Patients and tissue samples
The study was fully approved by the Institutional Medical Ethics Committee of Guangzhou Medical University and Suzhou University. Written informed consent forms were obtained from all participants. A total of 170 NSCLC and their adjacent non-tumor tissue specimens were obtained from First Affiliated Hospital of Guangzhou Medical University, Affiliated Tumor Hospital of Guangzhou Medical University, and First Affiliated Hospital of Suzhou University. No patients received any radiotherapy or chemotherapy before surgery. All clinical information including age, gender, clinical stage, smoking history, infiltration degree, lymph node metastasis and distant metastasis, of these patients were collected recorded in a database. In addition, the fresh surgically removed tissues were immediately preserved in RNA later Solution (Thermo Fisher Scientific, US) and stored at −80 °C refrigerator. The design route of this study was summarized in Fig. 1.
Cell culture
All the cell lines including A549, PC-9, NCI-H520, HCI-H460, L78, NCI-H292, GLC-82 human lung cancer cell lines, and HBE-pic, BEP-2D, BEAS-2B, 16HBE human Normal lung epithelial cell lines used in this study were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Science (Shanghai Institute of Cell Biology, China), and authenticated by STR typing. All cells were cultured in RPMI 1640 medium (Gibco, life technologies, California, USA) with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA). Cells were placed in a CO2 incubator (SANYO Electric Co., Ltd., Japan) with constant 90% humidity and 5% CO2.
Cell transfection
The full-length Lnc-LSAMP-1 cDNA sequence was synthesized by iGeneBio Co, Ltd, Company (Guangzhou, China) and cloned into the pEZ-Lv201 lentivirus expression vector (GeneCopoeia, China). The Lnc-LSAMP-1 vector or the empty vector were transfected into human 293 T cell to collect viral particles. The viral particles were then used to transfect lung cancer cell lines. After screening for 2 weeks using puromycin, the cells that stably expressed Lnc-LSAMP-1 were determined by RT-qPCR assay and cells that were infected with empty particles were used as control cells. Among the transfected lung cancer cell lines, A549 and PC-9 cells showed the best transfection efficiency, and they were chosen to perform subsequent functional experiments.
Real-time quantitative reverse transcription PCR (RT-qPCR)
Total RNA from 170 paired NSCLC tissues and 11 cell lines were extracted using TRIzol reagent (Invitrogen, Carlsbad, California, USA). The total RNA was then transcribed to cDNA using commercial kits according to the manufacturer’s instructions (TaKaRa, Japan). RT-qPCR reaction (DBI, Germany) was performed in the Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, CA, USA). β-actin was used as the endogenous control. The primers were synthesized by Sangon Biotech Ltd (Shanghai, China). The primer sequences used for RT-qPCR were presented in Additional file 4: Table S1. The 2−ΔΔCT was used to demonstrate the expression levels of Lnc-LSAMP-1 and LSAMP. All the experiments were conducted in triplicate.
Western blotting
Western blotting was performed as previously described [31]. Briefly, total protein lysates from lentivirus-transfected-A549 and PC-9 cells were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, then the membranes were washed and blocked. Primary antibodies of LSAMP purchased from Abcam (Cambridge, MA, USA) were applied to membranes, followed by horseradish-peroxidase-conjugated secondary antibodies according to the manufacturer's instructions.
Cell proliferation assay
Cell proliferation assay was performed with Cell Counting Kit-8 (CCK-8, Corning Corporation, USA). Logarithmic phase cells were seeded into 96-well plates and were cultured for 12, 24, 36 and 48 h, respectively [32]. The absorbance of each well was read on a Thermo Scientific™ VarioskanTM LUX plate reader (Thermo Instruments, USA) (detection wavelength was 450 nm and the reference wavelength was 600 nm). A dynamic cell monitoring was also performed using the Incucyte Zoom Live-Cell Imaging System (IncuCyte ZOOM, Essen BioScience Co., Ltd., USA). Cell confluence was calculated by phase-contrast images according to the manufacturer’s protocols.
Flow cytometric analysis of cell cycle and apoptosis
The flow cytometry analysis was used to identify whether Lnc-LSAMP-1 influences cell cycle and apoptosis. For cell cycle analysis, the stable transfected cells were trypsinized (without EDTA), washed with PBS, and fixed with 70% ethanol at 4 °C for 12 h and resuspended in staining buffer containing 450 µl propidium iodide (PI) and 50 µl RNaseA in the dark for 30 min at room temperature. Then the flow cytometric assays (FACScan; BD Biosciences, Shanghai, China) were performed according to the manufacturer's instructions.
For cell apoptosis analysis, Annexin V/7-AAD apoptosis kit (MultiSciences, HangZhou, China) was used to stain for early and late apoptotic cells according to the manufacturers' protocols. Cells were washed twice in PBS and re-suspended in 1 × Binding Buffer to achieve a cell concentration of 1.0 × 106 cells/ml. Subsequently, 10 ul of 7-AAD reagent and 5 ul of Annexin V reagent were added into cell suspension and stored for 30 min at room temperature in dark place. Apoptotic cells were examined and quantified using flow cytometry (Becton Dickinson, Lincoln Park, NJ, USA).
Colony-formation assay
The cells were trypsinized and seeded into 6-well plates at a density of 200 cells/well. After 10 days of culture, cell clones that had formed from individual cells were directly observed by eye and then the colonies were washed with PBS, fixed 5% paraformaldehyde and stained with 0.1% crystal violet solution, followed by air-drying. The stained colonies were photographed, and counted using ImageJ 8.0 software (National Institutes of Health).
Transwell assays
For the Transwell migration assay, the cells were trypsinized, adjusted to a concentration of 4 × 105/ml, and seeded into the upper chamber with a non-coated membrane with 200 µl per well (24-well insert, pore size 8 μm; Corning, NY, USA). Lower chambers were supplemented with 10% fetal bovine serum (600 μL). After being incubated for 24 h with 5% CO2 at 37 °C, the upper surface of the membrane was removed with a cotton tip, while the cells on the lower surface were fixed using formaldehyde and then stained with 0.1% crystal violet for 30 min. Ten fields were randomly selected under a 100 × microscope and the number of cells that migrated to the lower layer was counted. For the invasion assay, as it is identical to the migration experiment procedure with coating with matrigel chambers (BD Biosciences, San Jose, CA, USA) were carried out according to manufacturer’s instructions. Briefly, transfected Lnc-LSAMP-1 cells (2 × 104 cells/200 µl per well) were collected, resuspended in medium without serum, and then shifted to the hydrated matrigel chambers. The bottom chambers were incubated overnight in 600 μL culture medium with 10% FBS. The cells on the upper surface were scraped, whereas the invasive cells on the lower surface were fixed, colored and counted.
Tumorigenicity and metastasis assay in nude mice
0.2 mL of cells suspension that contained 1 × 107 cells was subcutaneously injected into the necks of 5 four-week-old female nude mice per group (Beijing Huarongkang Biotechnology Co. Ltd). Tumor growth was examined every 3 days for at least 1 month by measuring the length and width of the tumor mass. The experimental procedures for tumor metastasis model were similar to tumor growth model. 5 × 107 cells were injected into caudal vein of nude mice (five mice per group). All mice were kept until death due to the neoplastic progression or until the end of the experiment (6 weeks). After 6 weeks, the mice were euthanized. Mice were injected intraperitoneally with barbiturate in a dose of 150 µg/g (total injection volume, 0.4 mL). After approximately 30 min, the mice were then sacrificed and the lungs were collected to evaluate the number of pulmonary metastatic lesions. Hematoxylin and eosin (H&E) staining was performed for tissue morphology evaluation following relevant protocols and strict operating procedures after soaking and fixing with 4% paraformaldehyde. All experimental procedures were approved by the Animal Ethics Committee of Guangzhou Medical University.
Hematoxylin–eosin (HE) staining
The tumor masses from the nude mice were harvested and immersed in 4% paraformaldehyde for 4 h, and transferred to 70% ethanol. After that, the tissues were placed in processing cassettes, dehydrated through a serial alcohol gradient, and embedded in paraffin wax block, and then cut into 3-µm-thick sections that were baked at 45 °C for 5 h. Sections were then stained with HE (artificial hematoxylin and eosin) according to the following steps: 30 min of xylene dewaxing, treated with ethanol at different concentrations (100%, 90%, 70%), hydrated in distilled water, stained with hematoxylin (15 min), differentiated in hydrochloric acid ethanol and ammonia water, dehydrated with ethanol at 70% and 90% concentrations (10 min), stained with eosin ethanol (3 min), dehydrated with ethanol and cleared with xylene, and tumor tissue sections were then observed under a microscope.
Subcellular fractionation
Nuclear and cytoplasmic fractions were isolated from A549 and PC-9 cells using the nuclear/cytoplasmic isolation kit (Biovision, San Francisco, CA) according the manufacturer's protocols. The RNAs from cytoplasmic and nuclear were then extracted using TRIzol reagent (Invitrogen, Carlsbad, California, USA). At last, qRT-PCR was performed to assess the relative expressions of β-actin (cytoplasm control), U6 (nucleus control), and Lnc-LSAMP-1 in in each sample.
Actinomycin D inhibits RNA synthesis experiments
Over-expressed Lnc-LSAMP-1 or empty-control cells were seeded into 24-well plates at 5 × 104 per well. After 24 h, the cells were treated with actinomycin D at a concentration of 2 mg/L. After 30 min, 1 h, 2 h, 3 h and 4 h, the expression levels of LSAMP gene were detected by qRT-PCR.
Drug sensitivity test of lnc-LSAMP-1 on cisplatin and TKI (Tyrosine kinase inhibitors)
The IC50 of A549 and PC-9 cells was first screened using cisplatin concentration gradient (10 ug/ml, 5 ug/ml, 2.5 ug/ml, 1.25 ug/ml, 0.625 ug/ml, 0.3125 ug/ml), and finally determined as 5 ug/ml for A549 and 2.5 ug/ml for PC-9. In the preliminary experiments, several TKI drugs were also used to evaluate the effect of lnc-LSAMP-1 on therapeutic sensitivity, and found that only Nilotinib treatment were observed to have fulfilling inhibition rate changes induced by lnc-LSAMP-1. Nilotinib was then subsequently used as mean plasma drug concentrations such as Nilotinib = 3.6 μmol/L. High expression of lnc-LSAMP-1 and control cells were treated with cisplatin and Nilotinib. The cells were inoculated into a 96-well plate, and the number of cells per well was 5.0 × 103. Nilotinib was added after adhering to the wall and growing to about 10%, cisplatin was added up to 30–40%, and the 96-well plate was placed in a dynamic cell observer for cell proliferation detection (IncuCyte ZOOM. Essen BioScience Co. Ltd. USA). Proliferation inhibition rate = (experimental group (dosing)−experimental group)/(control group (dosing)−control group) × 100%.
Rescue experiment
The rescue experiment was performed to validate that Lnc-LSAMP-1 regulated lung cancer cell biological behaviors and enhanced the cell cytotoxicity induced by TKI treatment through targeting LSAMP. LSAMP inhibitor and blank inhibitor were transfected into A549 and PC-9 cells. The silencing efficiency of each siRNA targeting LSAMP was measured by qRT-PCR assay. The IncuCyte ZOOM long time live cell image monitoring system (Essen BioScience Co., Ltd., USA) was used to detect cell proliferations to evaluate the inhibition effect induce by LSAMP.
RNA pulldown assay
For RNA pulldown assays, Biotin-labeled Lnc-LSAMP-1 and its antisense were transcribed in vitro with the Biotin RNA Labeling Mix and T7 RNA polymerase (Roche, Basel, Switzerland), and then treated with RNase-free DNase I (Roche) and 0.2 M EDTA to stop the reaction. Biotinylated RNAs were mixed with streptavidin agarose beads (Life Technologies, Gaithersburg, MD) at 4 °C overnight. Total cell lysates were freshly prepared and added to each binding reaction with Protease/Phosphatase Inhibitor Cocktail and RNase inhibitor, incubated with rotation for 1 h at 4 °C. The RNA–protein binding mixture was separated using SDS-PAGE and the eluted proteins were detected by western blot.
RNA sequencing
The total RNA from over-expressed Lnc-LSAMP-1 and empty-control A549 cells were extracted with TRIZOL reagent. The RNA concentrations and purities were measured using NanoDrop 2000 spectrophotometer (Thermo Electron Corporation, USA). The transcriptome sequencing was prepared using HISAT2 for Illumina® according to the manufacturer protocol (Guangzhou, Promege Biotechnology Co., Ltd). Raw reads were aligned to the human genome GRCh38 by Bowtie2. Differentially expressed genes between the two cell groups were identified using the expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) method. The |log2(FoldChange)|> 1 and P value < 0.05 was as the threshold to judge the significance of gene expression differences.
Bioinformatics analysis
Differentially expressed mRNAs were further analyzed with Gene Ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway to investigate the functions and underlying mechanisms of Lnc-LSAMP-1 in NSCLC progression.
Statistical analysis
All statistical data were analyzed using the SPSS 16.0 software (SPSS, Chicago, USA). The differences of gene expression between lung cancer tissues and adjacent lung normal tissues were evaluated using paired-t test. The χ2 test was applied to analyze the distribution of gene expression between the demographics and clinical characteristics subgroup. Correlation between Lnc-LSAMP-1 level and LSAMP expression was tested with the Pearson correlation analysis. The Log-rank test and Cox regression analyses were used to assess the effect of Lnc-LSAMP-1 expression on lung cancer survival. Additionally, P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001).