Limits...
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

The 3′ UTR of uPAR isoform 2 (uPAR7b) differs from the 3′ UTR of uPAR isoforms 1, 3 and 43′ UTR sequences of uPAR isoforms 1, 2 (uPAR7b), 3 and 4 were analyzed using CLUSTALW2 software for multiple sequence alignment. (Potential) binding sites for GATA3 (underlined), for miR-221/-222 (ATGTAGC petrol blue in bold) and reverse primer (RP_uPAR7b, underlined) within 3′ UTR of uPAR7b (NM_001005376) are underlined or indicated. The reverse primer for uPAR isoforms 1, 3 and 4 is not located within the respective 3′ UTR sequences of these isoforms. Potential binding sites for HUR and hnRNPC (underlined 3 to 5 base pairs long gray or black sequences) and for HOXD10 (gray sequence) within 3′ UTR of isoforms 1, 3 and 4 are indicated. 60 to 392 demonstrate the length of nucleotide sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480738&req=5

Figure 5: The 3′ UTR of uPAR isoform 2 (uPAR7b) differs from the 3′ UTR of uPAR isoforms 1, 3 and 43′ UTR sequences of uPAR isoforms 1, 2 (uPAR7b), 3 and 4 were analyzed using CLUSTALW2 software for multiple sequence alignment. (Potential) binding sites for GATA3 (underlined), for miR-221/-222 (ATGTAGC petrol blue in bold) and reverse primer (RP_uPAR7b, underlined) within 3′ UTR of uPAR7b (NM_001005376) are underlined or indicated. The reverse primer for uPAR isoforms 1, 3 and 4 is not located within the respective 3′ UTR sequences of these isoforms. Potential binding sites for HUR and hnRNPC (underlined 3 to 5 base pairs long gray or black sequences) and for HOXD10 (gray sequence) within 3′ UTR of isoforms 1, 3 and 4 are indicated. 60 to 392 demonstrate the length of nucleotide sequence.

Mentions: However, mRNA expression of uPAR isoform 2 as well as of the uPAR isoforms 1, 3 and 4 were detected in miR-221-positive cells (Figure 4A). Ma et al., have shown that miR-10b is strongly overexpressed in MDA-MB-231 cells, regulating uPAR expression through translational inhibition of homeobox D10 gene (HOXD10) [26]. HOXD10 in turn represses expression of cell migration- and invasion-promoting markers, including (ras homolog family member C) RHOC and uPAR [27]. Therefore, expression of miR-10b was analyzed by qRT-PCR and that of RHOC by Western blotting. While we observe high miR-10b and moderate RHOC levels in miR-221-positive BT549 and MDA-MB-231 cells, the expression levels were significantly (miR-10b: p < 0.001, Figure 4A) or slightly (RHOC, Figure 4B) reduced following miR-221 inhibition in MDA-MB-231 cells. To support our theory that uPAR isoform 2 is directly regulated through miR-221 whereas the uPAR isoforms 1, 3 and 4 are indirectly regulated by miR-10b and miR-221 (Figure 4C), we conducted further in silico analyses. The induction of isoform 2 following miR-221 overexpression may be additionally controlled through the positive transcription regulator GATA3 (GATA binding protein 3) that is often overexpressed in breast cancer cells [28]. Using in silico analysis for putative binding sites for transcription factors [29], we have identified a putative target binding site for GATA3 only within the 3′ UTR of isoform 2 (uPAR7b) and for HOXD10 only within 3′ UTRs of isoforms 1, 3 and 4 (Figure 5). Moreover, HUR, an AU-/U-Rich Element-(ARE)-binding protein and hnRNPC positively regulate uPAR expression [17, 19] and show potential target binding sites only within 3′ UTR of uPAR isoforms 1, 3 and 4 (Figure 5).


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)

The 3′ UTR of uPAR isoform 2 (uPAR7b) differs from the 3′ UTR of uPAR isoforms 1, 3 and 43′ UTR sequences of uPAR isoforms 1, 2 (uPAR7b), 3 and 4 were analyzed using CLUSTALW2 software for multiple sequence alignment. (Potential) binding sites for GATA3 (underlined), for miR-221/-222 (ATGTAGC petrol blue in bold) and reverse primer (RP_uPAR7b, underlined) within 3′ UTR of uPAR7b (NM_001005376) are underlined or indicated. The reverse primer for uPAR isoforms 1, 3 and 4 is not located within the respective 3′ UTR sequences of these isoforms. Potential binding sites for HUR and hnRNPC (underlined 3 to 5 base pairs long gray or black sequences) and for HOXD10 (gray sequence) within 3′ UTR of isoforms 1, 3 and 4 are indicated. 60 to 392 demonstrate the length of nucleotide sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: The 3′ UTR of uPAR isoform 2 (uPAR7b) differs from the 3′ UTR of uPAR isoforms 1, 3 and 43′ UTR sequences of uPAR isoforms 1, 2 (uPAR7b), 3 and 4 were analyzed using CLUSTALW2 software for multiple sequence alignment. (Potential) binding sites for GATA3 (underlined), for miR-221/-222 (ATGTAGC petrol blue in bold) and reverse primer (RP_uPAR7b, underlined) within 3′ UTR of uPAR7b (NM_001005376) are underlined or indicated. The reverse primer for uPAR isoforms 1, 3 and 4 is not located within the respective 3′ UTR sequences of these isoforms. Potential binding sites for HUR and hnRNPC (underlined 3 to 5 base pairs long gray or black sequences) and for HOXD10 (gray sequence) within 3′ UTR of isoforms 1, 3 and 4 are indicated. 60 to 392 demonstrate the length of nucleotide sequence.
Mentions: However, mRNA expression of uPAR isoform 2 as well as of the uPAR isoforms 1, 3 and 4 were detected in miR-221-positive cells (Figure 4A). Ma et al., have shown that miR-10b is strongly overexpressed in MDA-MB-231 cells, regulating uPAR expression through translational inhibition of homeobox D10 gene (HOXD10) [26]. HOXD10 in turn represses expression of cell migration- and invasion-promoting markers, including (ras homolog family member C) RHOC and uPAR [27]. Therefore, expression of miR-10b was analyzed by qRT-PCR and that of RHOC by Western blotting. While we observe high miR-10b and moderate RHOC levels in miR-221-positive BT549 and MDA-MB-231 cells, the expression levels were significantly (miR-10b: p < 0.001, Figure 4A) or slightly (RHOC, Figure 4B) reduced following miR-221 inhibition in MDA-MB-231 cells. To support our theory that uPAR isoform 2 is directly regulated through miR-221 whereas the uPAR isoforms 1, 3 and 4 are indirectly regulated by miR-10b and miR-221 (Figure 4C), we conducted further in silico analyses. The induction of isoform 2 following miR-221 overexpression may be additionally controlled through the positive transcription regulator GATA3 (GATA binding protein 3) that is often overexpressed in breast cancer cells [28]. Using in silico analysis for putative binding sites for transcription factors [29], we have identified a putative target binding site for GATA3 only within the 3′ UTR of isoform 2 (uPAR7b) and for HOXD10 only within 3′ UTRs of isoforms 1, 3 and 4 (Figure 5). Moreover, HUR, an AU-/U-Rich Element-(ARE)-binding protein and hnRNPC positively regulate uPAR expression [17, 19] and show potential target binding sites only within 3′ UTR of uPAR isoforms 1, 3 and 4 (Figure 5).

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