MicroRNA-101-3p Suppresses mTOR and Causes Mitochondrial Fragmentation and Cell Degeneration in COPD

Background Cigarette smoke is assumed to cause the loss of airway wall structure in chronic obstructive pulmonary disease (COPD) by reducing airway smooth muscle cell (ASMC) function. It also modifies mTOR activity, microRNA (miR)-101-3p expression, and mitochondria function. Here, the link between miR-101-3p and mTOR-regulated mitochondria integrity and ASMC deterioration was assessed. Methods Disease-specific miR-101-3p expression was determined by RT-PCR in primary ASMC (non-COPD smokers: n = 6; COPD: n = 8; healthy: n = 6). The regulatory effect of miR-101-3p modification on mTOR expression, mitochondrial fragmentation, and remodeling properties (α-SMA, fibronectin, MTCO2, and p70S6 kinase) was assessed in ASMC (healthy nonsmokers: n = 3; COPD: n = 3) by Western blotting and immunofluorescence microscopy. MiR-101-3p was modified by specific mimics or inhibitors, in ASMC stimulated with TNF-α (10 ng/ml) or cigarette smoke extract (CSE). Results MiR-101-3p expression was significantly higher in ASMC of COPD patients, compared to ASMC of healthy or active smokers. MiR-101-3p expression was increased by TNF-α or CSE. TNF-α or miR-101-3p deteriorated ASMC and mitochondria, while decreasing mTOR signaling, α-SMA, fibronectin, and MTCO2. MiR-101-3p inhibition reduced ASMC deterioration and mitochondrial fragmentation. Conclusion Constitutive high miR-101-3p expression characterizes COPD-ASMC, causing increased mitochondrial fragmentation and ASMC deterioration. Thus, reactivation mTOR or blocking miR-101-3p presents a potential new strategy for COPD therapy.


Introduction
Chronic obstructive pulmonary disease (COPD) was the major cause of death of all chronic infammatory lung diseases in 2017 [1]. According to this latest analysis, the major cause of COPD is cigarette smoking and inhalation of fne dusts. COPD is characterized by irreversible, fxed airfow limitation, and declining forced expiratory volume in 1 second (FEV1). Additional factors such as air pollution, genetic or epigenetic predisposition, and chronic airway infections contribute to the severity of COPD [2,3]. With the progression of the disease, chronic lung infammation drives irreversible lung tissue deterioration and tissue remodeling in the small airways, leading to emphysema and chronic bronchiolitis [4].
Tumor necrosis factor alpha (TNF-α) plays a crucial role in the pathogenesis of COPD by modifying the function of lung resident tissue forming cell types, including epithelial cells, fbroblasts, and airway smooth muscle cells (ASMC), as well as immune cells [5]. Increased circulating TNF-α levels were reported in COPD patients [6]. High serum TNF-α levels correlated with the exacerbation frequency, GOLD staging (Global Initiative for Chronic Obstructive Lung Disease), and prediction of FEV1 decline in COPD [7]. TNFα in the sputum distinguished COPD patients from controls [8]. Te causative role of TNF-α in lung tissue restructuring was supported by animal models, where exposure to cigarette or environmental smoke caused infammation and remodeling [9][10][11].
In the lung tissue of COPD patients, miR-101-3p was elevated compared to control tissues specifcally in bronchial epithelial cells [17]. Comparing the expression of six different miRs in patients with tuberculosis versus other lung diseases, the expression of miR-101-3p was highest in COPD and lung cancer patients [18]. In non-small-cell lung cancer, the expression of miR-101-3p negatively correlated with drug resistance and epithelial to mesenchymal transition [19]. Te expression of miR-101-3p inhibited growth and metastasis of non-small-cell lung cancer cells by interfering with the Akt (serine/threonine kinase or protein kinase B)-mTOR signaling pathway [19]. Tese data suggest that TNFα might modify the airway and lung tissue structure through miR-101-3p in COPD.
In this study, the efect of TNF-α on the expression of miR-101-3p and its contribution to mitochondrial fragmentation was assessed in ASMC obtained from patients with COPD, active smokers without COPD, and nondiseased controls.

Primary Airway Smooth Muscle Cells.
Six primary human ASMC lines were purchased from Ruwag (Bettlach, Switzerland). Tree cell lines were originated from nonsmoking healthy controls and three from COPD patients. Tese cells were used for the analysis of cell intracellular signaling; the available patient information is presented in Table 1.
Additional primary ASMC lines were obtained from a local cell bank (Pneumology, University Hospital Basel) collected since 2002. Patients provided informed consent, and the study was approved by the local Ethics Board (Ethic commission of both Basels, Switzerland, EK: 05/06).

Cigarette Smoke Extract (CSE) Preparation.
Special standardized research cigarettes (1R6F, University of Kentucky, USA) were used to prepare CSE as previously described [22,23]. In brief, the cigarettes were smoked via a manual negative pressure syringe. A total of 300 ml of cigarette smoke was bubbled through 10 ml DMEM medium in a glass bottle and mixed by shaking and the pH was adjusted to 7.4. Te solution was passed through a 0.22 m flter and defned as 100% CSE. Working concentrations were made by diluting the 100% CSE with culture medium.

Statistical Analysis.
Te null-hypothesis was miR-101-3p, and TNF-α has no efect on ASMC. Data are presented as mean ± SEM and were analyzed by unpaired one-way ANOVA and subsequent paired Student's t-test. Statistical data analysis was performed by software GraphPad-Prism7 and P values < 0.05 were considered as statistically signifcant.
In regards to mitochondrial activity, TNF-α decreased the basal level of MTCO2 in ASMC of both nonsmokers and COPD patients, as shown by Western blotting and subsequent image analysis (Figure 3(b)). Compared to the basal level, the reducing efect of TNF-α was stronger in ASMC of nonsmokers than in COPD-ASMC (Figure 3(b), bar chart). Te reducing efect of TNF-α on MTCO2 expression was time-dependent and achieved signifcance after 6 hours in ASMC of nonsmokers, while it only became signifcant in COPD-ASMC after 48 hours (Figure 3(b)).
Te TNF-α-induced phosphorylation of p70S6K returned to the baseline level after 120 minutes (Figure 6(a)). However, when treated with TNF-α over 48 hours, a disease-specifc efect was observed. In ASMC of nonsmokers, but not COPD-ASMC, TNF-α signifcantly decreased the phosphorylation of p70S6K over time (Figures 6(b)-6(d)). TNF-α had a weaker reducing efect on total p70S6K expression in ASMC of nonsmokers, but time dependently and signifcantly reduced total p70S6K levels in COPD-ASMC (Figure 6(e)). Chemical inhibitors of the mTOR signaling pathway, rapamycin, were used to confrm the involvement of TNF-α-induced mTOR signaling on p70S6K phosphorylation (Figures 6(f) and 6(g)).

Te Additive Efect of TNF-α and miR-101-3p on ASMC
Deterioration. In ASMC of nonsmokers, miR-101-3p mimics modifed the expression of fbronectin and α-SMA in the presence and absence of TNF-α (Figure 7(a)). TNF-α reduced the baseline expression of fbronectin signifcantly in ASMC of nonsmokers, but not in cells of COPD patients as shown by Western blotting and subsequent image analysis (Figures 7(a) and 7(b)). MiR-101-3p mimics alone reduced the base line expression of fbronectin signifcantly in both ASMC of nonsmokers and COPD patients (Figures 7(a) and 7(b)). When combining TNF-α with miR-101-3p mimics, the inhibition of fbronectin was additive in ASMC of nonsmokers, but not in COPD-ASMC (Figures 7(a) and 7(b)).

Discussion
Tis study provided evidence that miR-101-3p is constitutively upregulated by over100 folds in ASMC of COPD patients when compared to cells of smokers and healthy   Canadian Respiratory Journal controls. Tis suggests an intrinsic lasting efect of cigarette smoking on miR-101-3p regulation. Increasing miR-101-3p levels reduced mTOR signaling, which impaired mitochondrial function. Consequently, the expression of α-SMA and fbronectin was reduced. COPD is one of the most prevalent chronic infammatory lung diseases worldwide and is responsible for signifcant morbidity and premature mortality (GOLD report 2019, https://goldcopd.org/gold-reports/). In COPD, the remodeling of small airways is a main pathology, and results in the irreversible decline of lung function [3,4]. Te destruction of the COPD lung tissue manifests as the breakdown of the small airways and alveolar wall attachment without fbrosis. Tese events lead to airspace enlargement, hyperinfation, and loss of pulmonary plasticity [25]. During the progression of COPD, small conducting airways disappear, before the emphysematous destruction of the parenchymal lung tissue [26]. In advanced emphysema, dysfunctional ASMC further contribute to ventilationperfusion (V/Q) inequality, and increased exacerbations [27,28].
TNF-α is highly expressed in lung tissues and the body fuids of COPD patients [6][7][8]. Infammatory cells and structural cells of the small airways of COPD patients responded to proinfammatory proteins including interleukin (IL)-1β, IL-6, TNF-α, and TGF-β [29]. ASMCs are a key cell type of the airway wall and contribute to the pathogenesis of COPD by acting as target cells and as initiators of chronic infammation and remodeling [30,31]. In an animal model, cigarette smoke upregulated miR-101-3p expression in the COPD lung tissue and in bronchial epithelial cells [17]. In this study, the increased expression of miR-101-3p was maintained in isolated ASMC of COPD patients, but not in ASMC of controls or active smokers. Te data presented above suggest that (i) the constitutive expression of miR-101-3p in COPD becomes independent from the exposure to cigarette smoke and is maintained in isolated ASMC; (ii) cigarette smoking alone is insufcient to imprint the high expression of miR-101-3p. Tus, future studies are needed to identify the cause of such event. However, the presented data suggest that TNF-α played the role in the upregulation of miR-101-3p, as it has been suggested in other conditions [32].
TNF-α was described as the main initiator or mediator of cigarette smoke-induced emphysema and therefore contributed to the degradation of the lung tissues in COPD [33]. In regards to ASMC remodeling, TNF-α and the mTOR signaling pathway played a central role of the de novo synthesis and the degradation of extracellular matrix components such as collagens and fbronectin [34]. Here, we showed that in ASMC from controls, the expression of miR-101-3p was induced by TNF-α, while in COPD-ASMC, such a stimulatory efect was not observed; most likely due to the fact that the miR-101-3p level was over 100 times higher compared to healthy controls and non-COPD smokers. Te literature indicates that miR-101-3p is a negative regulator of mTOR signaling, and thereby prevented autophagy [35]. Tis observation is in line with the downregulation of mTOR signaling by TNF-α in ASMC shown in this study. In COPD cells, TNF-α had no signifcant efect on the phosphorylation of p70S6K, while it reduced the phosphorylation of nonsmokers. Tis observation suggests a deregulation of TNF-α signaling in COPD. However, others reported that activation of mTOR corrected mitochondrial fssion and mitophagy in other cell types [36]. Tese opposing functions of mTOR on mitochondrial regulation might be disease specifc and needs to be further investigated. Mitochondrial dysfunction is a central pathology in various chronic infammatory lung diseases [37,38]. Infection, cigarette smoke, and environmental insults are known to afect mitochondria mass and mitochondrial activity. Mitochondria contributed to airway diseases by aberrant energy metabolism, excessive reactive oxidative species production, intracellular calcium overload, and decreased compensatory capacities to stress [37,38]. Te function and structure of mitochondria was regulated by mTOR signaling [39,40]. In an earlier study on asthma, mTOR signaling increased mitochondrial function in ASMC and thereby controlled remodeling [41]. In this study, the inhibition of mTOR signaling by TNF-α led to mitochondrial fragmentation and cell deterioration in COPD-ASMC. Te inhibition of the mTOR signaling by TNF-α also refected in a signifcant decrease of MTCO2, leading to mitochondrial fragmentation in isolated COPD-ASMC. Te same efects on mitochondria were induced by upregulation of miR-101-3p. Furthermore, the suppression of p70S6K, a key mTOR protein, resulted in mitochondrial fragmentation and ASMC deterioration. In contrast, miR-101-3p inhibition had the opposing efects.
Tis investigation has some limitations: the subject numbers were relatively small due to limited access to donor samples, the existence of a possible cell type diference for the above described efects of TNF-α and miR-101-3p was not investigated, and the increased expression of miR-101-3p was not confrmed in COPD tissue sections, due the lack of specially prepared tissue samples. Furthermore, the study did not compare if the upregulation of miR-101-3p correlated with the GOLD staging of COPD or if it occurred in nondiseased smokers. Tese topics need future investigations.
Te presented data indicate a COPD-specifc constitutively increased expression of miR-101-3p in ASMC isolated from COPD patients impairs mTOR signaling and leads to mitochondrial fragmentation-dependent ASMC deterioration. Reactivating mTOR or blocking miR-101-3p presents a potential new strategy for COPD therapy.

Data Availability
Te original data are available on request from the frst author.

Ethical Approval
Te study was conducted according to the guidelines of the Declaration of Helsinki and approved by the local Ethics Committee: Ethics Commission of both Basels (EK: 05/06).

Consent
Informed consent was obtained from all subjects involved in the study.

Conflicts of Interest
Te authors declare that they have no conficts of interest.