Smad3-related miRNAs regulated oncogenic TRIB2 promoter activity to effectively suppress lung adenocarcinoma growth

MicroRNAs (miRNAs) and Smad3, as key transcription factors in transforming growth factor-β1 (TGF-β1) signaling, help regulate various physiological and pathological processes. We investigated the roles of Smad3-regulated miRNAs with respect to lung adenocarcinoma cell apoptosis, proliferation, and metastasis. We observed that Smad3 and phospho-SMAD3 (p-Smad3) were decreased in miR-206- (or miR-140)-treated cells and there might be a feedback loop between miR-206 (or miR-140) and TGF-β1 expression. Smad3-related miRNAs affected tribbles homolog 2 (TRIB2) expression by regulating trib2 promoter activity through the CAGACA box. MiR-206 and miR-140 inhibited lung adenocarcinoma cell proliferation in vitro and in vivo by suppressing p-Smad3/Smad3 and TRIB2. Moreover, lung adenocarcinoma data supported a suppressive role for miR-206/miR-140 and an oncogenic role for TRIB2—patients with higher TRIB2 levels had poorer survival. In summary, miR-206 and miR-140, as tumor suppressors, induced lung adenocarcinoma cell death and inhibited cell proliferation by modifying oncogenic TRIB2 promoter activity through p-Smad3. MiR-206 and miR-140 also suppressed lung adenocarcinoma cell metastasis in vitro and in vivo by regulating EMT-related factors.

lung adenocarcinoma samples, and noted that miR-206 and miR-140 expression decreased in adenocarcinoma samples compared with para-carcinomas (n = 10, Figure 1a). In situ hybridization further proved that the expression of miR-206 and miR-140 significantly decreased in type 2 epithelial cells in lung adenocarcinoma samples compared with those in para-carcinomas ( Figure 1b). Next, we verified their roles in lung adenocarcinoma in vitro. We observed that miR-206 can inhibit lung adenocarcinoma cell (A549) proliferation ( Figure  1c and d) and FACS analysis confirmed that miR-206 treatment induced more apoptosis compared with NC-oligo control, whereas the inhibition of proliferation by miR-206 was abolished with Mu-206-control treatment (Figure 1e). The role of miR-206 in inducing A549 apoptosis was abolished after application of a miR-206 inhibitor (Aso-206, Figure 1e), supporting a tumor suppressive role of miR-206. Our results revealed that A549 cells in the G1 phase increased significantly after miR-206 treatment compared with control (oligo-treated) cultures (Figure 1f), suggesting that the suppressive role of miR-206 is relevant to regulating the cell cycle, most likely due to G1 phase inhibition.
Then we asked whether there is any relationship between miR-206 (or miR-140) and the TGF-β1 pathway or whether TGF-β1 levels are affected by miR-206 and miR-140 in turn? The results analyzed by online miRNA analysis software did not show that 3′-UTR of TGF-β1 was targeted by miR-206 (or  miR-206 and miR-140 regulate TRIB2 promoter activity through Smad3-binding 'CAGACA'. Previously, a Tribble family member TRIB3 was reported to interact with Smad3. 18 TRIB2, another Tribble family member, acts as an oncogene in acute myeloid and T-cell acute lymphoblastic leukemias 19 and some lung cancers, 20,21 but whether TRIB2 and Smad3 interact is unclear. Thus, we reduced p-Smad3 expression with miR-206 or miR-140, and noted that TRIB2 was reduced in miR-206-(or miR-140)-transfected A549 cells, and the TRIB2 levels inhibited by miR-206 (or miR-140) was abolished in the mutation mimics (Mu-206 or Mu-140)-treated cultures (Figure 4a,Supplementary Figure 4), indicating that TRIB2 was also regulated by miR-206 and miR-140.
SiRNA can be used to investigate gene function and signal pathways, so we used this method to study whether miR-206 and miR-140 regulated TRIB2 expression through Smad3. Using siRNA specifically designed for Smad3, we observed that this siRNA could inhibit Smad3 mRNA compared with siRNA control-treated cells (Figure 4b). Smad3 and p-Smad3 were lower in siRNA-treated A549 cells compared with control cells, indicating that siRNA decreased Smad3 expression (Figure 2d and e, Supplementary Figure 2). To understand whether these siRNAs (siRNA-Smad3, specific to Smad3) affected the expression of TRIB2, we observed that TRIB2 expression decreased in siRNA-Smad3-treated cells compared with control treatment (Figure 4a, Supplementary  Figure 4), an outcome similar to the effects of miR-206 and miR-140 on TRIB2 expression. siRNA experiments in LTEP-a-2 cells were also similar as well regarding TRIB2 expression. Therefore, Smad3-related miR-206, miR-140, and siRNA could regulate TRIB2 expression, suggesting that Smad3 may affect TRIB2 expression.
In the TGF-β1 pathway, p-Smad3 was reported to bind other Smad proteins together to form a protein complex, which then moves to the cell nucleus and promotes Smad3 responsive promoter activity to drive gene expression. 22 Therefore, we studied whether Smad3 could drive trib2 gene expression by affecting trib2 gene promoter. Then, different lengths of TRIB2 promoter luciferase plasmids were cloned ( Figure 4c) and Hela cells were treated with these plasmids. The 2.9 kb of the  Figure 4d). Activity of the 2.9 kb section of the TRIB2 promoter (WT1) induced by TGF-β1 was blocked in cells treated with 5 μmol/ml SB431542 or siRNA-Smad3 oligos ( Figure 4e). We further found that miR-206 and miR-140 also effectively blocked the activity of TRIB2 promoter (WT1) induced by TGF-β1 (Figure 4f), which proved that TGF-β1/ Smad3 promoted TRIB2 promoter activity. Moreover, experiments with cells treated mouse trib2 promoter-luciferase reporter also demonstrated that TGF-β1 treatment enhanced mouse TRIB2 promoter activity, whereas SB431542 treatment inhibited mouse TRIB2 promoter activity induced by TGF-β1  Figure 5), which further proved that TGF-β1/Smad3 could promote TRIB2 promoter activity obviously.
The activity of promoter induced by TGF-β1 might be related to the Smad3-binding consensus sequence CAGACA. 23 We found that there was a sequence box 'CAGACA' at position − 2698 and − 2692 of the human TRIB2 promoter and mutated this sequence to further prove whether TGF-β1/Smad3 promoted TRIB2 promoter activity through CAGACA. When the CAGACA box at position − 2698 was mutated in the Mutpromoter plasmid using site-directed Gene Mutagenesis Kit (Supplementary Figure 6), the luciferase expression decreased in Mut-promoter plasmid-treated cultures compared with wild type (WT1) cultures. Moreover, TGF-β1 treatment cannot increase the luciferase levels in Mutpromoter plasmid-treated cultures (Figure 4g), which proved that Smad3 promotes TRIB2 responsive promoter activity to drive TRIB2 expression by the 'CAGACA' box. To further investigate the role of Smad3 on TRIB2 responsive promoter activity, the transcriptional levels of TRIB2 mRNA were analyzed after TGF-β1 treatment. Our results showed that 10 ng/ml TGF-β significantly increased TRIB2 mRNA levels ( Figure 4h).
The CCAAT/enhancer-binding proteins α and β (C/EBPα and β) are reported to be downstream factors of TRIB2. 24,25 To investigate the effects of Smad3-related miRNAs on C/EBPα and β expression, lung adenocarcinoma cells were treated with miR-140 and miR-206. Western blot revealed that expression of C/EBPα and β increased in miR-206-and miR-140-treated A549 cells compared with control treatment (Figure 4i and j). Similar results occurred in siRNA oligo-treated cultures, indicating a regulatory role for miR-206 and miR-140 with respect to TRIB2 and its downstream factors.
miR-206 and miR-140 inhibited cell metastasis through Smad3. MiRNAs have been reported to inhibit tumor metastasis in hepatocellular or ovarian cancer cells. 26,27 In our studies to learn how miRNAs function in lung adenocarcinoma metastasis, we found that fewer cells migrated to the lower chamber in miR-206-(or miR-140)-treated cultures compared with scrambled oligo-treated cells (Figure 5a and b), suggesting that miR-206 or miR-140 can inhibit lung adenocarcinoma metastasis. Smad3, a target of miR-206 or miR-140, has a role in tumor cell metastasis and E-cadherin and α-SMA 12 expression, so we measured these proteins in miR-206-(or miR-140)-treated A549 cells. We found that E-cadherin was elevated, and α-SMA was decreased in miR-206-(or miR-140)-treated A549 cells, similar to data observed after SB431542 treatment (Figure 5c). Western blot confirmed that miR-206 and miR-140 increased E-cadherin and downregulated α-SMA expression in A549 cells (Figure 5d,Supplementary Figure 7). However, using TGF-β1 to induce p-Smad3 overexpression enhanced α-SMA and downregulated E-cadherin in TGF-β1-treated cells, which could be ameliorated in miR-206-(or miR-140)-treated cultures compared with control treatment (Figure 5c and d, Supplementary Figure 7). Because miR-206 and miR-140 also affects Smad3-related TRIB2 expression, we next investigated how TRIB2 contributes to cell migration.
Relatively fewer cells migrated to the lower chamber in siRNA (specific to TRIB2)-treated cultures compared with siRNA control-treated cells (Figure 5e and f), supporting that the inhibition to cell migration by miR-206 and miR-140 may also attribute to Smad3-related TRIB2 expression.
To study the effects of miR-206 and miR-140 on the metastasis of A549 cells in vivo, 2 × 10 6 GFP-positive A549 cells transfected with miRNAs were injected into male nude mice by tail vein. Seven weeks after injection, small animal in vivo imaging results showed that fewer GFP-positive A549 cells migrated to the lungs in nude mice treated with miR-206 or miR-140 compared with scrambled control treatment (Figure 6a). In addition, HE staining of lung sections also supported that miR-206 or miR-140 decreased migratory tumors in miRNAs-treated tumors (n = 3) compared with scrambled control treatment (n = 3, Figure 6b). As a cell adhesion molecule, human CD44 increases the migratory capacity of various cancers. 28 We further detected human CD44 expression to analyze the migratory capacity and number of A549 cells in oligo-treated metastatic nodules by using anti-human specific CD44 primary antibody. Interestingly, human CD44 expression decreased in miR-206-(or miR-140)-treated metastatic A549 cell nodules compared with that in scrambled control-treated metastatic nodules (n = 3, Figure 6c). Therefore, Smad3-related miR-206 and miR-140 could effectively inhibit lung cancer cell metastasis in vitro and in vivo.
miR-206 and miR-140 suppressed cell proliferation in vivo through TRIB2. To evaluate the roles of miR-206 and miR-140 in the regulation of cell proliferation in vivo, A549 lung cancer xenografts were established in BALB/C-nu mice. Tumor volumes and weights were smaller in miR-206-(or miR-140)-treated xenografts compared with scrambledoligo controls (Figure 7a and b). qRT-PCR data show that miR-206 (or miR-140) increased in miR-206-(or miR-140)treated xenografts compared with control tumors (Figure 7c). P-Smad3 (or Smad3), the target of miR-206 (or miR-140), was lower in tumors treated with miR-206 (or miR-140) compared with control tumors (Figure 7d). TRIB2 expression also decreased in miR-206-(or miR-140)-treated xenografts compared with control treatment (Figure 7d). The suppressive action of miRNA to tumorigenicity may be attributed to downregulation of TRIB2, which was supported by our previous study, 21 demonstrating that lower levels of TRIB2 lead to inhibiting lung adenocarcinoma cell growth in vivo.
Smad3 and TRIB2 expression in lung adenocarcinoma samples and clinical outcomes. We measured Smad3 and TRIB2 expression in lung adenocarcinoma samples. Different to lower levels of miR-206 and miR-140 in adenocarcinoma samples, we noted that p-Smad3, Smad3, and TRIB2 were higher in adenocarcinoma samples compared with para-carcinomas (n = 10, Figure 8a and b, Po0.01), and this was negatively correlated with miR-206 and miR-140 expression, which supporting that the suppressive roles of miR-206 (or miR-140) in regulating the expression of p-Smad3/Smad3 and TRIB2.
Kaplan-Meier survival analysis indicated that patients with greater TRIB2 had a poor survival (Figure 8d). TRIB2 was significantly positively correlated with Smad3 (n = 111, r s = 0.227, P = 0.016, Figure 8d), suggesting a positive regulation of Smad3 to TRIB2 promoter activity. These data with the luciferase experiments in vitro indicates that Smad3 can increase TRIB2 expression.

Discussion
MiRNAs are involved in cell proliferation, metastasis, apoptosis, and stress responses 29 and miR-206 and miR-140 are important suppressors of lung adenocarcinoma cell proliferation and metastasis. We observed there might be a negative feedback loop between miR-206 (or miR-140) and TGF-β1 whereby these two miRNAs, downregulated by TGF-β1, participated in Smad3-dependent TGF-β1 signaling and negatively regulated TGF-β1/Smad3 signals in lung adenocarcinoma (Figure 8e). After decreasing p-Smad3 expression, we observed that oncogenic TRIB2 was also regulated by miR-206/miR-140. As tumor suppressors, miR-206 and miR-140 can inhibit lung adenocarcinoma cell metastasis by increasing E-cadherin and decreasing α-SMA expression, and suppress lung adenocarcinoma cell growth in vivo by decreasing oncogenic TRIB2 promoter activity through Smad3.
MiR-140 was first identified (in chondrocytes) to play a role in cartilage development and homeostasis. 30 Compared with normal control tissues, miR-140 was downregulated in human ovarian cancer and basal cell carcinoma 31,32 and gastric cancer. MiR-140 overexpression inhibited HGC-27 cell viability and colony formation, and resulted in G0/G1 arrest by suppressing SOX4 expression. 33 In this study, we found that miR-140, as a novel miRNA directly regulating Smad3, acted as a tumor suppressor to inhibit lung adenocarcinoma proliferation and was downregulated in lung adenocarcinoma samples compared with para-carcinomas, a finding supported by Tan et al.'s study. 34 MiR-206 has also been reported to be a tumor suppressor that can block cell proliferation, migration, invasion, and tumorigenesis and induce apoptosis by regulating VEGF expression. 35 Recently, miR-206 was found to effectively inhibit stemness and metastasis of breast cancer by targeting MKL1/IL11 pathway. 36 Here we further explored the roles of miR-206 in lung adenocarcinoma and its new target. We found that miR-206 was down-expressed in lung adenocarcinoma samples and that miR-206 and miR-140 can inhibit lung adenocarcinoma cell proliferation in vitro and in vivo by downregulating new target-Smad3. Moreover, miR-206 and miR-140 can suppress A549 cell metastasis via regulating the expression of E-cadherin and α-SMA, a finding supported by Wang et al.'s group who reported that increasing miR-206 leads to cell proliferation arrest and weaker lung cancer cell invasiveness. 37 The TGF-β1 signaling pathway is critical to cell differentiation, development, proliferation, and migration. 38 Constitutive activation of TGF-β1 signaling seems to promote tumor progression through tumor-host cell interactions. 39,40 Smad3 is a central downstream modulator, which plays important roles in TGF-β1/Smad pathway. In this study, we further investigated the roles of miRNAs in lung adenocarcinoma through TGF-β1/Smad pathway, and found that miR-206 and miR-140, as signal factors in TGF-β1/Smad pathway, could inhibit lung adenocarcinoma cell proliferation and metastasis by downregulating p-Smad3/Smad3 and that this may be related to p-Smad3's promotion of cell growth or invasion 41 in TGF-β1 pathway. It was reported that p-Smad3 binds other Smad proteins into complexes to promote gene expression via regulating Smad3 responsive promoter activity. 22 Indeed, we constructed a luciferase vector driven by trib2 promoter to study whether p-Smad3 binds the promoter to activate TRIB2 transcription, and found that the 2.9 kb component of the trib2 promoter had the greatest activity for driving luciferase expression. Moreover, our results demonstrated that TGF-β1 promoted trib2 promoter activity, which was abolished by miR-206 and miR-140 treatment. By mutating the Smad-binding consensus sequence CAGACA, 23 we confirmed that p-Smad3 could bind CAGACA to regulate TRIB2 promoter activity.
Tribbles, which are inhibitors of mitosis, regulate cell proliferation, migration, and morphogenesis during development. In mammals, three Tribble homologs exist: TRIB1, TRIB2, and TRIB3, and all are associated with human malignancies. [42][43][44] Several studies indicated that TRIB2 can act as an oncogene involved in a mouse model of AML by inhibiting transcription factor C/EBPα. 24,45 In previous studies, we reported an oncogenic role of TRIB2 in lung adenocarcinoma, and proved that miR-511 and miR-1297 could suppress A549 cell proliferation in vitro and in vivo by suppressing TRIB2 and increasing C/EBPα expression. 20 Here we further demonstrated that TRIB2 were higher in adenocarcinoma samples and patients with greater TRIB2 had a poor survival. Downregulation of TRIB2 by miR-206 and miR-140 inhibited A549 cell migration in vitro and in vivo. TRIB3 can interact with Smad3 to modulate TGF-β1-Smad3 signaling and, as such, is important for tumor progression and metastasis. 18 These data suggest that TGF-β1/Smad3 may further affect TRIB2 expression in lung cancer. Indeed, we observed that TRIB2 expression decreased as p-Smad3/Smad3 was downregulated by miRNA or siRNA. Specifically, miR-206 and miR-140 suppressed lung adenocarcinoma proliferation in vitro and in vivo by decreasing TRIB2 through Smad3 in TGF-β1 pathway. Collectively, our results show that miR-206 or miR-140 can suppress lung cancer cell proliferation by reducing oncogenic TRIB2 through Smad3 regulating trib2 promoter. During tumor progression, EMT is critical for conversion of early stage tumors into invasive ones, because it promotes tumor cell infiltration into adjacent tissue and the formation of subsequent metastasis. 46 TGF-β1/Smad3 signaling regulates EMT through Smad3-dependent or -independent mechanisms. 47 During EMT, epithelial markers E-cadherin and zona occludin-1 are downregulated, whereas mesenchymal markers α-SMA and fibronectin are upregulated. 48,49 In this study, we investigated a miRNA-mediated mechanism of lung cancer cell migration and found that miR-206 and miR-140 could suppress A549 cell metastasis by regulating p-Smad3 and oncogenic TRIB2. Moreover, E-cadherin expression was upregulated, whereas α-SMA was downregulated in miR-206or miR-140-treated cells, suggesting that the mechanism of suppressing EMT by miR-206 and miR-140 might be related to regulating expression of E-cadherin and α-SMA.
Thus, we report that miR-206 and miR-140, as tumor suppressors, induce lung adenocarcinoma cell apoptosis and  111). (e) This study proposed model by which miR-206 (or miR-140) regulating Smad3 and TRIB2 in TGF-β1 pathway. p-Smad 3 binds to trib2 gene promoter to control its expression. miR-206 (or miR-140), negatively regulated by TGF-β1, can regulate the levels of Smad 3 in TGF-β1 pathway, which may in turn affect TGF-β1 levels Smad3-regulated miRNAs suppress lung cancer growth Y-X Zhang et al inhibit cell growth by reducing oncogenic trib2 promoter activity through Smad3 binding CAGACA box and that they suppress lung cancer metastasis by regulating EMT-related factors. Our work offers essential information about novel targets for the development of new therapeutics for treating lung cancers.

Materials and Methods
Lung adenocarcinoma samples. Fresh lung adenocarcinoma and paracarcinoma tissues from patients who underwent surgery at YanTaiShan Hospital were obtained after surgery and immediately prepared for pathological diagnosis, western blot or RNA analysis. All experiments were performed in accordance with relevant guidelines of the Medical Ethics Committee of Binzhou Medical University. Before study inclusion, patients provided written informed consent after study procedures were fully explained. RNAs were isolated with Trizol (Takara, Otsu, Shiga, Japan). cDNAs were synthesized using RT primer (Poly T) and forward primer to amplify Smad3 was: 5′-AGCACACAATAACTTGGACC-3′; reverse: 5′-TAAGACACACTGGAACAGCG GATG-3'. PCR conditions were 30 cycles of denaturation at 94°C for 45 s, annealing at 50°C for 45 s and elongation at 72°C for 45 s, performed in a PCR machine (Eppendorf, Hamburg, Germany).

RT-PCR
In situ hybridization. The paraffin sections were baked at 65°C for 2 h, then were dewaxed with Xylene and dehydrated in alcohol. Endogenous peroxidase activity in tissues was inhibited in 3% hydrogen peroxide solution in methanol for 10 min at room temperature. The sides were digested with protenase K for 30 min at 37°C. Following prehybridized at 37°C for 2 h, the sides were hybridized with hsa-miR-206/140-probes (1:50, Exon Biotech Inc., Guangzhou, China) overnight at 37°C. Then sides were rinsed and blocked at 37°C for 30 min. The sides were incubated with mouse anti-digoxingenin antibody (1 : 500, Abcam, Cambridge, UK) for 1 h. The Sides were incubated with SABC-POD at 37°C for 20 min, and then secondary antibody (PV-6000, ZSGB-Bio, Beijing, China) was applied for 30 min at room temperature. 3,3-Diaminobenzidine (DAB, Santa Cruz Biotechnology, Inc., Beijing, China) solution was used for staining. The sections were observed under a microscope (DM6000B, Leica, Dresden, Germany).
Cell proliferation or apoptosis. Cells proliferation was measured with an MTT assay (Sigma). Cells (1 × 10 4 ) in each well of 96-well flat bottom microtiter plates were treated with miR-140, miR-206, and control oligos for 48 h. At 4 h before the end of incubation, 10 μl MTT (5 mg/ml) was added into each well.
Construction of pcDNA-GFP-Smad-UTR vector. Smad3-3′-UTR was amplified by PCR from human genomic DNA. Forward primer: 5′-TGGAA CTCTACTCAACCCATTG-3′; reverse: 5′-TACATACGCCCAAAGCACCT-3′. PCR was carried out with 30 cycles of denaturation at 94°C for 45 s, annealing at 54°C for 45 s, and elongation at 72°C for 60 s, in a PCR machine (Eppendorf). The Smad3-3′-UTR was cloned into a T vector (Takara) to construct the T-Smad3 vector. The Smad3-3′-UTR was then cut from T-Smad3 and inserted downstream of the GFP gene in the pcDNA-GFP vector (described previously). 50 GFP assays. GFP-positive cells were observed 24 h after transfection. A549 and LTEP-a-2 cells were trypsinized and gently washed with serum-containing medium. Cells were then collected and centrifuged at 400 × g for 5 min. Then, GFP-positive cells were counted by FACS (Beckman).
Promoter and luciferase. Different lengths of TRIB2 (NM_021643) promoter elements were amplified by PCR (Primers appear in Supplementary Table 1). Promoters were cloned into the T vector (Takara) to construct T-promoter vectors. Then, promoters were cut from T-promoter vectors by KpnI/SalI, which were inserted before the luciferase sequence of pGL-basic (Promega, Madison,WI, USA) using KpnI/XhoI, constructing promoter-luciferase expression vectors. The mutated promoter-luciferase vector was constructed using a site-directed Gene Mutagenesis Kit (Beyotime). All constructs were confirmed by DNA sequencing.
The Hela cells were treated with both luciferase reporter plasmid and TGF-β1 or other factors. After 24 h, cells were collected and luciferase activity was measured with a Dual-Luciferase Reporter Assay according to the manufacturer's instructions (Promega).
Transwell cell migration assays. Transwell migration assays were performed using Corning Costar Transwell chambers with filter membranes of 8 μm pore size (Sigma). Cells treated with miRNAs or TGF-β1 were seeded into the upper chamber (10 4 cells per well in 100 μl 1640 medium, FBS-free). The lower chamber was filled with 600 μl 1640 medium supplemented with 10% calf serum. After 24 h, the liquid in the upper chamber were removed and the upper surface was carefully washed with PBS three times. In the upper chamber, − 20°C methanol was added for 10 min and samples were washed with PBS twice. Then, lower chamber cells were stained with 1% crystal violet (Sigma) in 2% ethanol for 20 min. Excess crystal violet was removed by quickly merging the insert in ddH 2 O for 3-4 s. Lower chamber cells were counted under a microscope (DM6000B, Leica). Each migration condition was tested three times.
Tail vein injection and migratory cell detection. GFP-positive A549 cells transfected with miRNAs or controls were collected from petri dish in 100 μl normal saline at 1.5 × 10 6 cells. Subsequently, these cells were injected into the tail veins of nude mice (5 weeks old). Seven weeks later, the migration of GFP-positive cells were observed using a Small Animal In vivo FX Pro (FX PRO, Bruker, Sweden).
Then the lungs were dissected, and hematoxylin-eosin staining (HE) and immunohistochemistry was performed as previously described. 51 The sections were incubated overnight with mouse anti-human CD44 primary antibodies (1:50, Boster Biological Technology Co., Wuhan, China) at 4°C, and secondary antibody (PV-6000) and DAB solution were used for detection. The sections were observed under a microscope ((DM6000B, Leica).
A549 lung adenocarcinoma cell xenografts. Briefly, after treatment with miR-140 or miR-206 for 48 h, A549 cells were cultured, collected, washed, and resuspended in culture medium (~2 × 10 7 /ml) and injected into the lower back of 6-8-week old female BALB/C-nu mice (nude mice, HFK Bio-Technology, Beijing, China). Once mice developed palpable tumors, tumor volume was measured with calipers daily. All mice were killed after 4 weeks and tumors were collected. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Binzhou Medical University.
Statistics. SPSS Statistics Client 22 (IBM) software was used to analyze the significance of all results. Group means comparisons were calculated using an unpaired, two-sided, Student's t-test. ANOVA was applied to compare different groups with respect to continuous variables. Array data of TRIB2 and Smad3 were downloaded from data link Data Link(s): http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc = GSE3141. Overall survival was determined using Kaplan-Meier survival analysis. Correlations were calculated with a Spearman rank test. P-values o0.05 were considered statistically significant differences.