Tissue samples
Lung adenocarcinoma tissues and normal lung tissues in our research were collected from 20 patients in the Edong Healthcare Group, Huangshi Central Hospital (Affiliated Hospital of Hubei Polytechnic University). The hospital Ethics Committee approved our study. The baseline characteristics of these patients were shown in Table 1.
Table 1
The relationship between TEK expression and clinicopathologic features from patients with lung cancer (n = 20).
| Number | TEK low | P value |
Age (years) | | | 0.892 |
≥ 45 | 8 | 9 | |
༜45 | 12 | 11 | |
Tumor size (cm) | | | 0.042 |
≥ 3 | 7 | 6 | |
༜3 | 13 | 14 | |
TNM stage | | | 0.691 |
I-II | 11 | 7 | |
III-IV | 9 | 13 | |
Lymph node metastasis | | | |
No | 13 | 5 | 0.039 |
Yes | 6 | 15 | |
Cell Culture And Transfection
The normal lung cell line BEAS-2B and lung adenocarcinoma cell lines A549, Calu-3, and H1975 were obtained from Chinese Academy of Sciences cell bank (Shanghai, China). DMEM (Gibco BRL, Grand Island, NY, USA) supplemented with 10% FBS (Gibco, Carlsbad, CA) was applied for cell culture, and the cell culture atmosphere was 37 °C and 5% CO2. The TEK overexpression (OE) plasmids,miR-19a-3p mimic and miR-19a-3p inhibitor were obtained from GeneChem (Shanghai, China). The Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) was employed for transfection.
Quantitative Real-time Pcr
Total RNA extracted by TRIzol (Invitrogen, CA, USA) was applied for reverse transcription and cDNA amplification using SYBR Green PCR kit on an ABI 7300 system (Applied Biosystems, USA). The 2−△△Ct method was employed to calculate the relative expression. The primer sequences for TEK, miR-19a-3p and the corresponding internal reference genes were provided in Table 2.
Table 2
The sequences of the primers in this study
Primer/Antibody | Sequences/Antibody information |
miR-19a-3p | |
Forward sequence | 5'-GTCCTCTGTTAGTTTTGCATAGTTG-3' |
Reverse sequence | 5'-GGCCACCATCAGTTTTGCATAG-3' |
TEK | |
Forward sequence | 5'-CCTTGGCTCTGCTGGAATGA-3' |
Reverse sequence | 5'-CACGTTTTGGAAGGCTTGGG-3' |
GAPDH | |
Forward sequence | 5'-GTCAAGGCTGAGAACGGGAA-3' |
Reverse sequence | 5'-AAATGAGCCCCAGCCTTCTC-3' |
U6 | |
Forward sequence | 5'-TGCGGGTGCTCGCTTCGGCAGC-3' |
Reverse sequence | 5'-CCAGTGCAGGGTCCGAGGT-3' |
TEK | Cat#: ab24859, Abcam, USA |
GAPDH | Cat#: ab9485, Abcam, USA |
Western Blot Assay
A549 and H1975 cells were harvested to extract proteins by lysis buffer (Thermo Scientific, Rockford, IL, USA). After measuring the concentration, proteins were firstly separated by electrophoresis, then transferred to PVDF membrane, and incubated with primary and secondary anti-bodies. Eventually, the protein band was detected and analyzed by BCA kit (Pierce, USA) and Image J Software (Bio-Rad Laboratories, San Diego, CA, USA). The anti-bodies were shown in Table 2.
3’utr Reporter Assay
The reporter plasmids contained PsiCHECK2-TEK-Mut1 (3'UTR 827–833), PsiCHECK2-TEK-Mut2 (3'UTR 892–898) and psiCHECK2-TEK-Wt, which were constructed with the psiCHECK2 plasmids (Promega, Madison, WI, USA). A549 and H1975 cells that were transfected with miR-19a-3p mimic or miR-NC were plated in 96-well plates for 48 h of incubation. Then luciferase activities were quantified using a luminometer.
Cell Proliferation And Cell Apoptosis Assay
CCK-8 and EDU fluorescence proliferation assay were used to determine cell proliferation. The CCK-8 kit (Sigma Aldrich Company Ltd, Poole, Dorset, UK) was firstly applied to determine the cell proliferation of A549 and H1975 cells according to the protocol. After incubation for 0, 24, 48, and 72 hours, each well was added with CCK-8 reagents to keep incubating for one hour. The optical absorbance of live A549 and H1975 cells at 450 nm was demonstrated by an automatic microplate reader (Bio-Rad, Hercules, CA, USA). For EDU fluorescence proliferation assay, 2 × 104 cells/well were firstly plated in 48-well plates. 100 µL of EDU solution (10 µM, Abcam, USA, #ab219801) was incubated with cells for 4 h at 37 °C. After washing with PBS three times, the cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized by the addition of 200 µl permeabilization buffer for 20 min. Thereafter, 100 µL of iFluor 488 staining solution was added to each well and incubated for 30 min in the dark at room temperature. After the solution was removed, 100 µL of DAPI (5 µg/mL) nuclear staining solution was added to each well and incubated for 10 min. Finally, the cells were visualized under a fluorescence microscope. The ratio of EDU-positive cells (green) to all DAPI-positive cells (blue) represents the proliferation ratio. The cell apoptosis rate of A549 and H1975 cells was measured by flow cytometry (BD Biosciences, San Jose, CA, USA). The cells in the well were stained with Annexin V/propidium iodide (PI) reagents (KeyGen Biotech). The sum of upper-right quadrant (Annexin V+/PI+) and lower-right quadrant (Annexin V+/PI−) represented the cell apoptosis rate.
Cell Migration And Adhesion Assays
For wound healing assay to detect cell migration, scratches on cell culture surfaces were made using plastic pipette tips. The cells were cultured without FBS for 48 hours, and the migrated lengths symbolized the cell migration ability. Cell adhesion mechanism is complex. It is involved in a variety of aggressive processes including cell migration and invasion and potential cell-cell communication. To determine the cell adhesion to extracellular matrix, a colorimetric method was employed. Briefly, 96-well plates were coated with collagen type I (50 lg/ml; Sigma Chemical Co., Steinheim, Germany) at 4 °C overnight. A549 and H1975 cells were allowed to adhere at 37 °C. Cell culture was washed with DMEM for three times. At 30 min and 60 min, unbound A549 and H1975 cells were washed away, and adherent cells were fixed with 4% paraformaldehyde, and stained with crystal violet (0.5%) for 10 minutes. After stain extraction, the relative cell attachment was determined using absorbance readings at 620 nm.
Statistical Analysis
The GraphPad PRISM Version 8.0.1 statistical program (San Diego, CA, USA) was employed for our data (expressed as mean ± standard deviation) analysis, and diagram construction. Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc tests for multiple-group data, and Student’s t test for two independent-group data. Probability smaller than 0.05 was regarded statistically significant. Each experiment was performed at least three times independently.