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Emerging reports have revealed that several microRNAs (miRNAs) are abnormally expressed in non-small cell lung cancer (NSCLC). miRNAs have been identified as oncogenes or tumor suppressors, and regulate various biological processes including oncogenesis and development. miR-802 is dysregulated in multiple types of human cancer, and exerts tumor-suppressive or promoting roles. However, the expression levels and functional roles of miR-802 in NSCLC remain largely unknown. In the present study, miR-802 expression was demonstrated to be decreased in NSCLC tissues and cell lines. A low miR-802 expression was significantly correlated with the tumor stage, lymph node metastasis and brain metastasis in NSCLC patients. Restoring miR-802 expression inhibited NSCLC cell proliferation and colony formation, induced cell apoptosis, decreased cell migration and invasion
Lung cancer is one of the most common human malignancies and is a leading cause of cancer-related death among both men and women around the world (
MicroRNAs (miRNAs) are a series of endogenous, short-length, single-stranded, and non-coding RNA molecules of ~21-25 nucleotides in length (
miR-802 is dysregulated in multiple types of human cancer, and exerts tumor-suppressive (
NSCLC tissues and paired normal adjacent tissues (NATs) were obtained from 52 patients undergoing surgical resection at The First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China) between August 2015 and July 2017. None of these patients received adjuvant therapy prior to surgery. All tissues were immediately frozen in liquid nitrogen and then stored at −80°C prior to RNA isolation. Written informed consent was obtained from all patients and this study was approved by the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China).
A total of five human NSCLC cell lines (A549, H522, H1299, H460, and SK-MES-1) and a non tumorigenic bronchial epithelium cell line, BEAS-2B, were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% v/v penicillin-streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C in a humidified atmosphere containing 5% CO2.
miR-802 mimics, miRNA mimic negative control (miR-NC), small interfering RNA (siRNA) against fibroblast growth factor receptor 1 (FGFR1; termed here si-FGFR1) and negative control siRNA (si-NC) were generated by GenePharma Co., Ltd. (Shanghai, China). The sequences were: miR-802 mimics, 5′-CAGUAACAAAGAUUCAUCCUUGU-3′; miR-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; si-FGFR1, 5′-GGAGGUGCUUCACUUAAGATT-3′; and si-NC, 5′-UUC UCCGAACGUGUCACGUTT-3′. FGFR1 overexpression vector pCMV-FGFR1 and empty pCMV plasmid were chemically synthesized by OriGene Technologies, Inc., (Beijing, China). Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for cell transfections according to the manufacturer’s protocol. After incubation for 6-8 h, the culture medium was discarded, and fresh DMEM containing 10% FBS was added into each well.
Total RNA was extracted from tissues and cells using the TRIzol reagent (Thermo Fisher Scientific, Inc.), and quantified using an ND-2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA), following the manufacturer’s protocol. The All-in-One miRNA RT-qPCR Detection kit (GeneCopoeia, Inc., Rockville, MD, USA) was used to determine miR-802 expression. To analyze FGFR1 mRNA expression, reverse transcription was performed using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). Subsequently, the relative expression of FGFR1 mRNA was detected with SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) using an Applied Biosystems 7900HT Real-Time PCR system (Thermo Fisher Scientific, Inc.). U6 snRNA and GAPDH were used as internal references for miR-802 and FGFR1, respectively. Relative expression levels were determined according to the 2−∆∆Cq method (
Transfected cells were harvested 24 h post-transfection and re-seeded in 96-well plates at a density of 3,000cells/well. Cells were then incubated at 37°C for 0, 24, 48 and 72 h. At each time point, 20
Transfected cells were collected at 24 h post-transfection and seeded into 6-well plates at a density of 1×103 cells/well in 2 ml of culture medium. After incubation at 37°C with 5% CO2, the colonies were washed with PBS (Gibco; Thermo Fisher Scientific, Inc.), fixed with 4% paraformaldehyde, and then stained with methyl violet (Beyotime Institute of Biotechnology, Inc., Shanghai, China). The number of colonies was counted under an inverted light microscope (Olympus Corporation, Tokyo, Japan).
Transfected cells were harvested at 48 h post-tansfection, washed with ice-cold PBS, and then stained with the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Biolegend, Inc., San Diego, CA, USA), which is used to detect cells undergoing apoptosis. In specific, transfected cells were resuspended in 100
Cell migration and invasion was determined using uncoated and matrigel-coated transwell chambers (8
A total of eight female BALB/c nude mice (20 g; aged 4-6 weeks) were obtained from the Shanghai Laboratory Animal Center (Shanghai, China). All nude mice were maintained under specific pathogen-free conditions (25°C; 50% humidity; 10-h light/14-h dark cycle). Cells were transfected with miR-802 mimics or miR-NC. Following incubation at 37°C with 5% CO2 for 24 h, the transfected cells were harvested and injected subcutaneously into the flank region of nude mice (n=4 for each group). The volume of tumor xenografts was calculated every four days using the following formula: length × width2 × 0.5. One month later, all nude mice were sacrificed, and the xenografts were removed and weighted. The animal experiments were approved by the Scientific Investigation Board of The First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China) and performed in accordance with the guidelines of the National Institutes of Health Guidance for the Care and Use of Laboratory Animals.
TargetScan (
For the luciferase reporter assay, the fragments of FGFR1 3′-UTR containing the wild-type (wt) and mutant (mut) miR-802 binding site were constructed by GenePharma Co., Ltd., and were then inserted into the pMIR-REPORT miRNA Expression Reporter vector (Ambion; Thermo Fisher Scientific, Inc.). The chemically synthesized luciferase reporter plasmids were termed pMIR-FGFR1-3′-UTR-wt and pMIR-FGFR1-3′-UTR-mut, respectively. Cells were plated into 24-well plates one night prior to transfection. miR-802 mimics or miR-NC in combination with pMIR-FGFR1-3′-UTR-wt or pMIR-FGFR1-3′-UTR-mut were cotransfected into cells using Lipofectamine 2000, according to the manufacturer’s instructions. Luciferase activity was detected 48 h post-transfection using a dual-luciferase reporter assay system (Promega Corporation, Madison, WI, USA).
Total protein was extracted from tissue samples and cells using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with protease and phosphatase inhibitors (Roche Applied Science, Mannheim, Germany). The protein concentration was measured using the BCA assay (KeyGen Biotech. Co., Ltd., Nanjing, China). Equal amounts of protein were separated on 10% SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF; Millipore, Billerica, MA, USA) membranes. After blocking in TBST containing 5% non fat dry milk, the membranes were incubated with primary antibodies at 4°C overnight. Afterwards, the membranes were washed with TBST followed by incubation with goat anti-rabbit (cat no. ab6721; 1:5,000 dilution) or goat anti-mouse (cat. no. ab6789; 1:5,000 dilution) horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, Cambridge, UK) at room temperature for 2 h. Finally, enhanced chemiluminescence reagents (ECL; Pierce; Thermo Fisher Scientific, Inc.) were used for protein band detection. Quantity One software version 4.62 (Bio Rad Laboratories, Inc.) was used for densitometry. The primary antibodies used in this study were as follows: rabbit anti-human monoclonal FGFR1 antibody (cat. no. ab76464; 1:1,000 dilution; Abcam), mouse anti-human monoclonal phosphoinositide 3-kinase (PI3K; cat. no. ab189403; 1:1,000 dilution; Abcam), rabbit anti-human polyclonal phosphorylated (p-) PI3K antibody (cat. no. ab182651; 1:500 dilution; Abcam), mouse anti-human monoclonal p-AKT serine/threonine kinase (Akt) antibody (cat. no. sc-81433; 1:1,000 dilution; Santa Cruz Biotechnology, Inc., Callas, TX, USA), mouse anti-human monoclonal Akt antibody (cat. no. sc-56878; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), rabbit anti-human monoclonal mammalian target of rapamycin (mTOR) antibody (cat. no. ab2732; 1:1,000 dilution; Abcam), rabbit anti-human monoclonal p-mTOR antibody (cat. no. ab109268; 1:1,000 dilution; Abcam), and rabbit anti-human monoclonal GAPDH antibody (cat. no. ab181603; 1:1,000 dilution; Abcam).
All data were presented as the mean ± standard deviation. SPSS 15.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The relationship between miR-802 and the clinicopathological variables of NSCLC patients was determined using the χ2 test. Student’s t-test was utilized to examine the differences between two groups. Significant differences between multiple groups were investigated using one-way analysis of variance, followed by a Bonferroni’s post hoc test. The association between miR-802 and FGFR1 mRNA levels in NSCLC tissues was analyzed using Spearman’s correlation analysis. P<0.05 was considered to indicate a statistically significant difference.
To uncover the expression pattern of miR-802 in NSCLC, RT-qPCR analysis was utilized to detect miR-802 expression in 52 pairs of NSCLC tissues and paired NATs. miR-802 was downregulated in NSCLC tissues as compared with NATs (
To determine the functional role of miR-802 in NSCLC cells, H522 and H460 cells were selected for transfection based on their lowest levels of miR-802 expression among the five NSCLC cell lines tested. miR-802 mimics or miR-NC were transfected into H522 and H460 cells and then RT-qPCR was performed to determine the miR-802 expression levels. RT-qPCR revealed that miR-802 was significantly increased in miR-802 mimics-transfected H522 and H460 cells, compared with the cells transfected with miR-NC (
Since miR-802 expression was strongly correlated with brain metastasis, cell migration and invasion assays were performed to examine whether miR-802 could affect NSCLC metastasis-related functions. The migration (
To investigate the underlying mechanism for the tumor-suppressing function of miR-802 in NSCLC cells, bioinformatics analysis was performed to search for the putative targets of miR-802. The analysis indicated that the 3′-UTR of FGFR1 contained a highly conserved binding site for miR-802 (
siRNA against FGFR1 (si-FGFR1) was introduced into H522 and H460 cells to knock down endogenous FGFR1 expression and examine the functions of FGFR1 in NSCLC cells. Western blot analysis verified that FGFR1 expression was efficiently suppressed in H522 and H460 cells following si-FGFR1 transfection (
The aforementioned results demonstrated that miR-802 decreased the growth and invasion of NSCLC cells
FGFR1 activates the PI3K/Akt/mTOR signaling pathway and contributes to the aggressive behaviors of NSCLC cells (
Finally, an
Emerging reports have revealed that several miRNAs are abnormally expressed in NSCLC (
miR-802 is downregulated in prostate cancer, and its downregulation is correlated with Gleason score, distant metastasis, and the pathological stage of the cancer (
miR-802 has been demonstrated to be a tumor suppressor in several human cancer types. For instance, miR-802 overexpression inhibits prostate cancer cell proliferation, promotes cell apoptosis
Multiple human genes, including flotillin-2 in human prostate cancer (
In summary, the present study demonstrated that miR-802 was downregulated in NSCLC and might contribute to the development of NSCLC. Mechanistically, miR-802 exerted its tumor-suppressive role, at least partially, by directly targeting the FGFR1-mediated PI3K/Akt/mTOR pathway in NSCLC. These observations help to improve our understanding of the mechanisms underlying the genesis and development of NSCLC, and may potentially promote the development of novel targeted therapies against this disease. However, a limitation of the present study is that miR-802 inhibitors were not used to knockdown endogenous miR-802 expression, and to examine the effects of miR-802 knockdown on the oncogenicity of NSCLC cells. Further studies will be required to fully elucidate the functions of miR-802 in NSCLC and its potential as a diagnostic or therapeutic target.
The present study was supported by grants from the Guangdong Science and Technology Project (grant no. 21040212).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
JL designed the present research. JZ and JL conducted the RT-qPCR, MTT assay and cell apoptosis analysis. The colony formation assay, cell migration and invasion assays and
The present study was approved by the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University, and was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University. Written informed consent was obtained from all patients for the use of their clinical tissues.
Not applicable.
The authors declare that they have no competing interests.
Not applicable.
miR-802 is weakly expressed in NSCLC tissues and cell lines. (A) miR-802 expression was determined in 52 pairs of NSCLC tissues and paired NATs by reverse transcription-quantitative polymerase chain reaction. *P<0.05 vs. NATs. (B) Expression levels of miR-802 in five human NSCLC cell lines (A549, H522, H1299, H460, and SK-MES-1) and a non tumorigenic bronchial epithelium cell line, BEAS-2B. *P<0.05 vs. BEAS-2B. NSCLC, non-small cell lung cancer; NATs, normal adjacent tissues.
miR-802 has an inhibitory role in the growth and invasion of H522 and H460 cells. H522 and H460 cells were transfected with miR-802 mimics or miR-NC. (A) Reverse transcription-quantitative polymerase chain reaction was performed to determine miR-802 expression in H522 and H460 cells transfected with miR-802 mimics or miR-NC. (B) The effects of miR-802 upregulation on H522 and H460 cell proliferation and (C) colony formation were evaluated using MTT and colony formation assays, respectively. (D) The apoptosis rate of H522 and H460 cells transfected with miR-802 mimics or miR-NC was assessed via Annexin V and PI staining. (E) Cell migration and (F) invasion assays were conducted to assess cellular migration and invasion of H522 and H460 cells following miR-802 overexpression (magnification, ×200). *P<0.05 vs. miR-NC. NC, negative control; PI, propidium iodide.
FGFR1 is a direct target gene of miR-802 in NSCLC cells. (A) The potential binding site of miR-802 in the 3′-UTR of the FGFR1 gene is shown. Mutant site in the 3′-UTR of FGFR1 is underlined. (B) Luciferase reporter assay was conducted in H522 and H460 cells that were co-transfected with miR-802 mimics or miR-NC and pMIR-FGFR1-3′-UTR-wt or pMIR-FGFR1-3′-UTR-mut. miR-802 upregulation reduced the luciferase activity of the plasmid containing the wt 3′-UTR of FGFR1, but not the mut 3′-UTR, in H522 and H460 cells. *P<0.05 vs. miR-NC. (C) The mRNA and (D) protein expression of FGFR1 was upregulated in NSCLC tissues compared with NATs. *P<0.05 vs. NATs. (E) The correlation between miR-802 and FGFR1 mRNA levels in NSCLC tissues was evaluated through Spearman’s correlation analysis. (F) mRNA levels and (G) protein levels of FGFR1 in H522 and H460 cells were measured following miR-802 mimics or miR-NC transfection. *P<0.05 vs. miR-NC. FGFR1, fibroblast growth factor receptor 1; NSCLC, non-small cell lung cancer; UTR, untranslated region; NC, negative control; wt, wild-type; mut, mutant; NATs, normal adjacent tissues.
Downregulation of FGFR1 suppresses the growth and invasion of H522 and H460 cells
miR-802 inhibition of FGFR1 expression is responsible for the tumor-suppressive effects of miR-802 in H522 and H460 cells. pCMV-FGFR1 or empty pCMV vector control were transfected into miR-802-overexpressing H522 and H460 cells to restore FGFR1 expression. (A) Protein levels of FGFR1 were confirmed by western blot analysis. (B) Proliferation, (C) colony formation, (D) apoptosis, (E) migration and (F) invasion of the aforementioned cells were assayed. *P<0.05 vs. miR-NC; #P<0.05 vs. miR-802 mimics + pCMV. FGFR1, fibroblast growth factor receptor 1; NC, negative control; PI, propidium iodide.
miR-802 inhibits the activation of the PI3K/Akt/mTOR pathway in H522 and H460 cells. miR-802 mimics along with pCMV-FGFR1 or pCMV were cotransfected into H522 and H460 cells. After 72 h, western blot analysis was conducted to measure the protein levels of important molecules within the PI3K/ Akt/mTOR pathway. *P<0.05 vs. miR-NC; #P<0.05 vs. miR-802 mimics + pCMV. PI3K, phosphoinositide 3-kinase; Akt, AKT serine/threonine kinase; mTOR, mammalian target of rapamycin; FGFR1, fibroblast growth factor receptor 1; NC, negative control; p-, phosphorylated.
miR-802 hinders tumor growth
Correlation between miR-802 expression and clinicopathological parameters of patients with non-small cell lung cancer.
Parameters | Cases | miR-802 expression level
|
P-value | |
---|---|---|---|---|
Low | High | |||
Sex | 0.388 | |||
Male | 33 | 15 | 18 | |
Female | 19 | 11 | 8 | |
Age (years) | 0.402 | |||
<60 | 29 | 13 | 16 | |
≥60 | 23 | 13 | 10 | |
Tumor size (cm) | 0.397 | |||
<3 | 31 | 17 | 14 | |
≥3 | 21 | 9 | 12 | |
Histologic grade | 0.532 | |||
Well/moderate | 38 | 20 | 18 | |
Poor | 14 | 6 | 8 | |
Tumor stage | 0.026 | |||
I-II | 24 | 8 | 16 | |
III-IV | 28 | 18 | 10 | |
Lymph node metastasis | 0.005 | |||
Negative | 28 | 9 | 19 | |
Positive | 24 | 17 | 7 | |
Brain metastasis | 0.004 | |||
NBM | 32 | 11 | 21 | |
BM | 20 | 15 | 5 |
BM, brain metastasis; NBM, non-brain metastasis.