LncRNA-CCAT1/miR-152-5p is Involved in CSE-Induced Inammation in HBE Cells Via Regulating ERK Signaling Pathway

Background: Emerging studies have noted that dysregulated long non-coding RNAs (lncRNAs) are implicated in the pathological processes of chronic obstructive pulmonary disease (COPD). LncRNA colon cancer-associated transcript 1 (CCAT1) plays well-dened roles in the inammatory progression. The study aims to gure out the effect and regulatory mechanism of CCAT1 in the cigarette smoke induced inammation in COPD. Methods: The expression levels of CCAT1 and miR-152-3p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The inammatory levels of IL-1β and IL-6 were evaluated by qRT-PCR and enzyme-linked immunosorbent assay (ELISA). Western blot was used for the measurement of ERK1/2, p-ERK1/2 protein levels. Luciferase reporter assay was performed for the target gene analysis. Results: CCAT1 was highly expressed in lung tissues of smokers with COPD compared with non-smokers without COPD samples. In human bronchial epithelial (HBE) cells, cigarette smoke extract (CSE) treatment led to an increase in CCAT1 expression in a dose- and time- dependent manner. Functional experiments showed that knockdown of CCAT1 ameliorated CSE-induced inammation. Mechanistically, CCAT1 directly targeted miR-152-3p, and miR-197-3p overexpression reversed the pro-inammatory effects of CCAT1 on HBE cells. Subsequently, miR-152-3p was found to regulate ERK signaling pathway. PD98059, ERK specic inhibitor, reversed miR-152-3p inhibition mediated inammation in HBE cells. In addition, CCAT1 acted as a sponge for miR-152-3p to positively regulate ERK signaling pathway. Conclusion: Current ndings suggest that CCAT1 promoted inammation by activating ERK signal pathway via sponging miR ‐ 152 ‐ 3p in CSE ‐ treated HBE cells. These results may provide a novel therapeutic target for alleviating cigarette smoke mediated airway inammation.


Background
Cigarette smoke is a complex aerosol consisting of more than 4500 identi ed chemical compounds, which have various toxic, mutagenic and carcinogenic effects [1]. The airway epithelium is the lung's rst line of defense and constitutes an essential protection to inhaled insults. Toxic particles of inhaled smoke induce airway in ammation, which plays an essential role in the occurrence and progress of multiple respiratory diseases, such as chronic obstructive pulmonary disease (COPD), and lung cancer [2]. Understanding the impact of airway in ammation induced by cigarette smoke and which mechanism modulates this process may provide new insight into signaling pathways and indicating novel therapeutic targets to control in ammation.
Long non-coding RNA (lncRNA) are a class of non-coding RNA molecules with a length of > 200 nucleotides that will not be translated into proteins, and they exert their physiological and pathological functions by interacting with genomic DNA, microRNAs (miRNAs), mRNAs, and proteins [3]. LncRNAs have been identi ed as essential regulators in numerous biological processes, such as cell proliferation, differentiation, apoptosis, and in ammatory response [4]. Several studies have recently shown that exposure to cigarette smoke in both humans and rats lead to global alterations in lncRNA expression [5,6], indicating the potential role of lncRNAs as a novel group of targets for the treatment of cigarette smoke-related lung diseases. Colon cancer-associated transcript 1 (CCAT1), a lncRNA of about 11 kb located on chromosome 8q24. 21, was one of the rst lncRNAs that were revealed to play functional roles in the pathogenesis of different types of human cancers, including lung cancer [7]. In cigarette smoke extract (CSE) induced human bronchial epithelial (HBE) cells, CCAT1 expression was signi cantly increased [8]. Current research has revealed that CCAT1 is associated with in ammatory response in intestinal epithelial cells [9]. Moreover, CCAT1 knockdown signi cantly alleviated LPS induced expression of pro-in ammatory factors in skin keratinocyte HaCaT cells. It is therefore rational to propose that CCAT1 might exhibit a role in cigarette smoke-induced airway in ammation.
In the present study, we used HBE cells under in vitro CSE conditions to mimic cigarette smoking insult to reveal the effects and molecular mechanisms of CCAT1 in the cigarette smoke induced airway in ammation. We assessed the expression level of CCAT1 in cell lines and examined its effects on the expression of in ammatory genes. Furthermore, we explored the target genes of CCAT1 and the underlying mechanism of its function. This study will provide a better understanding of the pathogenesis of cigarette smoke induced airway in ammation.

Lung tissue Acquirement
This study was approved by the Institutional Ethics Committee of the Second Xiangya Hospital of Central South University. We recruited consecutive patients from April 2018 to September 2019. Human lung tissue from patients with COPD (n = 10) and controls (n = 10) was obtained from patients undergoing surgery and who gave informed consent. All the 20 patients were enrolled with primary lung cancer of stage 1 and had the surgery of pulmonary segmentectomy or lobectomy. All the patients were clinically stable for 4 weeks without acute pulmonary infection, did not receive chemotherapy half a year before the study, and did not have obstructive atelectasis, metastasis, other pulmonary diseases, and severe diseases in other systems. The lung tissue 5 cm away from tumor margin was used in our studies. The pathological examination con rmed that these samples presented lung structure without metastasis or in ammation.
Cell lines and cell culture HBE cells were cultured in RPMI 1640 (Gibco, C11875500BT) supplemented with 10% fetal bovine serum (Gibco, 10082147), 100U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scienti c, Waltham, MA, USA) in 5% CO2 atmosphere at 37°C. Cells were passaged at 80% con uence and grown to full con uence for the experiments. All experiments were performed in triplicate and repeated independently at least three times. After serum starvation for 24 h, the HBE cells were treated with CSE at the indicated concentrations.
Preparation of CSE CSE was prepared as previously described [10]. Briefy, one non ltered Fu-Rong cigarette (Furong, Changde Cigarette Company, Hunan, China) was burned, and the smoke was passed through 20 ml of phosphate-bufered saline via a vacuum pump. This 100% CSE solution was adjusted to 7.2-7.4 and ltered through a 0.22 m membrane lter to remove large particles and bacteria before use. Then, CSE was diluted with PBS to obtain concentrations of 1%, 2.5%, and 5%. CSE was freshly prepared within the 30 min preceding each experiment.
Quantitative Real-Time polymerase chain reaction (qRT-PCR) Total RNA was isolated and extracted from different groups of lung tissues and cells by adding TRIzol Enzyme-Linked Immunosorbent Assay (ELISA) Following appropriate CSE treatment and indicated transfection, the concentrations of interleukin-1β (IL-1β) and IL-6 from the culture supernatants of HBE cells were assayed using commercial the ELISA kits (Proteintech, Wuhan, China) referring to the manufacturer's protocols. The results represent as the average of three independent replicates.

Dual-Luciferase Reporter Assay
The CCAT1 fragment with the predicted binding site of miR-152-3p was synthesized and cloned into the luciferase reporter gene to form the reporter vector CCAT1-wild-type (CCAT1-Wt). CCAT1-miR-152-3p binding site was mutated as instructed and named CCAT1 mutant (CCAT1-Mut). MiR-152-3p mimic and mimic-NC were co-transfected with CCAT1-Wt or CCAT1-Mut into HEK293T cells, respectively. After 48 h, the relative luciferase activity of each well was measured using the Dual Luciferase Reporter System (Promega, Madison, WI, United States).

Statistical analysis
Quantitative data were presented as mean ± SD taken from at least three independent experiments. For relative gene expression, the mean value of the vehicle control group was de ned as 1 or 100%. SPSS 21.0 (SPSS, Chicago, IL, USA) software was used for statistical analysis. Student's t-test and one-way analysis of variance were used for the comparison of the statistical differences between groups. Tukey's test or Dunnett's T3 test was used for post hoc multiple comparisons according to the homogeneity test of variance. Signi cance was de ned as p < 0.05.

CCAT1 expression was upregulated in lung tissues of COPD patients and CSE-induced HBE cells
To reveal the role of CCAT1 in COPD, we detected their expression levels in COPD patients and HBE cells. Results showed that CCAT1 expression was signi cantly upregulated in smokers with COPD as compared with non-smokers without COPD (Fig. 1A). Then, we detected the expression of CCAT1 in CSE treated HBE cells. After exposing HBE cells to CSE concentrations of 0.0%, 1.0%, 2.5%, and 5.0% for 24 h, the expression CCAT1 was increased in a dose-dependent manner (Fig. 1B). Finally, we incubated HBE cells with 5% CSE, a time-related increase of CCAT1 expression was observed from 6 h (Fig. 1C). These data suggest a potential role for CCAT1 in COPD pathogenesis.

CCAT1 mediates CSE induced in ammation
First, HBE cells were treated with different concentrations of CSE for different time. Gradually elevated expression and secretion of in ammatory factor were observed ( Fig. 2A-2H). Then, we examined the effect of CCAT1 on in ammation with a loss-of-function approach. The cells were transfected with si-CCAT1. The results shown that CCAT1 expression was increased after CSE treatment, whereas this effect was attenuated by si-CCAT1 transfection (Fig. 2I). In ammatory mediators IL-1β and IL-6 levels were upregulated in CSE-induced HBE cells, and these in uences were alleviated by si-CCAT1 (Fig. 2J-2M).

CCAT1 modulates miR-152-3p expression
We predicted the potential target miRNA of CCAT1 by online software StarBase, and found that there were binding sites of miR-152b-3p with CCAT1 (Fig. 3A). Luciferase reporter assay demonstrated that overexpression of miR-152b-3p signi cantly repressed the luciferase activity of CCAT1-Wt but not CCAT1-Mut (Fig. 3B). Next, miR-152-3p expression was further measured using qRT-PCR, which suggested that miR-152-3p expression was reduced in the lung tissues of smokers with COPD compared to non-smokers without COPD (Fig. 3C). In addition, CSE treatment led to a decrease in miR-152-3p expression in HBE cells in a dose-and time-dependent manner (Fig. 3D,3E). We further investigated the effects of CCAT1 upregulation on miR-152-3p expression in HBE cells. The results showed that CCAT1 upregulation reduced miR-152-3p expression (Fig. 3F,3G). These results con rmed that CCAT1 targeted miR-152-3p and negatively regulated its expression.
The proin ammatory effect of CCAT1 in HBE cells was dependent on miR-152-3p First, miR-152-3p mimics were transfected into HBE cells to investigate its role in in ammation. qRT-PCR analysis showed that miR-152-3p mimic signi cantly rescued the decrease of miR-152-3p expression induced by CSE (Fig. 4A). As the miR-152-3p level restored, the expression and secretion of in ammatory factors IL-1β and IL-6 decreased markedly (Fig. 4B-4E). Then, we explored whether miR-152-3p mediated the proin ammatory effect of CCAT1 on CSE treated HBE cells. CCAT1 overexpression vector and miR-152-3p mimic were co-transfected into HBE cells. As shown in the result, when CCAT1 vector was transfected, the level of CCAT1 increased signi cantly compared to CCAT1-NC group (Fig. 4F), meanwhile the in ammatory level increased. MiR-152-3p mimic rescued the decreased level of miR-152-3p induced by CCAT1 overexpression (Fig. 4G), and led to a corresponding reduction of in ammation (Fig. 4H-4K).
These results suggest that CCAT1 regulate in ammation at least partly via targeting miR-152-3p and restoration of miR-152-3p can relieve the pro-in ammatory effect of CCAT1 in HBE cells.

CSE triggers in ammation via extracellular signal-regulated kinase (ERK) signaling pathway
Our previous study has con rmed that ERK signaling pathway was activated in lung tissue of COPD patients [11]. The role of ERK signaling on CSE-induced in ammation in HBE cells was then elucidated. As expected, the expression level of p-ERK1/2 was substantially increased in HBE cells exposed to CSE, while this upregulation was inhibited by speci c ERK1/2 inhibitor, PD98059 (Fig. 5A,5B). After that, the in ammatory level was detected. The results showed that PD98059 signi cantly attenuated CSEtriggered elevation of IL-1β and IL-6 in 16HBE cell (Fig. 5C-5E).

CCAT1 activates ERK signaling pathway via targeting miR-152-3p
Finally, we detected whether CCAT1 could regulate ERK signaling pathway via targeting miR-152-3p. As shown in Fig. 7A and Fig. 7B, ERK signaling pathway was activated by CCAT1 overexpression, while these impacts were attenuated by miR-152-3p mimic in HBE cells, indicating that CCAT1 modulated ERK signaling pathway in an miR-152-3p-dependent manner. Altogether, this study demonstrated that CCAT1 activate ERK signaling pathway via targeting miR-152-3p to promote CSE-induced in ammation, thus involving in COPD process.

Discussion
Cigarette smoking is a major preventable risk factor for COPD. Chronic airway in ammation induced by cigarette smoke exposure was previously reported to be one of the key pathogenic mechanisms. However, the mechanism remains largely unknown, which is also the reason why this experiment is designed. In the current study, we observed that lncRNA CCAT1 was highly expressed in smokes with COPD and in CSE treated HBE cells. Silencing CCAT1 alleviated in ammation in HBE cells, suggesting a potential therapeutic target of CCAT1for CS-induced airway in ammation.
Emerging evidence has suggested that lncRNAs are abnormal expressed in several pulmonary disorders, implying the potential role of lncRNAs in the pathogenesis of these pulmonary diseases [12,13]. Recently, several studies had revealed that lncRNAs were identi ed as a pivotal modulator in the in ammatory process [14,15]. Therefore, research on lncRNA may help improve the diagnosis and treatment of lung in ammatory diseases, such as COPD. Furthermore, previous studies revealed that lncRNAs differentially expressed in lung tissue from non-smokers and smokers without or with COPD [5,16]. As a relatively wellinvestigated lncRNA, CCAT1 has been demonstrated to facilitate cell proliferation and inhibit apoptosis in lung cancer [17]. In addition, CCAT1 expression was upregulated in CSE treated HBE cells. Nonetheless, no further study on the function of CCAT1 in COPD have been studied. In the previous study, we showed that the expression of CCAT1 was obviously increased in COPD patients. Subsequently, we identi ed that CSE promoted CCAT1 expression in a dose-and time-dependent manner in HBE cells. Besides, we explored the effects of CCAT1 on in ammatory response. Results showed that knockdown of CCAT1 attenuated CSE-induced pro-in ammatory effects on HBE cells in vitro. These data suggested that CCAT1 was involved in COPD development and progression.
MiRNAs are a family of small non-coding RNAs with the length of 18-22 nucleotides. They can regulate the expression of one or more genes after transcription and participate in various physiological and pathological processes, which are closely related to the COPD [18]. Accumulating evidence has indicated that lncRNAs can play the role of endogenous miRNA sponge, and then inhibit miRNA expression, thereby modulating a variety of cellular biological activities [19]. In this study, we predicted that there were regulatory sites between miR-152-3p and CCAT1 through online software StarBase. Then we conducted a series of experiments to further con rm that CCAT1 could directly bind to miR-152-3p and negatively modulated its expression in HBE cells. Previous study has shown that miR-152 overexpression attenuated doxorubicin-induced in ammation, whereas miR-152 knockdown resulted in in ammation [20], suggesting a pro-in ammatory effect of miR-152. However, the role of miR-152 in COPD has never been investigated. Here, we found that the miR-152-3p abundance was decreased in the lung tissues of smoker with COPD. In CSE treated HBE cells, we also observed a decrease in miR-152-3p expression accompanied with increased in ammatory factor levels, while upregulation of miR-152-3p reduced CSEinduced in ammatory cytokines release. Moreover, miR-152-3p overexpression reversed the in uence of CCAT1 on in ammatory response in CSE-exposed HBE cells. These results indicate that CCAT1 might promote COPD development by sponging miR-152-3p.
ERK is a subclass of the mitogen-activated protein kinase family that plays critical roles in cellular signal transduction. The activated ERK was translocated to the nucleus and phosphorylates multiple substrates to regulate the activity of transcription factors and produce cellular effect [21]. It has been reported that ERK signaling pathway functions as a key mediator of various physiological processes, including in ammatory responses [22]. Besides, previous study has con rmed that miR-152 could inhibit cell proliferation, survival and migration via deactivating ERK signaling pathway [23]. Thus, we hypothesize that miR-152 may regulate CSE induced in ammation via modulating ERK signaling pathway. In the present study, we veri ed that ERK signaling pathway was activated after CSE exposure, and the inhibition of ERK signaling pathway reduced CSE-stimulated in ammation in HBE cells. Moreover, it was proved that overexpression of miR-152-3p exerted its anti-in ammatory effect on HBE cells by inhibiting ERK signaling pathway. Additionally, it is also demonstrated that CCAT1 could regulate ERK signaling pathway by sponging miR-152-3p in HBE cells.
Taken together, our study suggested that CCAT1 was overexpressed in lung tissues of smokers with COPD and in CSE treated HBE cells. CCAT1 knockdown alleviated CSE-induced in ammation in HBE cells by down-regulating ERK signaling pathway through sponging miR-197-3p. These results showed that CCAT1 acts as a potential therapeutic target for further treatment of COPD. Availability of data and materials All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests. Authors' contributions Every author contributed to the reviewing of the paper. ZDD and LXM performed the laboratory work, statistical analyses, and drafted the manuscript. LJH and LYJ performed part of the laboratory work and statistical analyses. OYRY supervised the study and helped to revise the manuscript. CY directed and corrected this research as corresponding author. All authors read and approved the nal manuscript. Figure 1 CCAT1 is highly expressed in smokers with COPD and in CSE-induced HBE cells. (A) qRT-PCR assay was used to detect the expression level of CCAT1 in lung tissues of non-smokers without COPD (control group) and smoker with COPD (COPD group). (B, C) The expression level of CCAT1 was measured by qRT-PCR assay in HBE cells treated with CSE at different concentrations for 24 h or exposed to 5% CSE for indicated times. Data shown are mean±SD and from three independent experiments. *P<0.05, **P<0.01.