Downregulation of Linc00173 increases BCL2 mRNA stability via the miR-1275/PROCA1/ZFP36L2 axis and induces acquired cisplatin resistance of lung adenocarcinoma

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 0.25 μM to the final concentration of 6 μM. 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 6 μM).

miRNA (mimics and inhibitor), siRNAs, plasmid, and lentivirus transfection

The LINC00173 Smart Silencer (RiboBio, Guangzhou, China) was a mixture of three small-interfering RNAs (siRNAs) with three antisense oligonucleotides that target different sites of the LINC00173 transcript. Compared with the LINC00173 Smart Silencer, the negative control Smart Silencer did not contain domain sequences homologous to humans. The other siRNAs (including three si-ZFP36L2 fragments) and microRNAs (miRNAs, including miR-1275 mimics and miR-1275 inhibitor) were also designed and synthesized by RiboBio. Transient transfections of all miRNAs and siRNAs were performed using the riboFECT™ CP reagent following the manufacturer’s instructions. PROCA1 plasmid was established by Vigene Bioscience, Inc. (Shandong, China). The human full-length LINC00173 was polymerase chain reaction (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). The transient transfection of plasmids was conducted using 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 are listed in Supplementary Table 1. All lentiviral particles were provided by Shanghai Genechem Co., Ltd. For lentiviral infection, 2 × 104 cells were seeded into 24-well plates and infected with proper lentivirus or control lentivirus (as a negative control) at a multiplicity of infection of 20. The stably transduced cells were screened using puromycin (3 μg/mL; Meilunbio, Dalian, China), and the infection efficiency was assessed using a fluorescence microscope (Nikon, Japan).

Patients and tissue specimens

Paraffin-embedded tissue samples from 129 patients with LUAD treated with platinum-based multidrug chemotherapy were used as tissue microarray (TMA), and were obtained from the Second Xiangya Hospital of Central South University (Hunan, China). The clinicopathological features of 129 patients with LUAD are summarized in Supplementary Table 2. Data regarding tumor stage was determined according to the pathological tumor-node-metastasis staging system (AJCC/UICC 2015). The histological types were determined according to the World Health Organization classification for LUAD. According to previous studies [21, 23, 24], the aforementioned patients with LUAD were defined as either platinum -resistant or platinum -sensitive.

In situ hybridization

The expression level of LINC00173 in 129 paraffin-embedded LUAD specimens was detected by in situ hybridization (ISH). The TMA sections were dewaxed in xylene, rehydrated through an ethanol gradient, and then treated with 3% H2O2 for 10 min. Subsequently, the sections were treated with pepsin dilution in 3% fresh citrate buffer at 37 °C for 30 min and then washed with phosphate-buffered saline (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 using LINC00173 probes at 75 °C for 5 min, quickly transferred to 42 °C, and incubated overnight. Then, the sections were incubated with anti-digoxygenin (HRP) Fab fragments at 37 °C for 1 h. All slides were scanned using the iViewer scanning system (UNIC, Beijing, China) and quantified using a 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; and 3: strong) [25, 26]. The staining index of each sample was scored by two independent pathologists and averaged.

Quantitative real-time reverse transcription–PCR

The cellular RNA was extracted from LUAD cells using the TRIzol reagent (Thermo Scientific, USA) and then reverse transcribed to cDNA using a Quantscript reverse transcription (RT) Kit (Takara, Japan). The quantitative RT-PCR (qRT-PCR) analysis was performed using an AriaMx Real-Time PCR (Agilent, USA) system following standard procedures. Glyceraldehyde-3-phosphate dehydrogenase (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 are listed in Supplementary Table 3.

Western blot analysis

The Western blot analysis was performed as described in a previous study [27]. The blotting bands were then incubated with primary antibodies overnight at 4 °C. The antibodies against the following proteins were used: Caspase-3 (Cell Signaling Technology #14220, 1:1000), PARP (Cell Signaling Technology #9542, 1:1000), BCL2 (Cell Signaling Technology #15071, 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). The internal reference antibodies were as follows: GAPDH (Cell Signaling Technology #5174, 1:1000) and β-tubulin (Proteintech #10068-1-AP, 1:1000). The secondary antibodies were as follows: HRP-conjugated Affinipure goat anti-rabbit immunoglobulin G (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 (IL, USA). The images were captured by chemiluminescence imaging (Bio-Rad, CA, USA) and quantitated using the Quantity One system (Bio-Rad).

Colony formation assay

The cells at a density of 200 cells/well were seeded in six-well plates. For the DDP-treated group, a medium containing 2 μM or 6 μM DDP was added, respectively, to the parental cells (A549 and PC9) or DDP-resistant cells (A549-DDP and PC9-DDP) after cell adhesion. As the transduction of inhibitors or mimics of miR-1275, plasmids of PROCA1 or LINC00173 was transient, we transducted them every three days in the 6-well plate to maintain their expression. The generated colonies were cultured for 10–14 days and then fixed with methanol for 30 min. Subsequently, the colonies were stained with a crystal violet solution. The colonies composed of more than 50 cells in a well were counted using a microscope.

Cell viability and half-maximal inhibitory concentration assays

For the cell viability assays, 2 × 104 LUAD cells were seeded into 96-well plates. DDP was added after cell attachment, and the cell viability was detected using a cell counting kit-8 (CCK-8) reagent after 72 h. The absorbance at 450 nm measured in cell-free wells was used as blank.

For half-maximal inhibitory concentration (IC50) assays, 1–2 × 104 LUAD cells were inoculated into a 96-well plate. A medium containing an increasing concentration of DDP (0 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, and 64 μM) was added to the wells after cell attachment. Then, the plate was incubated for 48 h in a humidified atmosphere containing 5% CO2 at 37 °C. The analysis was performed following the instructions of the CCK-8 manufacturer, and the IC50 value of DDP was calculated using the GraphPad Prism v5.0 software (GraphPad Software Inc., CA, USA).

Apoptosis

Annexin-V/propidium iodide (PI) was applied to quantify the number of apoptotic cells. The cells transfected with Smart Silencer for LINC00173 depletion or negative control were exposed to 3 μM DDP for 24 h. Both floated and adherent cells were collected, resuspended in binding buffer, and stained with FITC–Annexin-V and PI (BestBio, Shanghai, China). The stained cells were quantified using a flow cytometer (Biosciences, NJ, USA) following the manufacturer’s instructions.

Luciferase reporter assays

The online software DIANA (http://diana.imis.athena-innovation.gr/) predicted that miR-1275 bound to LINC00173, as shown in Fig. 3d. The RNA sequence of LINC00173 containing the putative wild-type (WT) binding sites or mutant-type (MUT) binding sites for miR-1275 was inserted into the psiCHECK-2 vector to construct the luciferase reporter vector psiCHECK-2-LINC00173-WT or psiCHECK-2-LINC00173-MUT (named as LINC00173-WT or LINC00173-MUT, respectively). PROCA1 was predicted to be directly regulated by miR-1275 using the prediction software (TargetScan), as shown in Fig. 3i. The predicted 3′ untranslated region (3′-UTR) fragment of PROCA1 recognized by miR-1275 was inserted into the psiCHECK-2 vector (named as “PROCA1-WT”). The GeneTailor system (Invitrogen) was used to perform the site-directed mutagenesis of the miR-1275-binding site at the PROCA1 3′-UTR (named as “PROCA1-MUT”). Subsequently, the cells were co-transfected with WT or MUT psi-CHECK2 vector and miR-1275 or miR-inhibitor. The 2000 bp upstream of LINC00173 transcriptional start site was 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 WT or MUT of c-Myc-binding sites were synthesized and constructed into the psiCHECK-2 vector (named as LINC00173-WT or LINC00173-MUT). Then, LINC00173-WT or LINC00173-MUT was, respectively, co-transfected into A549 cells together with the c-Myc or control plasmid. The luciferase activity was measured 48 h after transfection using the dual-luciferase reporter assay (Promega Corporation, WI, USA) kit following the manufacturer’s instructions. The detected relative luciferase activity was normalized to the Renilla luciferase activity.

RNA antisense purification and RNA immunoprecipitation assays

The RNA antisense purification (RAP) assay was performed using an RAP Kit (Axl-bio, Guangzhou, China). We cross-linked 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 negative control (NC) groups. A high-sensitivity DNA kit (Vazyme Biotech Co., Ltd, Q311) was used to examine the DNA fragment sizes. The extracted RNA was then verified by qRT-PCR.

The RNA immunoprecipitation (RIP) assay was performed using a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore, MA, USA; #MAGNARIP01). Then, 1 × 107 cells were cultured in 75-cm2 cell culture flasks and harvested using a RIP lysis buffer. The ZFP36L2 antibody (Santa Cruz #sc-365908) or normal mouse IgG (negative control) were pre-incubated with magnetic beads to form a magnetic bead–antibody complex. Subsequently, the 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 following the kit manufacturer’s instructions.

ChIP assay

A ChIP assay kit (Thermo, MA, USA, #26156) was used to determine whether c-Myc bound to the promoter of LINC00173 in A549 and A549-DDP cells, following the manufacturer’s instructions. The micrococcal nuclease was used to cut the cross-linked DNA to a length of 200–500 base pairs, which was then subjected to an immunoselection process requiring 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-IP assay

A Co-IP kit (Thermo, MA, USA, #26149) was used to detect whether PROCA1 bound to ZFP36L2, following the manufacturer’s instructions. The proteins were extracted from the parental cells (A549 and PC9) or DDP-resistant cells (A549-DDP and PC9-DDP), and their concentration was quantified. A total of 5 mg of protein was incubated with 10 μg 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.

Immunofluorescence

For the Immunofluorescence assay, 100–200 cells were seeded on a glass-bottom dish. After 48 h of culture, cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min. 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.

Animal experiments

Twenty female BALB/c nude mice (3–4 weeks old) were randomly divided into two groups. A549 NC control cells or A549 shLINC00173-1 cells (6 × 106 cells) in 50 μL of PBS mixed with 50 μL of Matrigel matrix (Corning, USA, #354234) were injected subcutaneously into the flanks. Subsequently, the mice were monitored for xenograft development every other day. Next, 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 5 mm in size and treated, respectively, with an intraperitoneal injection of DDP (3 mg/kg) or an equal volume (100 μL) of normal saline (N.S) every 2 days for 2 weeks. The mice were sacrificed by cervical dislocation, and the xenograft tumors resected from mice were weighed and examined with routine tissue processing. Animal experiments were performed in three independent replicates. 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.

Statistical analysis

All data were analyzed using the SPSS16.0 software (IBM Corp., NY, USA) and GraphPad Prism v7.0 software. 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).

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