lysates are shown. (C) Schematic illustration showing possible molecular mechanisms regulating expression of membrane anchored uPAR isoforms 1, 3 and 4 or the secreted uPAR isoform 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4480738&req=5

Figure 4: miR-221 directly regulates uPAR isoform 2 (uPAR7b) and indirectly uPAR isoforms 1, 3, 4 and miR-10b(A) qRT-PCR for expression analysis of miR-221 and miR-10b as well as of uPAR isoforms 1, 3, 4 and isoform 2 (uPAR7b). Data represent the means and ± SD (n = 3). The Student's t-test was used for statistical analysis: * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative Western blot analyses of protein expression of uPAR (in the lysate and supernatant) as well as of vimentin, RHOC and actin in the cell lysates are shown. (C) Schematic illustration showing possible molecular mechanisms regulating expression of membrane anchored uPAR isoforms 1, 3 and 4 or the secreted uPAR isoform 2.
Mentions: In ectopically miR-221- or miR-222-overexpressed HEK293T cells, the miR-221 overexpression stronger and more significantly affected luciferase activity than miR-222 (Figure 3C). Therefore, to investigate the association of overexpressed miR-221 with uPAR in more detail, Western blot and qRT-PCR analyses were performed in those cancer cells, which endogenously overexpress miR-221 or in miR-221-depleted MDA-MB-231 cells. When miR-221 was inhibited by anti-miR-221 in MDA-MB-231 cells (p < 0.001, Figure 4A), the mRNA expression of all four uPAR isoforms was almost unchanged compared with non-infected MDA-MB-231 cells (Figure 4A). Regarding protein expressions, a reduction of uPAR in the cell lysate and almost complete reduction of uPAR in the supernatant (indicating secreted uPAR7b) along with reduced levels of the tumor cell invasion marker vimentin following miR-221 inhibition were observed (Figure 4B) while uPAR mRNA levels were not affected. In addition, positive regulation and elevated uPAR protein expression was shown previously using Western blot analysis of ectopically miR-221-overexpressing SKBR3 cells [12] and elevated uPAR mRNA levels of all four isoforms were detected by qRT-PCR in miR-221-overexpressing SKBR3 cells (data not shown).

Bottom Line: For the first time, the uPAR isoform 2 was demonstrated as direct target for miR-221/-222.Inhibition of miR-221 reduced uPAR protein expression and expression of the tumor cell invasion markers vimentin and RHOC.These results demonstrate a direct and positive regulation of the secreted uPAR isoform 2 by miR-221, increasing its protein expression, a prerequisite for malignancy, while the other uPAR isoforms (1, 3 and 4) are indirectly regulated through miR-10b and miR-221/-222.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, German Research Center for Environmental Health, Neuherberg, Germany.

ABSTRACT
miR-221/-222 and components of the urokinase-type plasminogen activator system (uPAS) are associated with metastasis and poor prognosis in breast cancer, including the triple-negative subtype (TNBC). Modification of components of uPAS and involved miRNAs may contribute to targeted therapy for breast cancer patients. miR-221-/-222-overexpressing or miR-221-depleted cells were employed for qRT-PCR and Western blots to show associations of uPAR with miR-221/-222. To substantiate direct targeting of miR-221/-222 within 3' UTR of the uPAR isoform 2, in silico analysesand in vitro assays were conducted. Significant associations between miR-221 and uPAR isoform 2 expressions were observed at the mRNA and protein levels in breast cancer cells representing TNBC. For the first time, the uPAR isoform 2 was demonstrated as direct target for miR-221/-222. Inhibition of miR-221 reduced uPAR protein expression and expression of the tumor cell invasion markers vimentin and RHOC. These results demonstrate a direct and positive regulation of the secreted uPAR isoform 2 by miR-221, increasing its protein expression, a prerequisite for malignancy, while the other uPAR isoforms (1, 3 and 4) are indirectly regulated through miR-10b and miR-221/-222. By targeting uPAR isoforms and/or miRNA-221/-222, the diagnosis and therapy of breast cancer, in particular in TNBC, could be significantly improved.

No MeSH data available.


Related in: MedlinePlus