Dual-luciferase reporter assay
The analysis of miR-186 target gene was performed by using a biological prediction website (http://www.microrna.org/microrna/home.do), and the dual luciferase reporter assay was used to verify whether Shp2 is a direct target gene of miR-186. The 3'-UTR gene fragment of the Shp2 gene was cloned and amplified, and the PCR product was cloned into the multiple cloning site in downstream of the Luciferase gene of pmirGLO (Cat. E1330, Promega, USA), which were named Wt-Shp2. The predicted specific binding site of miR-186 and Shp2 was mutated to construct the Mut-Shp2 vector. The Renilla luciferase pRL-TK vector (Cat. E2241, Promega, USA) was used as an internal reference. The luciferase reporter vector was co-transfected with miR-186 mimic and NC mimic into 293T cells, respectively. The luciferase activity of each group was detected and compared.
Cell Culture
In this experimental study, Human lung adenocarcinoma cell line SPC-A-1 (purchased from ATCC, China) was cultured in a humidified 5% CO2 incubator (Thromo 3111, Jinan Besun Medical Devices Co., Ltd., Shandong, China) at 37 °C with RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 50 U/ml penicillin (Gibco, USA), 100 µg/ml streptomycin (Gibco, USA) for 2 days, and the cells were passaged every 3 to 4 days.
Preparation Of Liposome Complexes
After 2 µl of 50 nmol/l miR-186 mimic, miR-186 inhibitor, si-Shp2 or NC (Shanghai Jima, China) was rapidly centrifuged and mixed with 170 µl phosphate buffer (Thermo Fisher, USA), respectively, the samples were stood for 5 min and 30 µl of Lipofectamine 2000 (Thermo Fisher, USA) were added. Finally, the samples were mixed thoroughly by gentle shaking, and then incubated for 20 min at room temperature.
Cell Grouping And Transfection
SPC-A-1 cell line grown in log phase was inoculated into a 6-well culture plate at a density of 1*105 cells/well, and the cells were pretreated with DMEM medium without serum and antibiotics for one day before transfection. The cells were divided into 6 groups: Blank group (no treatment), NC group (negative control, transfected with 50 nM si-NC + 50 nM mi-NC), miR-186 mimic group (transfected with 50 nM miR-186 mimic), miR-186 inhibitor group (transfected with 50 nM miR-186 inhibitor), si-Shp2 group (transfected with 50 nM si-Shp2) and miR-186 inhibitor + si-Shp2 group (transfected with 50 nM miR-186 inhibitor and 50 nM si-Shp2). Transfection was carried out according to the instructions of Lipofectamine 2000. After 6 h of transfection, the culture medium was replaced with RPMI1640 medium containing 10% fetal bovine serum. After transfection for 48 h, cells were harvested for subsequent experiments.
Qrt-pcr
Total RNA was extracted by Trizol (Thermo Fisher Scientific, New York, USA) from each group of cells after transfection for 48 h. PrimeScript™ RT reagent Kit with gDNA Eraser kit (TaKaRa, Japan) was used for the reverse transcription synthesis of cDNA. SYBR® Premix Ex Taq™ II Kit (Xingzhi Biotechnology Co., Ltd., China) and ABI PRISM® 7300 (model Prism® 7300, Shanghai Kunke Instrument Equipment Co., Ltd., China) were used for fluorescent quantitative real-time PCR detection. The reaction system was listed as follows: 25 µl of SYBR® Premix Ex Taq™ II (2×), 2 µl of PCR upstream and downstream primers, l µl of ROX Reference Dye (50×), 4 µl of DNA template, 16 µl of ddH2O. The reaction conditions were as follows: pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 15 sec, annealing at 60 °C for 30 sec, 32 cycles, extended at 72 °C for 1 min. ∆Ct = CT (target gene) - CT (GAPDH), ∆∆Ct = ∆Ct (experimental group) - ∆Ct (control group). miR-186 used U6 as internal reference and other genes adopted GAPDH as internal reference, and 2−ΔΔCt represents the relative expression level of target gene. The sequence of primers is shown in Table 1.
Table 1
Gene | Sequence |
Shp2 | Upstream : 5'-CCTGGAGATTTTGTTCTTTCTG-3' |
Downstream : 5'-AGGGGCTGCTTGAGTTGTAGTA-3' |
miR-186 | Upstream : 5'-GGGCAAAGAATTCTCCTTT-3' |
Downstream : 5'-GTGCAGGGTCCGAGGT-3' |
PI3K | Upstream : 5'-CGAGAGTGTCGTCACAGTGTC-3' |
Downstream : 5'-TGTTCGCTTCCACAAACACAG-3' |
Akt | Upstream : 5'-CCCTGCTCCTAGTCCACCA-3' |
Downstream : 5'-TGTCTCTGTTTCAGTGGGCTC-3' |
mTOR | Upstream : 5'-TTGCACTATCCCTTCCCATTC-3' |
Downstream : 5'-TCGAGTGAGGACATCATGGT-3' |
E-cadherin | Upstream : 5'-CTCTGTGTATCAGGCTCG-3' |
Downstream : 5'-TAGACTGAGGGCCTCTCT-3' |
N-cadherin | Upstream : 5'-CCAGTAGCTACGTATGGAC-3' |
Downstream : 5'-CGTCATCACATAGTGGCAT-3' |
Bcl-2 | Upstream : 5'-TATAAGCTGTCGCAGAGGGG-3' |
Downstream : 5'-TGACGCTCTCCACACACATG-3' |
Bax | Upstream : 5'-TGCCAGCAAACTGGTGCTCA-3' |
Downstream : 5'-GCACTCCCGCCACAAAGATG-3' |
U6 | Upstream : 5'-GTGCTCGCTTCGGCAGCACATATAC-3′ |
| Downstream : 5′-AAAAATATGGAACGCTTCACGAATTTG-3′ |
GAPDH | Upstream : 5'-GGGCTGCTTTTAACTCTGGT-3' |
| Downstream : 5'-GCAGGTTTTTCTAGACGG-3' |
Shp2, protein tyrosine phosphatase |
Western Blot
After transfection for 48 h, the cells were digested and collected to extract total protein using RIPA lysate containing PMSF (R0010, Solarbio). The BCA kit (Thermo, Inc., USA) was used to measure the total protein concentration and deionized water was adopted to adjust the protein concentration. The sample was mixed with the loading buffer, and boiled in water for 10 min. Protein samples (30 µl for each well) were loaded in each well and electrophoresis was carried out for 2 h at a constant voltage of 80 V. The protein was then transferred to a PVDF membrane (ISEQ00010, Millipore, Billerica, MA, USA) at a voltage of 110 V for 2 h. The proteins were blocked by 5% skim milk at 4 °C for 2 h. Then, the milk was discarded and the membrane was washed once with TBST. Subsequently, rabbit anti human primary antibodies, including Shp2 (ab32083, 1:2,000, Abcam, UK), PI3K (ab86714, 1:10,000, Abcam, UK), p-PI3K (ab86714, 1:10,000, Abcam, UK), Akt (ab8805, 1:10,000, Abcam, UK), p-Akt phospho T308 (ab8805, 1:1,000, Abcam, UK), mTOR (ab2732, 1:2,000, Abcam, UK), p-mTOR phospho S2448 (ab131538, 1:1,000, Abcam, UK), E-cadherin (ab76055, 1:500, Abcam, UK), N-cadherin (ab18203, 1:1,000, Abcam, UK), Bax (ab32503, 1:1,000, Abcam, Cambridge, MA, UK), Bcl-2 (ab32124, 1:1,000, Abcam, UK), GAPDH (ab8226, 1:2,000, Abcam, UK) were added onto the membrane and the membrane was incubated at 4 °C overnight. After that, the membrane was washed by TBST for 3 times (10 min each time). Then, HRP-labeled goat anti-rabbit IgG antibody (Beijing Zhongshan Biotechnology Co., Ltd., diluted 1:5,000) was added and the membrane was incubated at 37 °C for 1 h. After rinsing with TBST, the membrane was place on a clean glass plate, developed by ECL Fluorescence Detection Kit (Cat. No. BB-3501, Ameshame, UK), photographed by Bio-Rad image analysis system (BIO-RAD, USA) and analyzed by Image J software. The relative protein content and protein phosphorylation level were calculated by the gray value of the corresponding protein band / the gray value of the GAPDH protein band and the amount of protein phosphorylated / total protein, respectively.
Mtt Assay
After transfection for 48 h, the cells were collected and inoculated into 96-well plates at density of 3–6 *103 cells/well. Each group had 6 duplicate wells. After transfection for 24 h, 48 h and 72 h, 20 µl of 5 mg/ml MTT solution (Gibco, USA) was added to each well, and the plates were incubated for 4 h in the dark. Then, 100 µl of DMSO was added to each well, and the optical density (OD) of each well was detected at 570 nm by Microplate Spectrophotometer (NYW-96M, Beijing Noah Instrument Co., Ltd., China). The cell viability curve was plotted with the time point as the abscissa and the OD value as the ordinate.
Flow Cytometry
Detection of cell cycle: Cells were collected after transfection for 48 h and washed for three times with PBS, then the cells were centrifuges at 1,000 xg for 20 min. After discarding the supernatant, cell concentration was adjusted to 1*105/ml by PBS and 1 ml of 75% ethanol which were pre-cold at -20 °C were added to fix cells at 4 °C for 1 h. Then, the samples were centrifuged at 200 xg for 5 min and washed twice with PBS. Subsequently, 100 µl of Rnase A (Siemo, USA) was added and the samples were incubated in a water bath at 37 °C for 30 min in the dark surrounding. After that, 400 µl of PI (Sigma, USA) were mixed into the samples to dye the cells for 30 min at 4 °C in the dark surrounding. Flow Cytometer (Beckman Coulter, USA) was used to record the red fluorescence detected at 488 nm to analyzed the cell cycle of cells in each group.
Detection of apoptosis: After transfection for 48 h, the cells were digested with trypsin (Semerfly, USA) without EDTA and collected in a flow tube, then the sample was centrifuged at 1,000 xg for 30 min, and then the supernatant was discarded. The cells were washed for 3 times with cold PBS, centrifuged at 1,000 xg for 15 min, and then the supernatant was discarded. HEPES buffer, Annexin-V-FITC and PI and (50:1:2) were mix as Annexin-V-FITC/PI dye solution according to the instruction of Annexin-V-FITC Apoptosis Detection Kit (Sigma, USA). The sample was added with 100 µl of the dye solution and the solution was mixed and stood at room temperature for 15 min. Then, 1 ml of HEPES buffer (Sermerfly, USA) were added and the solution was mixed by shaking. Apoptosis was detected by flow cytometer with an excitation wavelength at 488 nm.
Wound Healing Assay
After transfection for 48 h, the cells were collected and seeded at a density of 5*105 cells/well in a 6-well plate. When the cell growth fusion reached 90%, use a sterile tip to gently traverse the central axis of the well. The width of each scratch is consistent. The cells were continuously cultured by adding serum-free medium, and the cells were photographed at 0 h and 24 h after the scratches, and the cell migration distance was measured by Image-Pro Plus Analysis software (Media Cybernetics, USA), and multiple fields of view were randomly selected and photographed. Set 3 duplicate wells in each group.
Transwell Assay
Transwell chamber (Shanghai Kelton Bio, China) was placed in a 96-well plate, and the upper suface of the bottom membrane of the Transwell chamber was coated with Matrigel gel (Shanghai Qianchen Biotechnology, China) at 1:8 dilution, and air-dried at room temperature. Each group of cells was routinely digested and rinsed with PBS twice and resuspended by RPMI 1640 medium to adjusted the cell density to 1*105 cells/ml. Then, 200 µl of cell suspension was added to the upper surface of the bottom membrane of the Transwell chamber and 600 µl of RPMI 1640 medium containing 20% fetal bovine serum (Gibco, USA) was added to the lower chamber. After routine culture for 24 h, the Transwell chamber was taken out and the cells on the upper surface were wiped with a cotton swab and then the chamber were fixed with 4% paraformaldehyde (Beijing Reagan Bio, China) for 15 min and stained with 0.5% crystal violet solution (Beijing Solebao Bio, China) for 15 min. After washed for 3 times with PBS, the chamber was photographed with an inverted microscope and 5 fields (200×) were randomly selected to count transmembrane cells.
Statistical analysis
All data were processed by using SPSS 21.0 statistical software. The measurement data were expressed as mean ± standard deviation. One-way ANOVA were adopted for the comparison among groups and Tukey post-hoc test was used for pairwise comparison between groups. P < 0.05 indicated a significant difference.