Tissue samples
LUAD tissues and adjacent normal tissues were collected from 60 LUAD patients who were diagnosed at Nanjing First Hospital, Nanjing Medical University. Patients enrolled in this study were not treated with any type of therapies before surgical resection. Patients had signed written informed consent. This study has been approved by the Ethics Committee of Nanjing First Hospital, Nanjing Medical University.
Cell culture
LUAD cell lines including A549, Calu3 (Korean Cell Line Bank, Seoul, Korea), SPC-A1 (Chinese Academy of Sciences, Shanghai, China), and H1299 (University of Pennsylvania, Philadelphia, PA, USA) were cultured in RPMI-1640 medium (WelGENE, Seoul, Korea) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, USA). BEAS-2B cells were purchased from ATCC and cultured in DMEM medium containing 10% FBS and 1% Penicillin-Streptomycin. All cells were maintained in the incubator at 37℃ with 5% CO2.
Gene knockdown and overexpression
Small interfering RNAs (siRNAs) were obtained to generate siRNAs targeting LINC01468, SIX5, SERBP1, USP5, SERPINE1 and other 12 putative transcription factors of LINC01468. Short hairpin RNAs (shRNAs) were used to construct sh-LINC01468 and sh-SERPINE1 specially used in animal experiments. Additionally, the full-length of LINC01468, SIX5, SERBP1, USP5, SERPINE1 and other 13 putative transcription factors of LINC01468 was respectively cloned into pcDNA3.1 for overexpression of indicated genes. siCtrl, shCtrl and Ctrl were taken as negative controls. Lipofectamine 2000 was applied for plasmid transfection. Cells were collected for assays 48h post transfection.
Cell counting kit-8 (CCK-8) assay
For CCK-8 assays, cells at a density of 1000 cells/well were cultured in a 96-well plate. Plates were then incubated for 2 h after each well was added with 10 μl CCK-8 solution. Then, the optical density was measured at 450 nm for each sample. All the experiments were performed in triplicate.
Wound healing assay
Wound healing assay was done for assessment of cell migration. Cells at a density of 1×103 were seeded in 12-well plates and incubated in medium for 12 h. Then artificial wound was created using a 200 μL micropipette tip on the surface of plates. The wound was observed and pictures were taken at indicated time points (0 and 24 h).
Transwell invasion assay
Transwell chambers (with 8-μm pore size, Sigma-Aldrich, St. Louis, MO, USA) coated with matrigel were used in transwell assay to examine cell invasion. Briefly, 1×105 cells in serum-free RPMI-1640 medium were plated into the upper chamber. RPMI-1640 medium supplemented with 10% FBS was added to the lower chamber. After being cultured for 24 h, cells invaded to the lower chamber were fixed with methanol and stained with crystal violet. The number of invaded cells was counted from five fields which were randomly selected in the lower chamber.
Western blot
Firstly, RIPA lysis buffer (Beyotime Biotechnology, China) was utilized to extract total proteins. Protein concentration was then measured using BCA™ Protein Assay Kit (Pierce, Appleton, USA). Subsequently, protein extracts were separated using 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% skim milk for 2 h at room temperature, and then incubated overnight at 4℃ with diluted primary antibody. Following washed with TBST, the membranes were incubated with secondary antibody for 2 h at room temperature. Finally, the ECL chemiluminescent detection system (Thermo Fisher Scientific, Rochester, NY) was utilized to detect target bands. The primary antibodies used were as follows: anti-PAI1 (13801-1-AP, Proteintech, USA), anti-SERBP1 (14088-1-AP, Proteintech), anti-SIX5 (abcam, UK), anti-GAPDH (60004-1-Ig, Proteintech), anti-USP5 (ab154170, Abcam, Cambridge, MA, USA) and anti-Histone 3 (ab1791, Abcam, Cambridge). The original western blots were shown in Supplemental Material.
RNA extraction and reverse transcription quantitative real-time PCR (RT-qPCR)
Total RNA was extracted with Trizol reagent (Takara, Otsu, Japan) following the manufacturers’ instruction. For mRNA expression analysis, reverse transcription was performed using PrimeScript RT reagent kit (TaKaRa, Dalian, China). SYBR Green PCR Kit (Takara) was used to carry out Polymerase chain reaction (PCR). Thermal Cycler Dice Real-Time PCR System (Takara, Japan) was utilized to conduct Real-time PCR. GAPDH was used as endogenous controls. The fold change of genes was calculated using 2-ΔΔCt method. Primers used in this study were presented as follows: LINC01468: 5’-TTA CTG CCA CAC TCC CGT TC-3’ (Forward) and 5’-CAT GGG CAG ACC ACT CAT GT-3’ (Reverse), SERPINE1: 5’- CCC TCT ACT TCA ACG GCC AG-3’ (Forward) and 5’- GAG CTG GGC ACT CAG AAT GT-3’ (Reverse), SERBP1: 5’-CTG ACC GAC ATC GAA GGT GA-3’ (Forward) and 5’-AAG TGC AAT CCA TGG CTC CG-3’ (Reverse), SIX5: 5’- TGC AAG AGC GAG TCT GAT GG-3’ (Forward) and 5’- GGT CCC TGC CAG GAA TAT GG-3’ (Reverse), GAPDH: 5’-GAA GGT GAA GGT CGG AGT C-3’ (Forward) and 5’-GAA GAT GGT GAT GGG ATT TC-3’ (Reverse).
Dual-luciferase reporter assay
Wild-type and mutated LINC01468 promoter sequences were cloned into the pGL3 basic luciferase reporter vectors to construct pGL3/LINC01468-WT and pGL3/LINC01468-MUT (site 1)/(site 1&2). Then, HEK 293T, A549 and SPC-A1 cells were co-transfected with the abovementioned constructs and SIX5 overexpression plasmid or the control plasmid with Lipofectamine 2000. Luciferase reporter assay system (Promega, Madison WI, USA) was used to detect luciferase activities at 48 h post transfection.
RNA pull-down assay
RNA-Protein Pull down Kit (Thermo Fisher Scientific). Briefly, biotin-labeled RNAs were mixed with magnetic beads and then incubated with protein lysate at 4℃ with rotation for 4 h. After washing process (five times), the bound proteins were eluted and detected by western blot.
RNA binding protein immunoprecipitation assay (RIP)
RIP assay was conducted with Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) following the instructions. Briefly, cells were lysed using RIP lysis buffer, and then cell extract were incubated with magnetic beads which were coated with SERBP1 antibodies respectively. Input and normal IgG were used as controls. Immunoprecipitated RNAs were purified by proteinase K and detected by RT-qPCR.
Chromatin immunoprecipitation (ChIP) assay
SimpleChIP® Enzymatic Chromatin IP Kit (CST, USA) was utilized to conduct ChIP assay according to the manufacturers’ protocol. Firstly, enzymatic digestion was used to break cross-linked chromatin into 200 to 1000 bp fragments. The chromatin was immunoprecipitated using Anti-SIX5 or Anti-IgG (control) at 4℃ overnight with rotation. Subsequently, magnetic beads were added into tube and incubated at 4℃ with rotation for 2 h. Then the immunoprecipitated chromatin was washed with low salt solution three times and high salt solution one time. Finally, the immunoprecipitated chromatin was purified and analyzed by RT-qPCR.
Subcellular fractionation
In order to determine the cellular localization of LINC01468 and SERBP1, NUCLEI EZ PREP NUCLEI ISOLATION KIT (Sigma, USA) was used to isolate nuclear fraction from cytoplasm according to the instructions. Firstly, the cells were washed with ice-cold PBS twice. Then cells were lysed by ice cold Lysis Solution and harvested by a cell scraper on ice. Subsequently, cell lysate was centrifuged at 10000 rpm for 30 min at 4℃, the nuclear fraction was in the precipitate while the cytoplasmic fraction in the supernatant. Finally, the supernatant was carefully removed to a new 1.5 ml EP tube. The precipitate was washed by PBS twice and re-suspended by Nuclei EZ storage buffer. The level of LINC01468 was determined through RT-qPCR while the level of SERBP1 was measured via western blot.
Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) analysis
For FISH analysis of LINC01468 and SERPINE1, probes of LINC01468, and SERPINE1 were synthesized by Bersinbio Company (Guangzhou, China). Firstly, cell slides were fixed with 4% paraformaldehyde for 20 min. The slides were washed with PBS twice and dehydrated by ethanol. Thereafter, slides were denatured, added with 20 μL hybridization reaction solution (2 μL probes + 18 μL hybridization reaction) and hybridized at 37℃ overnight. After that, the slides were washed by 25% formamide/2 × Saline Sodium Citrate (SSC) twice. For IF analysis of SERBP1, SERBP1 antibody was utilized. Cells after fixation and permeablization were incubated with the primary antibody overnight and then incubated with secondary antibody for 1 h. Finally, the slides were stained with DAPI and subjected to fluorescent signal detection using Zeiss LSM800 confocal laser microscopy (Zeiss, Germany).
Co-immunoprecipitation (CoIP) analysis
Then total protein was extracted from cells and incubated with control anti-IgG, anti‑PAI1 antibody overnight at 4℃. Then the Protein A/G (Santa Cruz Biotechnology, Dallas, TX, USA) was added and incubated for overnight at 4℃. Immobilized protein complexes were extensively washed with the PBS, re-suspended in loading buffer, and separated on SDS-polyacrylamide gel electrophoresis.
Animal experiments
Animal experiments were done to investigate the influences of LINC01468 and SERPINE1 on LUAD progression under the approval of Nanjing First Hospital, Nanjing Medical University. Briefly, BALB/c nude mice obtained from Institute of Model Fauna, Nanjing University were casually divided into groups. SPC-A1 cells respectively transfected with shCtrl, sh-LINC01468#1 or sh-SERPINE1#1 were subcutaneously injected into mice. Tumor volumes were measured every 3 days since day 7 according to the formula: Volume= (length × width2)/2. At day 28, all mice were sacrificed and tumor weight was calculated. Additionally, the tumors were excised and collected for Immunohistochemistry (IHC) assay with anti-PCNA, anti-Ki67, Anti-E-cadherin and Anti-N-cadherin used.
Statistical analyses
Statistical analyses were carried out by SPSS 20.0 software (SPSS Inc., USA) and data collected from three independent experiments were shown as mean ± SD. The significant difference was analyzed by Student’s t-test and ANOVA as needed and presented as P< 0.05.