Mpared with control cells after a period of two weeks (Figure 6 A B). The miR-511 and 15900046 miR-1297 Tunicamycin site levels were found to be higher (Figure 6 C) and TRIB2 expression was decreased in miRNAtreated groups (Figure 6 D). These results demonstrated thattreatment with miR-511/1297 could also inhibit A549 cell proliferation in vivo by suppressing TRIB2 oncogene expression.DiscussionmiRNAs, together with partner proteins, bind to the 39 UTR region of their specific target mRNA to regulate target genes by degradation of target mRNAs or inhibition of gene expressionmiRNA Suppressing TRIB2 ExpressionFigure 2. The targeting sites of TRIB2?9UTR and structural map of the vector. (A) The targeting sites on TRIB2?9UTR. miR-511, miR-1297 and miR-26a are shown. Each miRNA is aligned with the mRNA of human TRIB2 with the nucleotide position on the TRIB2 mRNA indicated. Vertical lines indicate identity; gaps indicate mismatch. (B) Structural map of the pcDNA-GFP-TRIB2?9UTR vector. 39-UTR of TRIB2 is indicated downstream of the GFP gene. Pcmv: cmv promoter. GFP: green fluorescent protein. TRIB2?9UTR, 39-UTR of the TRIB2 gene. (C) Real-time PCR detection. 24 h after lung adenocarcinoma cells were treated with miRNAs, higher miR-511 and miR-1297 levels were found in the transfected cells than in the nontransfected cells. doi:10.1371/journal.pone.0046090.g[15]. We predicted the possible miRNAs (miR-511 and miR-1297, among others) which could possibly targeting TRIB2 by using microRNA analysis LED-209 site software online. A plasmid, containing the GFP reporter gene followed by the 39-UTR of TRIB2, was constructed to analyze the interaction between TRIB2 and miR511/1297. Our results showed that miR-511 and miR-1297 could downregulate the GFP expression compared with NC-treated cells. The expression of TRIB2 was further found to be suppressed by miR-511 and miR-1297. As the one factor known to be regulated by 
TRIB2 [13], C/EBPa expression was increased after miR-511 (or miR-1297) treatment. Our results demonstrate that miR-511 and miR-1297 could inhibit A549 cell proliferation by suppressing TRIB2 expression and thus increasing C/EBPa expression. Several previous studies showed that TRIB2 acted as an oncogene and increasing TRIB2 expression led to AML tumorTable 1. The sequences of chemically synthesized miRNA.aOligos miR-511 sense antisense miR-1297 sense antisense miR-26a sense antisense negative control(NC) sense antisense mut-miR-511 sense antisense mut-miR-1297 sense antisenseSequence (59R39) gugucuuuugcucugcaguca ugacugcagagcaaaagacacuu uucaaguaauucaggug caccugaauuacuugaauu uucaaguaauccaggauaggcu agccuauccuggauuacuugaauu caguacuuuuguguaguacaa Guacuacacaaaaguacuguu guauacuuugcucugcaguca* ugacugcagagcaaaguauacuu uugauauaauucaggug* caccugaauuauaucaauua The selected miRNAs were chemically synthesized in the form of small interfering RNA (siRNA) duplexes. *the bold and underlined letters were the mutation sites of miRNA. doi:10.1371/journal.pone.0046090.tigenesis [13,14]. The oncogenic role of TRIB2 was further confirmed in our study, which showed increased levels of TRIB2 in human lung adenocarcinoma tissue. We also showed that inhibition of TRIB2 expression by its relevant miRNA (miR-511 or miR-1297) could induce tumor cell apoptosis. Tribbles proteins promote ubiquitin-dependent degradation of their target proteins. 
TRIB2 promotes the degradation of C/EBPa and C/EBPb which have been described as transcription factors [13]. The intact Cterminal constitutive photomorphogenesis 1.Mpared with control cells after a period of two weeks (Figure 6 A B). The miR-511 and 15900046 miR-1297 levels were found to be higher (Figure 6 C) and TRIB2 expression was decreased in miRNAtreated groups (Figure 6 D). These results demonstrated thattreatment with miR-511/1297 could also inhibit A549 cell proliferation in vivo by suppressing TRIB2 oncogene expression.DiscussionmiRNAs, together with partner proteins, bind to the 39 UTR region of their specific target mRNA to regulate target genes by degradation of target mRNAs or inhibition of gene expressionmiRNA Suppressing TRIB2 ExpressionFigure 2. The targeting sites of TRIB2?9UTR and structural map of the vector. (A) The targeting sites on TRIB2?9UTR. miR-511, miR-1297 and miR-26a are shown. Each miRNA is aligned with the mRNA of human TRIB2 with the nucleotide position on the TRIB2 mRNA indicated. Vertical lines indicate identity; gaps indicate mismatch. (B) Structural map of the pcDNA-GFP-TRIB2?9UTR vector. 39-UTR of TRIB2 is indicated downstream of the GFP gene. Pcmv: cmv promoter. GFP: green fluorescent protein. TRIB2?9UTR, 39-UTR of the TRIB2 gene. (C) Real-time PCR detection. 24 h after lung adenocarcinoma cells were treated with miRNAs, higher miR-511 and miR-1297 levels were found in the transfected cells than in the nontransfected cells. doi:10.1371/journal.pone.0046090.g[15]. We predicted the possible miRNAs (miR-511 and miR-1297, among others) which could possibly targeting TRIB2 by using microRNA analysis software online. A plasmid, containing the GFP reporter gene followed by the 39-UTR of TRIB2, was constructed to analyze the interaction between TRIB2 and miR511/1297. Our results showed that miR-511 and miR-1297 could downregulate the GFP expression compared with NC-treated cells. The expression of TRIB2 was further found to be suppressed by miR-511 and miR-1297. As the one factor known to be regulated by TRIB2 [13], C/EBPa expression was increased after miR-511 (or miR-1297) treatment. Our results demonstrate that miR-511 and miR-1297 could inhibit A549 cell proliferation by suppressing TRIB2 expression and thus increasing C/EBPa expression. Several previous studies showed that TRIB2 acted as an oncogene and increasing TRIB2 expression led to AML tumorTable 1. The sequences of chemically synthesized miRNA.aOligos miR-511 sense antisense miR-1297 sense antisense miR-26a sense antisense negative control(NC) sense antisense mut-miR-511 sense antisense mut-miR-1297 sense antisenseSequence (59R39) gugucuuuugcucugcaguca ugacugcagagcaaaagacacuu uucaaguaauucaggug caccugaauuacuugaauu uucaaguaauccaggauaggcu agccuauccuggauuacuugaauu caguacuuuuguguaguacaa Guacuacacaaaaguacuguu guauacuuugcucugcaguca* ugacugcagagcaaaguauacuu uugauauaauucaggug* caccugaauuauaucaauua The selected miRNAs were chemically synthesized in the form of small interfering RNA (siRNA) duplexes. *the bold and underlined letters were the mutation sites of miRNA. doi:10.1371/journal.pone.0046090.tigenesis [13,14]. The oncogenic role of TRIB2 was further confirmed in our study, which showed increased levels of TRIB2 in human lung adenocarcinoma tissue. We also showed that inhibition of TRIB2 expression by its relevant miRNA (miR-511 or miR-1297) could induce tumor cell apoptosis. Tribbles proteins promote ubiquitin-dependent degradation of their target proteins. TRIB2 promotes the degradation of C/EBPa and C/EBPb which have been described as transcription factors [13]. The intact Cterminal constitutive photomorphogenesis 1.