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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

uPAR isoform 2 (uPAR7b) is a direct target of miR-221 and miR-222(A) A schematic overview of known transcript variants of the human PLAUR gene leading to four main isoforms of uPAR is shown. The nt (nucleotides), CDS (coding DNA sequence), 5′ and 3′ UTRs (untranslated region), binding sites for RP (reverse primer) and target site with complementary sequences for miR-221/-222 within 3′ UTR of uPAR isoform 2 (uPAR7b) are indicated. (B) A schematic illustration of control luciferase vector (ctrl_Luc) and miTarget™ 3′ UTR of uPAR7b luciferase vector (uPAR7b_Luc) encoding SV40 promoter, hLuc (gene for Firefly luciferase), 3′ UTR of uPAR7b, pA (poly A tail), CMV promoter, hRLuc (gene for Renilla luciferase), pUC Ori (origin of replication) and Kan/Neo® (kanamycin/neomycin resistance gene cassette as selection markers) is shown. (C) Luciferase assay for target identification of miR-221/-222 within 3′ UTR of uPAR7b was conducted in HEK293T cells that were transfected with miR-221 or miR-222 overexpression vectors and ctrl_Luc or uPAR7b_Luc. (D) qRT-PCR for analysis of miR-221 expression in MDA-MB-231 cells, which were infected with a control lentivirus (control vector) or infected with lentivirus for miR-221 inhibition (anti-miR-221). (E) MDA-MB-231 cells, cells infected with control vector or with anti-miR-221 encoding lentivirus (anti-miR-221) were transfected with 3′ UTR of uPAR7b luciferase vector (uPAR7b_Luc). Data represent means and ± SD (n = 3). The Student's t-test was used for statistical analysis.
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Figure 3: uPAR isoform 2 (uPAR7b) is a direct target of miR-221 and miR-222(A) A schematic overview of known transcript variants of the human PLAUR gene leading to four main isoforms of uPAR is shown. The nt (nucleotides), CDS (coding DNA sequence), 5′ and 3′ UTRs (untranslated region), binding sites for RP (reverse primer) and target site with complementary sequences for miR-221/-222 within 3′ UTR of uPAR isoform 2 (uPAR7b) are indicated. (B) A schematic illustration of control luciferase vector (ctrl_Luc) and miTarget™ 3′ UTR of uPAR7b luciferase vector (uPAR7b_Luc) encoding SV40 promoter, hLuc (gene for Firefly luciferase), 3′ UTR of uPAR7b, pA (poly A tail), CMV promoter, hRLuc (gene for Renilla luciferase), pUC Ori (origin of replication) and Kan/Neo® (kanamycin/neomycin resistance gene cassette as selection markers) is shown. (C) Luciferase assay for target identification of miR-221/-222 within 3′ UTR of uPAR7b was conducted in HEK293T cells that were transfected with miR-221 or miR-222 overexpression vectors and ctrl_Luc or uPAR7b_Luc. (D) qRT-PCR for analysis of miR-221 expression in MDA-MB-231 cells, which were infected with a control lentivirus (control vector) or infected with lentivirus for miR-221 inhibition (anti-miR-221). (E) MDA-MB-231 cells, cells infected with control vector or with anti-miR-221 encoding lentivirus (anti-miR-221) were transfected with 3′ UTR of uPAR7b luciferase vector (uPAR7b_Luc). Data represent means and ± SD (n = 3). The Student's t-test was used for statistical analysis.

Mentions: The gene PLAUR encoding the human glycoprotein uPAR is located on chromosome 19q13.1-q13.2 and consists of seven exons, separated by six introns [21]. While exon 1 encodes the 5′ UTR and a signal peptide, exons 2-3, 4-5 and 6-7 encode three homologous Ly-6 antigen/uPAR-like (LU) domains (DI, DII and DIII; Figure 2). According to the NCBI GenBank database, to date four main isoform sequences of uPAR are known, which mostly result from alternative splicing (Figure 2 and 3A). Further alternative splicing events, such as deletion of exon 4 and 5 with prognostic relevance in breast cancer [22] and post-translational modifications, such as glycosylation of uPAR leading to molecular weights between 35 and 60 kDa have been described [21]. The isoform 1 or full-length uPAR (NM_002659) [21] is the longest isoform consisting of three domains and is linked to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor at DIII (Figure 2). When pro-uPA binds to uPAR, it is activated and the uPAR-uPA complex is cleaved close to the GPI anchor and then released into the ECM [15]. The released uPAR-uPA complex has been demonstrated in multiple human diseases, including breast cancer [14]. Compared with full-length uPAR, isoform 2 (NM_001005376) is the shortest isoform arising from alternative splicing of exon 7 within DIII leading to loss of the GPI anchor region and a secreted uPAR (uPAR7b) [13, 14, 23] (Figure 2 and 3A). The physiological role of uPAR7b is not known in detail. Since these modifications do not affect major uPA binding sites within DI and DII, it may interact with and activate uPA and exhibit chemotactic properties leading to enhanced migration and invasion of cancer cells [14]. Isoform 3 (NM_001005377) lacks exon 5 in the coding region, encodes valine instead of isoleucine on the splice site (compared with full-length uPAR) and it is longer than isoform 2 (Figure 2 and 3A). These modifications are supposed to affect glycosylation pattern and folding of uPAR [24, 25]. Isoform 4 (NM_001301037) lacks exon 6 in the coding region and is shorter in comparison to variant 1 but longer than isoform 2 (Figure 2 and 3A).


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)

uPAR isoform 2 (uPAR7b) is a direct target of miR-221 and miR-222(A) A schematic overview of known transcript variants of the human PLAUR gene leading to four main isoforms of uPAR is shown. The nt (nucleotides), CDS (coding DNA sequence), 5′ and 3′ UTRs (untranslated region), binding sites for RP (reverse primer) and target site with complementary sequences for miR-221/-222 within 3′ UTR of uPAR isoform 2 (uPAR7b) are indicated. (B) A schematic illustration of control luciferase vector (ctrl_Luc) and miTarget™ 3′ UTR of uPAR7b luciferase vector (uPAR7b_Luc) encoding SV40 promoter, hLuc (gene for Firefly luciferase), 3′ UTR of uPAR7b, pA (poly A tail), CMV promoter, hRLuc (gene for Renilla luciferase), pUC Ori (origin of replication) and Kan/Neo® (kanamycin/neomycin resistance gene cassette as selection markers) is shown. (C) Luciferase assay for target identification of miR-221/-222 within 3′ UTR of uPAR7b was conducted in HEK293T cells that were transfected with miR-221 or miR-222 overexpression vectors and ctrl_Luc or uPAR7b_Luc. (D) qRT-PCR for analysis of miR-221 expression in MDA-MB-231 cells, which were infected with a control lentivirus (control vector) or infected with lentivirus for miR-221 inhibition (anti-miR-221). (E) MDA-MB-231 cells, cells infected with control vector or with anti-miR-221 encoding lentivirus (anti-miR-221) were transfected with 3′ UTR of uPAR7b luciferase vector (uPAR7b_Luc). Data represent means and ± SD (n = 3). The Student's t-test was used for statistical analysis.
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Figure 3: uPAR isoform 2 (uPAR7b) is a direct target of miR-221 and miR-222(A) A schematic overview of known transcript variants of the human PLAUR gene leading to four main isoforms of uPAR is shown. The nt (nucleotides), CDS (coding DNA sequence), 5′ and 3′ UTRs (untranslated region), binding sites for RP (reverse primer) and target site with complementary sequences for miR-221/-222 within 3′ UTR of uPAR isoform 2 (uPAR7b) are indicated. (B) A schematic illustration of control luciferase vector (ctrl_Luc) and miTarget™ 3′ UTR of uPAR7b luciferase vector (uPAR7b_Luc) encoding SV40 promoter, hLuc (gene for Firefly luciferase), 3′ UTR of uPAR7b, pA (poly A tail), CMV promoter, hRLuc (gene for Renilla luciferase), pUC Ori (origin of replication) and Kan/Neo® (kanamycin/neomycin resistance gene cassette as selection markers) is shown. (C) Luciferase assay for target identification of miR-221/-222 within 3′ UTR of uPAR7b was conducted in HEK293T cells that were transfected with miR-221 or miR-222 overexpression vectors and ctrl_Luc or uPAR7b_Luc. (D) qRT-PCR for analysis of miR-221 expression in MDA-MB-231 cells, which were infected with a control lentivirus (control vector) or infected with lentivirus for miR-221 inhibition (anti-miR-221). (E) MDA-MB-231 cells, cells infected with control vector or with anti-miR-221 encoding lentivirus (anti-miR-221) were transfected with 3′ UTR of uPAR7b luciferase vector (uPAR7b_Luc). Data represent means and ± SD (n = 3). The Student's t-test was used for statistical analysis.
Mentions: The gene PLAUR encoding the human glycoprotein uPAR is located on chromosome 19q13.1-q13.2 and consists of seven exons, separated by six introns [21]. While exon 1 encodes the 5′ UTR and a signal peptide, exons 2-3, 4-5 and 6-7 encode three homologous Ly-6 antigen/uPAR-like (LU) domains (DI, DII and DIII; Figure 2). According to the NCBI GenBank database, to date four main isoform sequences of uPAR are known, which mostly result from alternative splicing (Figure 2 and 3A). Further alternative splicing events, such as deletion of exon 4 and 5 with prognostic relevance in breast cancer [22] and post-translational modifications, such as glycosylation of uPAR leading to molecular weights between 35 and 60 kDa have been described [21]. The isoform 1 or full-length uPAR (NM_002659) [21] is the longest isoform consisting of three domains and is linked to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor at DIII (Figure 2). When pro-uPA binds to uPAR, it is activated and the uPAR-uPA complex is cleaved close to the GPI anchor and then released into the ECM [15]. The released uPAR-uPA complex has been demonstrated in multiple human diseases, including breast cancer [14]. Compared with full-length uPAR, isoform 2 (NM_001005376) is the shortest isoform arising from alternative splicing of exon 7 within DIII leading to loss of the GPI anchor region and a secreted uPAR (uPAR7b) [13, 14, 23] (Figure 2 and 3A). The physiological role of uPAR7b is not known in detail. Since these modifications do not affect major uPA binding sites within DI and DII, it may interact with and activate uPA and exhibit chemotactic properties leading to enhanced migration and invasion of cancer cells [14]. Isoform 3 (NM_001005377) lacks exon 5 in the coding region, encodes valine instead of isoleucine on the splice site (compared with full-length uPAR) and it is longer than isoform 2 (Figure 2 and 3A). These modifications are supposed to affect glycosylation pattern and folding of uPAR [24, 25]. Isoform 4 (NM_001301037) lacks exon 6 in the coding region and is shorter in comparison to variant 1 but longer than isoform 2 (Figure 2 and 3A).

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