This study was approved by the Human Research Ethics Committee of Xin Hua Hospital (NO: 2015-035). The NSCLC tissues and adjacent non-tumor lung tissues were obtained from six patients who underwent the primary surgical resection of NSCLC at the Xin Hua Hospital (Shanghai). All participants provided written informed consent. The samples contained well-documented clinicopathological information, including age, gender, tumor size and location, tumor differentiation, invasion depth, lymph node metastasis, distant metastasis, tumor stage, and follow-up data. Tissues were immediately frozen in liquid nitrogen after resection and stored at -80°C. Both NSCLC tissues and the adjacent non-tumor lung tissues were confirmed by a pathological examination.

The synthetic miR-26 mimics, miR-26 inhibitor oligonucleotides, as well as the control inhibitor oligonucleotides, were purchased from Sangon Biotech (Shanghai, China). The TGF-β1 inhibitor was purchased from Sigma (St. Louis, MO, United States). The TGF-β1 was obtained from R&D Systems (Minneapolis, MN, United States). The JNK inhibitor and Lipofectamine 2000, were purchased from Invitrogen (Carlsbad, CA, United States). The Apoptosis Detection Kit was acquired from BioLegend (San Diego, CA, United States).

The NSCLC cell lines A549, H1703, and 801D were purchased from the Shanghai Institute of Chinese Academy of Sciences (Shanghai, China). All cells were maintained in RPMI-1640 and supplemented with a 10% inactivated fetal bovine serum (Gibco, Grand Island, NY, United States), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco) in a humid environment at 37°C with 95% air and 5% CO₂.

The sequence of the JNK was designed to be amplified and cloned into a pCDNA3.1 expression vector (Invitrogen). Transfection was performed using the Lipofectamine 2000 reagent. Briefly, cells were inoculated into 6-well plates and a plasmid and liposomal transfection reagent was added to the cells.

The lentiviral expression systems were purchased from System Biosciences (SBI, Mountain View, CA, United States). Oligonucleotides of siRNA for Chop, ATF-4, Bip, XBP-1, and the control were obtained from Sangon Biotech (Shanghai). After co-transfection, the virus media was harvested. Cells were infected for 72 h with a lentivirus containing Chop, ATF-4, Bip, XBP-1, and control siRNA.

The activation of caspase-3, 9 was detected with a caspase activity assay. Briefly, cells in 96-well plates were treated with the miR-26 inhibitor or the control inhibitor. After incubation for 24 h, 20 μL of lysis buffer was added to each well. The cell lysate was incubated with 5 μL of a chromogenic substrate at room temperature in the dark for 20 min. The results were measured with a plate reader at 560 nm light length.

A TRIzol reagent (Invitrogen, Carlsbad, CA, United States) was used to extract the total RNA. The complementary DNA samples were subjected to denaturing at 95°C for 10 s, annealing at 55°C for 15 s, and extension at 72°C for 30 s, for 45 cycles using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc.). The following primer pairs were used (Table 1): Chop, ATF-4, Bip, XBP-1, DR5, BECLIN1, ATG5, ATG7, DAPK, and GAPDH. Relative gene expressions were quantified by real-time PCR, using the SYBR Premix Ex Taq^TM II (TaKaRa Bio, Dalian, China) on a Lightcycler 480 RealTime PCR System (Roche Diagnostics, Meylan, France).

Protein was resolved by an SDS-PAGE. Subsequently, gel-separated proteins were blotted. The membranes were probed with primary antibodies LC3, TGF-β1 (Abcam, Cambridge, MA, United States) and Bcl, Bax, BECLIN1, ATG5, ATG7, DAPK, JNK, Chop, ATF-4, Bip, and XBP-1 antibodies (Cell Signaling Technology, Beverly, MA, United States) were diluted according to the manufacturer’s instructions. The membranes were then probed with horseradish peroxidase-conjugating (HRP) secondary antibody (1:10000; GE Healthcare, Tokyo, Japan). The proteins in the blots were visualized using the ECL plus system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) to capture the images.

The slides were cut from paraffin-embedded tissue to evaluate the miRNA-26 expression by ISH. In brief, the slides were incubated at 60°C for 1 h, deparaffinized in xylene, and rehydrated with graded alcohol washes. Slides were washed and digested, then hybridized at 55°C for 2 h with 50 nmol/L locked nucleic acid -modified digoxigenin-labeled probes for miRNA-26 (Boster, Wuhan, China). Slides were placed in a blocking solution for 1 h at room temperature. An antibody signal was detected with a 4-nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolylphosphate substrate (Roche, Mannheim, Germany).

To detect cell apoptosis, transfected or treated cells were double stained with an annexin V-FITC/7-amino-actinomycin D (7-AAD) kit (Beckman Coulter) according to the manufacturer’s protocol. The stained cells were immediately analyzed by flow cytometry on the FACS calibur (BD Biosciences, CA, United States).

The cell cycle was assessed using the GENMED Universal periodic flow cytometry kit (Genmed Scientifics Inc., United States). Cells were seeded in 6-well plates and incubated with the miR-26 mimics at 37°C for 48 h in a humidified chamber containing 5% CO₂.

The promoter of the TGF-β1 was amplified and cloned into a pGL 3.0 luciferase reporter plasmid. Cells were then transfected with the pRL-CMV renilla luciferase reporter and the pGL 3.0 luciferase reporter plasmid. The activities of the luciferases were detected using a dual luciferase reporter assay system (Promega).

The Specific-pathogen-free (SPF)-grade nude mice (4–6 weeks of age) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China), and housed with a pathogen-free fodder, equipment, and environment. The control, miR-26 inhibitor, miR-26 inhibitor + TGF-β1 inhibitor, miR-26 inhibitor + JNK inhibitor treated A549 cells were subcutaneously injected at the inguinal region of the nude mice, in a SPF-grade ultraclean work station. Using the vernier calipers, tumor diameters were measured every 2 days after 2 weeks to calculate the tumor volume: TV (mm³) = d2 × D/2, where d and D represent the shortest and the longest diameters, respectively. The mice were sacrificed 30 days after the cell implantation, and the tumors were extracted.

Lungs cancer tissues were obtained from the sacrificed mice. The tissues were embedded in paraffin and sets of different consecutive 5-um-thick sections were acquired using an automatic microtome (SLEE Medical GmbH, Germany). The set of slides were processed for immunohistochemical staining using an anti-TGF-β1 antibody (1:100, Abcam).

After the mice were sacrificed, the lung cancer tissues were embedded, sectioned, and deparaffinized. The sections were incubated with proteinase K for 1 h at room temperature. Sections were then treated with 2% H₂O₂ in distilled water for 30 min at room temperature. After the enzymatic reaction, sections were washed with PBS and incubated with anti-digoxigenin peroxidase conjugate for 30 min at room temperature in a humidified chamber. Sections were stained with diaminobenzine and counterstained with hematoxylin and observed under a light microscope.

The data were analyzed using the SPSS 17.0 software (SPSS Inc., Chicago, IL, United States). The comparison between the two groups was analyzed by an unpaired Student’s t-test and multiple comparisons were compared by a one-way ANOVA analysis of variance followed by a Dunnett’s test. Statistical significance was defined as P < 0.05.

The in situ hybridization of miR-26 in adjacent non-tumor lung or NSCLC tissues was performed and the representative result is shown in Figures 1A,B. The expression level of miR-26 in NSCLC patients was relatively lower than the expression in adjacent non-tumor lung tissues (Figure 1C). In order to examine the effect of miR-26 on apoptosis in NSCLC cells, flow cytometry was performed in A549 cells after treatment with miR-26 mimics at a final concentration of 20 nM. In comparison with the non-treatment counterparts, miR-26 mimics treatment significantly increased the number of apoptotic cells (Figures 2A,B). Furthermore, we examined the activities of caspase-3 and caspase-9 in A549 cells. The results revealed that miR-26 mimics treatment significantly increased the activities of caspase-3 and caspase-9 in A549 cells, compared with the non-treatment counterparts (Figures 2C–E). The expression apoptosis related proteins was also examined and the results showed that Bcl was obviously decreased, while Bax was obviously increased following miR-26 mimics treatment in A549 cells (Figure 2F). In addition, to examine whether miR-26 mimics induces cell cycle arrest, we performed a flow cytometry analysis to investigate the cell cycle distribution after miR-26 mimics treatment. The result showed that the proportion of the cell cycle arrest in the G0/G1 phase and G2/M phase, obviously increased when treated with miR-26 mimics (Figure 2G). These results suggest that miR-26 induced apoptosis and the cell cycle arrest in NSCLC cells.

To understand the effect of miR-26 on the autophagy of NSCLC cells, we examined the expression of autophagy related molecules using Western blotting and RT-PCR in A549 cells. In comparison with the non-treatment counterparts, 50 nM of miR-26 inhibitor treatment significantly increased the protein expression of LC3 (Figure 3A). In addition, miR-26 inhibitor treatment also increased the protein expression of BECLIN1, ATG5, ATG7, and DAPK compared with the non-treatment counterparts (Figure 3B). Moreover, the mRNA expression of BECLIN1, ATG5, ATG7, and DAPK increased in the miR-26 inhibitor treatment group compared with the non-treatment counterparts in A549 cells (Figure 3C).

To verify whether miR-26 directly targets TGF-β1, we constructed luciferase-reporter plasmids containing the wt or mutant 3′-UTR segments of TGF-β1. The wt or mutant reporter plasmid was co-transfected into A549 cells along with the miR-26 or control. miR-26 significantly decreased the relative luciferase activity when co-transfected with the wt reporter plasmid. However, the mutant reporter plasmid reversed the miR-26 mediated decrease in luciferase activity (Figure 4B). The protein expression of TGF-β1 was significantly decreased following miR-26 treatment in A549 cells (Figure 4A), consistently. These findings suggest that miR-26 suppressed TGF-β1 by directly binding to the 3′-UTR of TGF-β1.

We assessed whether down-regulation of miR-26 affected JNK protein expression in A549 cells. Cells were treated with the miR-26 inhibitor, the TGF-β1 or TGF-β1 inhibitor. The increased JNK protein expression was detected in the miR-26 inhibitor treatment, which was further enhanced with TGF-β1 transfection. However, the TGF-β1 inhibitor notably reversed the increase of JNK protein expression induced by the miR-26 inhibitor (Figure 5A). We further tested the apoptosis in cells treated by miR-26 mimics and TGF-β1, JNK, or TGF-β1 inhibitor and JNK inhibitor. The results revealed that TGF-β1 or JNK treatment significantly decreased cell apoptosis induced by miR-26 mimics. Whereas, TGF-β1 inhibitor or the JNK inhibitor combined with miR-26 mimics treatment significantly increased apoptosis of A549 cells compared with the miR-26 mimics treatment alone (Figures 5B,D). Moreover, the protein expression of LC3 was notably increased in the TGF-β1 or JNK combined miR-26 inhibitor, while decreased in the TGF-β1 inhibitor or JNK inhibitor combined with the miR-26 inhibitor, compared with the miR-26 inhibitor treatment alone (Figures 5C,E).

We examined whether ERS signaling was involved in miR-26 regulated autophagy and apoptosis in NSCLC cells. It was found that the protein expression of Chop, ATF-4, Bip, and XBP-1 were up-regulated after treatment with miR-26 mimics (Figure 6A). We down-regulated Chop, ATF-4, Bip, and XBP-1 with siRNA transfection and the efficiency of the transfection was confirmed by a real-time PCR (Figure 6B). Furthermore, the apoptosis in cells treated with miR-26 mimics and Chop, ATF-4, Bip, and XBP-1 siRNA was detected. The results showed that miR-26 mimics combined with Bip, XBP-1, and Chop siRNA significantly decreased apoptosis of A549 cells (Figure 6E). The mRNA expression of DR5 was significantly decreased following siRNA of Chop, Bip, and XBP-1 combined with the miR-26 mimics treatment (Figure 6C). Consistently, the protein expression of Bcl was notably increased, while the Bax expression decreased following siRNA of Chop, Bip, and XBP-1 combined with the miR-26 mimics treatment (Figure 6D). However, the protein expression of LC3 was not obviously changed following siRNA of Bip, ATF-4, and XBP-1 combined with the miR-26 inhibitor treatment, except for the Chop siRNA treatment (Figure 6G). In addition, the mRNA expression of BECLIN1 did not change following siRNA of Bip, ATF-4, and XBP-1 combined with the miR-26 inhibitor treatment except for the Chop siRNA treatment (Figure 6F).

The effect of miR-26 on NSCLC growth was investigated in vivo. The tumor volume was measured in mouse xenografts treated with miR-26 combined with the TGF-β1 inhibitor or JNK inhibitor. The results showed that miR-26 significantly decreased the tumor volume compared with the control (Figure 7A). The protein expression of JNK, LC3 increased following the miR-26 inhibitor treatment, while treatment with the miR-26 inhibitor combined with the TGF-β1 inhibitor or JNK inhibitor reversed the protein expression of JNK and LC3 compared with the miR-26 inhibitor treatment alone (Figure 7B). To determine the role of miR-26 in cell apoptosis in vivo, a TUNEL assay was performed on tumor xenograft tissues. The results showed that TUNEL positive cells of the tumor tissue in the miR-26 treatment group, significantly increased in comparison with the control group, while the number of TUNEL positive cells of the tumor tissue remarkably decreased in the groups of the miR-26 combined with the TGF-β1 inhibitor or JNK inhibitor (Figure 7D). In addition, representative results of the immunohistochemical staining of TGF-β1 in the lung cancer tissues are shown in Figure 7C. The results showed that the immunoreactivity of TGF-β1 in the miR-26 inhibitor group decreased in comparison to the control group. Moreover, TGF-β1 intensity significantly attenuated after treatment with the miR-26 inhibitor combined with the TGF-β1 inhibitor in the lung cancer tissues.