The NSCLC cell lines A549 and H520, and the normal lung bronchus epithelial cell line 16HBE were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 µM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin sulfate and were cultured in a humidified chamber with 5% CO₂ at 37°C. All transfections were performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). In order to interrupt the MAP3K1 mediated signaling pathway, cells were treated with 30 µm SP100625 (Enzo Life Sciences, Inc., Farmingdale, NY, USA) for 24 h.

Targetscan (http://www.targetscan.org/vert_71/) is an online software for the prediction of the binding of miRNAs to target genes. miR-145-5p investigated using the software to search for its target genes.

The cells were placed in six-well plates at 70–90% confluence 24 h prior to transfection in serum-free media. The cells were transfected with plasmids, pcDNA3.1 or pcDNA3.1-MAP3K1 (2 µg/well; Fulengen, Guangzhou, China; http://www.fulengen.com/) or miRNA/small-interfering RNA (100 pmol/well) using Lipofectamine 2000, according to the manufacture's protocol. After 48 h, the cells were collected by trypsinization, washed with phosphate-buffered saline and used for subsequent experiments.

The entire 3′-untranslated region (UTR) of human mitogen-activated protein kinase kinase kinase 1 (MAP3K1) was cloned by Genscript (Nanjing, China; http://www.genscript.com.cn/). The entire 3′-UTR of MAP3K1 were inserted into the pMIR-reporter plasmid (Ambion; Thermo Fisher Scientific, Inc.). The insertion was confirmed to be correct by DNA sequencing. The sequences that interact with the seed sequences of miR-145-5p were mutated. Luciferase assays were performed using A549 cells. The cells were transfected with: miR-145-5p mimics, 5′-GUCCAGUUUUCCCAGGAAUCCCU-3′ or scrambled sequences, 5′-UUCUCCGAACGUGUCACGUTT-3′; GenePharm, Inc. (Sunnyvale, CA, USA) and Renilla luciferase plasmids (Ambion; Thermo Fisher Scientific, Inc.) in 24-well plates. The cells were lysed for luciferase assay 48 h following transfection. Luciferase assays were performed using dual luciferase assay kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol.

Total RNA was extracted from NSCLC cell lines and 16HBE cells using TRIzol (Thermo Fisher Scientific, Inc.). Complementary DNAs were synthesized by using specific-miRNA primers (GeneCopoeia Inc., Guangzhou, China). The primers of miR-145-5p (HmiRQP0192) were obtained from Fulengen. U6 snRNA was used as an internal control. Gene expression levels were measured by RT-qPCR using BioRad CFX (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and SYBR-Green kit (Takara Biotechnology Co., Ltd., Dalian, China). Reactions were incubated for 10 min at 95°C, followed by 40 cycles of 95°C for 10 sec; 60°C for 20 sec and 72°C for 10 sec. The primers of were as follows: MAP3K1 forward, 5′-TAGGGCCTAACTCTTTCCTGAT-3′ and reverse, 5′-GTTCTAGTTGAAACACCCGGA-3′; GAPDH forward, 5′-CAATGACCCCTTCATTGACC-3′ and reverse, 5′-GACAAGCTTCCCGTTCTCAG-3′. GAPDH was used as endogenous control to normalize the expression data. Reactions were incubated for 10 min at 95°C, followed by 40 cycles of 95°C for 10 sec; 60°C for 20 sec and 72°C for 15 sec. Each sample was analyzed in quadruplicate. The relative amount of miRNA was normalized to U6: Forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The data was calculated with the equation 2^−ΔΔCq, where ΔΔCq = (Cq_miRNA - Cq_U6) target - (Cq_miRNA - Cq_U6) control. Gene expression was calculated with the equation 2^−ΔΔCq, where ^ΔΔCq = (Cq_target - Cq_GAPDH) - (Cq_control - Cq_GAPDH) (19).

The cells were suspended in radioimmunoprecipitation assay protein lysis buffer (pH 7.4), containing 20 mM sodium phosphate, 150 mM sodium chloride, 5 mM EDTA, 5 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 1 µg/ml leupeptin, and 500 µM Na₃VO₄. Protein concentration was quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.). Total protein (50 µg) was resolved with 6% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Membranes were blocked in 5% non-fat milk diluted in TBST for 1 h at room temperature. The blots were probed with primary antibodies against MAP3K1 (cat. no. PA5-15085; Invitrogen; Thermo Fisher Scientific, Inc.), E-cadherin (cat. no. 14472), Vimentin (cat. no. 5741), JNK (cat. no. 9252), p-JNK (cat. no. 9255), GAPDH (cat. no. 5174) all purchased from Cell Signaling Technology, Inc., Danvers, MA, USA. All primary antibodies were diluted at 1:1,000 and incubated at 4°C overnight. After washing, membranes were probed with appropriate secondary antibodies, horseradish peroxidase conjugated-goat anti mouse IgG (cat. no. BA1051; Boster Biological Technology, Pleasanton, CA, USA) or horseradish peroxidase conjugated-goat anti rabbit IgG (cat. no. BA1054; Boster Biological Technology). Secondary antibodies were diluted at 1:5,000 and incubated at room temperature for 1 h. All membranes were stripped by incubating in Restore PLUS Western blot stripping buffer (Thermo Fisher Scientific, Inc.) for 15 min at room temperature and reprobed with anti-GAPDH antibody for loading control. The blots were visualized using enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA).

Statistical analysis was performed with SPSS17.0 (SPSS, Inc., Chicago, IL, USA). The Student's t-test was used to compare the different groups. P<0.05 was considered to indicate a statistically significant difference, and P-values are represented by an asterisk on the bars in the figures.

The effect of miR-145-5p mimics and inhibitor transfection was detected (Fig. 1A). It was demonstrated that ectopic expression of miR-145-5p markedly increased the expression of E-cadherin and suppressed the expression of vimentin in A549 and H520 cells, compared with mi-NC. By contrast, depletion of miR-145-5p induced E-cadherin downregulation and vimentin upregulation in A549 and H520 cells (Fig. 1B).

JNK is not required for initiation of NSCLC, but associated with EMT. JNK drives transcription of numerous key EMT genes (20). It has been reported that TGF-β1 induced JNK activation during EMT in idiopathic pulmonary fibrosis (21). In the present study, it was examined whether JNK signaling pathway has a critical role in the regulation of miR-145-5p-mediated EMT in NSCLC cells. The results revealed that overexpression of miR-145-5p was able to inhibit the expression of phosphorylated (p)-JNK, but has no effect on the level of total JNK protein (Fig. 2A). The cells were also treated with a combination of a specific JNK inhibitor (SP600125) and miR-145-5p. Western blotting analysis showed SP600125 was able to reverse the effect of miR-145-5p on EMT in A549 and H520 cells (Fig. 2B).

TargetScan online software was used to search for putative protein-coding gene targets of miR-145-5p. The results indicated that MAP3K1 is a putative target of miR-145-5p (Fig. 3A). The MAP3K1 3′UTR was cloned into a luciferase reporter vector and binding sites of the mutant vector. The A549 cells were co-transfected with either mutated pmiR-MAP3K1-3′UTR and miR-145-5p mimics or wild-type pmiR-MAP3K1-3′UTR and miR-145-5p mimics. The cells were also co-transfected with mutated pmiR-MAP3K1 and negative control or wild-type pmiR-MAP3K1 and negative control. Luciferase activity was measured 48 h following transfection.

The results showed that the cells co-transfected with wild-type pmiR-MAP3K1-3′UTR and miR-145-5p mimic exhibited significant reduction in luciferase activity compared with cells cotransfected with the control. By contrast, the luciferase activity in cells transfected with mutated pmiR-MAP3K1-3′UTR and miR-145-5p mimic was not significantly altered (Fig. 3B). Furthermore, RT-qPCR and western blotting analysis was used to investigate the expression of MAP3K1 regulated by miR-145-5p in A549 and H520 cells. The results showed that a marked reduction in mRNA and protein level of MAP3K1 in miR-145-5p-overexpressed A549 and H520 cells compared with the control (Fig. 3C and D).

Since MAP3K1 was a novel target of miR-145-5p in NSCLC cells, it was investigated whether the effect of miR-145 on EMT was mediated by MAP3K1. Fig. 4A showed that the knockdown of MAP3K1 markedly induced the expression of E-cadherin and inhibited the expression of vimentin in A549 and H520 cells, while overexpression of MAP3K1 treatment reversed this effect. The ectopic expression of MAP3K1 was able to partly reverse the downregulation of p-JNK induced by the miR-145-5p inhibitor compared to the vector group. While, the suppression of MAP3K1 was able to interrupt the upregulation of p-JNK induced by miR-145-5p mimics compared to the siRNA negative control group (Fig. 4B).