Specimen collection
We collected 30 LUAD tissues and matched adjacent normal tissues from the Biological Resource Center of Enze Medical Center, Taizhou Hospital. The specimens were staged according to the 8th manual of the American Joint Committee on Cancer Staging Manual. The time distribution of patients undergoing lung surgery was from 2004 to 2012, and the clinical data and follow-up information of the patients were fully recorded. This study was approved by the Medical Ethics Committee of Taizhou Hospital, and the informed consent of all patients was obtained.
Immunohistochemistry (IHC)
Paraffin sections were baked in a 60°C in an incubator at 60°C for 2h before processing, and then taken out and cooled to room temperature. After deparaffinization and rehydration, the tissue sections were heated with citrate buffer to perform antigen retrieval in an autoclave. Then, 3% H2O2 was added to the slices to block endogenous peroxidase. Normal sheep serum (Gibco) was used to block nonspecific binding sites. The sections were incubated with anti-EGFL6 antibody (1:100, ab140079, Abcam, MA, USA) overnight. After that, the secondary antibody was added to the sections. Finally, the tissue sections were sequentially treated with hydrochloric acid, hematoxylin counterstain, dehydration and sealing.
These sections were graded by professional pathologists and were based on the staining intensity of and the percentage of positively stained cells. The intensity was divided into four grades: 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The percentage of positive staining cells was graded as follows: 1 (0–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). We multiplied the two scores, the final score falling in the range of 0-6 was defined as low EGFL6 expression, and 7-12 was defined as high EGFL6 expression.
Cell culture
The human A549 LUAD cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The authenticity of A549 cells (RRID: CVCL_0023) has been verified using Short Tandem Repeat (STR) profiling within the last 3 years. A549 cells were cultured in RPMI 1640 medium containing 1% penicillin/streptomycin, 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA). Cells were maintained in an incubator at 37°C with a humidity of 5% CO2. All experiments were performed with cells without mycoplasma.
Cell transfection
The siRNA targeting EGFL6 (si-EGFL6) and the paired negative control (si-Ctrl) were synthesized by GenePharma (Shanghai, China). A549 cells were seeded in six-well plates and transfected with 5μl siRNA and 5μl Lipofectamine 2000 (Invitrogen). As for the overexpression of EGFL6, the LV-EGFL6-RNAi (sh-EGFL6) was obtained from GeneChem (Shanghai, China). A549 cells were grown in 24-well plates and transfected with lentivirus and 20μl HitransG according to the manufacturer's instructions.
Cell proliferation assay
Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was used to measure cell proliferation ability. The cells were cultured into 96-well plates, and 10μl CCK8 was added to each plate at 0, 24, 48, 72, 96h. Then, after 2h incubation at 37°C, the determination of cell proliferation was replaced by the absorbance value at 450nm.
Colony formation assay
The transfected cells were seeded in 6-well plates at a density of 1000 cells per well. After culturing for 10d, the colonies were fixed with 4% paraformaldehyde for 30min and then stained with 0.1% crystal violet for 30min. The number of colonies was counted and compared in each group.
Migration and invasion assay
A549 cells were transfected on the 24-well transwell plates (Corning Inc., Corning, NY). For migration assays, a serum free cell suspension (100μl) containing 5×104 cells was added into the upper chamber, and medium containing 10% FBS was added to the lower chamber. After incubating for 12h at 37°C, the cells at the bottom of the upper chamber were fixed with 4% paraformaldehyde for 30min and stained with 0.1% crystal violet for 30min. Then, a cotton swab was used to remove the non-migrating cells in the upper chamber. For the invasion assay, the upper chamber was pre-coated with Matrigel (BD Biosciences, San Jose, CA), and the cell number of the cell suspension was changed to 1×105. The remaining steps were the same as the migration assay. Finally, we counted and compared the number of cells of each group.
Wound healing assay
The wound healing assay was performed by culturing insert (ibidi GmbH, Martinsried, Germany) in 24-well plates. The cell suspension (70μl) at a density of 5×10^4 cells/ml was placed into each well of the culture-insert. After incubating at 37°C and 5% CO2 for 24h, we used sterile tweezers to gently remove the culture–insert and photographed under an optical microscope. Then, we added serum free RPMI 1640 medium (300μl) to each well and cultured for 24h. Time lapse images were captured at the same position at 0 and 24h.
Immunocytochemistry
A cell suspension containing 1×104 cells was seeded on a coverslip in 12-well plates. After the cells were attached to the coverslips, they were fixed with 4% paraformaldehyde for 30min. The cells were permeabilized in 0.5% Triton X‐100 for 10min, then blocked with 10% FBS for 30min. The primary antibodies E-cadherin, N-cadherin and Vimentin (CST, Beverly, MA, USA; diluted 1:250) were used to incubate with the cells overnight at 4°C. On the next day, the cells were cultured with the fluorescently labeled secondary antibody (diluted 1:250) for 1h in the dark. Later, the cells were stained with DAPI for 5min and then mounted. Images were captured under confocal microscope.
RNA isolation, reverse transcription and qPCR
Total RNA was extracted from transfected A549 cells by TRIzol reagent (Invitrogen) following the manufacturer's instructions. Then, the total RNA was reverse transcribed into cDNA using the HiFiScript cDNA Synthesis Kit (CWBIO, Beijing, China). Quantitative real-time PCR analysis was performed by an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) in triplicate. The GAPDH gene was utilized as housekeeping gene. All sequences were listed in Table S1.
Western blotting analysis
Total cellular protein of transfected cells was extracted using Radio-Immunoprecipitation Assay buffer (RIPA) containing protease inhibitor PMSF (100:1) and phosphatase inhibitor (Beyotime, Shanghai, China) (100:1). Quantitative of protein concentration was detected by BCA assay (Beyotime, Shanghai, China). The protein samples at the same quantity were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking with Western Blocking Buffer (Biosharp, China) for 2h, the PVDF membranes were incubated with primary antibodies at 4°C overnight. The membranes were washed by TBS-Tween and incubated with HRP-conjugated secondary antibodies for 1h at room temperature. Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA) and ImageQuant LAS 500 (GE Health Care, Fairfield, CT, USA) were used to observe the immunoreactivity. ImageJ software was utilized to quantify the intensity of the bands.
Quantitative analysis of medium EGFL6 level
An ELISA assay was used to verify that EGFL6 play a role through exocrine. The culture medium of the cells cultured for 2d was extracted and the concentration was detected by an ELISA kit (AMEKO, China). The optical density was tested at 450nm using the Multiskan FC Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, USA). The standard curve with OD value as the ordinate and standard concentration as the abscissa. The concentration of EGFL6 in each medium could be obtained by the measured OD value against the standard curve.
Osteoclast differentiation in vitro
BMMs were extracted from the femur and tibia of C57BL6 mice (8-week-old). After culturing for 4d or reaching 90% confluence in a 10cm cell culture dish, the BMMs were seeded into a 96-well plate at a density of 8000 cells/well in complete α-MEM containing 30ng/ml macrophage colonies stimulating factor (M-CSF). After 24h, we replaced the medium with complete α-MEM containing 30ng/ml M-CSF, 50ng/ml RANKL and supplemented with the culture medium of si-Ctrl, si-EGFL6, sh-Ctrl and sh-EGFL6 respectively. The osteoclast induction medium was changed every 2d until the 7th day. The cells were fixed by 4% paraformaldehyde for 1h, and then stained by TRAP activity kit (Sigma-Aldrich, St. Louis, MO, USA). ImageJ software was used to quantify the area (percentage of a well) of TRAP-positive cells (≥3 nuclei).
Osteoblast differentiation in vitro
Bone marrow mesenchymal stem cells (BMSCs) were extracted from the femur and tibia of C57BL6 mice (8 weeks old), and cultured in complete α-MEM for 6d or reached 90% confluence in a 10cm cell culture dish. For osteoblast differentiation, BMSCs were re-seeded into a 12-well plate at a density of 1×104 cells/well with osteogenic medium (Complete DMEM supplemented with 10mM β-glycerophosphate, 50μM ascorbic acid and 100nM dexamethasone). The medium was changed every 2d. On day 7 and 21, the cells were stained with alkaline phosphatase (ALP), and the mineralized bone nodules were stained with Alizarin Red.
Bone absorption assay in vitro
Bone marrow mononuclear macrophage (BMMs) were seeded into 100μm bovine bone slices (Rongzhi Haida Biotech Co., Ltd, Beijing, China) at a density of 1×10^4 cells/well in α‐MEM supplemented with 100 ng/ml RANKL and the culture medium of si-Ctrl, si-EGFL6, sh-Ctrl and sh-EGFL6 respectively. The osteoclast induction medium was changed every 2d until the 14th day. The cells were eliminated by mechanical agitation and sonication. Then, the resorption pits were examined by scanning electron microscope (Field Environmental Instruments Inc., Hillsboro, OR). ImageJ software was utilized to quantify the area of resorption pits.
Nude mouse tumor-induced bone destruction model in vivo
All animal experiments followed the guidance of the Institutional Animal Ethics Committee of Taizhou Hospital. 6-week-old female nude mice were randomly divided into si-Ctrl, si-EGFL6, sh-Ctrl and sh-EGFL6 group (6 mice/group). After induction of anesthesia, 20μl of transfected tumor cells (1×10^6 cells/ml) suspended in Phosphate Buffered Saline (PBS) were injected into the tibial marrow cavity through the tibial plateau of nude mice. All mice were euthanized on day 14. We removed the hind limbs of nude mice and compared the size of tumor. Then, the hind limbs were fixed in 4% paraformaldehyde and collected for further experiments.
Histological and histomorphometric analysis
After decalcification in 10% EDTA (ethylenediaminetetraacetic acid) for 14d, the fixed hind limbs were embedded in paraffin and then sectioned. The tissue slices were utilized for H&E and TRAP staining. We acquired images of each sample through a high-quality microscope (Olympus, Japan) and counted the number of TRAP-positive cells in each sample.
Statistical analysis
The significant differences between groups were analyzed by Student's t-test or one-way ANOVA or Kruskal Wallis test using GraphPad Prism® version 8.3.1. All in vitro experiments were performed independently at least 3 times. The relationship between the expression of EGFL6 and clinicopathological indexes was analyzed by χ2 or Fisher’s Exact Test by IBM SPSS Statistics 26.0 for Mac (IBM Corp, Armonk, NY, USA). Results were presented as mean ± SD. P <0.05 was considered statistically significant.