MicroRNAs (miRNAs) are frequently reported to be aberrantly expressed in non-small cell lung cancer (NSCLC) and are closely associated with aggressive tumor phenotypes. Hence, identification of cancer-related miRNAs in NSCLC may be helpful for improving the cure rate of NSCLC treatments. miR-889 has been demonstrated to be a novel cancer-associated miRNA that is aberrantly expressed and plays an important role in esophageal squamous cell carcinoma and hepatocellular carcinoma. However, the exact functions and precise molecular mechanisms through which miR-889 affects NSCLC progression are still unknown. In the present study, we report for the first time that miR-889 expression is low in NSCLC tissues and cell lines. Clinically, low miR-889 expression was found to be correlated with the TNM stage and distant metastasis in NSCLC patients. Functionally, miR-889 overexpression suppressed the proliferation and invasiveness of NSCLC cells
Lung cancer, a highly lethal malignant tumor, is a common cancer and represents a leading cause of cancer-related deaths worldwide (
MicroRNAs (miRNAs) are a family of noncoding, single-stranded, short RNA molecules that have been demonstrated to serve as an endogenous means of RNA interference (
Certain studies have identified miR-889 as a novel cancer-associated miRNA that is aberrantly expressed and plays an important role in esophageal squamous cell carcinoma (
The procedures of our present study were approved by the Ethics Committee of Weifang People's Hospital (Weifang, China) and written informed consent was also provided by all patients. Samples of primary NSCLC tissues and adjacent normal tissues were collected from 53 patients (30 males, 23 females; age range, 47–71 years) who had received surgical resection at Weifang People's Hospital between June 2016 and August 2017. None of these patients had been treated preoperatively with either radiotherapy or chemotherapy. All tissues were immediately snap-frozen in liquid nitrogen and then stored at −80°C for further RNA extraction.
A non-tumorigenic bronchial epithelium cell line, BEAS-2B, and four human NSCLC cell lines (A549, SK-MES-1, H522, and H460) were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin mixture (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2.
miR-889 mimics and corresponding miRNA mimics negative control (miR-NC), siRNA targeting the expression of TAB1 (si-TAB1) and negative control siRNA (si-NC) were constructed by GenePharma (Shanghai, China). The si-TAB1 sequence was 5′-GGAUGAGCUCUUCCGUCUUTT-3′ and the si-NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. TAB1 expression vector pcDNA3.1-TAB1 (pcTAB1) and empty vector (pcDNA3.1) was obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Before transfection, cells in logarithmic phase were plated into 6-well plates with a density of 4×105 cells/well. When the culture confluency reached 60–70%, the cells were transfected with the miR-889 mimics (100 pmol), miR-NC (100 pmol), si-TAB1 (100 pmol), si-NC (100 pmol), pcTAB1 (4 µg) or pcDNA3 (4 µg) using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). All of the transfection procedures were based on the product specifications. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Transwell Matrigel invasion assays were performed 48 h after transfection. The MTT assay and western blotting were performed at 24 and 72 h post-transfection, respectively.
Total RNA was extracted from tissue samples or cultured cell lines using a TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). All-in-One™ miRNA qRT-PCR Detection kit (GeneCopoeia, Inc., Rockville, MD, USA) was used to detect miR-889 expression. The thermocycling conditions were as follows: 95°C for 10 min, and 45 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 15 sec. To quantify TAB1 mRNA expression, total RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The temperature protocol for TAB1 mRNA reverse transcription was as follows: 37°C for 15 min and 85°C for 5 second. The quantitative PCR was carried out using an ABI 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and a SYBR Premix Ex Taq™ (Takara Biotechnology Co., Ltd). The thermocycling conditions for TAB1 mRNA qPCR was as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. U6 small nuclear RNA and GAPDH were used as the internal controls for miR-889 and TAB1 mRNA, respectively. All reactions were run in triplicate and relative gene expression was analyzed using the 2−∆∆Cq method (
An MTT assay was performed to determine cellular proliferation. In brief, transfected cells were inoculated in 96-well plates with a density of 3,000 cells/well. Then the cells were incubated at 37°C supplied with 5% CO2. for 0–3 days. The MTT assay was carried out every day by adding 20 µl of MTT reagent (5 mg/ml; Beyotime Institute of Biotechnology, Haimen, China) into each well. Following incubation at 37°C for an additional 4 h, the culture medium containing MTT solution was replaced with 150 µl of dimethyl sulfoxide (DMSO). A total of 15 min after incubation, the absorbance value of each well was detected at a 450 nm wavelength using an enzyme-linked immunosorbent assay reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Transwell insert chambers (8 µm pore size) covered with Matrigel (both from BD Biosciences, San Jose, CA, USA) were utilized to evaluate the invasive ability of NSCLC cells. Transfected cells were collected and suspended in FBS-free culture medium. A total of 5×104 cells were added into the upper chambers. In the lower chambers, 500 µl DMEM containing 20% FBS was added to serve as a chemoattractant. Subsequent to incubation at 37°C with 5% CO2. for 24 h, the non-invasive cells were gently wiped away with a cotton swab. The invasive cells that invaded through the pores were fixed with 100% methanol at room temperature for 30 min, stained with 0.5% crystal violet at room temperature for 30 min, and washed with phosphate buffer solution. Images of five different fields were captured from each insert, and the number of invasive cells was counted under an inverted light microscope (×200, magnification; Olympus Corporation, Tokyo, Japan).
A total of eight 6-week-old BALB/c male nude mice (20 g) were purchased from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China). All animals were maintained under specific pathogen-free conditions at 25°C, 50% relative humidity, a 10 h light/14 h dark cycle and had
Three online miRNA target prediction software, including miRDB (
The 3′-UTR region of TAB1 containing the predicted wild-type and mutant miR-889 binding sites was amplified by GenePharma and individually cloned into the pmiR-RB-REPORT™ vector (Promega Corporation, Madison, WI, USA). For the reporter assays, cells were plated into 24-well plates, and co-transfected with wild type or mutant reporter plasmid and miR-889 mimics or miR-NC using Lipofectamine™ 2000, according to the manufacturer's instructions. Forty-eight hours later, the luciferase activity of transfected cells was determined using a Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized against
Total protein was isolated from tissues or cells using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). The concentration of total protein was determined using a bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg) were separated by 10% sodium dodecyl sulfate polyacrylamide gels electrophoresis and transferred onto polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology), followed by blocking with 5% dried skimmed milk in Tris-buffered saline and 0.1% Tween-20 (TBS-T) at room temperature for 1 h. The membranes were subsequently incubated with the following primary antibodies at 4°C overnight: Rabbit anti-human TAB1 antibody (cat. no. ab76412; 1:1,000 dilution) and rabbit anti-human GAPDH antibody (cat. no. ab128915; 1:1,000 dilution; both from Abcam, Cambridge, UK). The membranes were then probed with horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; 1:5,000 dilution; Abcam,) for 2 h at room temperature. Finally, the protein bands were visualized using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was used as the internal control. Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.) was used for densitometry analysis.
All assays were repeated at least three times. All data are presented as the mean ± standard deviation, and analyzed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). All functional experiments were repeated three times. Differences between two groups were examined using an unpaired Student's t-test, or a paired Student's t-test to compare expression data from NSCLC and adjacent normal tissues. One-way ANOVA with Tukey's post-hoc test was used for the comparison between multiple groups. A χ2 test was performed to evaluate the association between miR-889 and the clinicopathological characteristics of NSCLC patients. A correlation between miR-889 and TAB1 mRNA expression was determined through Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.
In total, 53 pairs of NSCLC tissue samples and adjacent normal tissue samples were collected. Total RNA was isolated from these tissues, and RT-qPCR was performed to assess miR-889 expression. The data revealed that the expression of miR-889 was notably lower in NSCLC tissue samples than in adjacent normal tissues (P<0.05;
To clarify the possible clinical meaning of low miR-889 expression in NSCLC, all enrolled NSCLC patients were categorized into two subgroups, ‘low miR-889 expression’ and ‘high miR-889 expression’, based on the median value of miR-889 expression in NSCLC tissue samples. Statistical analysis revealed that underexpression of miR-889 was significantly correlated with TNM stage (P=0.019) and distant metastasis (P=0.020); however, no significant associations with other pathological characteristics were observed, including sex (P=0.477), age (P=0.685), tumor size (P=0.407), histological tumor type (P=0.449), or tumor differentiation status (P=0.317;
To assess whether miR-889 affects NSCLC progression, cell lines H522 and H460 (possessing relatively lower miR-889 expression among the four assessed NSCLC cell lines) were selected to conduct the following experiments. miR-889 in H522 and H460 cells was overexpressed by transfection with miR-889 mimics (P<0.05;
To illustrate the potential molecular mechanisms by which miR-889 exerts its influence on NSCLC progression, bioinformatics tools, including miRDB, miRanda, and TargetScan, were applied to find a potential target gene (i.e., target mRNA) of miR-889. This analysis indicated a possible binding site of miR-889 in the 3′-UTR of TAB1 (
To confirm that TAB1 contributes to NSCLC pathogenesis, RT-qPCR analysis was carried out to assess TAB1 mRNA expression in NSCLC tissue samples. The data indicated that the mRNA expression of TAB1 was notably higher in NSCLC tissue samples than in the adjacent normal tissues (P<0.05;
To precisely determine the involvement of TAB1 in NSCLC, endogenous TAB1 expression was knocked down in H522 and H460 cells by transfection with siRNA against TAB1 (si-TAB1). This transfection efficiently decreased TAB1 protein expression in H522 and H460 cells, as evidenced by western blot analysis (P<0.05;
To confirm that TAB1 is involved in the miR-889-induced tumor-suppressive effects in NSCLC cells, rescue experiments were conducted next by restoring TAB1 expression in miR-889 mimic-transfected H522 and H460 cells. Firstly, pcDNA3.1 or pc-TAB1 was introduced into H522 and H460 cells. RT-qPCR analysis confirmed that the expression level of TAB1 mRNA was increased in pc-TAB1-transfected H522 and H460 cells (P<0.05;
A xenograft experiment was next conducted to examine the influence of miR-889 overexpression on
miRNAs are frequently reported to be aberrantly expressed in NSCLC and closely related with aggressive phenotypes (
miR-889 has been revealed to be involved in tumor development and progression of esophageal squamous cell carcinoma (
Two genes, DOC-2/DAB2 interactive protein (
In summary, the significant downregulation of miR-889 was observed in NSCLC and it was demonstrated that miR-889 expression was associated with TNM stage and distant metastasis. It was further demonstrated that miR-889 suppressed NSCLC cell proliferation, invasion
Not applicable.
No funding was received.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
All authors significantly contributed to the findings and methods. XW designed this research and analyzed the data. ZD and BL performed all functional experiments. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
The present study was approved by the Ethics Committee of Weifang People's Hospital (Weifang, China), and was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of Weifang People's Hospital. Written informed consent was also provided by all patients.
Not applicable.
The authors declare that they have no competing interests.
miR-889 is downregulated in NSCLC tissues and cell lines. (A) The expression status of miR-889 was determined by RT-qPCR in 53 pairs of NSCLC tissues and adjacent normal tissues. *P<0.05 vs. adjacent normal tissues. (B) The expression level of miR-889 in four human NSCLC cell lines (A549, SK-MES-1, H522, and H460) and a non-tumorigenic bronchial epithelium BEAS-2B cell line was detected by RT-qPCR. *P<0.05 vs. BEAS-2B. NSCLC, non-small cell lung cancer.
miR-889 attenuates the proliferation and invasion of NSCLC cells. (A) Quantitation of the miR-889 expression was performed in H522 and H460 cells after transfection of miR-889 mimics or miR-NC. *P<0.05 vs. miR-NC. (B) The cell proliferation was assessed by MTT assay in H522 and H460 cells that were treated with miR-889 mimics or miR-NC. *P<0.05 vs. miR-NC. (C) Representative images and quantitation of the Transwell Matrigel invasion assay were carried out in miR-889 mimics- or miR-NC-transfected H522 and H460 cells (×200, magnification). *P<0.05 vs. miR-NC. NSCLC, non-small cell lung cancer.
TAB1 is a direct target of miR-889 in NSCLC cells. (A) The predicted miR-889 wild-type binding site in the 3′-UTR of TAB1 using bioinformatics tools and the mutated 3′-UTR is presented. (B) H522 and H460 cells were co-transfected with a luciferase reporter vector that contained the wild type or mutant binding site of TAB1-3′-UTR plus miR-889 mimics or miR-NC. Luciferase activity was detected 48 h after transfection. *P<0.05 vs. miR-NC. (C) The expression of TAB1 mRNA in NSCLC tissues was notably higher than that in adjacent normal tissues. *P<0.05 vs. adjacent normal tissues. (D) miR-889 expression was inversely correlated with the expression of TAB1 mRNA in NSCLC tissues. r=−0.5077, P=0.001. (E) Relative TAB1 mRNA expression was detected by RT-qPCR in H522 and H460 cells after miR-889 mimics or miR-NC transfection. *P<0.05 vs. miR-NC. (F) The protein level of TAB1 in H522 and H460 cells following transfection with miR-889 mimics or miR-NC was determined by western blot analysis. *P<0.05 vs. miR-NC. TAB1, TGF-β-activated kinase 1-binding protein 1; NSCLC, non-small cell lung cancer.
TAB1 inhibition suppresses cell proliferation and invasion in NSCLC. H522 and H460 cells were transfected with si-TAB1 or si-NC. (A) Following 72 h of incubation, western blot analysis was used to determine TAB1 proliferation. *P<0.05 vs. si-NC. (B) An MTT assay was performed to evaluate the proliferation of indicated cells. *P<0.05 vs. si-NC. (C) The effect of TAB1 silencing in H522 and H460 cells was assessed using Transwell Matrigel invasion assay (×200, magnification). *P<0.05 vs. si-NC. TAB1, TGF-β-activated kinase 1-binding protein 1; NSCLC, non-small cell lung cancer.
Recovered TAB1 expression reverses the antitumor effects of miR-889 in NSCLC cells. (A) RT-qPCR was utilized for the determination of TAB1 mRNA expression in H522 and H460 cells after pcDNA3.1 or pc-TAB1 transfection. *P<0.05 vs. pcDNA3.1. (B) TAB1 protein expression in H522 and H460 cells co-transfected with miR-889 mimics and pc-TAB1 or pcDNA3.1 was demonstrated by western blot analysis. *P<0.05 vs. miR-NC. **P<0.05 vs. miR-889 mimics+pcDNA3.1. (C and D) Cellular proliferation and invasion of aforementioned cells was determined by MTT and Transwell Matrigel invasion assays (×200, magnification), respectively. *P<0.05 vs. miR-NC. **P<0.05 vs. miR-889 mimics+pcDNA3.1. TAB1, TGF-β-activated kinase 1-binding protein 1; NSCLC, non-small cell lung cancer.
miR-889 upregulation decreases tumor growth
Correlation of miR-889 expression and clinicopathological characteristics of patients with non-small cell lung cancer.
miR-889 | |||
---|---|---|---|
Characteristics | Low expression | High expression | P-value |
Sex | 0.477 | ||
Male | 14 | 16 | |
Female | 13 | 10 | |
Age (years) | 0.685 | ||
<65 | 12 | 13 | |
≥65 | 15 | 13 | |
Tumor size (cm) | 0.407 | ||
<5 | 18 | 20 | |
≥5 | 9 | 6 | |
Histological tumor type | 0.449 | ||
Adenocarcinoma | 16 | 18 | |
Squamous cell carcinoma | 11 | 8 | |
Tumor differentiation | 0.317 | ||
I–II | 6 | 9 | |
III–IV | 21 | 17 | |
TNM stage | 0.019 |
||
I–II | 7 | 15 | |
III+IV | 20 | 11 | |
Distant metastasis | 0.020 |
||
Negative | 9 | 17 | |
Positive | 18 | 9 |
P<0.05. NSCLC, non-small cell lung cancer.