Background
Lung cancer is the leading cause of cancer-associated deaths worldwide. Approximately 80–85% of lung cancers are non-small cell lung cancers (NSCLCs), which are classified as adenocarcinomas, lung squamous cell carcinomas, and large-cell carcinomas. Despite the availability of multiple NSCLC clinical treatment options, such as surgery, chemotherapy, and radiotherapy, NSCLC prognosis is still very poor with a five-year survival rate less than 20%, which is mainly attributed to diagnosis at advanced stage, distant metastasis, and drug resistance. Consequently, effective treatment targets and therapies that improve the outcomes of NSCLC patients remain to be explored.
Metformin is a first-line drug for the treatment of type 2 diabetes mellitus. It lowers blood glucose predominantly by promoting insulin sensitivity, glucose uptake into peripheral tissues, and hepatic gluconeogenesis reduction. In recent years, the potential anti-cancer effect of metformin was reported in many cancer types, including NSCLC. There are two mechanisms associated with the anticancer effects of metformin: metformin lowers circulating insulin, which can bind to the highly expressed insulin receptor in cancer cells, thereby indirectly decreasing cell proliferation [
1]; and metformin directly activates AMPK and subsequently inhibits mammalian target of mTOR, leading to reduced cancer-cell proliferation [
2]. Recent studies demonstrated that AMPK activation induces Yes-associated protein (YAP) phosphorylation and inhibits YAP transcriptional activity [
3]. Additionally, LATS1/2 can be activated by an AMPK-dependent pathway to suppress YAP activity by phosphorylating YAP at S127, leading to the retention of YAP in the cytoplasm and the promotion of its degradation [
3]. In addition, our previous study indicated that metformin downregulates YAP by interfering with interferon regulatory factor 1 (IRF-1) binding to the
YAP promoter, inhibiting NSCLC growth and metastasis [
4]. Moreover, several studies showed that the use of metformin was associated with a lower risk of lung cancer among patients with diabetes and improved survival of NSCLC patients with diabetes [
5‐
7]. Furthermore, growing evidence indicates that metformin inhibits mammalian cancer growth and metastasis through regulation of microRNAs (miRNAs). For example, metformin prevents liver tumorigenesis by attenuating fibrosis in a transgenic mouse model of hepatocellular carcinoma [
8]; The treatment also suppresses melanoma cell growth and motility through modulation of miRNAs expression [
9]. Furthermore, metformin disrupts the metastasis associated lung adenocarcinoma transcript 1 (MALAT1)/miR-142-3p sponge, decreasing the invasion and migration of cervical cancer cells [
10]. However, whether other regulatory mechanisms underpin the effects of metformin in NSCLC, such as metformin-decreased YAP activity by miRNAs regulation, is currently unclear.
microRNAs (miRNAs), a cluster of endogenous small non-coding RNAs, play significant roles in multiple physiological and pathological processes, which maturation process includes catalysis, cleavage, and transport, resulting in three miRNA stages: pri-miRNA (1–3 k bp), pre-miRNA (60–70 bp), and mature miRNA (19–22 bp). The miRNAs biogenesis occurs in the nucleus and their effect is exerted in the cytoplasm. Here they cleave specific target mRNAs or repress the translation by binding to the 3′ untranslated region (UTR) of specific mRNAs with complementary sequences [
11]. Emerging evidence indicates that miRNAs have important regulatory effects in tumorigenicity and tumor progression, therefore being used as biomarkers for cancer diagnosis and prognosis as well as therapeutic targets. miR-381 has been reported to exert a tumor-suppressing role in various cancer types such breast [
12], pancreatic [
13], cervical [
14], and gastric [
15] cancers. It can also repressed cell proliferation, invasion, and migration of epithelial ovarian cancer cells [
16]. Furthermore, miR-381 overexpression inhibited xenograft growth in a nude mouse model of human pancreatic cancer [
13]. However, the underlying mechanism through which metformin-regulated miR-381 modulates these cellular processes has not been fully elucidated.
The transcriptional coactivator YAP is the crucial downstream effector of the Hippo signaling pathway, which plays important roles in organ size control, regeneration, and cancer [
17,
18]. This pathway is highly evolutionarily conserved. The main components of the mammalian MST-YAP signaling pathway are Mst1/2, LATS1/2, and YAP/TAZ. Following activation of the MST-YAP signaling pathway, Mst1/2, as the core component of this kinase chain, is activated and phosphorylates a component downstream of LATS1/2. LATS1/2 mainly inhibits proliferation and migration of tumor cells by blocking cell cycle progression and plays an important regulatory role in cell apoptosis. LATS1/2 phosphorylates YAP/TAZ, which inhibits YAP activity [
19]. The Hippo pathway, with a kinase cascade at its core, phosphorylates and inactivates YAP, leading to its sequestration or degradation in the cytoplasm by binding to 14–3-3 proteins or recruiting E3 ligase Skp Cullin F-box (SCF) β-TrCP [
20,
21]. When this pathway is inhibited, unphosphorylated YAP translocates into the nucleus and interacts with other transcription factors, mainly TEA domain transcription factors (TEADs), to turn on target gene expression, resulting in tissue overgrowth and tumorigenesis. Mounting evidence suggests that aberrant YAP expression or activity are involved in carcinogenesis, cancer progression, and metastasis in diverse types of cancer [
17,
22]. However, the regulatory mechanism of the Hippo signaling pathway, especially the regulatory mechanism of YAP at the level of transcription, remains unclear.
Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal phenotype with enhanced migratory and invasive abilities, accompanied by loss of cell polarity and cell-cell adhesion. EMT is a critical step in the cascade of events leading to cancer metastasis, which contributes to the majority of cancer-associated deaths, and is regulated by a set of transcription factors that include
snail family transcriptional repressor 1 (Snail),
snail family transcriptional repressor 2 (Slug),
zinc finger E-box binding homeobox 1 (ZEB1), and
wist family bHLH transcription factor 1 (Twist) [
23]. Snail suppresses the transcription of E-cadherin, an epithelial biomarker required for epithelial formation and maintenance, and drives EMT in a variety of human cancers [
24]. It was reported that Snail expression was significantly elevated in metastatic lesions of ovarian carcinoma [
25] and knockdown of Snail could reverse EMT and repress tumor growth and invasiveness [
26]. Accordingly, targeting Snail, and thereby interfering with EMT and further preventing metastasis, may represent a potential cancer therapy strategy. However, regulation of cancer cell EMT and metastasis by the miR-381/YAP axis was not reported in NSCLC. Therefore, in the current study, the molecular mechanism underpinning how metformin-induced upregulation of miR-381 directly targets YAP or its interactions with the epithelial-mesenchymal transition (EMT) marker protein Snail in NSCLC was explored.
Materials and methods
Molecular biology
Myc-tagged YAP constructs were made using the pcDNA 3.1 vector (Invitrogen, Carlsbad, CA, USA). Sequences encoding the Myc epitope (EQKLISEEDL) were added by PCR through replacement of the first Met-encoding codon in the respective cDNA clones.
Cell lines and culture
Human lung normal cell line HBEC and NSCLC cell lines A549, H1299, Calu6 and H520 were purchased from American Type Culture Collections (Manassas, VA). 95-D cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science (Shanghai, China). Cell lines were cultivated in RPMI-1640 medium supplemented with 10% FBS (Hyclone, USA), penicillin/streptomycin (100 mg/mL). Culture flasks were kept at 37 °C in a humid incubator with 5% CO2.
Over-expression and knockdown of genes
Overexpressing plasmids (2 μg) or siRNAs (1.5 μg) of indicated genes were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for over-expression and knockdown of indicated genes, followed by analysis 36 h later. The selected sequences for knockdown as follow:
siYAP-1 were: 5′-GGUGAUACUAUCAACCAAA-3′.
siYAP-2 were 5′-GACATCTTCTGGTCAGAGA-3′.
siSnail-1 were: 5′- AGUUUAUUGAUAUUCAAUA-3′.
siSnail-2 were: 5′-UGGUUAAUUUAUAUACU-3′.
siNC were: 5′-UUCUCCGAACGUGUCACGU-3′.
Transfection of miRNA
The miR-381 mimics, miR-381 inhibitors and another scramble oligonucleotide (negative control, NC) were synthesized by GenePharma biotechnology (Shanghai, China). The miRNAs (50 nM) transfection was performed with lipofectamine 2000 reagent (Invitrogen) in HBEC, A549 and 95-D cells for 36 h, according to the manufacturer’s instruction. The sequences of miR-381 mimics, miR-381 inhibitors and scramble oligonucleotide as follow:
miR-381 mimics: 5′-UAUACAAGGGCAAGCUCUCUGU-3′.
miR-381 inhibitors: 5′- ACAGAGAGCUUGCCCUUGUAUA-3′.
miR-381 mimics NC: 5′- UUGUACUACACAAAAGUACUG-3′.
miR-381 inhibitors NC: 5′- CAGUACUUUUGUGUAGUACAA-3′.
RNA isolation and reverse transcription (RT)-PCR assay
We used TRIzol reagent (TransGen Biotech, Beijing, China) to isolate total RNA from the samples. RNA was reverse transcribed into first-strand cDNA using a TransScript All-in-One First-Strand cDNA Synthesis Kit (TransGen Biotech). cDNAs were used in RT-PCR and RT-qPCR assay with the human GAPDH gene as an internal control. The final RT-qPCR reaction mix contained 10 μL Bestar® SYBR Green qPCR Master Mix, Amplification was performed as follows: a denaturation step at 94 °C for 5 min, followed by 40 cycles of amplification at 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s. The reaction was stopped at 25 °C for 5 min. The relative expression levels were detected and analyzed by ABI Prism 7900HT / FAST (Applied Biosystems, USA) based on the formula of 2-ΔΔct. We got the images of RT-PCR by Image Lab™ Software (ChemiDocTM XRS+, BiO-RAD) and these images were TIF with reversal color format. The RT-PCR primers were:
YAP forward primer: 5′-GGATTTCTGCCTTCCCTGAA-3′.
YAP reverse primer: 5′-GATAGCAGGGCGTGAGGAAC-3′.
CTGF forward primer: 5′-ACCGACTGGAAGACACGTTTG-3′.
CTGF reverse primer: 5′-CCAGGTCAGCTTCGCAAGG-3′.
Cyr61 forward primer: 5′-GGTCAAAGTTACCGGGCAGT-3′.
Cyr61 reverse primer: 5′-GGAGGCATCGAATCCCAGC-3′.
Snail forward primer: 5′-TACAAAAACCCACGCAGACA-3′.
Snail reverse primer: 5′-ACCCCACATCCTTCTCACTG-3′.
E-cadherin forward primer: 5′-ACCATTAACAGGAACACAGG − 3′.
E-cadherin reverse primer: 5′-CAGTCACTTTCAGTGTGGTG-3′.
Vimentin forward primer: 5′-CGCCAACTACATCGACAAGGTGC-3′.
Vimentin reverse primer: 5′-CTGGTCCACCTGCCGGCGCAG-3′.
GAPDH forward primer: 5′-CTCCTCCTGTTCGACAGTCAGC-3′.
GAPDH reverse primer: 5′-CCCAATACGACCAAATCCGTT-3′.
In situ hybridization
NSCLC tumor cells were seeded on glass coverslips, fixed with 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with 0.5% Triton X-100 (Biosharp, China) for 10 min at room temperature. The slides were then processed using a RiboTM Fluorescent In Situ Hybridization Kit (RiboBio, China). The corresponding FISH Probe Mix was also designed by RiboBio Co. The experiment was repeated three times in A549 cells. Images were obtained with a Zeiss Axio Imager Z1 Fluorescent Microscope (Zeiss, Oberkochen, Germany).
MTT and CCK8 assays
To assess the cellular viability and growth, 5 × 104 HBEC and NSCLC tumor cells were seeded onto 6-well plates with transfection of the relevant plasmids. Cell viability and growth were respectively determined using CCK8 and MTT assays in 96-well plates in a manner. Cells were transfected with the relevant plasmids culturing for 36 h, followed by incubation with CCK8 for 4 h. For MTT assay, MTT solution (5 mg/mL, Sigma, st. Louis, MO) of 5 μL was added to each well for another 4 h. The medium was removed and 100 μL DMSO was added into per well to oscillate for 10 min. Absorbance was read at 450 nm for CCK8 and 570 nm for MTT assay using a spectrophotometer (Tecan, Männedorf, Switzerland). Cell viability (%) = OD (treated cells)/OD (control cells) × 100.
Western blot analysis
Human lung cancer cells were transfected with the relevant plasmids and cultured for 36 h. For western blot analysis, the cells were lysed with NP-40 buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1 mM PMSF, and 0.5% NP-40) containing proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich) at 4 °C for 40 min. Following centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant was collected, and protein concentration was determined by BCA assay. Equal protein from the samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Millipore, Bredford, MA, USA). The membranes were blocked with 5% skim milk and then probed with corresponding primary antibodies. Following incubation with an HRP-conjugated secondary antibody (Beyotime), immonoreactive signals were detected using by Enhanced chemiluminescence (ECL) technique (Millipore). Tubulin was detected for equal loading control. The primary antibodies used in this study were 1:1000 Abcam (Cambridge, UK) antibody of anti-YAP (ab52771), anti-pYAP (ab62751), anti-CTGF (ab6992) and anti-Cyr61 (ab24448), anti-Ki67 (ab16667), anti-Snail (ab53519), anti-Vimentin (ab45939), anti-E-cadherin (ab1416), anti-cleaved Capase-3 (ab32042) and anti-Tubulin (ab6046).
The cells (200 cells/well) were inoculated into 6-well plates and cultured for 14 days. And then the cells were stained with 0.1% crystal violet. Colonies of more than 50 cells were counted by optical microscope (BX53, Olympus Corporation, Japan), and colony formation capacity was expressed by colony number/inoculated cell number × 100%.
Wound healing assays
To assess the cellular migration, 104 HBEC, A549 and 95-D cells were seeded onto 6-well plates with transfection of the relevant plasmids. These were then incubated in 5% CO2 at 37 °C for 48 h. A wound was scraped into the cells using a plastic 200 μL tip and then washed by PBS. The cells were then incubated in RPMI-1640 medium containing 2% FBS. Images were captured at the time points of 0 and 36 h after wounding. The relative distance of the scratches was observed under an optical microscope (IX53, Olympus, Tokyo, Japan) and assessed using the ImageJ software.
Transwell migration assays
Transwell migration assays were performed using a 24-well chamber (Costar 3422; Corning Inc., Corning, NY, USA). The lower and upper chambers were partitioned by a polycarbonate membrane (8-μm pore size). Lung cancer cells (5 × 103) were seeded into RPMI-1640 without FBS in the upper chamber. RPMI-1640 containing 10% FBS was added to the lower chamber. The cells were allowed to migrate for 36 h at 37 °C in a humidified atmosphere containing 5% CO2. Cells remaining on the upper side of the membrane were removed using PBS-soaked cotton swabs. The membrane was then fixed in 4% paraformaldehyde for 20 min at 37 °C and then stained with crystal violet. The number of randomly selected cells on the lower side of the membrane were counted under an Olympus light microscope (Olympus, Tokyo, Japan). The cells in the 5 non-repeating field was randomly selected and counted.
Immunofluorescent staining
To examine the protein expression and location by immunofluorescent staining, normal lung cell and lung cancer cells were seeded onto coverslips in a 24-well plate and left overnight. Cells were then fixed using 4% formaldehyde for 30 min at 25 °C and treated with 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 min. The coverslips were incubated with rabbit anti-YAP, Ki67, Annexin V, Snail, Vimentin and mouse anti-E-cadherin monoclonal antibody (Abcam) at 1:200 dilution in 3% BSA at 4 °C for overnight. Following incubation with Alexa-Fluor 467 (green, 1:500, A-11029; Invitrogen, USA) and 594 (red, 1:500, A-11032; Invitrogen, USA) tagged anti-rabbit or -mouse monoclonal secondary antibody in 3% BSA. Hoechst (3 μg/mL, cat. no. E607328; Sangon Biotech Co., Ltd.) was added for nuclear counterstaining. Images were obtained with a Zeiss Axio Imager Z1 Fluorescent Microscope (Zeiss, Oberkochen, Germany).
Analysis of publicly available datasets
To analyze correlation between miR-381 or YAP expression level and prognostic outcome of patients, Kaplan-Meier survival curves of NSCLC patients with low and high expression of miR-381 or YAP were generated using Kaplan-Meier Plotter (
www.kmplot.com/analysis and
www.oncolnc.org) [
27,
28].
Subcellular fraction
Transfected A549 cells were harvest in PBS and resuspended for 10 min on ice in 500 μL CLB Buffer (10 mM Hepes, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 5 mM EDTA, 1 mM CaCl2, 0.5 mM MgCl2). Thereafter, 50 μL of 2.5 M sucrose was added to restore isotonic conditions. The first round of centrifugation was performed at 6300 g for 5 min at 4 °C. The pellet washed with TSE buffer (10 mM Tris, 300 mM sucrose, 1 mM EDTA, 0.1% NP40, PH 7.5) at 1000 g for 5 min at 4 °C until the supernatant was clear. The resulting pellets were nucleus. The resulting supernatant from the first round was transferred and subjected to centrifugation at 14000 rpm for 30 min. The resulting pellets were membranes and the supernatant were cytoplasm.
Luciferase reporter assay
To construct the core region of Snail promoters, the indicated promoters of Snail was amplified by PCR from the human genomic DNA of A549 cells and were inserted into the upstream of the pGL3-Basic vector (Promega, Madison, WI, USA) via KpnI and XhoI sites to generate Snail luc. Thereafter, we use the Firefly Luciferase Reporter Gene Assay Kit (Beyotime, RG005) to detect the promoter activities using a spectrophotometer (Tecan, Männedorf, Switzerland). The primers of Snail are following:
Snail forward primer: 5′- CTCGACCACTATGCCGCGCTC-3′.
Snail reverse primer: 5′- CAAAGAGCAGCTCCAGGCAC-3′.
qPCR of MS2-GFP expression system
The Measurements of RNA-MS2-GFP were performed as described previously [
29]. In briefly, NSCLC cells with both MS2-GFP and transcript target plasmids were grown for 36 h at 37 °C in RPMI-1640 medium supplemented with 10% FBS (Hyclone, USA), penicillin/streptomycin (100 mg/mL). On the following day, cells were diluted in fresh medium plus antibiotics. To induce the production of MS2-GFP, 100 ng/ml anhydrotetracycline (Cat.no: 2–0401-001, IBA, Germany) was added to the diluted cellular culture. The expression of the target RNA was induced by the addition of IPTG and L-arabinose to the cultures. Cells were subsequently incubated with these inducers at 37 °C for 1 h with shaking to a final optical density (600 nm) of about 0.4. Then cell lysate immunoblotted by GFP antibody were detected by qPCR with the indicated primers.
CHIP assay
ChIP experiments were performed according to the laboratory manual. Immunoprecipitation was performed for 6 h or overnight at 4 °C with specific antibodies. After immunoprecipitation, 45 μL protein A-Sepharose and 2 μg of salmon sperm DNA were added and the incubation was continued for another 1 h. Precipitates were washed sequentially for 10 min each in TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Precipitates were then washed three times with TE buffer and extracted three times with 1% SDS, 0.1 M NaHCO3. Eluates were pooled and heated at 65 °C for at least 6 h to reverse the formaldehyde cross-linking. DNA fragments were purified with a QIAquick Spin Kit (Qiagen, CA). For PCR, 2 μL from a 5 mL extraction and 21–25 cycles of amplification were used. The sequences of the primers used are provided as follows:
Snail forward primer: 5′-GCCCTGGCTGCTACAAGGCCATG-3′.
Snail reverse primer: 5′-CTTAGCTGATGAAGAAAGTTTCTG-3′.
Drug treatment
HBEC and NSCLC cells were transfected with relevant plasmids and cultured for 36 h then treated with 10 mM metformin (D150959, Sigma, Saint Louis, USA) followed by analysis at indicated times. For Actinomycin D (129,935, Millipore, Massachusetts, USA) treatment, A549 cells were transfected with relevant plasmids and cultured for 36 h then treated with 5 μg/mL Actinomycin D followed by analysis at indicated times.
Human lung cancer specimen collection
All the human lung cancer and normal lung specimens were collected in Affiliated Hospital of Binzhou Medical College with written consents of patients and the approval from the Institute Research Ethics Committee.
In vivo experiments
To assess the in vivo effects of miR-381 and YAP, 3 to 5-week old female BALB/c athymic (NU/NU) nude mice were housed in a level 2 biosafety laboratory and raised according to the institutional animal guidelines of Binzhou Medical University. All animal experiments were carried out with the prior approval of the Binzhou Medical University Committee on Animal Care. For the experiments, mice were injected with 5 × 105 lung cancer cells with stably expression of relevant plasmids (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with Vehicle or Metformin (orally, 50 mg/kg per day) for three times a week and tumor volume were measured every second day. Tumor volume was estimated as 0.5 × a2 × b (where a and b represent a tumors short and long diameter, respectively). Mice were euthanized after 6 weeks and the tumors were measured a final time. Tumor and organ tissues were then collected from xenograft mice and analyzed by immunohistochemistry.
Immunohistochemical analysis
All specimens were formalin-fixed and paraffin-embedded. Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining were performed as described previously [
30]. In briefly, tumor tissues were fixed in 4% paraformaldehyde overnight and then embedded in paraffin wax. Four-micrometer thick sections were and stained using hematoxylin and eosin (H&E) for histological analysis. For IHC assay, the paraffin-embedded tissues were de-waxed and then antigen-repaired for 30 min. The activity of endogenous peroxidase was blocked by 0.3% H
2O
2 solution. The blocking solution containing 10% FBS (Gicbo, Life Technologies, Carlsbad, CA) was used to block the non-specific antigen at room temperature for 30 min. Then the primary antibodies of YAP and Snail were added to incubate the slide at 4 °C. PBS was used as a negative control instead of the primary antibody. Finally, DAB (Kanglang Biotechnology Co., Ltd., Shanghai, China) was used to develop the color. The scoring criteria for immunohistochemistry were developed by two pathologists in our hospital.
Statistical analysis
Each experiment was repeated at least three times. The statistical analyses of the experiment data were performed by using a two-tailed Student’s unpaired T-test and one-way ANOVA. Statistical significance was assessed at least three independent experiments and significance was considered at either P-value < 0.05 was considered statistically significant and highlighted an asterisk in the figures, while P-values < 0.01 were highlighted using two asterisks and P-values < 0.001 highlighted using three asterisks in the figures.
Discussion
Lung cancer has the highest incidence and mortality rates of all cancers, globally. With increases in environmental pollution and smoking, it has become the primary threat to people’s health. Currently, routine clinical treatment includes surgical resection, chemotherapy, and radiotherapy. However, chemotherapeutic drugs often present poor specificity, and induce side effects and tumor resistance. Moreover, cancer metastasis is the main cause of lung cancer clinical treatment failure, leading to very low cure and five-year survival rates [
34]. Therefore, finding new targets for the diagnosis and treatment of lung cancer has become an urgent clinical problem.
Our data show that miR-381 expression is downregulated in NSCLC cells and patient specimens, inhibiting cell proliferation, migration, invasion, and EMT, and promoting cell apoptosis (Fig.
1), opposite to YAP expression and YAP-mediated effects (Fig.
2). Moreover, miR-381 (GenePharma, Shanghai, China) bound to the 3’UTR of YAP mRNA and reduced YAP expression in a dose and time-dependent manner (Fig.
3). We also found that metformin decreased YAP activity via upregulating miR-381 and inhibited cell proliferation, migration, invasion, and EMT but induced apoptosis (Figs.
4 and
5). Furthermore, YAP bound to the promoter of
Snail and regulated its expression at the transcription level, therefore mediating the metformin-induced biological processes mentioned above (Figs.
6 and
7). Importantly, miR-381 inhibited lung cancer growth and metastasis in vivo by regulating YAP (Fig.
9). These data indicated that miR-381, YAP, and Snail constitute a signal transduction pathway, known as the miR-381-YAP-Snail signal axis, which was repressed by metformin, and reduce lung cancer cell invasiveness (which is regulated by EMT in lung cells) and migration (Fig.
9i).
Metformin is a first-line hypoglycemic drug that has been used in the clinic for more than half a century and has proven to be efficacious in treatment of type 2 diabetes mellitus. Recently published studies have shown that it can reduce the risk of tumor development, and data from case-control studies corroborated this [
35]. The anti-cancer effect of metformin results from its ability to interfere with several biological processes including: inhibition of tumor cell proliferation by activation of adenosine monophosphate-activated protein kinase [
36]; regulation of insulin/insulin-like growth factor 1 axis activity [
37]; induction of tumor cell cycle arrest and apoptosis [
38]; and regulation of energy metabolism [
39]. These data indicate that metformin has a clear role in tumor prevention and, therefore, the prolonged and increased use of metformin can significantly reduce the risk of malignant tumors. Moreover, our previously published research showed that metformin downregulates YAP by interfering with IRF-1 binding to the YAP promoter, and inhibits cancer growth and metastasis in NSCLC [
4]. Furthermore, recent studies have shown that metformin inhibits human cancer growth and metastasis by regulating miRNAs [
9,
10]. As demonstrated in this study, metformin upregulates the level of miR-381 but whether this regulatory effect occurs at the pri-, pre-miRNA, or post-transcriptional level remains unclear and needs to be further explored. Metformin treatment decreases the levels of miR-381 decay as demonstrated by our study (Fig.
4f, g). Therefore, these data indicated that metformin has potential as an effective drug for the treatment of human cancers, but further research is required to elucidate the underlying molecular mechanisms.
miRNAs extensively participate in cell growth, development, differentiation, metabolism, and defense mechanisms. Moreover, it has been found that miRNA, as a small molecular product regulated by gene expression, may be used as a tumor marker for early diagnosis of tumors and to determine the prognosis and recurrence of a tumor, revealing a correlation between miRNA expression and tumor occurrence [
40]. In addition, recent studies have shown that miR-381 has a very important role in tumor oncogenesis, development, metastasis and chemical resistance. For example, miR-381 inhibits the proliferation and invasion of prostate cancer cells via the regulation of UBE2C [
41]; miR-381 inhibits lung adenocarcinoma progression by directly targeting LMO3 through the regulation of the PI3K/Akt signaling pathway and EMT [
42]; up-regulation of miR-381 inhibits the NAD
+ salvage pathway and promotes apoptosis in breast cancer cells [
43]; miR-381 overcomes cisplatin resistance in breast cancer cells by targeting MDR1 [
44]. In line with these findings, metformin upregulated the level of miR-381 to inhibit NSCLC tumor growth and metastasis in our study. Therefore, these findings provide us with a new tool for the study of tumor pathogenesis and help us to find molecular markers for early tumor diagnosis and the establishment of an effective miRNA targeted therapy, which is greatly significant to the improvement of survival rates of cancer patients.
YAP is the main downstream effector of the Hippo pathway, which is highly conserved in mammals. The Hippo pathway can regulate the steady-state of tissues, cell proliferation, apoptosis, organ size, regeneration, and tumor formation [
17]. Some of the components of the Hippo pathway can inhibit cell proliferation, promote apoptosis, and regulate stem cell/progenitor cell expansion, thus playing an important role in regulating organ growth [
17]. Dysfunction of the core components of the Hippo pathway can cause overgrowth of an organ. For instance, if the Hippo pathway is inactivated, YAP dephosphorylation can be induced, and the expression of genes related to cell proliferation, reprogramming, stem cell activity, EMT, and anti-apoptosis can be upregulated [
45]. Moreover, YAP is overexpressed in a variety of tumors, such as NSCLC, liver, gastric, colorectal, and small cell lung cancer, and therefore can be used as a marker of poor prognosis in these tumors [
46]. Hence, it is urgent to clarify the correlation between YAP and human tumorigenesis and development, and its importance in cancer treatment efficiency, which directly impacts cancer patients quality of life and the economic burden caused by cancer [
46]. However, previous research mainly focused on the identification of new components and specific intermolecular mechanisms of known core proteins in the MST-YAP pathway. Additionally, there is little research on the regulation of YAP levels, particularly at the transcriptional level. In this study, we showed that miR-381 directly targets 3′UTR of YAP mRNA, consequently reducing the stability of YAP mRNA at the transcriptional level. Thus, our study provides a deeper insight in the relationship between YAP and human cancers.
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