Cell lines and clinical samples
The human lung cancer cell lines A549, H520 were obtained from ATCC (Manassas, VA), the H1299, SPC-A-1 cells and immortalized lung epithelial cell line HBE were purchased from Shanghai Cell Bank of Chinese Academy of Science (Shanghai, China). All cell lines were cultured in RPMI-1640 medium (Corning, NY, USA) containing 10% fetal bovine serum (FBS, GIBCO, South America, NY, USA) under a 5% CO
2 atmosphere at 37 °C. 15 matched non-small cell lung cancer (NSCLC) tissues and their adjacent noncancerous tissues (NCTs) were collected from Nanfang Hospital and stored in liquid nitrogen. All of the human samples were obtained with informed consents and histologically confirmed by hematoxylin and eosin (H&E) staining. This study project was approved by the Clinical Research Ethics Committee of Nanfang Hospital. Tissue chips consisted of 87 cases of human NSCLC were acquired from Outdo Biotech CO. Ltd. (Shanghai, China). The clinical pathological characteristics of 87 patients and 456 patients with NSCLC from The Cancer Genome Atlas (TCGA) were summarized in Supplementary Table 1 and Table 2.
In Situ Hybridization (ISH)
ISH experiments was performed according to the protocol described previously[6]. Briefly, probes for miR-744, positive control (U6) and negative control were purchased from Exiqon, Vedbek, Denmark. The intensity of cytoplasmic staining was scored manually semiquantitatively. The dominant staining intensity in tumor cells was scored as (S1 Fig): 0 = negative; 1 = weak; 2 = moderate; 3 = strong. A score of 3 was considered as high miR-744 expression and < 3 as low miR-744 expression.
Construction and stable transfection of lentivirus vector
The precursor sequence of miR-744 (MI0005559) was synthesized, annealed and then inserted into the AgeI/EcoR1 site of GV209 vector (GeneChem, Shanghai, China) to construct a vector continuously expressing miR-744, named as Lv-miR-744. The GFP vector was used for control. PLV-Luci-control was also constructed by GeneChem (Shanghai, China). A549 and H520 cells were co-transfected with Lv-miR-744 or Lv-control vector and pLV-Luci-control. The clones with GFP expression were isolated under puromycin (2 mg/mL) selection for 2 weeks.
Plasmid construction and transient transfection
AntagomiR744, antagomir negative control (antagomir-NC), agomiR744, agomir negative control (agomir-NC) were designed and synthesized by RiboBio (Guangzhou, China). Sic-FOS and sicontrol were designed and synthesized by Genepharma (Shanghai, China). The cells were seeded in 6-well plates at 30–50% density. Transient transfection was performed with Lipofectamine 3000 reagents according to the manufacturer’s instructions (Invitrogen, USA). For all the experiments, cells were collected at 48 h after transfection for further research.
RNA extraction and quantitative real-time PCR (qRT-PCR) analyses
Total RNA was extracted from cell lines using TRIzol® Reagent (Invitrogen, CA, USA) according to the manufacturer’s instruction. The reverse transcription of RNA for miR-744 and c-FOS mRNA was performed using SYBR® PrimeScript™ miRNA RT-PCR Kit (TaKaRa, Dalian, China) and PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China) separately. The real-time quantitative RT–PCR assay was carried on a Mx3005P real-time PCR instrument (Stratagene, USA) with SYBR® Premix Ex Taq™ kit (TaKaRa, Dalian, China). Small nuclear RNA U6 (U6 snRNA) or Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for miRNA or mRNA quantification respectively. The sequences of the primers used for qRT-PCR were listed in Supplementary Table 3.
Western Blot
Equal amounts of harvested proteins were first separated by 10% SDS-PAGE gels and then transferred to nitrocellulose membranes (Immobilon-P, Merck Millipore, Germany). The membranes were blocked with 5% nonfat milk and incubated with primary antibodies against GRB2, Raf1, Erk, p-Erk, c-FOS (1:2000, Abcam, Cambridge, USA) and GAPDH (1:2000, Santa Cruz Biotechnology, CA, USA) respectively at 4 °C overnight, and subsequently incubated for 2 h with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:5000; Santa Cruz Biotechnology, CA, USA) at room temperature. The protein complex was detected using enhanced chemiluminescence reagents (Immobilon Western, Merck Millipore, Germany). Endogenousβ-Actin protein was used as internal control.
Microarray analysis
Followed by RNA extraction with TRIzol reagent, cells transfected with antagomiR-744 or antagomir-NC were analyzed by Beijing CapitalBio Corporation with Affymetrix PrimeView™ Human Gene Expression Array.
EdU assay
After transfection for 48 h, cells were used to measure DNA synthesis with a CellLight™ EdU imaging detecting kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions.
CCK8 cell assay
After transfection, the cells were seeded in 96-well plates (5000 per well). After the cells were attached to the culture dish, paclitaxel was added to each well with the following concentration gradients (0–16 nmol/L or 0–32 nmol/L). 10 µl CCK8 was added into each well after 78 h and was incubated for 2 h. The absorbance values (OD) were measured at a wavelength of 450 nm using a microplate reader.
Colony formation assay
The cells were seeded at a density of 200-10000 cells/well in a 6-well plate. The cells were irradiated with different doses (0, 2, 4, 6 and 8 Gy) of X-rays. After 10 days incubation in RPMI-1640 medium with 20% FBS at 37 °C, 5% CO2, the cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet in 20% methanol. The colonies were counted. The survival curves were fitted by a linear-quadratic model [S = exp (-αD-βD2)] using GraphPad Prism 5.0 (GraphPad Prism, San Diego, CA, USA).
Cell migration and invasion assays
Cell invasion assay was performed using Transwell chamber (Corning, NY, USA) in a 24-well plate. Transfected cells (5–10 × 104 cells per 100 µl) in serum-free medium were seeded into the upper chamber with 12.5% matrigel-coated membrane (BD Bioscience, San Jose, CA), while the lower chamber was filled with 500 µl fresh complete medium. After being incubated for 18–24 h at 37℃ with 5% CO2, non-invading cells were removed from the upper surface of the filter by wet cotton swab. Invading cells that adhered to the lower surface of the chambers were fixed in methyl alcohol and stained with hematoxylin. The invading cells were manually counted at 200 × magnification under a inverted microscope. Five visual fields of each chamber were randomly chosen and photographed. Similar upper inserts without the matrigel precoating were used for the migration assay.
Wound healing assay
As described in previous study[4],when cells were grown to approximately 90% confluency (after 48 h of transfection), an artificial wound was created with a 10 µl pipette tip. The cells were then cultured in fresh medium without FBS. To visualize wound healing, images were taken at 0 h and 36 h. The relative percentage of wound healed was calculated as (the width of wound at 0 h - the width of wound at 36 h)/the width of wound at 0 h.
Promoter reporter construction and luciferase assays
The pGL3 vectors containing human c-FOS promoter with wild type or mutant putative miR-744 binding site were synthesized and confirmed with DNA sequencing by HuaAnPingKang Co., Ltd (Shenzhen, China). The sequences of these wild type or mutant promoter constructs were listed in Supplementary Table 4–7. The cells were plated onto 24-well plates at a density of 5 × 103 cells/well for transfection study. A mixture of 150 ng c-FOS promoter constructs (Luc-C-FOS-A, Luc-C-FOS-B, Luc-C-FOS-C, Luc-C-FOSmut−1, Luc-C-FOSmut−2, Luc-C-FOSmut−3, Luc-C-FOSmut−4) or responding control vectors, 100 nmol agomiR744 or agomir negative control (agoNC) and 5 ng pRL-TK vector (Promega, WI, USA) were co-transfected into cells for each well using Lipofectamine 3000, and then incubating for 48 hours. Dual luciferase reporter assay system (Promega, WI, USA) was used to perform the luciferase assay according to the manufacturer’s instruction.
Xenograft tumor growth assay
Fourtosix weeks old athymic male BALB/c nude mice were purchased from the Guangdong Medical Laboratory Animal Center (GDMLAC, Foshan, China), and were group-housed under specific pathogen-free (SPF) conditions. The experimental protocols were approved by the Institutional Animal Care and Use Committee at our University. A549 or H520 cells stably overexpressing miR744 or the control vector (8 × 105 cells) were injected subcutaneously into the dorsal flank of mice (n = 5 per group). A549 or H520 cells, transfected with antagomiR-744 or antagomir-NC, were also used for xenograft experiments following the procedures described above. After 8 days of implantation of tumor cells, tumor size was measured every 3 days and tumor volumes were calculated with the following formula: V= (L × W2)/2, where L is the length and W is the width of each tumor. At the end of experiments (about after 4–6 weeks), the mice were sacrificed and tumors were dissected and weighed.
In vivo metastasis assay
A549 or H520 cells (2 × 106) stably overexpressing miR744 or NC were injected into the tail veins of BALB/c nude mice (n = 5 per group). After 30 days, the lung colonization capacity of the cancer cells was analyzed based on the normalized bioluminescence imaging. Then the mice were sacrificed and the lung tissues were dissected and fixed in 4% formaldehyde overnight. The fixed samples were embedded in paraffin and stained with hematoxylin and eosin (HE). Lung metastasis index was calculated as our previous study described[4]: metastatic tumor areas/total lung areas.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA) and SPSS 13.0 (SPSS Inc., Chicago, USA). Each experiment was repeated at least three times and data were expressed as the mean ± SE. Two-tailed unpaired Student’s independent samples ttest was used to assess differences among groups in in vitro or in vivo studies. Brown-Forsythe method was used for the relative mRNA expression of each cell lines. Spearman’s correlation was used to analyze the relationship between miR744 and c-FOS mRNA expression. Kaplan Meier survival analysis and cox regression were performed to calculate overall survival (OS) and estimate the prognostic factors. P value < 0.05 was considered statistically, and *p < 0.05, **p < 0.01, ***p < 0.001.