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Abstract

LINC00599 has been reported to be upregulated in response to cigarette smoking. However, the effect and underlying mechanism of LINC00599 in chronic obstructive pulmonary disease (COPD) are still under exploration. In this study, LINC00599 was upregulated in the COPD patients and was of clinical value to distinguish COPD patients. COPD cell models were established using 16HBE cells under cigarette smoke extract (CSE) treatment. LINC00599 levels were elevated in a dose and time-dependent way in response to CSE stimulation. The effect of LINC00599 on CSE-induced 16HBE cells was explored. The results showed that LINC00599 deficiency reversed the CSE-induced inhibition on cell viability and proliferation, and rescued the CSE-induced enhancement on cell 16HBE cell apoptosis and inflammation response. Moreover, LINC00599 bound with miR-212-5p to upregulate the BASP1 (brain abundant membrane attached signal protein 1) expression. MiR-212-5p was expressed at a low level in the tissue samples of COPD patients, and its levels were upregulated in LINC00599 silenced cells. BASP1 was targeted by miR-212-5p and its upregulation was identified in the tissue samples of COPD patients and cell models. BASP1 levels were downregulated after miR-212-5p overexpression or LINC00599 silencing. Moreover, the rescue assays demonstrated that BASP1 overexpression reversed the effect of silenced LINC00599 on 16HBE cells after CSE treatment, which indicated that LINC00599 promoted the COPD development by regulating BASP1 expression. In conclusion, LINC00599 facilitated CSE-induced cell apoptosis and inflammation response, while inhibiting the cell viability and proliferation in COPD progression via modulating miR-212-5p/BASP1 axis.

Keywords

LINC00599 Chronic obstructive pulmonary disease(COPD)cigarette smoke extract(CSE)miR-212-5p BASP1

Introduction

Chronic obstructive pulmonary disease (COPD) is a common chronic lung respiratory disease with airflow limitation in the lungs, including chronic bronchitis and emphysema.¹ COPD is characterized by high morbidity and mortality globally. According to the Global Burden of Disease Study 2015, it is estimated that the global prevalence of COPD reached about 174 million cases with over three million death cases.² Risk factors for COPD include tobacco smoking, exposure to dust, harmful chemicals, and fumes from burning fuel, and lack of alpha-1-antitrypsin.³ Strategies such as smoking cessation, bronchodilator therapy, supplemental oxygen, pulmonary rehabilitation, and lung transplantation are recommended for the COPD treatment.⁴ However, the disease is still largely underdiagnosed and undertreated, and the underlying mechanisms in COPD remain to be fully explored.

Long non-coding RNAs (lncRNAs) represent a type of transcripts longer than 200 nucleotides without capacity to code proteins.⁵ LncRNAs also regulate gene expression at various levels, including epigenetic regulation, transcriptional regulation, and post-transcriptional regulation.⁶ The aberrant expression of many lncRNAs has been identified in various lung diseases,^7–9 including COPD.¹⁰ In the competing endogenous RNA (ceRNA) hypothesis, lncRNAs are indicated to regulate the downstream mRNA expression by competitively binding with miRNAs.¹¹ Numerous lncRNAs have been reported to act as ceRNAs in the pathogenesis of COPD. For example, lncRNA MIR155HG facilitates apoptosis and inflammation of cigarette smoke extract-induced human pulmonary microvascular endothelial cells in COPD by binding with miR-128-5p to upregulate BRD4.¹² OIP5-AS1 acts as a ceRNA against miR-410-3p to regulate IL-13 expression, enhancing the viability, and inhibiting the apoptosis as well as the inflammation response in cigarette smoke extract-treated16HBE cells in COPD.¹³ LncRNA MIAT promotes cigarette smoke-induced airway remodeling by binding with miR-29c-3p to regulate HIF3A.¹⁴ A previous study indicates that LINC00599 is differentially expressed lncRNA between current and former smokers,¹⁵ while its role in COPD remains unknown.

In this study, we aimed to investigate the biological function and underlying mechanisms of LINC00599 in COPD. We assumed that LINC00599 acted as a ceRNA by binding with miR-212-5p to upregulate BASP1 expression. The cigarette smoke extract-induced16HBE cells were used to mimic COPD in vitro. The findings of our study may provide clues for the targeting therapy for COPD patients.

Materials and methods

Clinical sample collection

The lung tissues were obtained from 90 patients with a solitary non-small cell lung cancer in Shanghai Pulmonary Hospital. Patients were divided into non-smokers (n = 30, no COPD), smokers (n = 30, no COPD) and smokers (n = 30, COPD). The patients were diagnosed with COPD following the Global Initiative for Chronic Obstructive Lung Disease (GOLD).¹⁶ Fresh tissue specimens were embedded in paraffin or snap-frozen, and maintained at −80°C. We have obtained the written informed consents from all participants. The study has been approved by the Ethics Committee of Shanghai Pulmonary Hospital.

Cell culture and treatment

The normal human bronchial epithelial cell line 16HBE provided by the American Type Culture Collection (ATCC, Manassas, VA, USA) were incubated in RPMI-1640 medium (HyClone) added with 1% penicillin/streptomycin in a humidified incubator with 5% CO₂ at 37°C. For the establishment of COPD cell models, different concentrations of cigarette smoke extract (CSE, 0.5%, 1%, 2%, 4%) were used to stimulate the 16HBE cells for 24 h or 2% CSE for different durations (0, 12, 24, 36, 48 h) as previously described.¹⁷ The same dose of PBS was used to treat the control groups.

Cell transfection

To knock down LINC00599, short hairpin RNAs against LINC00599 (sh-LINC00599) and sh-NC (negative control) were used. For miR-212-5p overexpression, miR-212-5p mimics and the NC mimics (negative controls) were constructed. For BASP1 overexpression, the pcDNA3.1/BASP1 was constructed and the empty pcDNA3.1 vectors were used as the negative controls. All plasmids were provided by the obtained RiboBio Co., Ltd. (Guangzhou, Guangdong, China) and transfected into the cells using the Lipofectamine® 3000 (Invitrogen).

RT-qPCR

The RNAs in the lung tissues of patients in indicated groups and 16HBE cells were collected using TRIzol reagent (Takara, Japan). Then a reverse reaction kit (Promega, Madison, WI, USA) was applied for RNA reverse transcription. Then PCR analysis was conducted using a SYBR Premix Ex Taq™ II kit (Takara) on an ABI 7500 Real-Time PCR System (Applied Biosystems). The 2^−ΔΔCt method was applied to calculate the relative levels of LINC00599, miR-212-5p and BASP1 normalized to GAPDH or U6. The primer sequences are presented as following: LINC00599: F, 5′-CTCTCATCCTCTGTCTCGC-3′, R, 5′-GTCCTCTCGCAGGTAAAGG-3′; miR-212-5p: F, 5′-ACCTTGGCTCTAGACTGCT-3′, R, 5′-GCAGGGTCCGAGGTATTC-3′; BASP1: F, 5′-GAATTCAGGAACTCCAAGATGG-3′, R, 5′-TCTCCTTGGCTTTCTCGTC-3′; GAPDH: F, 5′- CCTCCTGTTCGACAGTCAG-3′, R, 5′-CATACGACTGCAAAGACCC-3′; U6: F, 5′- CTTTGGCAGCACATATACCA-3′, R, 5′-CTCATTCAGAGGCCATGCT-3′.

Western blot

Protein extraction was conducted with RIPA buffer (Sigma). Then a BCA protein assay kit (Thermo Fisher Scientific, Inc.) was used to determine protein concentration. Then the protein (30 μg) was loaded on 12% SDS-PAGE before being transferred onto polyvinylidene fluoride membranes (Bio-Rad, USA). Next, the 5% skim milk was used to block the membranes which were then cultured overnight at 4°C with primary antibodies, including anti-BASP1 (#PA5-78,320, 1:1000, Invitrogen), anti-IL-6 (#P620, 1:1000, Invitrogen), anti-TNF-α (#ab215188, 1:1000, Abcam), anti-IL-1β (#ab216995, 1:1000, Abcam), followed by TBST washing and addition of corresponding secondary antibodies and incubation for 2 h in dark. ECL reagents (CoWin Biosciences, China) were used to visualize the protein bands, and the band density was calculated with the ImageJ software. GAPDH was used for normalization

Cell viability

After the indicated transfections, 16HBE cells were cultured in a 96-well plate at 5 × 10³ cells/well for 48 h. Then CCK-8 reagent was incubated with the plate at 37°C for 2 h. The viability was measured using a microplate reader (Bio-Rad, USA) under 450 nm wavelength.

Cell proliferation

The 16HBE cells after indicated transfection were seeded in 6-well plates (5 × 10³ cells/well) for 2 weeks. After washing twice with PBS, the cells were fixed with methanol for 20 min, and dyed with crystal violet for another 20 min. Five randomly chosen visual fields were photographed by an inverted microscope to manually count the number of colonies.

Flow cytometry analysis

Following indicated transfection, 16HBE cells were assessed with an Annexin V-FITC/PI Apoptosis Detection kit (BD Biosciences) in accordance with the manufacturer’s protocol. Brifely, 16HBE cells were harvested and stained using 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) for 15 min at room temperature in dark. Cell apoptosis was detected by a FACS-calibur Flow Cytometry (BD Biosciences, USA).

Enzyme-linked immunosorbent assay

The inflammation response of 16HBE cells to CSE treatment was determined by the secretion levels of IL-6, tumor necrosis factor (TNF)-α, and IL-1β using an ELISA assay as previously described.¹⁸ The concentration of these inflammatory cytokines in the supernatants of 16HBE cells was measured by the ELISA kits (Cusabio Biotech, China).

Luciferase reporter assay

The LINC00599 sequences or the BASP1 3′UTR fragments with potential binding sites of miR-212-5p was cloned into the pmirGLO vectors for the construction of wild-type LINC00599 (LINC00599-WT) or BASP1 (BASP1-WT) vectors, respectively. The mutant LINC00599 (LINC00599-MUT) or BASP1 (BASP1-MUT) vectors were constructed via directed-mutation by Genepharma (Shanghai, China). Then Lipofectamine 2000 was used for the cotransfection of these plasmids with miR-212-5p mimics or NC mimics (negative control) into the 16HBE cells. The relative luciferase activity was measured with the Dual Luciferase Reporter Assay System (Yeasen, Shanghai, China).

RNA pull down

LINC00599 was biotinylated to create bio-LINC00599 by GenePharma, and incubated with the cell lysates of 16HBE added with M-280 streptavidin magnetic beads (Sigma) at room temperature for 30 min. After washing the cells with buffer for three times, the target RNAs enriched in the complex were eluted and then detected using RT-qPCR.

RNA-binding protein immunoprecipitation assay

A RIP kit (Sigma) was used to explore the interaction among LINC00599, miR-212-5p and BASP1 following manufacturer’s instructions. The 16HBE cells were harvested and lysed, and protein A/G beads (40 μL) were added and cocultured with the cell lysate at 4°C for 1 h for the complex. Finally, the RNA was separated and subject to RT-qPCR analyses for quantification.

Immunohistochemistry assay

The sectioned lung tissues were embedded in paraffin at 4 μm, then baked, dewaxed with xylene, and subject to alcohol treatment. Then water washing for 30 min, and the sodium citrate repair solution (PH = 6.0, 0.01 M) repaired for 10 min using medium fire. After the treatment using hydrogen peroxide solution (3%) for 15 min, 1% FBS was used to seal the sections 30 min, followed by coincubation with the primary anti-BASP1 antibody (#PA5-78,320, 1:1000, Invitrogen) overnight at 4°C. Subsequently, the secondary antibodies were supplemented to incubate for 1 h. After DAB staining for 5 min, the alcohol was used to dehydrate sections which were then blocked with neutral gum. The images were photographed using a microscope.

Statistical analysis

All data are presented as the mean ± SD of three independent experiments and were analyzed using SPSS version 19.0 (IBM Corp.). Student t-test was used to analyze the difference between two groups and one-way analysis of variance was used to determine the difference among multiple groups. The clinical significance of LINC00599 was predicted with the ROC curve. p < 0.05 was regarded as statistical significance.

Results

The expression and prognostic value of LINC00599 in COPD patients

According to the results of RT-qPCR analysis, LINC00599 was highly expressed in the tissue sections of smoker than non-smoker, especially in COPD smoker (Figure 1(a)). The area under the curve (AUC) was applied for the exploration of the prognostic value of LINC00599 in COPD. AUC of 0.8961 showed that LINC00599 may be an indicator to distinguish COPD patients (Figure 1(b)). Moreover, we established COPD cell models with cigarette smoke extract (CSE) stimulation. The levels of LINC00599 were increased after CSE stimulation as the concentration and treatment time increased (Figures 1(c) and (d)). COPD cell models were used with 2% CSE treatment in the following assays.

Figure 1.

The expression and prognostic value of LINC00599 in COPD patients. (a) LINC00599 expression in the lung tissues of non-smokers (n = 30, no COPD), non-COPD smokers (n = 30) and COPD smokers (n = 30). (b) The diagnostic value of LINC00599 for distinguishing COPD patients. (c) The LINC00599 levels in 16HBE cells after various CSE stimulation (0, 0.5, 1, 2, 4%). (d) LINC00599 levels after treatment with 2% CSE for 0, 12, 24, 36, 48 h. *p < 0.05, **p < 0.01, ***p < 0.001.

The effect of LINC00599 knockdown on CSE-stimulated 16HBE cells

The knockdown efficiency of LINC00599 was detected in 16HBE cells without CSE treatments. The results of RT-qPCR analysis showed that the LINC00599 expression was significantly decreased after the transfection of sh-LINC00599 (Figure 2(a)). In COPD cell models, LINC00599 expression was significantly reduced after transfection of sh-LINC00599 (Figure 2(b)). The viability of 16HBE cells showed significant decrease after the CSE treatment, which was partially reversed by LINC00599 knockdown (Figure 2(c)). Colony formation assay revealed that the CSE-induced reduction in colony numbers of 16HBE cells was rescued by LINC00599 deficiency (Figure 2(d)). According to flow cytometry analysis, the apoptosis rate of CSE-treated 16HBE cells was significantly elevated in comparison with the control group, which was offset by silenced LINC00599 (Figure 2(e)). Then the impact of LINC00599 on the inflammation response of 16HBE cells was investigated. The levels of inflammation cytokines (IL-6, TNF-α, IL-1β) were elevated after the CSE treatment compared with the control group, and showed a reduction in the CSE + sh-LINC00599 group in comparison with the CSE + sh-NC group (Figure 2(f)).

Figure 2.

The impact of LINC00599 knockdown on COPD cell models. (a) LINC00599 levels in 16HBE cells after indicated transfection. (b) LINC00599 levels in 16HBE cells with CSE treatments after the transfection of sh-NC or sh-LINC00599. (c) The 16HBE cell viability under CSE treatment alone or in combination with the transfection of sh-NC or sh-LINC00599. (d) Colony formation assays were conducted to determine the cell proliferation after indicated treatments. (e) The apoptosis rate of CSE-treated 16HBE cells transfected with sh-LINC00599 was subject to flow cytometry. (f) The levels of inflammation cytokines in 16HBE cells after indicated treatments were measured using western blot and ELISA assays. *p < 0.05, **p < 0.01, ***p < 0.001.

LINC00599 bound with miR-212-5p

The downstream regulatory mechanism of LINC00599 was further explored. The subcellular fractionation assay was conducted to detect the cellular localization of LINC00599. LINC00599 was largely distributed in the cell cytoplasm (Figure 3(a)). We selected the potential miRNAs with binding sites to LINC00599 based on the starBase database. Seven potential miRNAs were screened out and subject to RNA pull down assays (Figures 3(b) and (c)). The results showed that only miR-212-5p was abundantly enriched in the complex of bio-LINC00599 compared with the bio-NC groups (Figure 3(c)). Moreover, miR-212-5p downregulation was identified in smoker patient tissues in comparison with the non-smoker, and expressed at a lower level in COPD smokers than non-COPD smokers (Figure 3(d)). Then expression levels of miR-212-5p in the CSE-treated 16HBE cells were detected and showed downregulation in 16HBE cells as the CSE concentration and treatment time (Figures 3(e) and (f)). The starBase predicted the binding sequence between LINC00599 and miR-212-5p (Figure 3(g)). The overexpression efficiency of miR-212-5p was identified using RT-qPCR analysis (Figure 3(h)). Luciferase reporter assay revealed that the relative luciferase reporter activity of LINC00599-WT was significantly reduced after miR-212-5p overexpression. In the LINC00599-MUT groups, the relative luciferase reporter activity was not significantly changed (Figure 3(i)). Moreover, we also detected the effect of LINC00599 silencing on the levels of miR-212-5p in 16HBE cells. The result showed that LINC00599 downregulated miR-212-5p in 16HBE cells (Figure 3(j)).

Figure 3.

LINC00599 bound with miR-212-5p. (a) The cellular localization of LINC00599 in 16HBE cells was detected by subcellular fractionation assay. (b) Seven potential miRNAs with binding sites to LINC00599 were predicted by starBase database (https://starbase.sysu.edu.cn/). (c) RNA pull down assays was performed to explore the binding relationship of LINC00599 and the seven candidate miRNAs. (d) MiR-212-5p levels in non-smokers (n = 30, no COPD), non-COPD smokers (n = 30) and COPD smokers (n = 30). (e) MiR-212-5p levels in 16HBE cells under treatment og various concentrations of CSE (0, 0.5, 1, 2, 4%). (f) MiR-212-5p levels in 2% CSE stimulated 16HBE cells for different time durations. (g) The binding sequence between LINC00599 and miR-212-5p was obtained from the starBase. (h) The miR-212-5p upregulation efficiency was measured with RT-qPCR analysis. (i) Luciferase reporter assay was applied for the binding relationship detection of LINC00599 and miR-212-5p. (j) The levels of miR-212-5p in 16HBE cells transected with sh-NC or sh- LINC00599. *p < 0.05, **p < 0.01, ***p < 0.001.

BASP1 was directly targeted by miR-212-5p

We further explored the downstream targets for miR-212-5p. Based on the prediction on starBase, seven mRNAs were selected with CLIP-Data ≥5, Degradome-Data ≥3, program: miRmap, TargetScan (Figure 4(a)). Only BASP1 exhibited significant downregulation after transfecting miR-212-5p mimics in 16HBE cells (Figure 4(b)). BASP1 was upregulated in the smokers and most highly expressed in COPD smokers (Figure 4(c)). Immunohistochemistry further demonstrated that the elevated levels of BASP1 from non-smokers, non-COPD smokers and COPD smokers (Figure 4(d)). We also detected the levels of BASP1 in COPD cell models. As shown in Figures 4(e) and (f), BASP1 levels were increased as the concentration and treatment time of CSE increased. The binding sequence of BASP1 and miR-212-5p was shown in Figure 4(g). Moreover, miR-212-5p overexpression significantly reduced the luciferase reporter activity of BASP1-WT, while no effect was observed on the BASP1-MUT group (Figure 4(h)). According to the results of RIP assays, LINC00599, miR-212-5p and BASP1 were all significantly enriched in the precipitates of anti-AGO2, which indicated that LINC00599, miR-212-5p and BASP1 were coexisted in the RNA-induced silencing-complex (RISC) (Figure 4(i)). The effect of LINC00599 on BASP1 expression was detected and LINC00599 downregulated BASP1 in 16HBE cells (Figure 4(j)).

Figure 4.

BASP1 was a target gene of miR-212-5p. (a) StarBase predicted seven downstream targets of miR-212-5p with CLIP-Data≥5, Degradome-Data≥3, program: miRmap, TargetScan. (b) The levels of selected mRNAs in miR-212-5p overexpressed 16HBE cells. (c-d) RT-qPCR and IHC were used to detect BASP1 levels in the tissue samples of non-smokers (n = 30, no COPD), smokers (n = 30, no COPD) and smokers with COPD (n = 30). (e) BASP1 expression in 16HBE cells. (f) The levels of BASP1 in 16HBE cells treated with 2% CSE for different time periods. (g) The binding site between BASP1 and miR-212-5p. (h) The interaction of BASP1 and miR-212-5p was explored using luciferase reporter assays. (i) RIP assay was conducted to further investigate the interaction of LINC00599, miR-212-5p and BASP1. (j) The BASP1 expression in LINC00599 downregulated 16HBE cells. *p < 0.05, **p < 0.01, ***p < 0.001.

LINC00599 promoted COPD progression by regulating BASP1

The rescue assays were conducted for the impact of BASP1 on LINC00599 silencing mediated inhibition on COPD. The BASP1 upregulation efficiency was confirmed by RT-qPCR and western blot (Figures 5(a) and (b)). Then we detected BASP1 levels after LINC00599 knockdown and BASP1 overexpression. The BASP1 mRNA and protein level showed reduction after the LINC00599 silencing, and showed an increase after BASP1 overexpression (Figures 5(c) and (d)). LINC00599 knockdown also enhanced the viability and proliferation of CSE-treated 16HBE cells, which was rescued by BASP1 upregulation (Figures 5(e) and (f)). Moreover, the LINC00599 knockdown-induced inhibition on cell apoptosis after BASP1 upregulation (Figure 5(g)). The levels of inflammation cytokines (IL-6, TNF-α, IL-1β) were elevated after LINC00599 knockdown and BASP1 overexpression, and inflammation response was reversed after BASP1 overexpression (Figure 2(h)).

Figure 5.

LINC00599 promoted COPD progression by regulating BASP1. (a-b) The BASP1 overexpression efficiency was determined using RT-qPCR and western blot. (c-d) The mRNA and protein expression of BASP1 in 16HBE cells after indicated transfection. (e-f) The viability and proliferation of 16HBE cells after the transfection of sh-NC, sh-LINC00599 and sh-LINC00599 + BASP1. (g-h) The apoptosis and inflammation response of 16HBE cells after indicated transfections. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

COPD is characterized by partially reversible airflow obstruction, airway inflammation, and other systemic effects.¹⁹ In recent years, the important role of chronic pulmonary inflammation in the development of pulmonary hypertension has been studied.²⁰ When complications occur in patients with COPD, it will lead to heart failure, corpulmonale, and so on.²¹ Therefore, it is urgent to study the molecular mechanism of COPD.

There is increasing evidence that lncRNAs are involved in the pathogenesis of COPD through various signals. For instance, LASI lncRNA may represent a novel target to control the smoke-mediated dysregulation in airway responses and COPD exacerbations.²² lncRNA GAS5 promotes pyroptosis in COPD by functioning as a ceRNA to regulate the miR-223-3p/NLRP3 axis.²³ Many studies have also confirmed the expression profile of lncRNA in the lung tissues of non-smokers and smokers, with or without COPD,²⁴ which will help to find early biomarkers and treatment targets associated with COPD.¹³

Differential gene expression of LINC00599 has been reported in current smokers as compared to former smokers, and up-regulation of LINC00599 was indicated in response to cigarette smoking.¹⁵ The current study demonstrated that LINC00599 levels were conspicuously increased in smokers, particularly with COPD, which provided that LINC00599 may be used as an early biomarker of COPD occurrence and development. Besides, the AUC of 0.8961 indicated that LINC00599 was of prognostic value to distinguish COPD patients.

In order to provide theoretical guidance for the early diagnosis of COPD, this paper discusses the influence mechanism of LINC00599 on the development of COPD. The expression of LINC00599 was raised as CSE treatment concentration and time increased in 16HBE cells. The CSE treatment significantly inhibited the viability and proliferation potential of 16HBE cells while promoting cell apoptosis. A previous study has reported that lncRNA RNCR3 facilitates the proliferation and secretion of inflammatory cytokines such as IL-6, IL-1β, and TNF-α in endothelial cells by binding with miR-185-5p to regulate cyclin D2 expression.²⁵ In our study, we also found that LINC00599 silencing reversed the CSE-induced increase in the secretion of inflammation cytokines, including TNF-α, IL-6 and IL-1β.

As far as we know, lncRNA can inhibit the expression of miRNA to regulate gene expression and regulate physiological and pathological processes.²⁶ In our investigation, based on bioinformatics analysis, miR-212-5p was screened and verified to bind with LINC00599 in 16HBE cells. As an important member of the miR family, miR-212-5p has been reported to be dysregulated in a variety of human diseases, such as non-alcoholic fatty liver disease,²⁷ nasopharyngeal carcinoma,²⁸ and ovarian cancer.²⁹ For example, circ_0,099630 curbed periodontal ligament cells proliferation and osteogenic differentiation through elevating SPRY1 expression via sponging miR-212-5p in periodontitis.³⁰ Besides, miR-212-5p attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents.³¹ Interestingly, miR-212-5p downregulation has been identified in COPD patients and inhibited the CSE-induced increase in the expression of inflammation and COPD related genes in NCI-H292 cells, suggesting that miR-212-5p may serve as a potential therapeutic target for COPD.³² In the present study, we demonstrated that the expression of miR-212-5p was remarkably decreased in COPD smokers, suggesting that miR-212-5p might play a significant role in the progression of COPD. In the COPD cell models, the miR-212-5p expression showed decrease as the concentration of CSE increased, while the absence of LINC00599 reversed the restricted miR-410-3p expression in 16HBE cells. The interaction of LINC00599 and miR-212-5p has also been verified and miR-212-5p was revealed to be negatively modulated by LINC00599.

In the current publication, BASP1 was predicted as a downstream gene of miR-212-5p, and verified by luciferase and RIP. Besides, BASP1 overexpression was demonstrated to be upregulated in COPD patients and its expression was elevated as the CSE concentration and treatment time increased. BASP1, the downstream target for miR-212-5p, has been indicated to be critically implicated in the progression of lung diseases. Previous studies suggest that high BASP1 levels are related to poor prognosis and facilitates the development of human lung adenocarcinoma.³³ The interaction of BASP1 and EGFR promotes EGFR signaling in brain metastatic lung cancer and serves as targeting positive feedback between BASP1 and EGFR as a therapeutic strategy for lung cancer.³⁴ Mechanistically, BASP1 overexpression rescued the impact exerted by silenced LINC00599 on cell proliferation, apoptosis and inflammation.

In conclusion, LINC00599 was upregulated in COPD patients and CSE-treated 16HBE cells. LINC00599 facilitated the COPD progression in vitro by binding with miR-212-5p to modulate BASP1. Considering LINC00599 is associated with cell apoptosis and inflammation in COPD patients, it may be used as a biomarker to predict the prognosis of the patients, and the response to the treatment, and it may also be a potential targeting therapy for COPD.

Footnotes

Acknowledgements

We appreciate technical support from Shanghai Pulmonary Hospital, Tongji University School of Medicine (Shanghai, China).

Author contributions

Liyun Xu and Zhiyi Dong made majority contribution to the conception of this study, carried out all of experiments and analysed the data, Liyun Xu prepared the first draft of manuscript and Zhiyi Dong agreed the final design of this work and revised this manuscript critically. All authors have read and approved the final manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Data availability

The datasets used in our work are available from all the corresponding authors on reasonable request.

ORCID iD

Zhiyi Dong

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LINC00599 influences smoke-related chronic obstructive pulmonary disease and regulates CSE-induced epithelial cell apoptosis and inflammation by targeting miR-212-5p/BASP1 axis

Abstract

Keywords

Introduction

Materials and methods

Clinical sample collection

Cell culture and treatment

Cell transfection

RT-qPCR

Western blot

Cell viability

Cell proliferation

Flow cytometry analysis

Enzyme-linked immunosorbent assay

Luciferase reporter assay

RNA pull down

RNA-binding protein immunoprecipitation assay

Immunohistochemistry assay

Statistical analysis

Results

The expression and prognostic value of LINC00599 in COPD patients

The effect of LINC00599 knockdown on CSE-stimulated 16HBE cells

LINC00599 bound with miR-212-5p

BASP1 was directly targeted by miR-212-5p

LINC00599 promoted COPD progression by regulating BASP1

Discussion

Footnotes

Acknowledgements

Author contributions

Declaration of conflicting interests

Funding

Data availability

ORCID iD

References