Redicted using microRNA analysis software, which showed that the 39UTR of TRIB2 might be targeted by miR-511, miR-1297, et al (Figure 2A), which were not published before. The pcDNA-GFP-TRIB2?9UTR vector was then constructed (Figure 2B). These miRNAs, negative control, and mutation miRNAs were chemically synthesized in the form of small interfering RNA (siRNA) duplexes according to Park’s study (Park SY et al., 2009) (Table 1). After the reporter plasmid (pcDNA-GFP-TRIB2?9UTR vector) was constructed, miRNA was co-transfected with the reporter plasmid into A549 cells, and higher miRNAs were detected in the miRNA-treated cells than in the untreated cells (Figure 2C). The GFP expression levels were then estimated by examination under fluorescence microscopy and 
by flow cytometry. The intensities of fluorescence from the miRNA-treated cultures were all decreased and the number of GFP-positive cells was reduced in comparison to the control cultures (Figure 3A), indicating a partial knock-down of expression of the TRIB2-GFP reporter by these miRNA molecules tested. Particularly, the intensity of fluorescence in the miR-511- and KDM5A-IN-1 site miR-1297-treated cells provided the strongest inhibitory effect. For example, the percentage of GFP-positive cells in the miR-511 (or miR-1297)-treated culture was 29.7 (or 25.8 ), much lower than NC control culture (40.8 ) (Figure 3B), the other miRNAs (miR-26a, miR-125a, miR-132) did not inhibit GFP expression appreciably compared with the control cultures (data not shown). When we mutated the seed sequences of miR511/1297, the expression of GFP was not decreased obviously in mut-miR-511- or mut-miR-1297-treated cells compared with miR-511- or miR-1297-treated cells (Figure S1).C/EBPa expression affected by the miR-511/1297 suppression pathwayAccording to previous studies, the expression of C/EBPa, a transcription factor downstream of TRIB2, can also be regulated by factors that affect TRIB2 expression [13]. Therefore, we examined the expression of C/EBPa by western blot after A549 cells were treated with miR-511/1297. The results showed that C/ EBPa was increased in miR-511- and miR-1297-treated cells compared with NC-treated cultures after miR-511/1297 inhibiting TRIB2 expression, while C/EBPa was decreased after overexpression TRIB2 by transfecting pcDNA-TRIB2 vector (Figure 4E, F). Our results showed that miR-511 and miR-1297 could 
inhibit A549 cell proliferation by downregulation of TRIB2 and upregulation of C/EBPa.miR-511/1297 inhibiting lung adenocarcinoma cell proliferation in nude miceAfter miR-511/1297 transfection, a xenograft of A549 cells was subcutaneously injected into the K162 web dorsal flank of nude mice. TumormiRNA Suppressing TRIB2 ExpressionFigure 1. The expression of TRIB2 and miR-511/1297 on control tissue and adenocarcinoma of lung. (A,B) Immunohistochemistry and IOD analysis of TRIB2. Upper row: Scale bar = 200 1326631 mm. Lower row: Scale bar = 20 mm. Control, the para-carcinoma tissues. Carcinoma, adenocarcinoma of lung. TRIB2 expression (Fig. 1 A) and its IOD in the adenocarcinoma tissue (Fig. 1 B) were higher than that of the control tissue (p,0.01). (C) Realtime PCR showed that the expression of miR-511 and miR-1297 was much lower in the lung adenocarcinoma than that of control tissue (p,0.05). doi:10.1371/journal.pone.0046090.gvolumes were calculated after the mice developed palpable tumors. The volumes of xenografts were found to be smaller in mice which received miR-511- and miR-1297-treated cells co.Redicted using microRNA analysis software, which showed that the 39UTR of TRIB2 might be targeted by miR-511, miR-1297, et al (Figure 2A), which were not published before. The pcDNA-GFP-TRIB2?9UTR vector was then constructed (Figure 2B). These miRNAs, negative control, and mutation miRNAs were chemically synthesized in the form of small interfering RNA (siRNA) duplexes according to Park’s study (Park SY et al., 2009) (Table 1). After the reporter plasmid (pcDNA-GFP-TRIB2?9UTR vector) was constructed, miRNA was co-transfected with the reporter plasmid into A549 cells, and higher miRNAs were detected in the miRNA-treated cells than in the untreated cells (Figure 2C). The GFP expression levels were then estimated by examination under fluorescence microscopy and by flow cytometry. The intensities of fluorescence from the miRNA-treated cultures were all decreased and the number of GFP-positive cells was reduced in comparison to the control cultures (Figure 3A), indicating a partial knock-down of expression of the TRIB2-GFP reporter by these miRNA molecules tested. Particularly, the intensity of fluorescence in the miR-511- and miR-1297-treated cells provided the strongest inhibitory effect. For example, the percentage of GFP-positive cells in the miR-511 (or miR-1297)-treated culture was 29.7 (or 25.8 ), much lower than NC control culture (40.8 ) (Figure 3B), the other miRNAs (miR-26a, miR-125a, miR-132) did not inhibit GFP expression appreciably compared with the control cultures (data not shown). When we mutated the seed sequences of miR511/1297, the expression of GFP was not decreased obviously in mut-miR-511- or mut-miR-1297-treated cells compared with miR-511- or miR-1297-treated cells (Figure S1).C/EBPa expression affected by the miR-511/1297 suppression pathwayAccording to previous studies, the expression of C/EBPa, a transcription factor downstream of TRIB2, can also be regulated by factors that affect TRIB2 expression [13]. Therefore, we examined the expression of C/EBPa by western blot after A549 cells were treated with miR-511/1297. The results showed that C/ EBPa was increased in miR-511- and miR-1297-treated cells compared with NC-treated cultures after miR-511/1297 inhibiting TRIB2 expression, while C/EBPa was decreased after overexpression TRIB2 by transfecting pcDNA-TRIB2 vector (Figure 4E, F). Our results showed that miR-511 and miR-1297 could inhibit A549 cell proliferation by downregulation of TRIB2 and upregulation of C/EBPa.miR-511/1297 inhibiting lung adenocarcinoma cell proliferation in nude miceAfter miR-511/1297 transfection, a xenograft of A549 cells was subcutaneously injected into the dorsal flank of nude mice. TumormiRNA Suppressing TRIB2 ExpressionFigure 1. The expression of TRIB2 and miR-511/1297 on control tissue and adenocarcinoma of lung. (A,B) Immunohistochemistry and IOD analysis of TRIB2. Upper row: Scale bar = 200 1326631 mm. Lower row: Scale bar = 20 mm. Control, the para-carcinoma tissues. Carcinoma, adenocarcinoma of lung. TRIB2 expression (Fig. 1 A) and its IOD in the adenocarcinoma tissue (Fig. 1 B) were higher than that of the control tissue (p,0.01). (C) Realtime PCR showed that the expression of miR-511 and miR-1297 was much lower in the lung adenocarcinoma than that of control tissue (p,0.05). doi:10.1371/journal.pone.0046090.gvolumes were calculated after the mice developed palpable tumors. The volumes of xenografts were found to be smaller in mice which received miR-511- and miR-1297-treated cells co.
