Cell lines and cell culture
The five human LUAD cell lines (H1299, H1650, H1975, A549, SPCA1) used in this study were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The PC9 cell line was kindly provided by Sun Yat-Sen University Cancer Center (Guangzhou, China). Immortalized non-malignant human bronchial epithelial cells 16HBE were obtained from the Cancer Research Institute of Southern Medical University (Guangzhou, China). In this study, all the cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, PAN, USA), 100 units/mL penicillin, and 100 mg/mL streptomycin and kept in a humidified atmosphere containing 5% CO2 at 37°C. To establish DDP-resistant A549 and PC9 cell lines, cells were treated with increasing concentrations of DDP (Sigma) from the initial concentration of 0.25μM to the final concentration of 6μM. DDP was added to the RPMI-1640 culture media for A549/DDP cells and PC9/DDP cells to maintain their DDP-resistant phenotype (with a final concentration of 6μM).
miRNA (mimics and inhibitor), siRNAs, plasmid, and lentivirus transfection
LINC00173 Smart Silencer (RiboBio, Guangzhou, China) was a mixture of 3 siRNAs with 3 antisense oligonucleotides that target different sites of the LINC00173 transcript. Compared with LINC00173 Smart Silencer, the negative control Smart Silencer did not contain domains sequences homologous to humans. The other siRNAs (including three si-ZFP36L2 fragments) and miRNAs (including miR-1275 mimics and miR-1275 inhibitor) were also designed and synthesized by RiboBio (Guangzhou, China). Transient transfections of all miRNAs and siRNAs were performed with riboFECTTM CP reagent according to the manufacturer’s instructions. PROCA1 plasmid was established by Vigene Bioscience, Inc (Shandong, China). The human full-length LINC00173 was PCR-amplified from cDNA and cloned into the CMV-MCS-IRES-EGFP-SV40-Neomycin overexpression vector (GV146, Genechem, China). All reporter plasmids used in the luciferase reporter assay were obtained from IGE Biotechnology LTD (Guangzhou, China). Transient transfection of plasmids was conducted by Lipofectamine 3000 (Invitrogen, USA). The short hairpin RNA (shRNA) for human LINC00173 was cloned into a hU6-MCS-Ubiquitin-EGFP-IRES-puromycin lentiviral vector (GV248, Genechem, China). The sequences of smart-silencer, siRNAs or shRNA, miR-1275 mimics, miR-1275 inhibitor and their corresponding negative control were listed in Supplementary Table 1. All lentiviral particles were provided by Shanghai Genechem Co., LTD. For lentivirus infection, 2×104 cells were seeded into 24 well plates and infected with the proper lentivirus or control lentivirus (as negative control) at a multiplicity of infection (MOI) of 20. Puromycin (Meilunbio, Dalian, China, 3μg/ml) was used to screen the stably transduced cells and the infection efficiency was assessed under a fluorescence microscope (Nikon, Japan).
Patients and tissue specimens
Paraffin-embedded tissue samples from 129 LUAD patients treated with platinum-based multi-drug chemotherapy were made as tissue microarray (TMA) and obtained from the Second Xiangya Hospital of Central South University (Hunan, China). The clinicopathological features of 129 LUAD patients were summarized in Supplemental Table 2. Data regarding tumor stage was determined according to the pathology Tumor-Node-Metastasis (pTNM) system (AJCC/UICC 2015). Histological types were determined according to the World Health Organization classification for LUAD. According to previous studies [13,15,16], the above LUAD patients were defined as either platinum-resistant or platinum-sensitive.
In situ hybridization (ISH)
The expression level of LINC00173 in 129 paraffin-embedded LUAD specimens was detected by in situ hybridization. The TMA sections were dewaxed in xylene, rehydrated through an ethanol gradient, and then treated with 3% H2O2 for 10 min. Subsequently, sections were treated with pepsin dilution in 3% fresh citrate buffer at 37℃ for 30min and then washed with PBS. LINC00173 digoxygenin-labeled probes were designed and synthesized by BersinBio (Bersin Biotechnology Co. Ltd, Guangzhou, China) (Probe sequence: 5’-Dig-CGTGATCTGAGTACATGTAGGATAAATGACCCCAGGCAAGGC-3’). Further, hybridization was performed with LINC00173 probes at 75℃ for 5min and quickly transferred to 42℃ and incubated overnight. Then sections were incubated with anti- digoxygenin HRP Fab fragments at 37℃ for 1h. All slides were scanned using the iViewer scanning system (UNIC, Beijing, China) and quantified with staining index (ranging from 0 to 9), which was evaluated by multiplying the percentage of positively stained cells (0: 0-25%; 1: 26-50%; 2: 51-75%; 3: 76-100%) and staining intensity (0: negative; 1: weak; 2: moderate; 3: strong)[17,18]. The staining index of each sample was scored by two independent pathologists and averaged.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Cellular RNA was extracted from LUAD cells using TRIzol reagent (Thermo Scientific, USA) and then reverse transcribed to cDNA using a Quantscript RT Kit (Takara, Japan). The qRT-PCR analysis was performed on an AriaMx Real-Time PCR (Agilent, USA) system using standard procedures. GAPDH was used as a loading control, and the primer sequence was as follows: forward: 5’-CGGAGTCAACGGATTTGGTCGTAT-3’, reverse: 5’-AGCCTTCTCCATGGTGGTGAAGAC-3’. The other primers used for the qRT-PCR analyses were listed in Supplementary Table 3.
Western blot analysis
Western blotting was performed as described previously . Blotting bands were then incubated with primary antibodies overnight at 4°C. Antibodies against the following proteins were used: c-PARP (Cell Signaling Technology #5625, 1:1000), PROCA1 (Biorbyt #orb1703, 1:1000), ZFP36L2 (Santa Cruz #sc-365908, 1:1000), c-Myc (Cell Signaling Technology #9402, 1:1000), phosphor-AKT (Cell Signaling Technology #4060, 1:1000), AKT (Cell Signaling Technology #4691, 1:1000), phosphor-PI3K (Cell Signaling Technology #17366, 1:1000), and PI3K (Cell Signaling Technology #4292, 1:1000). Internal reference antibodies were used: GAPDH (Cell Signaling Technology #5174, 1:1000), β-Tubulin (Proteintech #10068-1-AP, 1:1000). Secondary antibodies used as follows: HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (Proteintech #SA00001-2, 1:5000) and HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L) (Proteintech #SA00001-1, 1:5000) were purchased from Proteintech (Rosemont, IL, USA). Images were captured by chemiluminescence (Bio-rad, Hercules, California) and quantitated using a Quantity One system (Bio-Rad, Hercules, CA, USA).
Colony formation assay
Cells at a density of 200 cells/well were seeded in six-well plates. For the DDP-treated group, a medium containing DDP of 2μM or 6μM was added respectively to the parental cells (A549 and PC9) or DDP-resistant cells (A549-DDP and PC9-DDP) after cell adhesion. The generated colonies were fixed with methanol for 30 min after culturing for 10-14 days. Subsequently, colonies were stained with a crystal violet solution. The colonies composed of more than 50 cells in a well were counted under a microscope.
Cell viability and IC50 assays
For cell viability assays, 2×104 LUAD cells were seeded into 96-well plates, DDP was added after cell attachment and cell viability was detected by CCK8 reagent after 72h. The absorbance at 450 nm measured in cell-free wells was used as blank.
For IC50 assays, 1-2×104 LUAD cells were inoculated into a 96-well plate. Medium containing an increasing concentration of DDP (0μM, 0.25μM, 0.5μM, 1μM, 2μM, 4μM, 8μM, 16μM, 32μM, 64μM) was added to the wells after cell attachment. Then, the plate was incubated for 48h in a humidified atmosphere containing 5% CO2 at 37°C. Analysis was performed according to the instructions of the CCK8 manufacturer, and the IC50 value to DDP was calculated using GraphPad Prism v5.0 software (GraphPad Software Inc., La Jolla, CA, USA).
Annexin-V/propidium iodide (PI) was applied to quantify the number of apoptotic cells. Cells transfected with smart-silencer for LINC00173 depletion or negative control were exposed to 3μM DDP for 24h. Both floated and adherent cells were collected and then resuspended in binding buffer and stained with FITC-Annexin-V and PI (BestBio, Shanghai, China). Stained cells were quantified by flow cytometer (Biosciences, NJ, USA) according to the manufacturer’s directions.
Luciferase reporter assays
Online software DIANA (http://diana.imis.athena-innovation.gr/) predicted that miR-1275 binds to LINC00173 as shown in Figure 3d. The RNA sequence of LINC00173 containing the putative wild-type binding sites or mutant binding sites for miR-1275 was inserted into the psiCHECK-2 vector to construct the luciferase reporter vector psi-CHECK-2-LINC00173-WT or psi-CHECK-2-LINC00173-MUT (named respectively as LINC00173-WT or LINC00173-MUT). PROCA1 was predicted to be directly regulated by miR-1275 using prediction software (TargetScan), as shown in Fig. 3i. The predicted 3’UTR fragment of PROCA1 recognized by miR-1275 was inserted into the psiCHECK-2 vector (named as ‘PROCA1-WT’). GeneTailor system (Invitrogen) was used to perform site-directed mutagenesis of the miR-1275 binding site at the PROCA1 3’UTR (named as ‘PROCA1-MUT’). Subsequently, cells were co-transfected with WT or MUT psi-CHECK2 vector and miR-1275 or miR-inhibitor. The 2000bp upstream of LINC00173 transcriptional start site were analyzed by PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=T-F_8.3) to identify the possible binding transcriptional factors. A c-Myc binding site was found in the promoter region of LINC00173 (Fig. 7a). The LINC00173 promoter sequences containing wild or mutant types of c-Myc binding sites were synthesized and constructed into the psi-CHECK-2 vector (named as LINC00173-WT or LINC00173-MUT). Then, LINC00173-WT or LINC00173-MUT were respectively co-transfected into A549 cells together with the c-Myc or control plasmid. Luciferase activity was measured at 48h after transfection using the dual-luciferase reporter assay (Promega Corporation, Madison, WI, USA) kit according to the manufacturer’s instructions. The detected relative luciferase activity was normalized to the Renilla luciferase activity.
RNA antisense purification (RAP) and RNA immunoprecipitation (RIP) assay
RAP assay was performed with the RAP Kit (Axl-bio, Guangzhou, China). We crosslinked 1×108 cells to fix endogenous RNA complexes and then purified these complexes through hybrid capture with biotinylated antisense oligos. According to the number of probes (probe sequence: AGCGTGTGCAGGACTTAGCTTTG CTCTTGCACTGAGA; ATAGAAGGTCCCCCACGGGGACTTGGGAAGGCAC AGAGAACGCTCCCT; ATAAACAAGCTGTGACAGGTGATCATTCATCATAT TCCGCTGAACGTTCCA), streptavidin beads were prepared for RAP and NC groups. A high-Sensitivity DNA kit （Vazyme, Q311）was used to examine DNA fragment sizes. The extracted RNA was then verified by qRT-PCR.
RIP assays were performed using a Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore, MA, USA, #MAGNARIP01). 1×107 cells were cultured in 75 cm2 cell culture flasks and then harvested using RIP lysis buffer. ZFP36L2 antibody (Santa Cruz #sc-365908) or normal mouse lgG (Negative control) were pre-incubated with Magnetic beads to form a magnetic bead–antibody complex. Subsequently, cell lysates were incubated with the bead–antibody complex overnight at 4°C. The extracted RNA was then verified by qRT-PCR. All steps of RAP and RIP assays were performed according to the kit manufacturer’s instructions.
Chromatin immunoprecipitation (ChIP) assay
According to the manufacturer’s instructions, a ChIP assay kit (Thermo, MA, USA, #26156) was used to determine whether c-Myc binds to the promoter of LINC00173 in A549 and A549/DDP cells. Micrococcal nuclease was used to cut the cross-linked DNA to a length of 200–500 base pairs which was then subjected to an immuno-selection process, which requires the use of an anti-c-Myc antibody (Cell Signaling Technology #13987). Ultimately, the agarose gel electrophoresis results demonstrated whether the DNA fragment of the putative c-Myc binding site was present in the LINC00173 promoter.
Co-immunoprecipitation (Co-IP) assay
According to the manufacturer’s instructions, a Co-IP kit (Thermo, MA, USA, #26149) was used to detect whether PROCA1 binds to ZFP36L2. Proteins were extracted from A549 or A549/DDP cells, and their concentration was quantified. A total of 300μg of protein was incubated with 10μg of specific ZFP36L2 antibody (Santa Cruz #sc-365908) or IgG control overnight at 4°C. After elution, the proteins binding with ZFP36L2 were subjected to Western blot analysis. IgG was used as a negative control. One percent of the sample was used as input.
For the Immunofluorescence assay, 100-200 cells were seeded on a glass-bottom dish. After 48 hours of culture, cells were washed with PBS and fixed in 4% paraformaldehyde for 15min. The cells were permeabilized using 0.1% Triton X-100 in PBS before antibody incubation. Images were captured using a Zeiss confocal fluorescence microscope (LSM 800, Oberkochen, Germany) and ZenPro software 2011 (AZoM.com Limited, UK). Antibodies used in this assay were listed in the methods for Western Blot analysis.
Twenty female BALB/c nude mice (3-4 weeks old) were randomly divided into two groups and injected subcutaneously into the flanks with A549 NC control cells or A549 shLINC00173-1 cells (6×106 cells), respectively, in 50μl PBS mixed with 50μl matrigel matrix (Corning, USA, #354234). Subsequently, mice were monitored and recorded for xenograft development every other day. Next, the 10 mice in the A549 NC group or A549 shLINC00173-1 group were randomly divided into two subgroups when the tumors reached a diameter of 5mm in size and treated respectively with an intraperitoneal injection of DDP (3mg/kg) or an equal volume (100μl) of normal saline (NS) every 2 days for 2 weeks. The mice were sacrificed by cervical dislocation and the xenograft tumors resected from mice were weighted and then examined with routine tissue processing. All animal experiments were conducted according to standards regarding the use of laboratory animals. The protocols for animal experiments complied with the requirements of the Institutional Animal Ethical Committee, Experimental Animal Center of Guangzhou Medical University, China.
All data were analyzed using the SPSS16.0 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism v5.0 software (GraphPad Software Inc., La Jolla, CA, USA). Data were presented as mean ±SD. Statistical significance was determined using the Student’s two-tailed t-test between two groups and one-way analysis of variance (ANOVA) for multiple groups. ISH analysis results were analyzed by the chi-square (χ2) test. Survival analysis was performed using the Kaplan–Meier method. P value< 0.05 indicated statistical significance (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).