Role of miR-29b on the regulation of the extracellular matrix in human trabecular meshwork cells under chronic oxidative stress

Cell cultures and oxidative stress conditions

HTM cell cultures were generated from cadaver eyes, with no history of eye disease, within 48 h post mortem, as previously reported [20]. All procedures involving human tissue were conducted in accordance with the tenets of the Declaration of Helsinski. Cell cultures were maintained at 37 ^oC in 5% CO₂ in media (low glucose Dulbecco’s Modified Eagle Medium with L-glutamine, 110 mg/ml sodium pyruvate, 10% fetal bovine serum, 100 µM nonessential amino acids, 100 units/ml penicillin,100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B). All the reagents were obtained from Invitrogen Corporation (Carlsbad, CA). Oxidative stress was induced by incubation at 40% oxygen 5% CO₂ for 4–5 days. Cells incubated at 40% oxygen were compared to cells incubated at an oxygen concentration close to the physiological concentration reported in the aqueous humor (oxygen partial pressure PO₂ 5%) [21].

Transfections

HTM cells were plated 24 h before transfection with hsa-miR-29b mimic or control mimic (scramble; 20-40pmolar [Dharmacon, Chicago, IL] with Lipofectamine 2000 [Invitrogen] or amaxa nucleofactor kit [Lonza, Basel, Switzerland]) following the manufacturer’s instructions. Briefly, for nucleofection, cells were transfected at density of 4×10⁶ using an endothelial nucleofactor Kit and the program T23 in the Nucleofector, following the basic protocol for primary mammalian endothelial cells and 20 picomolar of miRNAs (Amaxa, scientific-support.US@amaxa.com).  For lipofectamine transfections, cells were seeded at 2-3×10⁵ cells in 12 well plates or 5-6×10⁵ cells in 6 well plates using 40 picomolar miRNAs and 1 or 2 µl of lipofectamine (for 12 and 6 well plates, respectively) and OPTI-MEM I  (Invitrogen). Cotransfections of 293A cells with luciferase 3’untranslated region (UTR) constructs (0.3 µg), miR-29b mimic, or control mimic (20 pmolar) were accomplished using Effectene (Qiagen, Valencia, CA).

Gene microarray analysis

Gene array analysis was conducted in three independent sets of transfections with either miR-29b mimic or mimic control of the same HTM cell line. Total RNA was extracted three days post transfection using RNeasy kit (Qiagen), amplified (one round amplification) using one cycle target labeling and control reagents (Affymetrix, Santa Clara, CA), and hybridized to human genome U133A2 arrays (Affymetrix) at the Duke University Microarray facility. Raw data were normalized and analyzed using GeneSpring 7.3 (Silicon Genetics, Santa Clara, CA). Genes were filtered to their intensities in the control channel. Pre-mixed polyadenylated prokaryotic sequences from the One cycle target labeling and control reagents kit (Affymetrix) were spiked directly into the samples before target labeling and used as controls. Raw data values below 100 were considered as unreliable. Intensity-dependent normalization was performed per spot and per chip (LOWESS normalization). ANOVA test was performed (p-values ≤0.05 were considered significant) for genes differentially expressed, using the Benjamin and Hochberg False Discovery Rate correction test. The list of genes were compared to three databases that predict targets for miRNAs: Microcosm, TargetScan, and PicTar-Vert. To study the potential biological significance of the changes observed in the arrays, we performed network analysis of differentially expressed genes, using Metacore pathway analysis (GeneGo, St. Joseph, MI).

Analysis of miR-29b interaction with 3’ untranslated regions

The entire 3’UTRs from bone morphogenic protein 1 (BMP1), ADAM metallopeptidase domain 12 (ADAM12), and NFKB inhibitor-interacting Ras-like protein 2 (NKIRAS2) were amplified using primers in Table 1, with carried XhoI and NotI restriction sites in the forward or the reverse position. PCR amplifications from 3’UTRs and the complementary sequences were confirmed by sequencing and cloned into XhoI and NotI sites downstream of Renilla luciferase in the psiCheck2 vector (Promega, Madison, WI). For analysis of luciferase activity, 293A cells were seeded in 12-well culture dishes 24 h before transfection and transfected with psicheck 3’UTR, or the complementary sequence from BMP1, ADAM12, or NKIRAS2 (300 ng), and miRNAs for 29b mimic or control mimic. Luciferase was measured using the Dual Luciferase Kit (Promega, Madison, WI) following manufacturer’s instructions and read in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). In this assay, the activities of Photinus pyralis and Renilla reniformis luciferases are measured sequentially from a single sample. The firefly reporter is measured first by adding Luciferase Assay Reagent II to generate a luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube.

RNA isolation and Quantitative PCR

Total RNA was isolated using an RNeasy kit (Qiagen Inc.) or Trizol (Invitrogen) extraction, according to the manufacturer’s instructions. RNA yields were measured using RiboGreen fluorescent dye (Invitrogen). First-strand cDNA was synthesized from total RNA (1 µg) by reverse transcription using oligodT and Superscript II reverse transcriptase (Invitrogen), according to the manufacturer’s instructions. Quantitative (Q)-PCR reactions were performed in a 20-µl mixture containing 1 µl of the cDNA preparation, 1X iQ SYBR Green Supermix (Bio-Rad, Hercules, CA), using the following PCR parameters: 95 ^°C for 5 min followed by 50 cycles of 95 ^°C for 15 s, 65 ^°C for 15 s and 72 ^°C for 15 s. β-Actin was used as an internal standard of mRNA expression. The absence of nonspecific products was confirmed by both the analysis of the melt curves and by electrophoresis in 3% Super AcrylAgarose gels (DNA technology, Risskov, Denmark). The primers used for Q-PCR amplification are shown in Table 2. MicroRNAS were extracted using RT² qPCR-Grade miRNA isolation kit (SABiosciences, Frederick, MD) from total RNA extracted with Trizol. miRNAs cDNA (25 ng) were amplified using TaqMan microRNA reverse transcription Kit (Applied Biosystems, Foster City, CA) and specific primers for miR-29b and U6B (Applied Biosystems), as a standard. Q-PCR products were amplified using TaqMan® Universal PCR Master Mix (Applied Biosystems), following manufacturer’s instructions. Briefly, TaqMan quantification is a two step process, in the reverse transcription step cDNA was reverse transcribed from RNA enriched with miRNAs using miR29b and U6B specific primers. PCR products were amplified from these cDNAs using 29b and U6b probes and the recommended PCR parameters: 95 ^°C for 10 min followed by 40 cycles of 95 ^°C for 15 s and 60 ^°C for 1 min. During PCR, the probe anneals specifically to a complementary sequence between the forward and reverse primer sites, when the probe is intact the proximity of the reporter dye to the quencher dye results in suppression of reporter fluorescence. The DNA polymerase cleaves only probes that are hybridized to the target, the cleavage separate the reporter from the quencher and this separation results in increased fluorescence by the reporter. The fluorescence threshold value (C_t) was calculated using the iCycle system software (Bio-Rad). The results were expressed as mean value± SE (standard error) in three independent experiments.

Cell viability assay

To evaluate changes in cell viability induced by miR-29b, cells were transfected with control mimic or miR-29b mimic. Cell viability was assayed after 5 days at 40% and 5% oxygen, by measuring the lactate dehydrogenase released to the culture media as a result of plasma membrane damage using the Cito Tox 96® Non-Radioactive Cytotoxicity assay (Promega) following the manufacturers instructions. Briefly, LDH was measured in culture supernatants and in cell lysates from each well, in a 30-min coupled enzymatic reaction, which results in the conversion of a tetrazolium salt (INT) into a red formazan product. The amount of color formed is proportional to the number of lysed cells. LDH in the supernatant was corrected for the total LDH (Supernatant/Supernatant + cell lysis). For each sample the quantification was performed in duplicate. The results were expressed as mean value±SE in three independent experiments.

Changes in gene expression induced by miR-29b in human trabecular meshwork cells

Differences in gene expression induced by miR-29b were evaluated by gene array analysis using Affymetrix U133A2 chips. HTM cells were transfected with miR-29b mimic and gene expression was compared to that in cell cultures transfected with a control mimic. One hundred sixteen genes represented by 181 probes were significantly (p≤0.05) upregulated or downregulated more than 1.5-fold. Thirty-one percent of these transcripts were predicted in at least one of the three miRNA databases as putative targets for miR-29b. MiR-29b downregulated several ECM structural proteins, as collagens (COL5A2, COL5A1, COL4A1, COL3A1, COL1A1, and COL1A2) laminin C, fibrillin 1, and microfibrillar-associated protein 3; and extracellular matrix regulators, such as MMP14, LOXL2, SERPINH1, SPARC, TNFAIP6, and ADAM 12. Other matrix regulators, such as plasminogen activator inhibitor 2, RECK, and TIMP3, showed upregulation. Table 3 shows transcripts upregulated or downregulated by more than twofold and some selected genes related to ECM that were significantly downregulated or upregulated between 1.5- and 1.9-fold.

To validate Affymetrix microarray data, changes in expression of 14 genes were analyzed by Q-PCR in three independent HTM cell lines different from the one used for array hybridization (Table 4).

Functional network analysis of gene expression changes induced by miR-29b

In order to identify the pathways and regulatory elements more likely associated with the changes in gene expression induced by miR-29b, genes significantly (p<0.5) upregulated or downregulated by 1.5-fold in the array analysis were further analyzed using MetaCore algorithms. The three canonical pathways most significantly affected by miR-29b are represented in Figure 1 and include cell adhesion–ECM remodeling (p=1.5 E-107); cytoskeleton remodeling (p=2 E-105), and cell adhesion–integrin-mediated cell adhesion and migration (p=4 E-105; Figure 1). Analysis of transcription factor regulation identified SP1 as the transcription factor most significantly (p=4.82 E-106) involved in the regulation of genes affected by miR-29b with 46 nodes (Figure 2).

Targeting of the 3’ untranslated regions of BMP1, ADAM12, and NKIRAS2 mRNA by miR-29b

Computational predictions indicate that miR-29b shares complementarity with sequences in the 3’UTR of three genes found to be downregulated by miR-29b according to the gene array analysis: BMP1, ADAM12, and NKIRAS2 (Figure 3A). We investigated whether miR-29b could interact with the 3’UTRs of these genes by using the psiCheck2 luciferase assay system. MiR-29b mimic significantly reduced luciferase expression in cells cotransfected with the 3’UTR of BMP1, ADAM12, or NKIRAS2 compared to mimic control (scramble). The decrease in luciferase activity was completely or significantly prevented when the 3’UTR complementary sequences were used (Figure 3B). Downregulation of these gene transcripts by miR-29b was confirmed by Q-PCR in three HTM cells lines (Figure 3C).

Effects of chronic oxidative stress on the expression of miR-29b.

Changes in expression of miR-29b induced by chronic oxidative stress were analyzed by Q-PCR in three independent HTM lines after 4 days at 40% oxygen compared to parallel cultures incubated at 5% oxygen. In these conditions miR-29b decreased significantly between 2- and 2.5-fold in two of the three cell lines analyzed and showed no significant change in a third line (Figure 4).

Role of miR-29b on changes in expression of extracellular matrix genes induced by chronic oxidative stress.

To investigate whether the downregulation of miR-29b observed in two cell lines could mediate alterations in gene expression induced by chronic oxidative stress, the effects of incubation at 40% oxygen on the expression of six genes known to be regulated by miR-29b (COL1A2, COL5A1, COL3A1, COL1A1, LAMC, and SPARC) were analyzed in the same three HTM cell lines used in the previous experiment. The experiments were conducted in cells transfected with either control mimic or miR-29b mimic. Although there was a high level of variability in the effects of chronic oxidative stress on the expression of the selected genes, the two cell lines where miR-29b had been found to be downregulated more than twofold under oxidative stress conditions showed a significant increase in the expression of several genes regulated by miR-29b. In contrast, incubation at 40% oxygen had little effect on the expression of these genes in the cell line where miR-29b had not been found to be altered by chronic oxidative stress. In all cell lines, transfection with miR-29b mimic led to either a significant downregulation or decrease in the upregulation mediated by chronic oxidative stress of all genes analyzed compared to cultures transfected with control mimic (Figure 5).

Effects of miR-29b on cytotoxicity

HTM cells transfected with control mimic or miR-29b mimic were subjected to 40% or 5% oxygen and analyzed for cytotoxicity after 5 days. Cells transfected with miR-29b mimic showed a significant decrease in cytotoxicity compared to the control in both oxygen concentrations (5 and 40%) except for HTM cell line 3 at 40% O₂, which showed a nonsignificant decrease in cytotoxicity (Figure 6).