- Open Access
Long non-coding RNA CASC2 improved acute lung injury by regulating miR-144-3p/AQP1 axis to reduce lung epithelial cell apoptosis
© The Author(s) 2018
- Received: 5 September 2017
- Accepted: 20 January 2018
- Published: 26 February 2018
Background and objective
Apoptosis of lung epithelial cell is implicated in the pathogenesis of acute lung injury (ALI). To study the protective effect and mechanism of cancer susceptibility candidate 2 (CASC2) on reducing lung epithelial cell apoptosis after LPS inducing acute lung injury in mice.
Methods and results
The ALI mice model was performed by intratracheally instilling with lipopolysaccharide (LPS). The CASC2 expression detected by quantitative real-time polymerase chain reaction was significantly decreased in LPS-induced A549 cell and ALI mice model. LPS induced A549 cell apoptosis, while transfection with pcDNA-CASC2 reversed the increased cell apoptosis, suggesting overexpression of CASC2 inhibited LPS-induced A549 cell apoptosis. In addition, we found that miR-144-3p expression were opposite to CASC2, while Aquaporin-1 (AQP1) expression was opposite to miR-144-3p in LPS-induced A549 cell and ALI mice model. The RNA immunoprecipitation and RNA pull-down assay demonstrated that CASC2 could function as a miR-144-3p decoy. The luciferase reporter assay revealed that AQP1 was a target of miR-144-3p in A549 cell. And then, further in vitro studied showed that CASC2 controlled AQP1 expression by regulating miR-144-3p, and LPS induced A549 cell apoptosis by regulating CASC2/miR-144-3p/AQP1 axis. At last, after injection with lentivirus-expressing CASC2 or control lentivirus, the mice were intratracheally instilled with LPS. Comparing to the mice injected with pcDNA, the mice injected with pcDNA-CASC2 had a significantly reduced lung wet–dry weight ratio.
Long non-coding RNA CASC2 improved acute lung injury by regulating miR-144-3p/AQP1 axis to reduce lung epithelial cell apoptosis.
- lncRNA CASC2
- Acute lung injury
- Lung epithelial cells apoptosis
Acute lung injury (ALI) is a life-threatening syndrome that can cause acute hypoxemic respiratory failure, which is characterized by diffuse alveolar damage, increased alveolar capillary membrane permeability, edema, excessive pulmonary inflammation and apoptosis of alveolar epithelial cells . Studies have shown that excessive apoptosis of type II alveolar epithelial cells can injury the epithelial barrier thus lead to ALI [2, 3]. Lipopolysaccharide (LPS) is the component of the cell wall of Gram-negative bacteria, which is closely related to alveolar epithelial cell apoptosis [4–6]. Thus, we used LPS to induce apoptosis of alveolar epithelial cells.
Aquaporin-1 (AQP1) is protein that functioned as water transport across cell membranes that mainly expressed in alveolus capillary endothelial cells. A number of studies have shown that AQP1 might play an important role in the pathogenesis of ALI, and the decreased lung AQP1 expression was closely related to the development of ALI, while the upregulated the expression of AQP1 could ameliorate ALI [7, 8].
Recent evidence showed that microRNAs (miRNAs) played an important regulatory role in the development of ALI [9, 10]. For example, Tao  and Tang  reported that overexpression of miR-454 and miR-126-5p may alleviate ALI. Li et al.  found that miR-181a inhibition can significantly protected mice from LPS-induced ALI. These studies indicated that miRNAs might be a potential candidate for the therapy against ALI. MiR-144-3p was a tumor suppressive miRNA in many cancer cells [13, 14], while the expression and the role of miR-144-3p in LPS-induced ALI and lung epithelial cells has not been reported. In addition, bioinformatics software predicted AQP1 was one of the putative target genes of miR-144-3p. Thus, this study raised a question regarding whether miR-144-3p participated in LPS-induced ALI and lung epithelial cells apoptosis by regulating AQP1.
Long noncoding RNA cancer susceptibility candidate 2 (CASC2) has been demonstrated as playing crucial regulatory role in many cancers, such as lung adenocarcinoma, thyroid carcinoma, and hepatocellular carcinoma, etc. However, the expression of CASC2 in ALI and molecular mechanisms underlying CASC2-mediated ALI remain unknown. Bioinformatics software predicted that there were binding sites between CASC2 and miR-144-3p. Thus, we speculated that CASC2 might be involved in ALI by regulating miR-144-3p/AQP1 axis to affect lung epithelial cell apoptosis.
In this study, we first assessed the CASC2 expression in LPS-induced ALI mice and A549 cell. Further experiments were conducted to investigate the biological regulation function of CASC2 with respect to the miR-144-3p and AQP1 expression and cell apoptosis. Additionally, mechanism analysis revealed that CASC2 might function as a ceRNA to regulate AQP1 expression by sponging miR-144-3p, thus affecting LPS-induced lung epithelial cell apoptosis and playing a critical role in the pathobiology of ALI.
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 . 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.
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.
Decreased expression of CASC2 in ALI mice
Decreased expression of CASC2 in LPS-induced A549 cell
Overexpression of CASC2 inhibited LPS-induced A549 cell apoptosis
CASC2 functioned as a decoy for miR-144-3p in A549 cell
CASC2 controlled AQP1 expression by regulating miR-144-3p
AQP1 was a target of miR-144-3p
LPS induced A549 cell apoptosis by regulating CASC2/miR-144-3p/AQP1 axis
Overexpression of CASC2 alleviated LPS-induced acute lung injury in mice
ALI is a serious disease with high morbidity and mortality rate. Although there are effective strategies for treatment of ALI, a widely accepted specific pharmacotherapy has not yet established . Conventional research on ALI often involved investigations on gene expression and regulation, specific protein or protein related signaling pathways, etc. The discovery of lncRNA reveals a new insight on the pathogenesis of ALI which will open a new era for lung researchers to develop specific effective therapeutics and diagnostics. A recent study have identified a highly up-regulated mammalian lncRNA, FOXD3-AS1, in the setting of hyperoxia/ROS-induced lung injury . Another study demonstrated that the lncRNA-NANCI was decreased in hyperoxia-induced lung injury mice and involved in the development of immature lung tissues . Lipopolysaccharide (LPS) is one of the major factors that induce acute lung injury and lung endothelial apoptosis. To date, the study of lncRNA on LPS-induced ALI has not been reported. The novel lncRNA CASC2 is located on chromosome 10 in humans and has been characterized as a tumor suppressor in human malignancies . In this study, we found that LPS induced a downregulated CASC2 in mice lung tissue and lung epithelial cell (Figs. 1a and 2a). Overexpression of CASC2 inhibited LPS-induced lung epithelial cell apoptosis (Fig. 3), and reduced lung wet–dry weight ratio in LPS-induced ALI mice (Fig. 8). Thus, CASC2 played an important role in LPS-induced ALI.
Aquaporin-1 water channels are membrane proteins that control the permeability of endothelial and epithelial barriers by facilitating water movement across cell membranes. In addition to be responsible for the high vascular permeability and interstitial fluid pressure in tumors of the brain, colon, breast and pancreas, AQP1 was reported to play a key role in the pathogenesis of ALI. This study revealed that AQP1 expression was decreased in LPS-induced ALI mice lung tissue and lung epithelial cell (Figs. 1c and 2c), and overexpression of AQP1 reduced cell apoptosis, which was identical with those reported in literature [7, 8]. There are a few miRNA, such as miR-320 , miR-666 and miR-708 , which have identified as potential modulator of AQP1. In this study, the miR-144-3p expression was opposite to AQP1 expression in LPS-induced ALI mice lung tissue and lung epithelial cell (Figs. 1b and 2b), and the luciferase reporter assay revealed that AQP1 was a target of miR-144-3p (Fig. 6). Previous study showed that the miR-144-3p expression was increased in A549 cell, and miR-144-3p mimic suppressed cell proliferation . In the present study, we found that miR-144-3p expression was increased in LPS-induced ALI mice lung tissue and A549 cell, and miR-144-3p mimic promoted cell apoptosis. The results presented in current study are in agreement with the previous study. Further, we found that pcDNA-AQP1 reversed the effect of miR-144-3p mimic on cell apoptosis. Thus, miR-144-3p participated in LPS-induced ALI and lung epithelial cells apoptosis by regulating AQP1.
Studies have also shown that lncRNAs functioned as competing endogenous RNAs or as molecular sponges in modulating the concentration and biological functions of miRNAs . For example, Fan et al.  reported the ability of PTCSC3 to bind hsa-miR-574-5p as ceRNA. H19 triggers EMT progression by binding miR-138-5p and miR-200a-3p, antagonizing their functions and leading to the increase of their endogenous targets . LncRNA CASC2 have been demonstrated as playing crucial regulatory roles in a few of cancers, and functioned as endogenous RNA by sponging miRNAs, such as miR-18a , miR-367 , miR-21 , etc., so whether the CASC2 could function as a miR-144-3p decoy was verified by the RNA immunoprecipitation (RIP) and RNA pull-down assay in this study. And then, the mechanism of CASC2 on AQP1 expression was investigated. Co-transfection with pcDNA-CASC2 and miR-144-3p mimic reversed the pcDNA-CASC2-upregulated AQP1 expression (Fig. 5), which fully demonstrated CASC2 controlled AQP1 expression by regulating miR-144-3p. At last, whether CASC2/miR-144-3p/AQP1 axis was involved the LPS-induced lung epithelial cell apoptosis was investigated. The pcDNA-CASC2 transfection reversed LPS-induced cell apoptosis, miR-144-3p mimic again reversed pcDNA-CASC2-reduced cell apoptosis, and pcDNA-AQP1 reversed again the effect of pcDNA-CASC2 combined miR-144-3p mimic on cell apoptosis, which implied LPS induced A549 cell apoptosis through regulating CASC2/miR-144-3p/AQP1 axis.
In summary, the expression of CASC2 was decreased, while miR-144-3p was increased in LPS-induced ALI mice and lung epithelial cell. Further, CASC2 and AQP1 were targets of miR-144-3p and CASC2 can indirectly regulate AQP1 expression via miR-144-3p. Moreover, CASC2 regulated A549 cell apoptosis through regulating miR-144-3p and its target, AQP1. The results concerning CASC2 functionality and molecular mechanisms relevant to LPS-induced ALI provided a new perspective for lncRNA-directed therapeutic target for LPS-induced ALI.
HL conceived and designed the study and drafted the manuscript. RS designed and completed the experiments. HS collected the data and MG analyzed the data. NM interpreted the data. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
Consent for publication
The study was undertaken with the consent of the First Affiliated Hospital of Zhengzhou University.
Ethics approval and consent to participate
The study was approved by ethics committee of the First Affiliated Hospital of Zhengzhou University.
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