MicroRNA-338-3p suppresses cell proliferation and induces apoptosis of non-small-cell lung cancer by targeting sphingosine kinase 2

This study was approved by the Ethics Committee of ZhengZhou University (ZhengZhou, China) and full informed consent was provided by all of the patients involved prior to sample collection.

A total of 34 patients diagnosed with primary NSCLC at the Henan Tumor Hospital (Zhengzhou, China) between August of 2015 and June of 2016 were included in this study. No patient received chemotherapy or radiotherapy prior to surgery. Tumor and corresponding non-tumor lung tissue samples were collected and rapidly frozen in liquid nitrogen and stored at −80 °C. Tumors were classified according to World Health Organization classification. Data for patient age, gender, smoking history, differentiation, and TNM stage were obtained from patient records (see Table 1).

Normal human bronchial epithelial cell line NHBE and human lung cancer cell lines H460, H1299, A549, SPC-A-1 and Calu-3 were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified cell incubator with 5% CO₂.

Total RNA was isolated from tissue samples and cell lines using the Qiagen RNeasy kit (Valencia, CA) according to the manufacturer’s instructions. RNA quality and quantity were assessed by standard electrophoretic and spectrophotometric methods. Mature miR-338-3p expression was measured by qRT-PCR according to the Taqman MicroRNA Assays protocol (Applied Biosystems, Carlsbad, CA) and normalized using U6 small nuclear RNA (RNU6B; Applied Biosystems, Carlsbad, CA) with the 2^−ΔΔCt method.

Total protein was extracted from tissue samples and cell lines using RIPA buffer containing PMSF. A BCA protein assay kit (Beyotime, Haimen, China) was used to measure total protein. Proteins were electrophoresed via SDS-PAGE and transferred onto PVDF membranes. After blocking, membranes were washed four times with TBST at room temperature and then incubated overnight at 4 °C with diluted primary antibody. Following extensive washing, the membranes were incubated with secondary antibody (HRP-conjugated goat anti-rabbit IgG, 1:3000; Santa Cruz Biotechnology, Santa Cruz, CA). Signals were visualized using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). Antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) served as an endogenous reference. Protein intensity was scanned on Typhoon PhosphorImager (GE Healthcare, Pittsburgh, PA) for fluorescent signal. Experiments were performed in triplicate.

The miR-338-3p mimics (GMR-miR MicroRNA-338-3p mimics) used in this study were synthesized by Shanghai GenePharma Co. Ltd. Human NSCLC cells A549 and H1299 were seeded into six-well plates (2 × 10⁵ cells/well) and allowed to settle overnight until they were 50–80% confluent. Cells were transfected with miR-338-3p mimics using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Three groups were generated for the ensuing experiments: non-transfected group (blank control), scrambled miRNA transfected group (negative control, NC) and miR-338-3p mimics transfected group (inhibitor). Then, 24–48 h after the initial transfection, the cells were harvested for further experiments.

SphK2 coding sequences lacking the 3′-UTR were cloned into the pcDNA3.1 vector (Invitrogen) to generate the pcDNA3.1-SphK2 expression vector. Cell lines were grown at 37 °C in a humidified atmosphere with 5% CO₂. For transfection, cells were cultured to 70% confluence and transfected with plasmids using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations.

For growth curve experiments, different experimental groups of A549 and H1299 cells were plated in 96-well plates at 1 × 10⁴ cells per well and incubated for 48 h after transfection. Optical density (OD) was measured using water-soluble tetrazolium salt assay and microplate computer software (Bio-Rad Laboratories, Hercules, CA) according to Cell Counting Kit-8 (CCK-8) assay kit instructions (Dojindo Laboratories, Japan). Absorbance at 450 nm was read on a microplate reader (168–1000 Model 680, Bio-Rad), and proliferation curves were plotted. Cell proliferation was measured using the ratio of OD of transfected cells in each group to ODs of blank control cells in each group. Data were expressed as percents of control.

To measure colony-forming activity, three groups of A549 and H1299 cells were counted and seeded into 12-well plates (100 cells/well). Culture medium was replaced every 3 days. Twelve days after seeding, colonies containing more than 50 cells were counted.

The 3′-UTR of the SphK2 fragment was PCR-amplified from human genomic DNA and inserted into the pmiR-GLO control vector (Promega, Madison, WI) at the XhoI and XbaI sites 3′ to the luciferase gene. Primer sequences used for PCR amplification were as follows: forward 5′-AUGGGACCAGACGUGAUGCUGGA-3′, reverse 5′-GUUGUUUUAGUGACUACGACCU-3′. The 3′-UTR of SphK2 was confirmed with sequencing and named pmiR-GLO-WT. Site-directed mutagenesis of the miR-338-3p target site in the SphK2 3′-UTR (pmiR-GLO-mut) was carried out using a Quikchange site-directed mutagenesis kit (Promega, Madison, WI), with pmiR-GLO-WT as the template. For the luciferase reporter assay, A549 and H1299 cells were cultured in 96-well plates. Then, using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA), they were each cotransfected with wildtype or mutant reporter plasmid (100 nM) and microRNA (100 nM). At 48 h after transfection, luciferase activity was measured using the dual-luciferase assay system (Promega, Madison, WI).

A549 and H1299 cells were harvested 48 h after transfection and cell concentration was adjusted to 1 × 10⁶ cells. Annexin V-FITC/PI Apoptosis Detetion Kit Ι (BestBio, Shanghai, China) was used to measure Annexin V. Results were obtained using FACScan Flow Cytometer (BD Biosciences, San Jose, CA). Tests were repeated in triplicate. Data were analyzed with Cell Quest software.

Male athymic nude mice (6–8 weeks-of-age) were obtained from the Animal Experimental Center of ZhengZhou University and were acclimated for 2 weeks. This study was conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of ZhengZhou University. The protocol was approved by the Committee on the Ethics of Animal Experiments of ZhengZhou University.

For in vivo tumorigenesis assays, all surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. NSCLC A549 and H1299 cell line stably expressing luciferase infected with lentivirus packaged with lentiviral vectors LV6-miR-338-3p or LV6 empty vector (GenePharma, Shanghai, China), and cells were collected and injected into the flanks of nude mice. Thirty minutes after injection, luciferase substrate was added (150 mg/kg) and luciferase activity was measured every 5 days using the same protocol. Live tumor images were measured every 5 days for 3 weeks and monitored with bioluminescent imaging (PerkinElmer, Fremont, CA).

Statistical analysis was performed using SPSS software version 16.0. All data are presented as mean ± SD where applicable. Differences were analyzed with the Student’s t test. Differences were considered significant when p < 0.05.

Analysis of patient data indicated that SphK2 expression was significantly correlated with NSCLC TNM stage (p = 0.017), and expression of miR-338-3p was also significantly correlated with NSCLC TNM stage (p = 0.023). Western blot confirmed that, compared to the distant normal tissues, SphK2 protein expression in NSCLC cancer tissues were higher (Fig. 1a). The results of half quantitative analysis for protein by Western blot indicated that the relative expression of SphK2 in NSCLC tumor tissues was much higher than in normal tissues (p < 0.001; Fig. 1b), and the relative expression of SphK2 in NSCLC I + II stage tumor tissues was lower than in NSCLC III stage tumor tissues (p = 0.017; Fig. 1c). qRT-PCR results showed that the relative expression of miR-338-3p in NSCLC tumor tissues was much lower than in normal tissues (p < 0.001; Fig. 1d), and the relative expression of miR-338-3p in I + II stage tumor tissues was higher than in III stage tumor tissues of NSCLC (p = 0.023; Fig. 1e). Thus, relative expression of SphK2 and miR-338-3p in NSCLC was negatively correlated (Fig. 1f). Results of western blot indicated that protein expression level of SphK2 in human lung cancer cell lines H460, H1299, A549, SPC-A-1, Calu-3 was much higher than that in NHBE, and more obvious in H1299 and A549 cell lines (Fig. 1g). qRT-PCR of the relative expression of miR-338-3p showed the opposite results, in human lung cancer cell lines H460, H1299, A549, SPC-A-1 and Calu-3, it was much lower than that in NHBE, and also more obvious in H1299 and A549 cell lines (Fig. 1h). Thus, the lung cancer cell lines used in the following experiments are H1299 and A549.

Bioinformatic analysis using TargetScan and miRanda indicated that the 3′-UTR of SphK2 contains a predicted binding site for miR-338-3p (Fig. 2a). Western blot and luciferase reporter assays were used to determine whether SphK2 is regulated by miR-338-3p. Western blot indicated that SphK2 expression was downregulated in A549 and H1299 cells after transfection with miR-338-3p (Fig. 2b). A dual-luciferase reporter system with luciferase reporter vectors containing either the wild-type or the mutant 3′-UTR of SphK2 verified whether SphK2 is a direct target of miR-338-3p. Co-transfection of miR-338-3p significantly decreased luciferase activity of the reporter containing wild-type 3′-UTR, but did not decrease that of the mutant 3′-UTR reporter (*p < 0.05) in A549 cells (Fig. 2c). Also, co-transfection of miR-338-3p significantly decreased luciferase activity of the reporter containing the wild-type 3′-UTR reporter in H1299 cells (*p < 0.05; Fig. 2d). Thus, SphK2 is a direct functional target of miR-338-3p, which negatively regulates SphK2 expression by directly binding to its putative binding site in the 3′-UTR sequence.

CCK-8 assays and colony formation assays were performed in NSCLC cells to evaluate the effect of miR-338-3p on cell proliferation. Data show that compared with NCs cell viability of the miR-338-3p group decreased from 24 h onwards in A549 (Fig. 3a) and H1299 (Fig. 3b) cells. Also, colony formation in the miR-338-3p group was much lower than that of NCs in A549 and H1299 cells (Fig. 3c). Thus, miR-338-3p suppresses viability of NSCLC A549 and H1299 cells.

Flow cytometry assay indicate that, after A549 and H1299 cells were transfected with miR-338-3p for 48 h, Annexin-FITC positive A549 cells were significantly increased compared with NCs, and results of H1299 cells were similar (Fig. 4a). TUNEL staining to measure NSCLC apoptosis indicated that apoptosis increased compared to NCs (Fig. 4b).

Studies with human NSCLC xenografts in nude mice indicated that bioluminescence in the miR-338-3p group less than in NCs and tumor growth curves for mice in the miR-338-3p group was less than NCs (Fig. 5a). H1299 cells were similar (Fig. 5b). Thus, miR-338-3p inhibited tumorigenicity of NSCLC A549 and H1299 cells in a nude mouse xenograft model.

Western blot indicated that transfection of SphK2-siRNA and miR-338-3p inhibited expression of SphK2, respectively (Fig. 6a, b). CCK-8 assay showed that SphK2-siRNA inhibited proliferation of A549 and H1299 cells compared to NCs, and this was similar to cells transfected with miR-338-3p (Fig. 6c, d). A colony formation assay indicated that SphK2-siRNA reduced colonies of A549 and H1299 cells compared to NCs, and these reductions were similar to cells transfected with miR-338-3p (Fig. 6e). Flow cytometry confirmed that SphK2-siRNA induced apoptosis of A549 and H1299 cells compared to NCs, and activation was similar to cells transfected with miR-338-3p (Fig. 6f). Thus, miR-338-3p inhibited NSCLC biological effects by down-regulating SphK2.

To investigate whether the effects of miR-338-3p on the cell proliferation and apoptosis of NSCLC cells was mediated by SphK2 repression, we overexpressed SphK2 lacking the 3′-UTR in NSCLC cell lines and co-transfected with miR-338-3p. Results of western blot showed that expression level of SphK2 protein was downregulated in A549 cells after transfected with miR-338-3p, and overexpressed both in cells transfected with pcDNA3.1-SphK2 (without the 3′-UTR) alone and co-transfected with miR-338-3p. In addition, expression level of SphK2 protein showed no significant difference between the later two groups (Fig. 7a). Results of CCK-8 assay and colony formation assay showed that the proliferation inhibitory effects of miR-338-3p on A549 cells were partly restored by pcDNA3.1-SphK2 lacking the 3′-UTR (Fig. 7b, c), and the apoptosis promoted effects of miR-338-3p were also partly restored (Fig. 7d). These results indicated that the effects of miR-338-3p on NSCLC cell proliferation and apoptosis were restored by SphK2 lacking the 3′-UTR, suggesting that miR-338-3p suppress NSCLC cell proliferation and induce apoptosis by targeting the 3′-UTR of SphK2.