Ptosis by targeting the oncogene TRIB2. Study of the TRIB2 oncogene and its related miRNAs miR-511 and miR-1297) may provide new targets for lung cancer therapy.embedded in paraffin, and sectioned. Sections were deparaffinized and rehydrated in alcohol, incubated in hydrogen peroxide, followed by 10 normal goat serum (Bei Jing Zhong Shan-Golden Bridge Technology CO, LTD, China). Sections were then incubated with anti-TRIB2 primary antibodies (1:300, dilution, Santa Cruz Biotechnology, Inc. USA), and were exposed to the biotin-conjugated goat anti-rabbit IgG (1:300, dilution, Santa Cruz Biotechnology, Inc. USA). TRIB2 expression was examined under the Olympus BX51 AX-70 microscope (Olympus, Japan). Image analysis was used by the Image-Pro Plus software. Parameters include positive expression area, mean density and integral optical density (IOD). Brown regions represent Rubusoside protein positive expression. Then, the data of each group was analyzed.Construction of pcDNA-GFP-TRIB2?9UTR vectorThe relationship between TRIB2?9-UTR and its targeted miRNAs was predicted using microRNA analysis software online (http://www.microrna.org/microrna/getMirnaForm.do, or http://www.targetscan.org/index.html). These websites provide a comprehensive analysis of the targeting genes of miRNAs. The 39-UTR (1739 bp) of TRIB2 gene was cloned by PCR using the following Primers: Tubastatin-A biological activity forward 59-TGGTGCTAAGGAAGTGTC-39 and reverse 59-CTGGTTACGAAGGGTGAA-39. Amplification conditions were as follows: 5 min initial denaturation at 95uC followed by 28 cycles of 45 sec denaturation at 95uC, 45 sec annealing at 54uC, 2 min elongation at 72uC. The 39-UTR was cloned into the T vector (Takara Bio Inc, Japan) to construct TTRIB2-UTR vector. The 39-UTR of TRIB2 was cut from T-Materials and Methods ImmunohistochemistryLung adenocarcinoma tissue samples (obtained from the Affiliated Hospital to Binzhou Medical University after a curative operation, with approval 15755315 from the Medical Ethics Committee of Binzhou Medical University. Written informed consent of each patient was obstained.) were fixed in 4 paraformaldehydemiRNA Suppressing TRIB2 ExpressionFigure 4. Detection of protein by western blotting. (A, B) lung adenocarcinoma A549 cells were treated with miRNAs and their controls, TRIB2 expression was detected and the results showed that its expression in the miR-511- and miR-1297-treated cultures was much lower than that of NC(or mutation miRNA)-treated cultures (*p,0.01). Relative values for TRIB2 vs GAPDH are indicated to the right of the gel (Fig. 4B). (C, D) Another lung adenocarcinoma LTEP-a-2 cells were treated with miRNAs and their controls, TRIB2 expression was aso much lower in 
the miR-511- and miR-1297treated cells than that of NC-(or mutation miRNA)-treated cultures (*p,0.01). Relative values for TRIB2 vs GAPDH are shown to the right of the gel (Fig. 4D). (E, F) C/EBPa expression was analyzed and the results showed that its expression was increased in the miR-511- and miR-1297-treated cells than that of the control cells (NC group, *p,0.05). Relative values for TRIB2 vs GAPDH are indicated to the right of the gel (Fig. 4F). N, negative control cells. NC, miR-511, miR-1297, mut-miR-511, mut-miR-1297, and pcDNA-TRIB2, cells treated with NC, 
miR-511, miR-1297, mut-miR-511, mutmiR-1297, and pcDNA-TRIB2 vector, respectively. doi:10.1371/journal.pone.0046090.gTRIB2-UTR vector and inserted to the downstream of the GFP gene in the pcDNA-GFP vector (described previously) [32] by KpnI/HindI.Ptosis by targeting the oncogene TRIB2. Study of the TRIB2 oncogene and its related miRNAs miR-511 and miR-1297) may provide new targets for lung cancer therapy.embedded in paraffin, and sectioned. Sections were deparaffinized and rehydrated in alcohol, incubated in hydrogen peroxide, followed by 10 normal goat serum (Bei Jing Zhong Shan-Golden Bridge Technology CO, LTD, China). Sections were then incubated with anti-TRIB2 primary antibodies (1:300, dilution, Santa Cruz Biotechnology, Inc. USA), and were exposed to the biotin-conjugated goat anti-rabbit IgG (1:300, dilution, Santa Cruz Biotechnology, Inc. USA). TRIB2 expression was examined under the Olympus BX51 AX-70 microscope (Olympus, Japan). Image analysis was used by the Image-Pro Plus software. Parameters include positive expression area, mean density and integral optical density (IOD). Brown regions represent protein positive expression. Then, the data of each group was analyzed.Construction of pcDNA-GFP-TRIB2?9UTR vectorThe relationship between TRIB2?9-UTR and its targeted miRNAs was predicted using microRNA analysis software online (http://www.microrna.org/microrna/getMirnaForm.do, or http://www.targetscan.org/index.html). These websites provide a comprehensive analysis of the targeting genes of miRNAs. The 39-UTR (1739 bp) of TRIB2 gene was cloned by PCR using the following Primers: forward 59-TGGTGCTAAGGAAGTGTC-39 and reverse 59-CTGGTTACGAAGGGTGAA-39. Amplification conditions were as follows: 5 min initial denaturation at 95uC followed by 28 cycles of 45 sec denaturation at 95uC, 45 sec annealing at 54uC, 2 min elongation at 72uC. The 39-UTR was cloned into the T vector (Takara Bio Inc, Japan) to construct TTRIB2-UTR vector. The 39-UTR of TRIB2 was cut from T-Materials and Methods ImmunohistochemistryLung adenocarcinoma tissue samples (obtained from the Affiliated Hospital to Binzhou Medical University after a curative operation, with approval 15755315 from the Medical Ethics Committee of Binzhou Medical University. Written informed consent of each patient was obstained.) were fixed in 4 paraformaldehydemiRNA Suppressing TRIB2 ExpressionFigure 4. Detection of protein by western blotting. (A, B) lung adenocarcinoma A549 cells were treated with miRNAs and their controls, TRIB2 expression was detected and the results showed that its expression in the miR-511- and miR-1297-treated cultures was much lower than that of NC(or mutation miRNA)-treated cultures (*p,0.01). Relative values for TRIB2 vs GAPDH are indicated to the right of the gel (Fig. 4B). (C, D) Another lung adenocarcinoma LTEP-a-2 cells were treated with miRNAs and their controls, TRIB2 expression was aso much lower in the miR-511- and miR-1297treated cells than that of NC-(or mutation miRNA)-treated cultures (*p,0.01). Relative values for TRIB2 vs GAPDH are shown to the right of the gel (Fig. 4D). (E, F) C/EBPa expression was analyzed and the results showed that its expression was increased in the miR-511- and miR-1297-treated cells than that of the control cells (NC group, *p,0.05). Relative values for TRIB2 vs GAPDH are indicated to the right of the gel (Fig. 4F). N, negative control cells. NC, miR-511, miR-1297, mut-miR-511, mut-miR-1297, and pcDNA-TRIB2, cells treated with NC, miR-511, miR-1297, mut-miR-511, mutmiR-1297, and pcDNA-TRIB2 vector, respectively. doi:10.1371/journal.pone.0046090.gTRIB2-UTR vector and inserted to the downstream of the GFP gene in the pcDNA-GFP vector (described previously) [32] by KpnI/HindI.
