Clinical data
The data of 513 NSCLC tissues and 46 pair-matched normal tissues were downloaded from The Cancer Genome Atlas (TCGA) database (https://genome-cancer.ucsc.edu). The data included the overall survival (OS) time and status, recurrence time and status, and miRNA and mRNA expression levels. The target genes of miR-182-5p were identified using the prediction tools PITA [18], miRmap [19], DIANA-microT [20], miRanda [21], PicTar [22], and TargetScan [23]. Analysis of the differentially expressed genes in NSCLC was performed using Gene Expression Profiling Interactive Analysis 2 (GEPIA2) [24]. The correlation analysis between miR-182-5p and the target genes was performed using ENCORI [25]. Disease-free survival (DFS) was defined as the time between surgery and the first event of either disease recurrence or death from any cause. OS was defined as the time between the date of diagnosis and death from any cause.
Cell culture
NSCLC cell lines (NCI-H1975, NCI-H460, A549, 95-D), 16HBE, and BEAS-2B were purchased from the Chinese Academy of Sciences Cell Bank. The cells were cultured in RPMI 1640 medium (Corning, USA) supplemented with 10% Fetal Bovine Serum(FBS) (Gibco, USA) and 100 units per ml of penicillin‐streptomycin solution. The 293T cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS (Gibco, USA). Culturing was done in a humidified atmosphere containing 5% CO2 at 37°C. The mycoplasma test of all cells was negative.
miRNA and mRNA quantitative real-time PCR
Total RNA of NSCLC cells was extracted using the TRIzol reagent [26]. Briefly, 2 μg of extracted RNA was reverse transcribed to cDNA with the application of reverse transcriptase (Thermo Fisher, USA). The qRT-PCR reactions were performed with Power SYBR Green PCR Master Mix (Kapa biosystems, USA), and GAPDH was used as an internal control. Then miRNAs were isolated from the total RNA using a High Pure miRNA isolation kit (TIANGEN, KR211-01, China) followed by transcribing them through RT-PCR using a TaqMan MicroRNA Reverse Transcription kit (TIANGEN Technologies, FP411-01, China). Data were analyzed using the comparative Ct method (2−ΔΔCt). Three experiments were performed for each clone. The primer sequences used are described in the supplementary information (Table 1 & 2).
Table 1. List of qPCR primers used for mRNA expression
Gene name
|
|
Sequence
|
E-cadherin
|
forward
|
CCTTGTGATCCGCCTGCCTTG
|
|
reverse
|
CTGCCTGCCTGCCTTCTGATTAC
|
N-cadherin
|
forward
|
CAGAATCGTGTCTCAGGCTCCAAG
|
|
reverse
|
CTGCGTTCCAGGCTGGTGTATG
|
Twist
|
forward
|
CGACGACAGCCTGAGCAACAG
|
|
reverse
|
TCCTCGTAAGACTGCGGACTCC
|
Snail
|
forward
|
TTACCTTCCAGCAGCCCTAC
|
|
reverse
|
CTTTCGAGCCTGGAGATCCT
|
MMP-2
|
forward
|
TTCCAAGTCTGGAGCGATGT
|
|
reverse
|
CAGAAGCCGTACTTGCCATC
|
MMP-9
|
forward
|
GCCACTACTGTGCCTTTGAG
|
|
reverse
|
TCAAAGACCGAGTCCAGCTT
|
Vimentin
|
forward
|
CGCCAACTACATCGACAAGG
|
|
reverse
|
TGAAGCATCTCCTCCTGCAA
|
GAPDH
|
forward
|
CAGACCACAGTCCATGCCATCAC
|
|
reverse
|
GACGCCTGCTTCACCACCTTC
|
Table 2. Sequences of miR-182-5p mimic, inhibitor, and NC.
Name
|
Sequences(5’ to 3’)
|
miR-182-5p NC
|
UUCUCCGAACGUGUCACGUTT
|
miR-182-5p mimic
|
UUUGGCAAUGGUAGAACUCACACU
|
miR-182-5p NC
|
CAG UAC UUU UGU GUA GUA CAA
|
miR-182-5p inhibitor
|
AGUGUGAGUUCUACCAUUGCCAAA
|
Western blot analysis
Cells were lysed by RIPA buffer (Sangon, China) containing Proteinase inhibitor (Roche, Switzerland) and Pierce phosphatase inhibitor (Thermo Fisher, USA) [26]. The protein extracts were boiled in a loading buffer followed by separation on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis platform (SDS-PAGE). The separated protein bands were transferred onto polyvinylidene fluoride membranes. The primary antibodies against EPAS1 (7096s, CST, USA), GAPDH (2118S, CST, USA), N-cadherin (13116S, CST, USA), Vimentin (5741S, CST, USA), E-cadherin (3195S, CST, USA), MMP-9 (13667S, CST, USA), MMP-2 (40994S, CST, USA), Snail (3879S, CST, USA), and Twist (69366S, CST, USA) were diluted in the ratio of 1:1000 in accordance with the manufacturer’s instructions. The membranes were then probed with goat anti-rabbit IgG highly cross‐adsorbed secondary antibody (HSA0003, maibio, China) for 2 hours at room temperature. The membranes were then exposed to the enhanced chemiluminescence (ECL) reagent for visualization.
Luciferase reporter assay
The 293T cells were cultured in 24-well plates. The cells were then divided into four groups for transfection studies using the Lip3000 transfection kit (Thermo Fisher Scientific, USA). The plasmids’ sequences are described in the supplementary information (Table 3). In each group, co-transfection was done using a control plasmid of 40 ng, a recombinant plasmid of 800 ng, and a final mimics concentration of 20 nmol/L. Each group was set with 5 multiple pores. The cells were lysed, and positive transfection was detected using a double luciferase assay kit (Biology of Hanheng, China) after 48 h of transfection. The blank hole's basal signal was subtracted from the data of each hole after measuring using a chemiluminescence instrument. The firefly fluorescence signal generated by the plasmid was used as the control. The relative fluorescence activity was divided by the sea kidney's fluorescence signal and then normalized for each group.
Table 3. Sequence of plasmids
Name
|
Sequence (5 'to 3')
|
miR-182-5p NC
|
UUCUCCGAACGUGUCACGUTT
ACGUGACACGUUCGGAGAATT
|
miR-182-5p mimics
|
UUUGGCAAUGGUAGAACUCACACU
UGUGAGUUCUACCAUUGCCAAAUU
|
EPAS1-WT-hsa-miR-182-5p
|
GGAUAGACUUAUUGCCAA
UUGGCAAUG-GUAGAACU
|
EPAS1-MUT-hsa-miR-182-5p
|
GCAAAGTGUUTAACGGTT
UUGGCAAUG-GUAGAACU
|
Data analysis
The univariate Cox regression analysis was performed to identify the primary prognostic factors. he multivariate Cox regression analysis and construction of the K–M survival curve were performed to establish the risk score model and identify the independent prognostic factors. Rceiver operating characteristic curve (ROC curve) analysis was performed to estimate the prognostic power of the risk score model. The GraphPad Prism 7.0 (GraphPad Software, USA) was used to analyze data regarding the function of miR-82-5p and presented as means ± standard deviation (SD). The differences between groups were determined using the GraphPad Prism software. P less than 0.05 (P< 0.05) indicated that there significant differences between groups.