Matrix metalloproteinase 9 expression: new regulatory elements

Abstract

Retinal ganglion cells apoptosis is linked to matrix metalloproteinase 9 (MMP-9) controlled changes of extracellular matrix. Abnormal expression of MMP-9 is associated with glaucomatous alterations. Thus, the knowledge of MMP-9 regulation is important for the understanding the pathogenesis of glaucoma. Here, we investigated the role of 3'-untranslated regions (3'-UTR) and microRNAs in MMP-9 regulation. We used in vitro mutagenesis and Luc reporter system to identify regulatory elements in the 3'-UTR of MMP-9. microRNAs were analyzed by qRT-PCR, and their role was investigated with inhibitors and mimics. We identified targets for miRNAs in 3'-UTR of MMP-9 involved in the regulation of MMP-9 expression. We then isolated miRNAs from the optic nerve A7 astrocytes and 293 T cells and confirmed the role of mi340 in the regulation using specific inhibitors and mimics. The results obtained show a new miRNA-mediated mechanism of MMP-9 expression regulation.

Keywords: Extracellular matrix; Matrix metalloproteinases; MicroRNA; Retinal ganglion cells; Untranslated region.

Fig. 1

a Nucleotide sequence of the rat 3′-UTR of MMP-9 gene downstream of the stop codon (3′-UTR446). Polyadenylation signal AATAAA (arrow) and AT-rich regulatory elements ATTTA (ARE1 and ARE2) are underlined. Putative miRNA targets for miR328 and miR132 are shown in small letters. Putative miRNA targets for miR340, miR130b, and miR540 are underlined. b pGL3-Control Vector Map showing the sites where 3′-UTR446 was inserted in distal (dist) and proximal (prox) position. MCS multiple cloning site. c Alignment of nucleotide sequences of MMP-9 3′-UTR corresponding to the targets for mi340 from several mammalian species. A high level of conservation suggests a functional role for these sequences

Fig. 2

Effect of the 3′-UTR fragment of the MMP-9 gene and its fragments on the efficiency of the reporter luciferase gene expression. The UTR446 shown in Fig. 1a was amplified using cDNA as a template. The forward primer was directly upstream of the stop codon and the reverse primer 446 bp downstream of the stop codon (primer's sequences are presented in Table 1, I). All constructions were amplified in E. coli, and transfected into A7 (gray bars) or 293 T cells (black bars). Luc activity was determined using Dual-Luciferase Reporter Assay System (Promega). Y axe ratio of Firefly to Renila luminescence was first calculated in arbitrary units and then expressed in percent. The expression in a–c is driven by SV40 promoter, in d by MMP-9 promoter. In b–d the putative regulatory sequences were inserted in a proximal position. Columns represent the mean of at least three individual experiments; bars are SD. a The UTR446 was inserted into proximal, distal and in both positions of pGL3 Control vector. b The effect of deletion of ARE1 (-ARE1) and ARE2 (-ARE2) fragments from UTR446 on its inhibitory activity. All fragments were inserted into proximal position. pGL3 control; +UTR indicates that the UTR446 is inserted; -ARE1 signifies that the UTR446 with deleted ARE1 is inserted; -ARE2 indicates that UTR446 with deleted ARE2 is inserted. c The effect of truncated forms of UTR446 on Luc expression. The following fragments have been inserted using primers shown in Table 1, I. +UTR indicates that 3′-UTR446 has been inserted; +t340 signifies that putative target for mi340 has been inserted; UTR-6 means that UTR446 with deleted six nucleotides has been inserted; UTR-12 means that UTR446 with deleted 12 nucleotides has been inserted; UTR-19 means that UTR446 with deleted 19 nucleotides has been used. d Luc expression driven by the basic pGL3 vector with human MMP-9 promoter 2.2 kb fragment. pGL3 control value of Luc expression; +t340 putative target for miR340 has been inserted in a proximal position

Fig. 3

The predicted binding sites of rat miRNAs (blue) 340-3p, 328, 130b, and 132 in the 3′-UTR of MMP-9 mRNA identified with miRanda algorithm [28, 29]. The number below the green bar indicates the position relative to the UGA stop codon of the MMP-9 mRNA (accession number 031055)

Fig. 4

Quantification of luciferase firefly mRNA by qRT-PCR using Taqman assay. The quantity of luciferase mRNA in A7 cells transfected by pGL3 control vector with inserted UTR or t340 fragments was compared with the quantity in cells transfected with intact pGL3 vector which was considered to be 100%. Y axe as in Fig. 1. Columns represent the mean of at least three individual experiments; bars are SD

Fig. 5

Relative expression of microRNAs in A7 astrocytes and T293 cells are shown in duplicate for each point. Functional validity of all Taqman miRNA assays were confirmed using Human total RNA pool or Rat tissue LMW RNA pool as positive control (not shown). Differential microRNA expression is seen between A7 cells and 293 T cells for miR-130b, miR-132, miR-212, miR-296, miR-328, miR-340, and miR-877

Fig. 6

Effect of mi340 mimics and inhibitors on the efficiency of expression. a Target validation of miRNA340 by Luc assay using mimics and inhibitors. The A7 astrocytes cell line was transfected with 20–400 nM of mimic rno-340-3p (left panel, mimics) or 1 nM of anti-miR340 (right panel, inhibitors). The cells were cotransfected for 72 h with Luc reporter constructs bearing a 3′-UTR446 target sequence and assayed for luciferase activity. Left panel 1 pGL3 with UTR446; 2 the same as 1 +20 nM mimic-340; 3 the same as 1 + 100 nM mimic-340; 4 the same as 1 + 200 nM mimic-340; 5 the same as 1 + 400 nM mimic-340; 6 mimic negative control. Right panel 1 pGL3 with UTR446 + 1 nM of the mi340 inhibitor; 2 inhibitor negative control. The use of mimics and inhibitors of mi340 showed that the luciferase activity of cells transfected with mimic was decreased and that transfected with anti-miRNA340 was increased. Y axe is as in Fig. 2. Columns represent the mean of at least three individual experiments; bars are SD. These results confirm that mi340 is able to bind to its target in the 3′-UTR446. B. Western blotting of A7 cells transfected by mimic miR340. 1 Control sample, 2 200 nM of mimic-340; 3 400 nM of mimic-340. Arrow corresponds to 92 kDa protein. c Scan of the Western blot shown on b