Acute lung injury mice model
The BALB/c mice were housed in a room maintained at 25 °C with a light/dark cycle of 12 h/12 h, and then they were randomly divided into two groups (n = 10/group): The control group and LPS group. The ALI mice model was performed by intratracheally instilling with 10 μg LPS in 50 μL of PBS, and the control mice were given an equal volume of PBS. Six hours after the infusion of LPS or PBS, the mice were sacrificed, and lung tissues were harvested for RT-qPCR and western blot. All experimental procedures were approved by the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Measurement of wet-to-dry ratio of the lungs
Thirty minutes before LPS treatment, the BALB/c mice were injected with 100 μL lentivirus-expressing CASC2 or the control lentivirus (MOI = 5 * 107 TU/mL, GeneChem, Shanghai, China) by tail vein, and then treated with LPS as described in ALI mice model. At 6 h after treatment with LPS, mice were euthanized, the right lung was then removed for detection of mRNA and protein, and determination of wet weight. Subsequently, the lungs were incubated at 60 °C for 3–4 days to remove all moisture, then the dry weight was measured and the ratio of wet-to-dry weight calculated.
Cell culture and transfection
The human lung adenocarcinoma A549 cell line was purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were grown in DMEM (Sigma-Aldrich) supplemented with 5% FBS (HyClone, Logan, USA) and antibiotics (penicillin, 100 U/mL; streptomycin 100 μg/mL) in a 5% CO2 atmosphere at 37 °C. MiR-144-3p inhibitor/mimic, pcDNA-CASC2 and their respective negative control/vector was transfected or co-transfected into A549 cells by Lipofectamine 2000 according to the instructions. After transfection for 48 h, the cell was collected and used to detect mRNA and protein expression and cell apoptosis.
Quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA samples were extracted from lung tissue or A549 cells using Trizol (Invitrogen) according to the manufacturer’s instructions. The Taqman microRNA Reverse Transcription Kit and Taqman Universal Master Mix II with the TaqMan MicroRNA Assay of miRNAs (Applied Biosystems, Foster City, USA) were used for testing the miR-144-3P expression level. The level of AQP1 and CASC2 was calculated relative to internal control using the 2−ΔΔCt method using real-time PCR system according to manufacturer’s instructions in SYBR green master mix (Applied Biosystems).
RNA immunoprecipitation (RIP)
RNA immunoprecipitation assay was performed by the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) and the AGO2 antibody according to the manufacturer’s protocol. The AGO2 was detected by immunoprecipitation-western, and RT-qPCR detected CASC2 and miR-144-3p in the precipitates. The IgG antibody group as control.
Western blot analysis
Tissue and cells were collected and lysed in protein lysis buffer. Proteins samples (30 μg) were separated on SDS-12% PAGE and then PAGE transfer onto PVDF membranes (Thermo, USA). Primary mouse monoclonal antibodies against AQP1 and β-actin (Abcam, UK), and secondary antibody peroxidase-conjugated rabbit anti-IgG (Sigma) were used in western blot analysis.
Apoptosis by flow cytometry assay
Annexin V/PI staining was performed according to previous procedures [15]. Cells from different groups were collected, centrifuged at 1500×g for 5 min, and washed with PBS for three times. 1× Annexin V binding buffer was added to make a final concentration of 2 × 105/mL. Annexin-V and PI (propidium iodide) solution (100 μg, 1 μg/ml) were added for staining at room temperature for 15 min, then flow cytometry was used to evaluate cell apoptosis. The apoptotic cells were detected by flow cytometry (FACS 420, BD Biosciences, USA). Percentage of apoptosis rate (%) = (number of apoptotic cells/number of all cells) × 100%.
Luciferase reporter assay
The 3′UTR of AQP1 including conserved binding sites for miR-144-3p was amplified from human cDNA by PCR. A mutant 3′UTR fragment of AQP1 which the mutations was in conserved binding sites for miR-144-3p, was also generated. The fragments including the 3′UTR regions (3′UTR-WT) or mutant 3′UTR regions (3′UTR-Mut) of AQP1 were inserted into vector p-Luc-UTR. Then the miR-144-3p mimic/inhibitor and their respective control were transfected into AQP1-overexpressing A549 cells, respectively. After 24 h, cells were collected, and the firefly luciferase activities were determined using a luciferase reporter assay system (Promega, WI) according to the manufacturer’s instructions.
RNA pull down
The RNA precipitation assay was performed to determine whether CASC2 is coupled with the RISC complex, using synthesized CASC2 as a probe and then detected AGO2 from the precipitation complex using western blot and miR-144-3p using qRT-PCR. The biotin-labeled lncRNA-CASC2 RNA was transcribed in vitro with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), recycled with QIA quick Nucleotide Removal Kit (Qiagen) and purified with the RNeasy Mini Kit (Qiagen). Loc285194 was also cloned as a negative control and used in precipitation experiments for comparison. A549 cell proteins were mixed with biotin-labeled lncRNA-CASC2 RNAs incubated at 4 °C for 1 h. The streptavidin agarose beads (Invitrogen) were added to each binding reaction and incubated at room temperature for 1 h. Western blot was performed to detect AGO2, and the three group of precipitates were used for detecting miR-144-3p expression by RT-PCR according to the standard procedures.
Statistical analysis
The SPSS 17.0 software (SPSS Inc., USA) was applied for statistical analyses. All experiments were repeated three times, and all data were presented as mean ± standard deviation. The differences between groups were assessed by Student’s t test with a significance level of P < 0.05.