Patients and tissue samples
We obtained twenty primary lung adenocarcinoma tissues and adjacent normal tissues (ANTs) from the First Affiliated Hospital of Soochow University (Additional file 2: Table S2). There are no patients who received radiotherapy or chemotherapy before surgery. Two pathologists diagnosed the histological characteristics of the tissue, independently. And the study was approved by the Human Research Ethics Committee of the First Affiliated Hospital of Soochow University. Moreover, we obtained informed consent from all patients. Lung adenocarcinoma samples from The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/. Accessed 2 May 2020.) were also included in this study.
We screened the differentially expressed lncRNAs from the TCGA database with R version 3.63 (fold change=1.0, P<0.05). The survival status and survival time of the patients were downloaded from the TCGA database (https://portal.gdc.cancer.gov/. Accessed 2 May 2020.) to obtain a prognostic model. Next, we combined the information about 309 patients with complete clinical data on TCGA-LUAD with the risk score, and the independent prognostic factor was analyzed by using the “survival” package of R software.
The Cerna Network
We used the Cytoscape (v3.6.0) to construct the network of the LINC00518-miRNA-target gene to visualize their interactions. And we predicted the LINC00518/miRNA interaction by using miRcode (https://www.mircode.org/. Accessed 2 May 2020.), and identified the target genes of the miRNAs by using miRDB (https://www.mirdb.org/. Accessed 2 May 2020.), TargetScan (https://www.targetscan.org/. Accessed 2 May 2020.), and miRTarBase (https://mirtarbase.mbc.nctu.edu.tw/. Accessed 2 May 2020.).
We purchased the cells from the Cell Bank of the Chinese Academy of Sciences in Shanghai, China, including A549, H1299 (lung adenocarcinoma), H460 (large cell carcinoma), H226 (lung squamous cell carcinoma), and BEAS-2B (human immortalized normal epithelial cells) cells. And cultured them in RPM1 1640 medium with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA) at 37 ℃ in a 5% CO2 atmosphere.
MiR-335-3p mimics, miR-335-3p inhibitors, and the related negative control (NC) were purchased from GenePharma (Suzhou, China). The GenePharma (Suzhou, China) directly synthesized the small interfering RNA (siRNA) and sequences corresponding to the target sequences for us. The siRNA constructs were as follows: si-NC: 5′-UUCUCCGAACGUGUCAC GUTT-3′; si-LINC00518–1: 5′-CCGGGCTAGATGGAACCTTAGTGAACTCGAGTTCACTAA GGTTCCATCTAGCTTTTT-3′; si-LINC00518–2: 5′-CCGGCACCTCCAAAGTGACGACTTAC TCGAGTAAGTCGTCACTTTGGAGGTGTTTTT-3′; si-CTHRC1–1: 5′-CACAUUCAAUGGAG CUGAATT-3′; si-CTHRC1–2: 5′-CGGAGUGUACAUUUACAAATT-3′. Next, insert short hairpin RNA (shRNA) and its corresponding control sequences into the lentivirus vector (GenePharma, Suzhou, China). To obtain cells stably expressing miR-335-3p or LINC00518 shRNA, lung adenocarcinoma cells were infected with lentiviruses. Then, we performed with Lipofectamine 2000 to transfect siRNA into cells according to the instructions of the manufacturer.
Quantitative Real-time Pcr Analysis
As we described previously, the detailed processes were performed. And we listed the primers used in the study in Additional file 1: Table S1. We normalized the CT values of the gene mRNA levels to those of β-actin, the internal control. The △△Ct method was applied to calculate the relative quantities of these mRNAs.
Rna Binding Protein Immunoprecipitation (Rip)
According to the manufacturer's protocol, the RIP kit (BersinBio, Guangzhou, China) was used to accomplish RIP analysis. Firstly, lyse A549 and H1299 cells were lysed by RIP lysis buffer. Secondly, we incubated the lysate products, pre-conjugated with anti-IgG or anti-Ago2 antibody, with magnetic beads at 4°C for 12–16 hours or overnight. Then, to obtain purified RNA, we used protease K to eliminate proteins. Last, the expression level of LINC00518 and miR-335-3p were determined by qRT–PCR. We performed each experiment in triplicate.
Cell Proliferation Analysis
Using Cell Counting Kit-8 (Dojindo, Shanghai, China) to examine cell proliferation. First, we cultivated LUAD cells into 96-well plates (3000 cells per well) treated with a short interfering RNA (siRNA) or plasmid and for 24, 48, and 72 h further grown under normal culture conditions. According to the manufacturer’s instructions, we determined cell viability. Moreover, we used the clonogenic assay to detect cell proliferation. In brief, we diluted cells transfected with si-LINC00518, si-CTHRC1, si-NC, miR-335-3p mimics, or miR-NC in the complete culture medium, and reseeded 200 cells into a 60-mm plate. Depending on the cell growth rate, after about 7–10 days incubating, foci formed by at least 50 cells were stained with Giemsa and counted.
To fulfill cell migration and invasion assays, transwell inserts in 8.0 µm in size (Corning, New York, NY, USA) were used. For the migration assay, firstly, we added 800 µl RPMI 1640 medium with 10 % FBS to each lower chamber of the insert. Secondly, trypsinize the stable cells, and then we seeded 4×104 cells with medium containing 1% FBS into the upper chamber and incubated them at 37 ℃ for 24 h. Furthermore, fix the cells migrated onto the lower surface of the insert with 100% methanol for 20 min, air-dried for 10 min, stained with 0.1% crystal violet overnight and washed with 1×PBS twice. Last, photograph and count the cells. For the invasion assay, coat the inserts with Matrigel matrix (BD Science, Sparks, MD, USA) diluted in serum-free medium and then incubate them at 37 ℃ for 2h. The following steps were the same as the migration assay. We performed each experiment in triplicate.
Wound Healing Assay
We seeded the stable cells of A549/H1299 into 6-well tissue culture plates at a density of ~ 70–80 % confluence as a monolayer. And then scratch the monolayer. We used a fresh 10-µl pipette tip, gently and slowly, to scratch across the centre of the well, so as to a resulting gap distance equivalent to the outer diameter of the end of the tip. Next, make another scratch perpendicular to the first to create a cross. Two gentle washes with 1 × PBS should be done to remove the detached cells. Fresh medium was replenished within the wells, and cells were cultured for an additional 24 h. A microscope (CKX41, Olympus) was used to observe and image the cells at the same magnification and settings.
Cell Cycle Analysis
We purchased the cell cycle analysis kit from Beyotime Biotechnology (Shanghai, China). After suspending in 80% ethanol at 4 ℃ overnight, the cells were stained with a PI/RNase mixture, particularly in the dark, at 37 ℃ for 30 min to the cell cycle analysis. Then, we detected the stained cells in a FACSCalibur system (Beckman Coulter, Brea, CA, USA).
Western Blot Analysis
We lysed the transfected cells and tissues by using 1× RIPA buffer with phosphatase inhibitor and protease inhibitor (Apexbio). Afterward put them on ice for 30 min to shake, and then centrifuge them at 4°C and 12,000 g for 15 min. Next, we used the 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) to separate the extracted proteins and then transfer them to NC membranes (Millipore, Billerica, MA, USA). The following antibodies were used in the study: anti-CTHRC1 (ab85739), anti-CyclinD1 (ab226977), anti-Integrin-αV (ab179475) (Abcam, London, UK), anti-MMP-9 (13677), anti-Fak (13009), anti-p-Fak (Ser397) (8556) (Cell Signaling Technology), and anti-Integrin β3 (A19073) (Abclonal, Wuhan, China). Anti-β-actin (CW0096M), anti-rabbit (CW0103), and anti-mouse (CW0102) secondary antibodies were purchased from Cowin.
Luciferase Reporter Assay
We inserted the fragments of the CTHRC1 3’-UTR and LINC00518 containing the binding site of miR-335-3p into the pMIR-REPORT vector. Then, we cotransfected with luciferase reporter plasmids and related oligonucleotides in A549 and H1299 cells. Plasmids with mutation-binding sites were used as controls. To detect the luciferase activity of reporter plasmids, we used the Dual Luciferase Reporter Assay System purchased from the Promega .
Co-immunoprecipitation (Co-ip) Assay
Lysed the cell for 30 min with 1 ml of RIPA buffer purchased from the Cell Signaling Technology (Danvers, MA, USA). Next, scraping to collect the cells and the protein in the lysates was incubated for more than 12 h with anti-CTHRC1 (Abcam, London, UK) or normal rabbit IgG antibody at 4 ℃ with rotation. Overnight, incubate the mixture with protein A/G beads or anti-c-Myc magnetic beads at 4 ℃ for 4 h. Boil the beads in the 2×SDS protein loading buffer, after washing with lysis buffer triples, and then subject them to Western blot analysis.
We obtained the female BALB/c nude mice (about 3–4 weeks old) from the Experimental Animal Center of Soochow University and which bred under pathogen-free conditions. We carried out all animal experiments in accordance with the Guide for the Care and Use of Experimental Animals Center of Soochow University. Aim to find the xenograft model of lung carcinoma, we subcutaneously inoculated a total of 3.0×106 A549 cells into the flanks, and the female mice were randomly divided into three groups (five mice per group): LV-NC, sh-LINC00518–1, sh-LINC00518–2. Finally, determine the tumour volume (V) by using the Vernier caliper to measure the width (W) and length (L) and apply the following formula: V = (L × W2) × 0.5.
Fixed all specimens with formalin and embedded in paraffin. It was deparaffinized with xylene after the specimen was sectioned and then hydrated with alcohol. Then incubate the sections of specimens with primary antibodies against CTHRC1 (Abcam, London, UK) and integrin β3 (Abclonal, Wuhan, China) at 4°C overnight. Next, we incubated the sections of specimens with the corresponding biotinylated secondary antibodies. As we described before, the reactions were developed using the DAB kit (BD Bioscience, San Jose, CA, USA), and the sections were counterstained with hematoxylin .
Membranous PSMA quantification for each sample was determined by a pathologist blinded to clinical and molecular data using modified H-Score(H-SCORE=∑༈pi×i)=༈percentage of weak intensity area ×1)+(percentage of moderate intensity area ×2)+(percentage of strong intensity area ×3), to determine the overall percentage of mPSMA positivity across the entire stained tumour sample, yielding a range from 0 to 300 [35, 36].
Use the paired Student’s t-test to assess the differences between lung adenocarcinoma tissues and adjacent normal tissues for LINC00518 analysis. Use the unpaired Student’s t-test to assess the differences between the two groups. All results were presented as the means ±SD. We performed the statistical analyses using GraphPad Prism 8 software (GraphPad, San Diego, CA, USA). Differences for which P<0.05 were considered significant.