2.1 Reagents and cell cultures

Antibodies specific for total PDPK1 were purchased from Cell Signaling Technology. miR-214-3p mimics and inhibitors were purchased from RiboBio Co. Ltd. miR-214-3p, U6 primers and HOTAIR siRNAs were provided by GenePharma. HOTAIR and GAPDH primers were obtained from Life Technologies. Lipofectamine 3000 reagent was supplied by Life Technologies. SM was purchased from Chengdu Must Bio-technology Company, which was freshly diluted to the final concentration with culture medium before experiment. A549 and PC9 cells and BEAS-2B cells were obtained from the Chinese Academy of Sciences Cell Bank of Type Culture Collection and authenticated for the absence of mycoplasma, genotypes and morphology by using a commercial kit provided by Guangzhou Cellcook Biotech Co. Ltd. Cells were cultured in RPMI 1640 (Life Technologies) supplemented with 10% foetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. A549-Luc and A549-PDPK1(+/+)-Luc cells were provided by Guangzhou Land Technology Co and cultured in a medium containing geneticin Sulfate (Life Technologies). Cells at 70% confluence were trypsinized with 0.25% trypsin and used in all in vitro experiments.

2.2 Cell viability assay

The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to measure the cell viability as described previously.32 NSCLC cells (5 × 10³ cells/well) were counted and seeded into 96-well microtiter plates, and treated with indicated doses of SM for up to 72 hours. After adding MTT solution (5 mg/mL), plates were incubated at 37°C for 4 hours followed by the addition of dimethyl sulfoxide. Absorbance was measured at 570 nm on a microplate reader (Perkin Elmer, Victor X5). Cell viability was calculated as the ratio of absorbance of sample/control. The cells treated with vehicle only (0.1% DMSO) served as a negative control and the control values were set to 1 by default.

2.3 EdU assay for cell proliferation

Non-small cell lung cancer cell proliferation was assessed by Cell-Light EdU Apollo 488 In Vitro Imaging Kit (RiboBio) according to instructions from the manufacturer. Briefly, after treatment with SM in 96-well plates for 24 hours, the cells were incubated with EdU reagent for 2 hours and fixed in 4% paraformaldehyde for 30 minutes. Thereafter, cells were washed in glycine and incubated in 0.2% Trion X-100 for 10 minutes followed by the addition of 1× Apollo reaction buffer. After washing in 0.5% Triton X-100, the cells were stained with Hoechst (5 mg/mL). Images were taken under a BX53+DP72 Microscope (Olympus Corporation) and evaluated by Image-Pro plus 6.0 image analysis software (Media Cybernetics, Inc). Per cent cell proliferation was calculated as: (EdU positive cells/Hoechst stained cells) × 100.

2.4 Flow cytometric analysis

Cell apoptosis was detected using Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's protocol. Briefly, the cells were treated with SM for 24 hours, washed with pre-cooled phosphate-buffered saline (PBS), and mildly trypsinized without the use of EDTA. Subsequently, the cells were harvested, resuspended in 500 μL of the binding buffer and incubated with 5 μL Annexin V-FITC reagent for 15 minutes and 10 μL PI for 5 minutes at room temperature (RT) in the dark. Cell apoptosis was measured by flow cytometry (FC500, Beckman Coulter), and data were analysed by GraphPad Prism 5.0 software.

2.5 Cell invasion assay

The potential invasiveness of lung cancer cells was assessed using transwell plates with 10 mm diameter and 8 μm pore size polycarbonate membrane coated with a film of Matrigel (BD Biosciences) as reported previously.33 The upper compartment was seeded with 0.5 × 10⁶ cells and treated with or without SM. The lower compartment was filled with culture medium. After 24 hours incubation, non-migrated cells were removed and migrated cells were fixed in 4% paraformaldehyde, and stained with crystal violet. Stained cells were counted and images were taken under Ts2RFL microscope (Nikon). The number of invaded cells is represented as percentage of control.

2.6 Quantitative real-time RT-PCR

First-strand cDNA was synthesized from total RNA (2 μg) by reverse transcription using oligo-dT primers and Superscript II reverse transcriptase (Invitrogen). A quantitative real-time RT-PCR (qRT-PCR) assay was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems) for the quantification of HOTAIR and miR-214-3p transcripts. The sequences of the primers used were as follows: HOTAIR-Forward: 5′-GGTAGAAAAAGCAACCACGAAGC-3′; Reverse: 5′-ACATAAACCTCTGTCTGTGAGTGCC-3′; miR-214-3p-Forward: 5′-CAATACTGACAGCAGGCACA-3′ Reverse: 5′-TATGGTTGTTCACGACTCCTTCAC-3′ U6: Forward: 5′-ATTGGAACGATACAGAGAAGATT-3′ Reverse: 5′-GGAACGCTTCACGAATTTG-3′ GAPDH-Forward: 5′-AAGCCTGCCGGTGACTAAC-3′; Reverse: 5′-GCGCCCAATACGACCAAATC-3′. The PCR conditions were as follows: 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C. Threshold values for each sample/primer pair are represented as mean ± standard error.

2.7 Determination of amplification efficiency

Reverse-transcribed cDNA was diluted by 5 10-fold serial dilutions. qRT-PCR was performed to detect the expression of HOTAIR and miR-214-3p with the corresponding primers, and each sample was processed three times. A standard curve was generated by plotting the log values of dilution multiples on the X-axis and the corresponding CT values on the Y-axis. The amplification efficiency of the primers, E, was calculated as: E = 10^−1/slope−1. The amplification efficiency in the range from 90% to 110% is generally considered to be adequate. Our results showed that amplification efficiency for HOTAIR and miR-214-3p was 100.99% and 100.34%, respectively.

2.8 Western blot analysis

Equal amounts of protein from whole cell lysates were dissolved in SDS-sample buffers and separated on 10% SDS polyacrylamide gels. Membranes (Millipore) were incubated with antibodies against PDPK1 for 2 hours, washed and incubated with secondary rabbit IgG antibody conjugated to horseradish peroxidase (Cell Signaling Technology). The membranes were washed again and transferred to a freshly prepared enhanced chemiluminescence solution (Immobilon Western; Millipore). Protein bands were observed using the Gel Imagine System (Bio-Rad). ImageJ software (National Institutes of Health) was used to quantify and compare the intensity of single band between the control and proteins of interest.

2.9 Transient transfection assays

Cells were seeded in 6-well or 96-well culture plates and grown to 60% confluency before transfection with the control or HOTAIR (pcDNA3.1-HOTAIR) and PDPK1 plasmids (pCMV6-PDPK1) using Lipofectamine 3000 reagent. miR-214-3p mimics or inhibitors were transfected with RiboFect CP reagent (RiboBio Co.) according to the manufacturer's instructions. Briefly, Lipofectamine 3000 was incubated with Opti-MEM medium (Invitrogen) for 10 minutes followed by respective siRNA (up to 25 nmol/L) for an additional 15 minutes before adding to the cells. RiboFect CP regent was incubated the miRNA mimics or inhibitors for 15 minutes. After culturing the cells for up to 48 hours, the cells were washed and resuspended in fresh media in the presence or absence of SM for an additional 24 hours.

2.10 Luciferase reporter assay

The binding sites of HOTAIR and PDPK1 for miR-214-3p were obtained using bioinformatics prediction databases (TargetScan, http://www.targetscan.org/vert_72/, MiRBase, http://www.mirbase.org/, and MiRWalk http://mirwalk.umm.uni-heidelberg.de/). 3′-UTR cDNA fragment of PDPK1 containing the wild-type and mutated miR-214-3p binding sites, named pEZX-MT05-Luc-PDPK1-WT or pEZX-MT05-Luc-PDPK1-Mut and 3′-UTR cDNA fragments of HOTAIR containing the wild-type and mutated miR-214-3p binding sites, named pEZX-MT05-Luc-HOTAIR-WT or pEZX-MT05-Luc-HOTAIR-Mut were ordered from GeneCopoeia, Inc. The cells were transfected with the plasmids (1.25 μg/mL each) for 24 hours using Lipofectamine 3000 reagent and then treated with miR-214-3p mimics for an additional 48 hours. The preparation of cell extracts and measurement of luciferase activities were done by using Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia, Inc) and were normalized with SEAP activity within each sample.

2.11 Cell immunofluorescence

Cells were fixed in paraformaldehyde and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) followed by incubation in normal goat serum for 30 minutes, and incubation with anti-human PDPK1 antibody (1:100) overnight. After washing, the cells were incubated with secondary anti-Rabbit IgG (H+L) antibody conjugated to Alexa Fluor 594 (A21442, Invitrogen, ThermoFisher) followed by washing and counterstaining with DAPI. Slides were mounted with FluorSave (Calbiochem). Fluorescence was detected under an Axio Observer Z1 immunofluorescence microscope (Carl Zeiss Inc).

2.12 RNA immunoprecipitation (RIP) assay

RNA immunoprecipitation (RIP) assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) following instruction from the manufacturer. Briefly, after washing in PBS, the cells were lysed in RNA lysis buffer containing RNase inhibitor. The cells lysates were incubated with magnetic beads coated with the specific PDPK1 antibody (Abcam), anti-Ago2 antibody (Millipore) or the negative control IgG (Millipore). Finally, the expression of HOTAIR, miR-214-3p, PDPK1 and Ago2 was measured by qRT-PCR as well as Western blot.

2.13 Xenograft tumours and bioluminescent imaging

Animal studies were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Guangdong Provincial Hospital of Chinese Medicine (Ethics Approval Number 2017037) and the National Institutes of Health guidelines for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Nude mice (aged 4-6 weeks) purchased from Beijing Vital River Experimental Animal Co. Ltd. were maintained at the Animal Center of Guangdong Provincial Hospital of Chinese Medicine. A549-Luc or A549PDPK1+/+-Luc (2 × 10⁶) were injected subcutaneously into the nude mice. Xenografts were allowed to grow when the initial measurements were made with calipers. For bioluminescence imaging (BLI), mice were anesthetized by inhalation of 2% isoflurane. The substrate D-luciferin (Caliper Life Sciences) was injected into the peritoneal cavity of the mice at a dose of 150 mg/kg. The intensity of the BLI signal was determined using the IVIS-200 Imaging System (Xenogen/Caliper). Mice were then randomly divided into the control and SM groups (n = 10/group), and SM was injected intraperitoneally daily at a dose of 8 mg/kg for up to 25 days based on our previous reports and others,7, 34, 35 which showed significant inhibitory effect of SM on tumour growth without apparent toxicity. Tumour volume was calculated using the formula for a spheroid: volume = (width² × length) × 0.5. Bioluminescence is expressed as photons/s. Body weights of mice were measured once a week. All mice were sacrificed on the 25th day in accordance with the Guidelines for the Care and Use of Laboratory Animals. At the end of the experiments, xenograft tumours were isolated, and expression of HOTAIR, miR-214-3p and PDPK1 was determined by qRT-PCR as well as Western blot.

2.14 Statistical analysis

All in vitro experiments were performed at least three times. Statistical analysis was carried out using GraphPad Prism version 5.04 for Windows (GraphPad Software). Pair wise comparisons were done by paired two-tailed t test, Mann-Whitney test or Fisher exact test. The data in most graphs are presented relative to the control. P values <.05 were considered significant.

3.1 SM-inhibited proliferation of NSCLC cells via inhibition of HOTAIR

Previous reports showed that SM significantly inhibited the growth of NSCLC cells via several mechanisms.7, 34 In the current study, we demonstrated that percentage of EdU positive NSCLC cells was significantly reduced in the SM-treated group compared with the control group (Figure 1A). This further confirmed the inhibitory effect of SM on the growth of NSCLC cells. Moreover, SM induced a high magnitude of apoptosis, as determined by staining with Annexin V/PI and flow cytometry analysis (Figure 1B).

Studies have demonstrated the important roles of lncRNAs such as HOTAIR in growth and progression of cancers.13, 36 Herein, the results unveiled that SM significantly inhibited the expression as well as the promoter activity of HOTAIR (Figure 1C,D). Moreover, siRNA mediated silencing HOTAIR significantly inhibited the growth and invasion of A549 and PC9 cells as determined by MTT and in vitro invasion assays (Figure 1E,F), respectively. Overexpression of HOTAIR, on the other hand, diminished the inhibitory effect of SM on cell growth (Figure 1G). Taken together, our results demonstrated that HOTAIR may be an important target of SM and that inhibition of HOTAIR is involved in the SM-mediated inhibition of lung cancer cells.

3.2 SM increased miR-214-3p expression in NSCLC cells

To further dissect the mechanism of the inhibitory effect of SM on lung cancer cells, we searched for potential miRNAs that may link HOTAIR and other target genes. We observed reduced transcript abundance of miR-214-3p in A549 and PC9 cells compared with normal human bronchial epithelial cell (BEAS-2B; Figure 2A), however, in the presence of SM there was a significant increase in expression of miR-214-3p (Figure 2B). Furthermore, miR-214-3p mimics significantly inhibited the growth and invasiveness of A549 and PC9 cells as compared with the control group (Figure 2C,D). These results confirmed that miR-214-3p functions as a tumour suppressor role and suggested that miR-214-3p may enhance the SM-mediated inhibition of NSCLC cell growth.

3.3 Interaction between HOTAIR and miR-214-3p was involved in SM-inhibited cell growth

Next, we began to test the biological significance of the interaction between HOTAIR and miR-214-3p in mediating the effect of SM. Results showed that the expression of miR-214-3p was significantly higher in cells with silenced HOTAIR compared with the control siRNA-transfected cells (Figure 3A). Conversely, mimics of miR-214-3p had no effect on HOTAIR expression (Figure 3B). Moreover, we found that silencing HOTAIR further strengthened SM-induced miR-214-3p expression (Figure 3C), while overexpression of HOTAIR reversed SM-induced miR-214-3p expression (Figure 3D).

By utilizing bioinformatics prediction databases, we found that miR-214-3p has a classical and conservative binding site in the 3′-UTR region of HOTAIR (Figure 3E). We generated a 3′-UTR cDNA fragment of HOTAIR containing the wild-type and mutated miR-214-3p binding sites (Figure 3E). We found that the interaction of miR-214-3p with the wild-type 3′-UTR cDNA fragment of HOTAIR significantly decreased the luciferase activities of HOTAIR in A549 and PC9 cells compared with the negative control group (Figure 3E) suggesting a direct inhibitory effect of miR-214-3p on HOTAIR expression. Furthermore, RIP assay showed that Ago2 protein was successful pulled down by Ago2 antibody-coated magnetic beads (Figure 3F). And qRT-PCR analysis revealed that both HOTAIR and miR-214-3p were enriched in the Ago2-containing beads and were higher compared with the input group (Figure 3F). Moreover, the physical binding of HOTAIR to miR-214-3p was also significantly affected in the presence of SM (Figure 3F). These results suggested that the physical interaction between HOTAIR and miR-214-3p may also play an additional role in mediating the anti-carcinogenic effect of SM.

3.4 SM-inhibited PDPK1 expression via correlation and interaction of HOTAIR and miR-214-3p

Previously, we demonstrated an important role of PDPK1 in cancer growth with the implication that targeting PDPK1 may be a potential treatment of lung cancer.33, 37 In line with this, overexpression of PDPK1 in A549PDPK1+/+-Luc cells not only showed more growth regression, it also significantly resisted the SM-inhibited growth as compared with the wild-type A549-Luc cells (Figure 4A). Both Western blot and immunofluorescence confirmed that SM reduced PDPK1 protein expression (Figure 4B,C).

In order to validate our findings and further identify the relevant targets of HOTAIR and miR-214-3p association, we treated the NSCLC cells with miR-214-3p mimics and determined PDPK1 protein expression. Results showed that miR-214-3p mimics reduced whereas miR-214-3p inhibitors increased PDPK1 protein expression (Figure 5A,B). While over-expressing miR-214-3p had no effect, silencing HOTAIR resulted in decreased PDPK1 promoter activity (Figure 5C,D). Note that the latter also suppressed PDPK1 protein expression (Figure 5E). Together, these results suggested that while miR-214-3p may regulate PDPK1 post-transcriptionally, HOTAIR may regulate both transcriptionally and translationally.

To further delineate the functional role of miR-214-3p and whether PDPK1 is its downstream target, we searched bioinformatics prediction databases and found a classical and conservative binding site for miR-214-3p in 3′-UTR region of PDPK1 (Figure 5F). We observed that the binding of miR-214-3p mimics to the wild-type 3′-UTR cDNA fragment of PDPK1 significantly reduced the luciferase activity in comparison with the mutated 3′UTR or negative control/ scrambled mimics (Figure 5F). The results suggested that PDPK1 is a downstream target of miR-214-3p. Finally, RIP assay demonstrated a physical interaction between HOTAIR and PDPK1 protein which was inhibited by SM (Figure 5G). These results suggested that SM exerts its effect on the interaction between HOTAIR and PDPK1 protein by suppressing PDPK1 expression.

3.5 Overexpression of PDPK1 neutralized the effect of SM on HOTAIR and miR-214-3p expressions, and cell growth

In order to confirm the role PDPK1 in the interaction of HOTAIR and miR-214-3p, we showed that overexpression of PDPK1 neutralized the effects of SM on miR-214-3p, but not HOTAIR, expression (Figure 6A,B). In addition, overexpressed PDPK1 overcame the SM-inhibited growth in NSCLC cells (Figure 6C). Together, the findings indicate the presence of a feedback loop between PDPK1 and miR-214-3p, and the interactive regulatory axis among HOTAIR, miR214-3p and PDPK1 contributing to the inhibitory activity of SM on growth of NSCLC cells.

3.6 SM-inhibited tumour growth and regulated the expressions of HOTAIR, miR-214-3p and PDPK1 in vivo

We used A549-Luc and A549-PDPK1 (+/+)-Luc cells to examine whether overexpression of PDPK1 resisted the effect of SM on tumour growth in vivo. We found that SM significantly inhibited tumour growth (Figure 7A). However, the inhibitory effect was reduced with A549-PDPK1 (+/+)-Luc cells (Figure 7A). In addition, SM caused significant decrease in tumour weight and sizes in the established A549 xenograft tumours compared with that of the vehicle-treated control animals (Figure 7B-D). Note that there was less effect with A549-PDPK1 (+/+)-Luc cells (Figure 7B-D). Moreover, consistent with the in vitro data, SM-induced miR-214-3p expression, while reduced the expressions of HOTAIR and PDPK1 in fresh tumours harvested from the aforementioned experiments as compared with the control group (Figure 7E-G). As seen before, xenograft tumours with A549-PDPK1 (+/+)-Luc cells showed less inhibitory effects (Figure 7E-G). Taken together, these results indicated that both in vitro and in vivo studies show similar effects of SM on lung tumour growth and expressions of relevant molecules.