Sodium new houttuyfonate suppresses metastasis in NSCLC cells through the Linc00668/miR-147a/slug axis
As most lung cancer patients present with invasive, metastatic disease, it is vital to investigate anti-metastatic treatments for non-small cell lung cancer (NSCLC). Houttuynia cordata is commonly used as a Chinese anticancer medicine in the clinic, and sodium new houttuyfonate (SNH), a main compound of this herb, has long been found to have antibiotic effects, although its anticancer effects have not been investigated. Here, we tried to address this lack of research from the perspective of the competing endogenous RNA (ceRNA) theory.
The effects of SNH on NSCLC cells were analysed with Cell Counting Kit-8 assays and colony formation assays. In addition, transwell assays and wound healing assays were used to determine the effects of SNH on migration and invasion in NSCLC cells. The levels of key genes and proteins were examined by quantitative real-time PCR, western blotting, immunofluorescence staining and IHC staining. Through transcriptome screening and digital gene expression profiling, Linc00668 was identified to be regulated by SNH. Dual-luciferase reporter assays and RNA immunoprecipitation assays verified the binding efficiency between miR-147a and Linc00668 or Slug.
In the present study, SNH regulated NSCLC cells in multiple ways, the most prominent of which was suppressing the expression of Linc00668, which was indicated to promote migration and invasion in NSCLC cells. Functional studies demonstrated that Linc00668 acted as a ceRNA by sponging miR-147a to further regulate Slug mRNA levels, thereby influencing the progression of the epithelial-mesenchymal transition. Consistently, the results of in vivo animal models showed that SNH depressed Linc00668 and suppressed the metastasis of NSCLC.
SNH suppressed metastasis of NSCLC cells and the mechanism may involve with the Linc00668/miR-147a/Slug axis.
KeywordsSodium new houttuyfonate (SNH) Linc00668 miR-147a NSCLC Metastasis ceRNA
Competing endogenous RNA
- IHC assay
Long non-coding RNAs
Non-small cell lung cancer
Quantitative real-time PCR
- RIP assay
RNA immunoprecipitation assay
Sodium new houttuyfonate
The Cancer Genome Atlas
Lung cancer is the most common cause of cancer death and was responsible for one-quarter of cancer deaths last year . Despite improvements in the development of targeted drugs and advances in treatment measures, the overall 5-year survival rate for lung carcinoma is still less than 15% due to limited therapeutic options, tumour metastasis, and recurrence . Given the vicious biological behaviour of lung cancer, it is vital to seek out additional measures to control the disease.
Metastasis, which is responsible for 90% of cancer deaths worldwide, is one of the primary characteristics of lung cancer . During metastasis, epithelial tumour cells invade the nearby extracellular matrix (ECM), expand into the systemic circulation and contribute to secondary tumours at distant sites . The epithelial-mesenchymal transition (EMT) is a process in which stationary epithelial cells lose their epithelial characteristics and gain a mesenchymal morphology with the ability to migrate and invade . Several EMT drivers, such as Snail, Slug and TCF8/ZEB1, have been closely associated with recurrence and survival in patients with breast, colorectal and ovarian cancer, indicating that the EMT process leads to poor clinical outcomes .
Natural products (NPs) have been used as traditional medicines since antiquity . Elucidating the mechanisms of the compounds from traditional medicine is important to support clinical usage and inspire treatment strategies. Houttuynia cordata Thunb. is a traditional Chinese herb that has been used to treat lung diseases for thousands of years. Researches of Houttuynia cordata Thunb on lung cancer is obviously. Han K et al. declared that Houttuynia cordata is of potential value in the treatment of lung cancer, although the underlying mechanisms need to be further confirmed . The main ingredient of Houttuynia cordata, sodium houttuyfonate (SH), has been used for the treatment of purulent skin infections and respiratory tract infections . Because of the chemical instability of houttuynin, its addition product, sodium new houttuyfonate (SNH), has been synthesized for improved stability. Previous studies have indicated that SNH/SH can significantly repress various kinds of bacteria, including Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa [10, 11, 12]. Recent studies have even revealed that SNH inhibits the inflammatory response through NF-κB-associated signalling pathways such as the TLR4/NF-κB and MAPKs/NF-κB pathways [13, 14]. However, although Houttuynia cordata Thunb. is frequently used to treat lung cancer in Chinese clinics, there have been no further in-depth studies on its mechanisms.
A large proportion of the human genome is transcribed as noncoding RNAs (ncRNAs) . Long ncRNAs (lncRNAs) demonstrate multiple functions, including nuclear sequestration, modulation of chromosomal interactions, chromatin looping, gene methylation and chromatin modification, in various malignant tumours such as lung adenocarcinoma, breast carcinoma, gastric cancer and hepatocellular carcinoma [16, 17, 18, 19]. Among the potential mechanisms, the competing endogenous RNA (ceRNA) theory has received much recognition based on mounting evidence . In the ceRNA theory, lncRNAs communicate or co-regulate by competing with or binding with shared microRNAs, which are small ncRNAs that play important roles in the post-transcriptional regulation .
In this study, we first confirmed that SNH could restrain NSCLC progression in multiple ways, especially by regulating migration and invasion. Then, we attempted to explain the mechanism with ceRNA theory. We found that Linc00668 was suppressed dramatically by SNH treatment in NSCLC cells, and upon further investigation, a Linc00668/miR-147a/Slug axis was discovered that could markedly modulate migration, invasion and the EMT in NSCLC cells.
Materials and methods
SNH (MW: 330.41, purity≥98%) was purchased from Shanghai Yuanye Bio-technology Co. Ltd. (Shanghai, China). SNH was dissolved in 75 °C ddH2O as a 16 mmol/l stock solution and stored at 4 °C. Staurosporine (a PKC inhibitor) was purchased from Beyotime Biotech Inc. (Shanghai, China).
NCI-H1299, A549, NCI-H460 and 293 T cells were obtained from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). SK-MES-1, SPC-A1 and HBE cells were kindly provided by Technology Transfer Center, NJUCM. 293 T, A549, SK-MES-1 and HBE cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and F12 medium (Gibco, Australia), and NCI-H1299, NCI-H460 and SPC-A1 cells were cultured in RPMI 1640 medium (Gibco, Australia) with 10% foetal bovine serum (FBS; Gibco, Australia) supplemented with a 1% penicillin/streptomycin solution (Gibco, Australia). All of the cells were maintained at 37 °C in a humidified atmosphere with 5% CO2.
Plasmid construction and cell transfection
A Linc00668 overexpression plasmid (p-Linc00668) and a negative control (NC) plasmid (p-NC) were designed by Realgene Biotech Co. (Nanjing, China). Three individual short hairpin RNA plasmids for Linc00668 (sh-Linc00668–1, sh-Linc00668–2, and sh-Linc00668–3) and a negative control (sh-NC) were purchased from Sangon Biotech (Shanghai, China) Co., Ltd. (Additional file 1: Table S2.). Hsa-miR-147a and NC mimics were purchased from Realgene Biotech Co. (Nanjing, China). Plasmids including the binding sites for miR-147a on Linc00668 and Slug mRNA were also designed by Realgene Biotech Co. (Nanjing, China). Cell transfection was performed with Lipofectamine 2000 transfection reagent (Invitrogen, US) according to the manufacturer’s protocol.
Cell counting Kit-8 assay
The viability of NSCLC cells was assayed by Cell Counting Kit-8 (CCK-8; Dojindo, Beijing, China) assay. First, 5 × 103 cells/well were added to a 96-well plate, with 100 μl of suspension in each well. Twenty-four hours after seeding, the medium was replaced with media containing different concentrations of SNH created through serial dilution. After culturing the cells for 24 H, 48 H, or 72 H, 10 μl of CCK-8 solution was added to each well, and the cells were incubated for 30 min. Curves of the cell death rates at the different SNH concentrations were calculated after measurement of the absorbance at each time point at a wavelength of 450 nm. The half maximal inhibitory concentration (IC50) values were also determined from these data.
Transwell invasion assay and wound scratch assay
For the transwell cell invasion assay, 8.0 μm Transwell Permeable Supports 3422 (Corning, US) were used. The upper chambers were coated with Matrigel Basement Membrane Matrix 354,234 (Corning, US), and transfected or normal cells were seeded at a density of 5 × 103 in the upper chambers with media containing different concentrations of SNH. A volume of 500 μl of medium containing 10% FBS was added to the lower chambers. After incubation for 24 H, the Matrigel and the cells on upper chambers were removed, and the cells on the bottom surface were fixed with 4% paraformaldehyde for 20 min. The cells were then stained with 0.3% crystal violet dye for 30 min. The invading cells were imaged using a digital light microscope (Leica, Germany).
For the wound scratch assay, cells were seeded at a density of 5 × 105 cells/well in 6-well plates and exposed to the indicated treatments. After the cells reached 100% confluence, a sterilized 200 μl pipette tip was used to make a straight scratch in the middle of each well. Images were obtained by using digital light microscopy at each indicated time.
Western blot analysis
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Solarbio, US) with 1% phenylmethanesulfonyl fluoride (PMSF). The lysates were suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime, Shanghai, China), separated on 10–15% SDS-PA gels (Beyotime, Shanghai, China) and transferred onto pure nitrocellulose blotting membranes (Pall, US). After the membranes were blocked with 5% non-fat milk, they were incubated at 4 °C overnight with primary antibodies against Slug (1:400, CST, US), E-cadherin (1:500, CST, US), N-cadherin (1:500, CST, US), Vimentin (1:1000, CST, US), and β-actin (1:2000, Santa Cruz, US). After incubation with horseradish peroxidase (HRP)-linked anti-rabbit (1:2000, CST, US) or anti-mouse (1:2000, CST, US) secondary antibodies for 2 h at room temperature, the bands were detected with a Gel Doc™ XR+ Gel Documentation System (Bio-Rad, US) with enhanced chemiluminescence (ECL) reagents (Bio-Rad, US).
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from cells and tissues using TRIzol reagent (Invitrogen, US) according to the manufacturer’s procedural guidelines. For mRNA and lncRNA quantification, the RNA was reverse transcribed into cDNA with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, US). EvaGreen 2X qRT-PCR MasterMix-Low ROX (abm, Canada) was used for quantitation with specific primers for the mRNA and lncRNA. GAPDH was used as an internal control. For miRNA quantification, reverse transcription was performed using an miRNA First Strand cDNA Synthesis (Stem-loop Method) Kit (Sangon Biotech, Shanghai, China). A MicroRNA qRT-PCR Kit (SYBR Green Method) (Sangon Biotech, Shanghai, China) was used for quantitation with specific primers for miRNA. U6 was used as an internal control. The primer sequences are listed in Additional file 2: Table S1. All quantitative real-time PCR experiments were performed with an Agilent Technologies Stratagene Mx3000P system (Agilent Technologies, US). The data were processed with the 2-ΔΔCt method, and the fold changes were normalized to the expression of the internal controls.
Cells at a density of 1 × 104 cells/well were seeded into 96-well plates. After exposure to the indicated treatments, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the cells were blocked with 5% goat serum and 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 1 h. After the blocking solution was aspirated, the cells were incubated with a primary antibody against E-cadherin (1:200, CST, US), N-cadherin(1:200, CST, US), and Vimentin (1:100, CST, US) in antibody dilution buffer (ADB; 1X PBS/1% bovine serum albumin (BSA)/0.3% Triton X-100) overnight at 4 °C. The next day, the cells were incubated with a fluorochrome-conjugated anti-rabbit secondary antibody (1:1000, CST, US) in ADB for 2 h at room temperature in the dark. Subsequently, the cells were stained with DAPI (1 μg/ml, CST, US) for 5 min. Images were obtained under a fluorescence microscope.
When frozen sections were used for immunofluorescence, the sections were first blocked with goat serum; the rest of the procedure was the same as that for the goat serum-blocked cells.
Dual-luciferase reporter assays
The target genes were inserted downstream of the firefly luciferase gene in the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, US). 293 T, NCI-H1299, and A549 cells were seeded in 24-well plates at a density of 1 × 104 cells per well 24 H before transfection. The cells were co-transfected with 0.5 μg of pmirGLO vector and 20 pmol of mimics with 2 μl of Lipofectamine 2000 reagent (Invitrogen, US). After 36 h of transfection, the cells were lysed, and the firefly and Renilla luciferase activity was measured with a SpectraMax i3x microplate reader (Molecular Devices, US). The Renilla luciferase activity was used as an internal control.
RNA immunoprecipitation (RIP) assay
The EZ-Magna RNA immunoprecipitation kit (Millipore, US) was used according to the manufacturer’s specifications. A549 and NCI-H1299 cells were lysed in complete RIP lysis buffer. The cell extract was then incubated with magnetic beads conjugated with Ago2 antibody (abcam, UK) or control IgG (Millipore, US) overnight at 4 °C. The beads were then washed and incubated with 0.1% SDS/0.5 mg/ml proteinase K for 30 min at 55 °C to remove proteins. Finally, the immunoprecipitated RNA was analysed by qRT-PCR analysis.
Immunohistochemical (IHC) analysis
Tissue sections were dewaxed, dehydrated, and rehydrated. Then, citrate buffer was used for antigen retrieval, and hydrogen peroxide (3.0%) was used to block endogenous peroxidase activity. After blocking with 10% goat plasma, primary antibodies, including an antibody against Slug (1:400, ImmunoWay, US), were added to the sections and incubated at 4 °C overnight. SignalStain Antibody Diluent (CST, US) was used to detect the primary antibodies. Counterstaining was performed using haematoxylin.
Lung metastasis in vivo
To assess the influence of SNH on the metastatic ability of NSCLC in vivo, we established a NSCLC orthotopic xenograft tumour model. First, we established the luciferase-expressing NCI-H1299 cell line with lentivirus (Ubi-MCS-firefly_Luciferase-IRES-Puromycin, GENE, Shanghai, China), and then NCI-H1299-luc cells (5 × 106 in 0.2 ml medium of a 1:1 mixture of RPMI 1640 and Matrigel 354,248) were injected into left lung parenchyma of BALA/c nude mice. Two weeks later, the NSCLC model mice were identified and randomly divided into three groups that were treated with SNH (18.75, 37.5 or 75 mg/kg orally (p.o.) daily; n = 10) and a control group that was treated with olive oil (control, 100 μl, p.o. daily; n = 10) for 14 consecutive days. During this period, we monitored metastasis by luciferase imaging of live animals using an IVIS Spectrum bioluminescence imaging system (PerkinElmer, US) and the intraperitoneal injection of 200 μl D-Luciferin substrate (15 mg/ml in DPBS, PerkinElmer). Then, we collected half number of mice’ lungs in each group. The collected lung tissues were imaged with the IVIS system in the D-Luciferin substrate (150 μg/ml in DPBS) and analysed the expression of Linc00668 and slug with qRT-PCR. For the rest half number of mice, after the mice were subjected to vascular perfusion-fixation, the lungs were fixed with buffered formaldehyde solution. After paraffin-embedding the lungs, routine haematoxylin-eosin (H&E) staining and immunohistochemistry staining were performed to detect the basic lung morphology and target protein expression. Immunofluorescence of frozen sections was performed to evaluate the lung metastases. The protocols for the animal experiments were approved by the Ethics Review Committee of Nanjing University of Chinese Medicine.
The data from the experiments are presented as the mean ± standard deviation (SD) of 3 independent experiments. Student’s t-test (two-tailed, with p < 0.05 considered significant) was used to assess differences between two groups. One-way analysis of variance, paired t-tests, χ2 tests or Spearman correlations were used to analyse multiple comparisons. We performed statistical analyses with GraphPad Prism 6 software.
SNH beneficially reduces EMT progression in NSCLC cells
Linc00668 is a potential target of SNH
Linc00668 is required for the EMT in NSCLC cells
Linc00668 functions as a ceRNA and sponges miR-147a in NSCLC cells
MiR-147a directly targets slug to mediate EMT progression in NSCLC cells
Linc00668 regulated slug expression indirectly via sponging miR-147a
Verification that SNH suppresses the EMT in vivo
An increasing number of research studies have focused on the anticancer mechanisms of NPs in recent years . In an analysis of all approved anticancer drugs worldwide, 74.9% were found to be either NPs per se or naturally inspired agents . Compared to chemically synthesized compounds, NPs are more frequently clinically used . SNH, a naturally inspired compound of Houttuynia cordata, has traditionally been used to treat lung disease in Chinese medicine. For instance, Chen YF et al. demonstrated that Houttuynia cordata Thunb. induced apoptosis in A549 cells .
Metastasis is a basic characteristic of lung cancer. Recent studies have indicated that EMT progression is part of a classic theory of metastasis . Studies on the NPs that regulate EMT progress in lung cancer are currently ongoing. Avtanski DB et al. reported the specific pathways of HNK (Honokiol, a natural phenolic compound)-mediated EMT inhibition for breast carcinoma . Because we successfully confirmed that SNH could repress EMT progression, a further study of the mechanism was needed to support this phenomenon.
The sheer number and diversity of lncRNAs juxtaposed with their complex molecular functions in editing, modification, retrotransposition and inheritance  suggests that scientists are far from understanding the interconnected relationships among lncRNAs and biological processes. Considering that an increasing number of lncRNAs have been identified as regulators of migration and invasion in NSCLC cells, we strived to determine whether there was an intimate relationship between lncRNAs and changes in metastasis after SNH treatment in NSCLC cells. After the NSCLC cells were treated with SNH, a screening of the lncRNA and mRNA sequences was performed, and GO and KEGG analyses confirmed that the EMT-associated pathway was influenced and the expression of Linc00668 was a potential target. Genomics and survival analyses with the TCGA database showed that Linc00668 expression was significantly elevated in NSCLC samples and was associated with poor prognosis. Thus, we proposed that Linc00668 might be a tumour promoter in NSCLC.
Recent studies on Linc00668 focused on proliferation and poor prognosis. Zhang CZ observed that LINC00668 exerts its oncogenic effects in oral squamous cell carcinoma (OSCC) partially via sponging miR-297 and activating VEGFA, which may be a negative prognostic factor . Zhang E et al. declared that LINC00668 was a directly regulated target of E2F1, which may be a downstream effector that binds to PRC2 in gastric cancer (GC) . To further investigate the biological function of Linc00668, we constructed knockdown and overexpression models. The results showed that Linc00668 could encourage the metastasis of NSCLC cells. However, when cells overexpressing Linc00668 were treated with SNH, migration and invasion were diminished, as was the expression of Linc00668, which confirmed that SNH suppresses metastasis by inhibiting Linc00668.
The ceRNA theory of lncRNA has been extensively accepted by increasing numbers of studies [31, 32, 33, 34]. In bioinformatics prediction of Linc00668, 4 possible binding miRNAs were identified, and miR-147a was considered the most likely candidate. Confirmation of the ability of Linc00668/miR-147a to bind to the Ago2 protein by RIP assay further illuminated the role of miR-147a. Convincingly, miR-147a-associated metastasis experiments showed that overexpression of miR-147a promoted cell migration and invasion. Recent studies on miR-147a have focused only on proliferation [35, 36]. Bertero T et al. reported that miR-147a appeared to be a potent inhibitor of cell proliferation and migration , but there is a lack of subsequent data to explain the mechanism of miR-147a in metastasis. Encouragingly, in the current study, the 3 ‘UTR region of Slug was predicted to partly bind with miR-147a. Slug is a common zinc finger transcriptional repressor that downregulates the expression of E-cadherin and triggers metastasis . The qRT-PCR and western blot analyses confirmed that Slug was downregulated by SNH. Overall, we verified that SNH negatively regulates metastasis of NSCLC in vitro and in vivo by the Linc00668/miR-147a/Slug axis.
We thank Prof. Haian Fu (School of Medicine, Emory University) and Prof. Peng Cao (Nanjing University of Chinese Medicine) for helpful advice and critical reading of the manuscript.
This work was supported by grants from the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_1317) (to R.J.), National Natural Science Foundation of China (81503374) (to M.C.), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) (to X.Z.), Natural Science Foundation of Jiangsu Province (BK20151003) (to Y.G.), Research Foundation of Education Bureau of Jiangsu Province (16KJA360001) (to X.Z.), People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007–2013/ under REA (PIRSES-GA-2013-612589) (to X.Z.).
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
XZ, MJC and RLJ conceived the study and provided the project direction. RLJ guided and performed the experiments, analyzed the data and wrote the manuscript. CH, ZYC and YYL completed the cell experiments. QL, LG and HXL assisted in performing the animal experiments. RLJ, MJC and YYG made contributions to the bioinformatics results. QRL and KL gave valuable suggestions to the experiments. XZ, MJC, LG and QL revised the manuscript. All authors approved the manuscript and they are informed of this submission.
Ethics approval and consent to participate
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics Review Committee of Nanjing University of Chinese Medicine (201804A005).
Consent for publication
The authors declare that they have no competing interests.
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