Patient and clinical information
Eighty paired NSCLC tissues and paratumor tissues were collected from the surgery specimen archives of the Second Affiliated Hospital of Soochow University. Patient follow-up continued to December 2020. No recruited patients received preoperative chemotherapy or radiotherapy. This research was approved by the institutional clinical research ethics committee of the Second Affiliated Hospital of Soochow University. Patient stratification was based on the new TNM staging criteria in lung cancer (8th edition).
Cell culture and treatment
The human normal lung epithelial cell line HBE and four human NSCLC cell lines (A549, PC9, H1650, H1299) were obtained from ATCC (Manassas, VA, UA) and cultured in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C, in a humid 5% CO2 incubator. Two small interfering RNA (siRNA) constructs (GenePharma, Shanghai, China) were designed to knock down AL355338 expression, and non-specific siRNA served as the negative control; all siRNAs were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). For ectopic AL355338, ENO1, and EGFR expression, the appropriate cDNA was amplified with reverse transcription polymerase chain reaction (RT-PCR) and subcloned into a pcDNA-4/TO vector. Neofect transfection reagent (Neofect biotech, Beijing, China) was used for plasmid transfection. The sh-AL355338, sh-ENO1 and sh-NC lentiviruses were purchased from GenePharma. The siRNA and sh-RNA sequences are listed in Table S1.
Total RNA isolation and quantitative RT-PCR (qRT-PCR) assays
Total RNA was extracted from cells or tissues with TRIzol reagent (Omega, Norcross, GA, USA) according to the manufacturer’s instructions. RNA quality and quantity were assessed with the NanoDrop2000 platform (Thermo Fisher Scientific, Waltham, MA, USA). cDNA Synthesis kits (TaKaRa, Otsu, Japan) were used for mRNA reverse transcription. Real-time PCR analysis was performed with SYBR Green PCR Kits (TaKaRa) on the 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The amplified transcript levels of genes were normalized to β-actin with the optimized comparative 2-ΔΔCt value method. The primer sequences are in Table S1.
The nuclear and cytoplasmic fractions were generated with a PARIS™ Kit (Ambion, Austin, TX, USA) according to the manufacturer protocol. Nuclear and cytoplasmic RNA levels were normalized to U6 and β-actin, respectively using real-time PCR.
Fluorescence in situ hybridization (FISH)
FISH analyses were performed with RNA FISH kits (RiboBio, Guangzhou, China) using a previously described method. The AL35538 probe sequence was 5’-DIG-ACTTCGAGACCAGCATGGCCAACATGGTGAAGCCC-DIG-3’. Nuclei were stained with DAPI, and high-resolution images were taken with a confocal microscope (ZEISS, Oberkochen, Germany). Percentages of positive stained cells were quantified as: 1 = 0-25%, 2 = 26-50%, 3 = 51-75%, and 4 = 76-100%. Staining intensity scores were: 0 = none, 1 = weak, 2 = moderate, and 3 = strong. The final scores were calculated by multiplying the percentage and intensity scores of positive cells. Samples were divided into low (score 0-6) and high (score 7-12) expression groups.
Flow cytometry assay
Transfected cells were harvested and fixed in pre-cooled 70% ethanol for 1 h on ice. Then cells were washed with cold phosphate-buffered saline (PBS) three times and incubated with RNase for 30 min at 37°C. After staining the cells with propidium Iodide for 15 minutes at room temperature, they were analyzed with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The percentages of cells in the G0/G1, S, and G2/M phases were calculated using ModFit software (Verity Software, Topsham, ME, USA).
Cell proliferation assay
Cell proliferation rates were measured with Cell Counting Kit-8 (bimake, Shanghai, China) according to the manufacturer’s protocol. Briefly, 5 × 103 cells per well were seeded into 96-well plates. At 0, 24, 48, and 72 h after cell transfection, 10 μL CCK-8 solution was added to each well. After incubating he plates for 2 h at 37°C, the optical density at 450 nm was measured for each well. For colony formation assays, transfected or control A549 and PC9 cells were harvested 24 h after transfection, and 500 cells per well were seeded into 6-well plates. After 2 weeks of incubation, the colonies were fixed in 4% paraformaldehyde and stained with crystal violet solution. Cell proliferation was also assessed using ethynyldeoxyuridine (EdU) analyses (RiboBio) according to the manufacturer’s instructions as previously reported.
Cell migration and invasion assay
Wound healing assays were carried out as previously described . Transwell assays were performed using Boyden chambers containing transwell membrane filters (Corning, Corning, NY, USA). The detailed protocol was published elsewhere. Images were collected with an inverted microscope (Olympus, Tokyo, Japan) and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). At least five random fields of view were analyzed in each chamber.
Measurement of glucose uptake, lactate production, and ATP
As a glucose analogue, 18F-FDG uptake reflects the intracellular glucose uptake level. In vitro experiments were performed as previously reported. Lactate production was measured in cells that were seeded into 12-well plates in triplicate for 24 h and then refreshed with 1 mM glucose culture medium overnight. Culture supernatants were collected to measure lactate concentrations (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and lysed cell pellets were used to measure ATP levels (Nanjing Jiancheng) using the manufacturer’s instructions. 18F-FDG uptake and lactate production were normalized to number of cells in each sample; ATP levels were normalized to cell protein mass.
Western blotting was performed following a standard protocol. The antibodies used are listed in Table S2. Immunoreactive bands were detected using ECL western blot kits (Amersham Biosciences, Little Chalfont, UK).
IF was performed as described elsewhere. Protein expression and localization were observed under a confocal microscope (ZEISS). Antibodies used for IF are listed in Table S2.
RNA pull-down assay
Biotin-RNA pull-down assays were performed as described elsewhere. Briefly, full-length sense, antisense, and serial deletion sequences of AL355338 were amplified with a T7-containing primer and reverse transcribed using MAXIscript™ T7 Transcription Kits (Thermo Fisher Scientific). Pierce™ RNA 3’ End Desthiobiotinylation Kits (Thermo Fisher Scientific) were used to label targeted RNA. The biotin-labeled AL355338 probe was incubated with total cell lysates of A549 cells using Pierce™ Magnetic RNA-Protein Pull-Down Kits (Thermo Fisher Scientific). Eluted proteins were purified by silver staining and subjected to mass spectrometry (MS) or western blotting.
RNA immunoprecipitation (RIP) assay
Magna RNA-binding protein immunoprecipitation kits (Millipore, Billerica, MA, USA) were used to determine specific protein binding with targeted RNA according to the manufacturer’s protocol and as described previously. Brieﬂy, cells were lysed with RIP lysis buffer, and lysates were incubated overnight at 4°C with magnetic beads conjugated with human anti-ENO1 antibody or anti-mouse immunoglobulin G (IgG). The next day, co-immunoprecipitated RNA was extracted for qRT-PCR analyses. Total RNA (input controls) and IgG controls were simultaneously assayed to confirm that the RNA specifically bound to ENO1.
Reciprocal exogenous and endogenous co-IP assays were performed as described elsewhere repor. IP samples and input controls were analyzed by western blot. The co-IP antibodies are listed in Table S2.
Proximity ligation assay (PLA)
PLA (Duolink®, Sigma-Aldrich, St. Louis, MO, USA) was performed to evaluate interactions between ENO1 and EGFR as previously reported. Briefly, A549 and PC9 cells were grown on glass coverslips in 24-well plates at 37 °C overnight. The next day, cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 5 min. After blocking for 30 min, the cells were incubated with anti-ENO1 and anti-EGFR antibodies diluted in blocking buffer at 4°C overnight. The next day, cells were incubated with species-specific PLA probes at 37°C for 1 h, then 1× ligase for 30 min, and finally 1× polymerase diluted with amplification stock solution at 37°C for 100 min to amplify the fluorescence signal. Finally, coverslips were mounted on slides with anti-fluorescence quenching sealing liquid containing DAPI. Images were acquired under confocal microscopy (ZEISS).
In vivo xenograft assays
Four-week-old male BALB/c nude mice were purchased from the Animal Center of Soochow University. Animal experiments were carried out according to the protocol approved by the Animal Care and Ethics Committee of the Second Affiliated Hospital of Soochow University. A total of 20 mice were subcutaneously injected with 5 × 106 A549 cells in the right flank with sh-AL355339, and sh-NC was given in the left flank to establish an NSCLC xenograft model. Tumor volume was measured every fifth day and calculated according to the equation V = length × width2 × 1/2. Micro-PET/CT (Super Nova® PET/CT, Pingseng, China) was performed 3 weeks after cell injection to measure 18F-FDG uptake by xenograft tumors. All tumor-bearing mice were fasted for 8 h prior to PET/CT. 18F-FDG (0.1 mL, 7.4 MBq) was injected via tail vein. After 30 min, they were anesthetized with 2% isoflurane in oxygen and immobilized for 15-min scans. 18F-FDG uptake was measured as maximum standard uptake (SUVmax) in the regions of interest covering the tumors. After 3 weeks, mice were humanely sacrificed by euthanasia. The mice were inhaled with CO2 for 5 min. The CO2 was gradually filled into an airtight chamber at 30% of the chamber volume/min rate. The xenograft tumors were harvested and measured.
In vivo metastasis assays
Stably transfected A549 cells (1 × 106/0.2 ml PBS) were injected into the tail vein of nude mice. Micro-CT scans were performed 5 weeks later to observe lung metastasis. After 5 weeks, mice were humanely sacrificed by euthanasia. Following sacrifice, the lungs were surgically dissected and embedded in paraffin for hematoxylin and eosin (HE) staining. The numbers of pulmonary metastatic nodules were compared between groups.
IHC was performed and quantified as previously described.
All data are expressed as mean ± standard deviation. Statistical analysis was performed with SPSS (version 22.0, IBM Corp., Armonk, NY, USA) and graphs were generated with GraphPad Prism software (version 8.0, GraphPad Inc., San Diego, CA, USA). Student’s t-tests or one-way analyses of variance were used to assess significant differences between groups. Categorical data were compared with Chi-square tests. Survival analyses were performed using Kaplan-Meier analyses.