NSCLC cell lines (A549, H1975, HCC827) and normal lung cell line (NL20) were purchased from the American Type Culture Collection (ATCC). The cells were maintained in 5% CO₂ at 37°C, in pH 7.4 DMEM with 10% FBS, penicillin (100 U/ml)/streptomycin (100 µg/ml) antibiotic mixture. Mito Tracker Red was obtained from Invitrogen; Thermo Fisher Scientific, Inc. Hoechst 33342 was purchased from Sigma-Aldrich; Merck KGaA.

Tissue specimens from adjacent tissue and matched primary lung adenocarcinoma tissues (median age was 56 years old) were collected from 15 patients between September 2017 and September 2018 at the First Affiliated Hospital of Zhengzhou University (Henan, China). Samples were immediately stored in liquid nitrogen. All samples were obtained after written informed consent was provided from each patient and approval for the study was obtained from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

Tissue samples were fixed in 10% (v/v) formaldehyde at room temperature for 24 h, embedded in paraffin and cut into 5-µm sections. IHC staining was performed according to a previous protocol (23) using ORP8 (dilution 1:200; product code ab99069; Abcam) antibody. The relative expression was assessed by the Image-Pro Premier software (v9.0) program (Media Cybernetics, Inc.).

Lentiviruses carrying ORP8-cDNA (accession no. NM_001003712), miR-421 mimic and miR-421 inhibitor were obtained from Shanghai GenePharma Co., Ltd. The miR-421 mimic sequence was 5′-CGCGGGUUAAUUACAGACAACUA-3′. The miR-421 mimic negative control (NC) sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. The miR-421 inhibitor sequence was 5′-GCGCCCAAUUAAUGUCUGUUGAU-3′. The miR-421 inhibitor negative control (NC) sequence was 5′-CAGUACUUUUGUGUAGUACAA-3′. For lentiviral infection, 1×10⁶ cells were resuspended in 100 µl DMEM containing Polybrene (5 µg/ml) and lentivirus (MOI=10) in a 24-well plate. After 6 h of infection at 5% CO₂ and 37°C, 400 µl DMEM was added. The infection efficiency was detected using western blotting or qPCR after 3 days of infection.

Total RNA was extracted from cell lines using TRIzol reagent with a well-established method. The RNA samples were reverse-transcribed with the PrimeScript™ RT Reagent kit (DRR047A; Takara Bio, Inc.). qPCR was performed with the SYBR Advantage qPCR Premix (Takara Bio, Inc.) kit, following the manufacturer's protocol. qPCR was detected using the ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, inc.). The qPCR conditions were 95°C for 2 min followed by 40 cycles at 95°C for 15 sec and 60°C for 40 sec and a final dissociation stage. The relative expression levels were calculated using the 2^−∆∆Cq method (24), and U6 or actin was used as the endogenous controls. The primer sequences are listed in Table I.

The cells were harvested and lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using a BCA kit (Beyotime Institute of Biotechnology). The amount of protein loaded per lane was 20 µg. The protein samples were separated by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc.). Membranes were blocked with bovine serum albumin (5%) for 1 h at room temperature with gentle shaking, incubated with the primary antibodies (1:1,000) at 4°C for 12–16 h, and then incubated with the HRP-labeled secondary antibodies (goat anti-mouse 1:1,000 and goat anti-rabbit 1:3,000) for 1 h at 25°C. Finally, the membranes were detected by enhanced chemiluminescence (ECL) plus western blot detection reagents (Thermo Fisher Scientific, Inc.). Proteins were quantified by a gel imager (Tanon 5200; Tanon Science and Technology). The corresponding primary antibodies were as follows: ORP8 (product code ab99069; Abcam), actin (product no. 3700), caspase-3 (product no. 14220), caspase-9 (product no. 9502), cytochrome c (product no. 12963), COX 4 (product no. 4844; all from Cell Signaling Technology, Inc.). The HRP-conjugated secondary antibodies (product nos. 7076 and 7074) were obtained from Cell Signaling Technology, Inc.

To detect cell proliferation, H1975, A549 and HCC827 cells (1×10³ cells/well) stably expressing a control or ORP8 were seeded in 96-well plates. For each well, 20 µl of the MTS solution (Promega Corp.) was added. After 1 h of incubation, 25 µl of 10% SDS solution was added, and the absorbance was detected at 492 and 690 nm.

Cells (8×10³/ml/well) were suspended and added to the upper layer of solidified 1 ml DMEM/10% FBS/0.3% agar with 2 ml of DMEM/10% FBS/0.6% in the bottom layer of the agar in each well of a 6-well plate. After maintenance for 10–14 days in 5% CO₂ at 37°C, the colonies were counted using the Image-Pro Plus software (version 6.2; Media Cybernetics, Inc.). The diameter of each colony was >25 µm.

Cell apoptosis analysis was performed with Hoechst 33342 staining in H1975, A549, and HCC827 cells. Cells (1×10⁶) were collected, washed with cold PBS, stained with Hoechst 33342 (20 µg/ml) at 37°C for 10 min, resuspended in 20 µl PBS, and immediately imaged using an Olympus BX53 fluorescence microscope (Olympus Corp.) at an ×400 magnification. The number of apoptotic cells in each group were counted in 10 random fields with >500 cells.

H1975 and A549 cells (1×10⁷) were collected and washed with PBS, and cytochrome c release was determined using a mitochondrial isolation kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol.

To detect the intracellular cytochrome c distribution, confocal microscopy was used as previously described (25). Cells were incubated with Mito Tracker dye (Mito Tracker Red; Molecular Probes; Thermo Fisher Scientific, Inc.) for 10–15 min in an incubator in the dark. Then the slides were fixed with 4% formaldehyde at room tempetature for 30 min. The fixed slides were stained with an anti-cytochrome c antibody at 4°C for 12–16 h, then with a FITC-labeled antibody at 37°C for 30 min. The cytochrome c antibody (product no. 12963) was purchased from Cell Signaling Technology, the FITC-labeled antibody (product no. A-21202) was purchased from Thermo Fisher Scientific, Inc. Images were obtained using different excitation filters and merged.

The potential targets of miR-421 were predicted by miRanda (http://www.microrna.org/microrna/home.do), miRDB (http://mirdb.org/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/) and TargetScan (http://www.targetscan.org/vert_72/). The 3′-UTR segments of miR-421 binding sites of ORP8 were inserted into the Dual-Glo luciferase assay system plasmid, pGLO (Promega Corporation). pGLO vectors and mimic-miR-421 were co-transfected into cells. Then, the cells were subjected to luciferase activity detection with a luciferase assay kit (Promega Corporation) according to the manufacturer's protocol. Renilla fluorescence was used as the internal standard.

The results are presented as the mean ± standard deviation (SD) of 3 independent experiments, and each dose or treatment was tested in triplicate. For the statistical analyses, all data were analyzed by Student's t-test for pairwise comparisons or one-way analysis of variance (ANOVA) followed by Bonferroni test for multivariate analysis. Differences were considered to be statistically significant when the P-value was <0.05.

NSCLC is the most common type of lung cancer, and numerous oncogenes are associated with this disease. ORP8 is a member of the oxysterol-binding protein-related protein (ORP) family of proteins that are expressed in the brain, liver, lung, spleen, and kidney in mice (15). However, ORP8 expression in human NSCLC has not been well defined. Initially, the expression of ORP8 was investigated in 15-paired NSCLC specimens and lung cancer cell lines. The results indicated that ORP8 was expressed at lower levels in the NSCLC tissues than in the adjacent non-tumor tissues (Fig. 1A). Similarly, ORP8 expression was downregulated in several NSCLC cell lines (HCC827, A549 and H1975) compared with that of the normal NL20 lung cell line both at the mRNA and protein levels (Fig. 1B and C). These results indicated that ORP8 downregulation may promote NSCLC progression.

Aberrant cell proliferation is a hallmark of cancer, and the elimination of uncontrolled cell differentiation contributes to cancer treatment (26). In the present study, it was hypothesized that ORP8 may play a crucial role in cell growth. To assess the function of ORP8 in NSCLC, stable ORP8-overexpressing cells were generated. The overexpression efficiency was verified in H1975, A549 and HCC827 cells using western blotting (Figs. 2A and S1A and B). MTS and anchorage-independent cell growth assays were investigated to evaluate the effect of ORP8 expression. The results revealed that ORP8 overexpression inhibited proliferation in H1975, A549 and HCC827 cells (Fig. 2B, D and F). In addition, colony formation was reduced in ORP8-overexpressing cells (Fig. 2C, E and G), which indicated that increased ORP8 expression reduced cell transformation.

During the progression of transformation, cell growth is regulated not only by proliferation but also by cell death or apoptosis. Studies have revealed that ORP8 plays a key role in coordinating apoptosis in liver and gastric cancer (20,22). In the present study, the apoptosis assay results indicated that overexpression of ORP8 resulted in increased apoptosis in H1975, A549 and HCC827 lung cancer cell lines stained with Hoechst 33342 (Fig. 3A). The protein levels of cleaved caspase-3 and caspase-9 were analyzed using western blot analysis. The cleavage of caspases was significantly enhanced, indicating that apoptosis was induced in ORP8-overexpressing cells (Figs. 3B and S1C). Overall, the present data revealed that ORP8 was involved in modulating cell survival and apoptosis.

In liver cancer cells and gastric cancer cells, studies have shown that ORP8 overexpression could induce ER stress responses (20–22) and that ORP8 localizes to ER-mitochondria contact sites (14). To determine whether ORP8 overexpression induced ER stress in NSCLC cells, the mRNA levels of Chop and Bip, which are the central components of ER stress responses, were detected (27). Results revealed that the expression of Chop and Bip was barely altered at both the mRNA and protein levels in H1975 and A549 cells (Figs. 4A and B and S2A and B). Mitochondria are the intracellular organelles that have critical roles in the apoptotic pathway (28), and the release of cytochrome c from the mitochondria into the cytoplasm is crucial to initiate the apoptotic cascade (8). Thus, to better understand the role of ORP8 in inducing cell apoptosis, cytochrome c release from mitochondria was assessed. Immunofluorescence staining was employed to observe the localization of cytochrome c relative to mitochondria. The present results revealed that cytochrome c co-localized with the mitochondrial marker (Mito Tracker) in the control H1975 and A549 cells (Fig. 4C and D, upper images), while cytosolic cytochrome c was increased in the ORP8-overexpressing cells (Fig. 4C and D, lower images). Western blot analysis similarly revealed that cytosolic cytochrome c levels were significantly enhanced after ORP8 overexpression in H1975, A549 and HCC827 cells (Figs. 4E and S2C and D). These data indicated that ORP8 upregulation promoted the release of cytochrome c from mitochondria, which induced cancer cell apoptosis.

Studies have revealed that upregulated miR-421 expression is associated with poor prognosis in NSCLC (29). Therefore, it was assessed whether the downregulation of ORP8 in NSCLC was due to miR-421 dysregulation. Notably, miR-421 was revealed to target ORP8 by using four different analytical programs (Fig. 5A). In addition, miR-421 was upregulated in NSCLC cells, compared with NL20 cells, in contrast to ORP8 downregulation in NSCLC (Fig. 5B). When cells were infected with miR-421 mimic or inhibitor, miR-421 expression was significantly increased or decreased, respectively (Fig. S3A and B). Moreover, ORP8 protein expression was significantly increased in H1975 and A549 cells treated with the miR-421 inhibitor (Figs. 5C and S3C), whereas ORP8 expression was significantly inhibited in H1975 and A549 cells treated with a miR-421 mimic (Figs. 5D and S3D). Moreover, H1975 cells were transfected with the mutant (MUT) and wild-type (WT) 3′-UTRs of ORP8. The luciferase activity was inhibited by miR-421 when transfected with the WT 3′-UTR, however, the activity was restored by the MUT 3′-UTR transfection (Fig. 5E). In addition, the inhibition of cell proliferation with ORP8 overexpression was rescued by the miR-421 mimic (Fig. 5F). These data indicated thatmiR-421 could be a potential target for ORP8 downregulation in NSCLC patients.