ABSTRACT
Chlamydia psittaci is a human pathogen that causes atypical pneumonia after zoonotic transmission. We confirmed that C. psittaci infection induces oxidative stress in human bronchial epithelial (HBEs) cells and explored how this is regulated through miR-184 and the Wnt/β-catenin signaling pathway. miR-184 mimic, miR-184 inhibitor, FOXO1 siRNA, or negative control sequence was transfected into HBE cells cultured in serum-free medium using Lipofectamine 2000. Then, prior to the cells were infected with C. psittaci 6BC, and the cells were treated with or without 30 µM Wnt/β-catenin inhibitor ICG-001. Quantification of reactive oxygen species, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione was carried out according to the manufacturer’s protocol using a corresponding assay kit. The outcome of both protein and gene was measured by western blotting or real-time fluorescence quantitative PCR. In C. psittaci-infected HBE cells, miR-184 was upregulated, while one of its target genes, FOXO1, was downregulated. ROS and MDA levels increased, while SOD and GSH contents decreased after C. psittaci infection. When miR-184 expression was downregulated, the level of oxidative stress caused by C. psittaci infection was reduced, and the Wnt/β-catenin signaling pathway was inhibited. The opposite results were seen when miR-184 mimic was used. Transfecting with FOXO1 siRNA reversed the effect of miR-184 inhibitor. Moreover, when the Wnt/β-catenin-specific inhibitor ICG-001 was used, the level of oxidative stress induced by C. psittaci infection was significantly suppressed. miR-184 can target FOXO1 to promote oxidative stress in HBE cells following C. psittaci infection by activation of the Wnt/β-catenin signaling pathway.
KEYWORDS: Chlamydia psittaci, oxidative stress, MiR-184, FOXO1, Wnt/β-catenin signaling pathway
INTRODUCTION
Chlamydia psittaci is a human pathogen that causes atypical pneumonia, bacteremia, arthritis, and even lymphoma after zoonotic transmission, mainly in the form of aerosols, which are taken up via the respiratory tract (1). C. psittaci infections, which are often facilitated by increased drug resistance due to long-term abuse of antibiotics, can be occult and persistent, causing progressive and irreversible pathological damage to the host (2 – 4). Therefore, it is very important to clarify the pathogenic mechanisms of C. psittaci, which should allow the identification of possible therapeutic targets, thereby safeguarding human health and allowing improvements in animal husbandry.
MicroRNAs (miRNAs) are a class of conserved endogenous non-coding single-stranded RNAs, which exert their biological functions by targeting the 3′-untranslated regions of mRNAs to transcriptionally repress gene expression (5). As regulators of gene expression, miRNAs play an important role in several essential cellular functions, including cell migration, proliferation, invasion, autophagy, oxidative stress, apoptosis, and differentiation (6 – 8). At present, research on the role of miRNAs in chlamydia infection mainly focuses on disease caused by C. trachomatis and C. pneumoniae. For example, Derrick et al. found that nine miRNAs were differentially expressed in children with trachoma and follicular conjunctivitis caused by C. trachomatis. Among these, miR-155 was upregulated and miR-184 was downregulated, and the changes were positively correlated with the degree of trachoma inflammation (9, 10). Batteiger et al. found that women infected with C. trachomatis and showing signs and/or symptoms of disease have miRNA profiles distinct from those of asymptomatic infected women (11). Another miRNA, miR-33, plays a regulatory role in atherosclerosis induced by C. pneumoniae infection (12). Although all chlamydia share some biological characteristics, the tissue tropism and pathogenic spectrum of different species vary greatly. At present, there are few reports on the role of miRNAs in C. psittaci infection. In a previous study, we used small RNA sequencing technology to identify 151 differentially expressed miRNAs in C. psittaci-infected human bronchial epithelial (HBE) cells; six of these miRNAs, including hsa-miR-184, were verified as significantly upregulated by real-time quantitative PCR (RT-qPCR) (13).
Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and the protective activity of the endogenous antioxidant defense system (14, 15). Under normal circumstances, the moderate levels of ROS produced in cells are easily detoxified by the antioxidant defense system. However, when cells experience adverse conditions, the antioxidant defense system is not able to deal with the excessive levels of ROS generated, which can lead to the oxidation of nucleic acids, proteins, and lipids, resulting in cellular damage and the development of pathology (16, 17). Two defense systems in the body are involved in preventing uncontrolled ROS generation: the first is an enzymatic antioxidant system involving superoxide dismutase (SOD), and the second is non-enzymic, involving glutathione (GSH) (18). When cells are under oxidative stress, intracellular polyunsaturated fatty acids undergo lipid peroxidation, and the main end-product is malondialdehyde (MDA). Therefore, measuring the content of MDA can indirectly reflect the degree of cell damage.
MiRNAs play a regulatory role in the cellular oxidative-stress response, and thus, their function has an effect on the occurrence and development of many diseases. For example, the expression level of miR-494 is upregulated in rats with sepsis-related acute respiratory distress syndrome and negatively regulates the NQO1 gene. This blocks the Nrf2 signaling pathway, which enhances the oxidative-stress response in rat tissues and aggravates the inflammatory response, thus increasing the chance of acute lung injury (19).
Studies have found that tissue or cell dysfunction caused by chlamydia infection is closely associated with host oxidative stress. For example, in vascular endothelial cells infected with C. pneumoniae, the NADPH oxidases NOX-2 and NOX-4 are upregulated, while the expression levels of thioredoxin-1 and SOD-1 are strongly downregulated, pointing to a redox imbalance within C. pneumoniae-infected cells (20). Tošić-Pajić et al. found that the oxidative stress index (i.e., the ratio of the sum of superoxide anion and nitrite to glutathione) in infertile patients with persistent C. trachomatis infection was significantly higher than that in normal childbearing women, indicating that C. trachomatis infection affects the pro-oxidant/antioxidant balance, resulting in oxidative stress, which impacts the childbearing potential of the women concerned (21). Whether miRNAs participate in the pathogenic processes resulting from C. psittaci infection by regulating oxidative stress in host cells is currently unclear, however, and deserves further investigation.
MiR-184, which is widely acknowledged to be involved in the regulation of the oxidative stress response, is one of the miRNAs that are significantly upregulated in C. psittaci-infected HBE cells, as reported in our previous study. Liu et al. found that miR-184 can promote aging in glomerular mesangial cells by enhancing oxidative damage (22). Furthermore, inhibition of miR-184 expression markedly reduced oxidative stress in H2O2-treated H9c2 cells (23). FOXO1, a member of the FOXO family that can participate in the regulation of cellular oxidative stress by promoting or inhibiting gene transcription, has been confirmed as one of the target genes of miR-184 (24 – 26).
The Wnt/β-catenin pathway is an important intracellular signaling pathway that plays a key role in organ development, tissue, and cell damage repair. Wnt and β-catenin are related proteins and represent key components of the pathway. Wnt binds to the membrane receptor protein FZD and activates the intracellular protein DVL, thereby inhibiting the activity of the β-catenin degradation complex, which stabilizes free β-catenin protein in the cytoplasm. When β-catenin accumulates to a certain level in the cytoplasm, it enters the nucleus, where it binds to members of the LEF/TCF transcription factor family and triggers the transcription of downstream target genes, such as c-myc and cyclin D1. Oxidative stress can promote the binding of FOXO protein to β-catenin, resulting in competitive inhibition of Wnt signaling; as a result, transcription of ROS clearance genes, including SOD genes, is initiated (27). Almeida et al. found that in colon cancer cells and osteoblasts, FOXO1 can bind to β-catenin during oxidative stress, thereby repressing transcriptional regulation mediated by TCF/LEF transcription factors and ultimately inhibiting the Wnt signaling pathway (27). It has also been reported that inhibiting the Wnt/β-catenin signaling pathway can significantly attenuate the oxidative stress mediated by renal ischemia-reperfusion injury (28).
In this study, we examined the effects of miR-184 and FOXO1 on oxidative stress in HBE cells infected with C. psittaci and investigated the regulatory role of the Wnt/β-catenin signaling pathway in this process.
RESULTS
C. psittaci induces oxidative stress and regulates the expression of miR-184 and FOXO1 in HBE cells
In order to detect the effects of C. psittaci on oxidative stress, the oxidative or antioxidant indexes were examined. Compared with uninfected HBE cells, the levels of ROS and MDA were significantly higher, while SOD and GSH levels were lower, following C. psittaci infection (Fig. 1), indicating that C. psittaci can induce oxidative stress in HBE cells.
Fig 1.
Oxidative stress levels in C. psittaci-infected HBE cells. HBE cells were infected with C. psittaci at a multiplicity of infection (MOI) of 3 for 44 h, and the levels of ROS (A), MDA (B), SOD (C), and GSH (D) were measured with commercial kits. Experiments were repeated three times. *P < 0.05, **P < 0.01.
In our previous study, we found miR-184 was significantly upregulated in C. psittaci-infected HBE cells, and FOXO1 has been confirmed as one of the target genes of miR-184 (24 – 26). In this experiment, miR-184 was significantly upregulated (Fig. 2A), while the expression of FOXO1 was decreased at both the mRNA and protein levels (Fig. 2B and C). We also found, either with or without C. psittaci infection, the miR-184 levels significantly decreased and both the mRNA and protein levels of FOXO1 increased after transfection with the miR-184 inhibitor; when the miR-184 mimic was used, the opposite changes in miR-184 and FOXO1 expression levels were observed (Fig. 3A through D).
Fig 2.
Expression levels of miR-184 and FOXO1 in C. psittaci-infected HBE cells. HBE cells were infected with C. psittaci, and the expression of miR-184 (A) and FOXO1 mRNA (B) was measured by RT-qPCR, while the protein expression level of FOXO1 was assessed by western blotting (C). Experiments were repeated three times. *P < 0.05, **P < 0.01.
Fig 3.
Negative correlation between miR-184 and FOXO1 levels in HBE cells. HBE cells were transfected with miR-184 inhibitor or miR-184 mimic individually or cotransfected with miR-184 inhibitor and FOXO1 siRNA. The expression levels of miR-184 and FOXO1 mRNA (A, C, E) were measured by RT-qPCR, while the protein expression level of FOXO1 was assessed by western blotting (B, D, F). Experiments were carried out without (A and B) or with (C–F) C. psittaci infection and were repeated three times. **P < 0.01 vs the corresponding negative control (NC) groups; ##P < 0.01 vs the miR-184 inhibitor + NC siRNA + Cps group.
To further explore the relationship between miR-184 and FOXO1, miR-184 inhibitor was transfected into HBE cells, with or without FOXO1 siRNA, and the cells were then infected with C. psittaci. As shown in Fig. 3E and F, the FOXO1 mRNA and protein expression levels in cells treated with the miR-184 inhibitor significantly increased, while co-transfection with FOXO1 siRNA could partially reverse this effect. These results suggest that there is a negative correlation between miR-184 and FOXO1 expression levels in HBE cells with or without C. psittaci infection.
The effects of miR-184 on oxidative stress induced by C. psittaci in HBE cells
To explore the effect of miR-184 on oxidative stress induced by C. psittaci, miR-184 inhibitor or miR-184 mimic was transfected into HBE cells, and the cells were then infected with C. psittaci. Compared with negative control (NC) inhibitor-treated cells, the level of ROS and the content of MDA in the miR-184 inhibitor-treated cells significantly decreased, while the activity of SOD and the content of GSH significantly increased. However, treatment with the miR-184 mimic produced the opposite effects (Fig. 4A). These results indicate that miR-184 can promote C. psittaci-induced oxidative stress in HBE cells.
Fig 4.
The effect of miR-184 on oxidative stress in C. psittaci-infected HBE cells involves targeting of FOXO1. HBE cells were transfected with miR-184 inhibitor and miR-184 mimic individually (A) or cotransfected with miR-184 inhibitor and FOXO1 siRNA (B) and then infected with C. psittaci. The levels of ROS, MDA, SOD, and GSH were measured with commercial kits. *P < 0.05, **P < 0.01 vs the NC inhibitor + Cps group or the NC mimic + Cps group, ##P < 0.01 vs the miR-184 inhibitor + NC siRNA + Cps group.
To further ask whether the effect of miR-184 on oxidative stress is targeted through FOXO1, miR-184 inhibitor was transfected with or without FOXO1 siRNA into HBE cells. It was found that FOXO1 siRNA could reverse the amelioration effect of miR-184 inhibitor on C. psittaci-induced oxidative stress in HBE cells (Fig. 4B).
MiR-184 targeting of FOXO1 activates the Wnt/β-catenin signaling pathway in C. psittaci-infected HBE cells
To determine whether miR-184 targeting of FOXO1 promotes the activity of the Wnt/β-catenin signaling pathway in C. psittaci-infected HBE cells, miR-184 inhibitor or miR-184 mimic individually or miR-184 inhibitor and FOXO1 siRNA together were transfected into HBE cells, and then levels of proteins or mRNA in the Wnt/β-catenin signaling pathway were assessed. The results show that either the mRNA levels of c-myc and cyclin D1 or the protein levels of Wnt3a, β-catenin, c-myc, and cyclin D1 in miR-184 mimic-treated cells were significantly higher than those in NC mimic-treated cells. However, in cells treated with miR-184 inhibitor, the expression levels of Wnt3a, β-catenin, c-myc, and cyclin D1 were much lower than in NC inhibitor-treated cells (Fig. 5A and B). These results indicate that miR-184 can promote Wnt/β-catenin signaling pathway activation in C. psittaci-infected HBE cells. After co-transfection with miR-184 inhibitor and FOXO1 siRNA, the expression levels of Wnt3a, β-catenin, c-myc, and cyclin D1 were significantly higher than these in cells treated with miR-184 inhibitor only (Fig. 5C and D), indicating that FOXO1 siRNA can reverse the blocking effect of miR-184 inhibitor on the Wnt/β-catenin signaling pathway in C. psittaci-infected HBE cells.
Fig 5.
Effect of miR-184 on activation of the Wnt/β-catenin signaling pathway in C. psittaci-infected HBE cells. HBE cells were transfected with miR-184 inhibitor or miR-184 mimic individually (A and B) or miR-184 inhibitor and FOXO1 siRNA together (C and D) and then infected with C. psittaci. The expression levels of c-myc and cyclin D1 mRNA were measured by RT-qPCR, and the expression of Wnt3a, β-catenin, c-myc, and cyclin D1 protein was measured by western blotting. **P < 0.01 vs the NC inhibitor + Cps group or the NC mimic + Cps group, #P < 0.05, ##P < 0.01 vs the miR-184 inhibitor + NC siRNA + Cps group.
Inhibition of the Wnt/β-catenin signaling pathway reduces oxidative stress in C. psittaci-infected HBE cells
HBE cells were treated with different concentrations (0, 5, 10, 20, 30 µmol/L) of the Wnt/β-catenin signaling pathway inhibitor ICG-001 for 2 h and then infected with C. psittaci. The results showed that, with increasing ICG-001 concentration, the expression levels of Wnt3a, β-catenin, c-myc, and cyclin D1 gradually decreased and decreased to the lowest level at 30 µmol/L ICG-001 (Fig. 6A and B). This concentration (30 µmol/L ICG-001) was, therefore, used in subsequent experiments.
Fig 6.
Inhibition of the Wnt/β-catenin signaling pathway blocks the stimulatory effect of miR-184 mimic or FOXO1 siRNA on oxidative stress in C. psittaci-infected HBE cells. HBE cells were treated with 30 µmol/L ICG-001 after transfection with miR-184 mimic or FOXO1 siRNA and then infected with C. psittaci. The protein expression levels of Wnt3a, β-catenin, c-myc, and cyclin D1 (A, C, E) were measured by western blotting; the mRNA levels of c-myc and cyclin D1 (B, D, F) were measured by RT-qPCR; and the levels of ROS, MDA, SOD, and GSH (G and H) were measured using commercial kits. *P < 0.05, **P < 0.01 vs the miR-184 mimic + Cps group or the FOXO1 siRNA + Cps group.
The earlier experiments showed that, by targeting FOXO1, miR-184 can regulate C. psittaci-induced oxidative stress in HBE cells. We, therefore, further explored the role of the Wnt/β-catenin signaling pathway in this process. The results showed that, after ICG-001 treatment, not only were the levels of Wnt3a, β-catenin, c-myc, and cyclin D1 reduced in miR-184 mimic-infected cells, but also that the levels of ROS and MDA decreased, while the activity of SOD and the content of GSH increased (Fig. 6C, D, and G). Similar results were found in cells transfected with FOXO1 siRNA (Fig. 6E, F, and H). The data, therefore, suggest that inhibition of the Wnt/β-catenin signaling pathway can reverse the stimulatory effect of miR-184 mimic or FOXO1 siRNA on oxidative stress in C. psittaci-infected HBE cells.
DISCUSSION
C. psittaci efficiently disseminates in host organisms causing pulmonary and systemic disease, which occasionally can take a fulminant course. Oxidative stress is a state in which the cellular antioxidant defense system is not able to detoxify excess ROS, resulting in a redox imbalance. Based on the literature, we speculated that oxidative stress may be involved in the pathogenesis caused by C. psittaci.
We first confirmed that C. psittaci did, indeed, induce oxidative stress in HBE cells by assessing the levels of ROS, MDA, SOD, and GSH. This finding is consistent with other reports. For example, ROS produced by C. trachomatis infection can promote lipid peroxidation of the sperm membrane, reducing sperm motility and affecting fertility (29). In the late stages of C. pneumoniae infection, the level of GSH in T cells decreases significantly, which perturbs the redox homeostasis of cells, leading to an accumulation of ROS and, finally, to cell death (30). We previously found that the Pgp3 protein alleviated oxidative stress to promote the infectivity of C. trachomatis (31). The main reason resulting the difference might be the whole live chlamydia organism was used in this experiment, while the purified recombinant Pgp3 protein was used to stimulate the cells in our last study. As we know, Chlamydia is composed of many proteins, and different proteins perform different functions.
In our previous study, we found that miR-184 was significantly upregulated in C. psittaci-infected HBE cells, so we initiated the current investigation to further understand the role of this miRNA in the oxidative stress associated with the infection. We found that downregulation of miR-184 could ameliorate C. psittaci-induced oxidative stress, whereas overexpression of miR-184 increased this stress. This indicates that miR-184 can promote C. psittaci-induced oxidative stress in HBE cells, which is consistent with a recent report showing that inhibition of miR-184 markedly inhibits oxidative stress in H2O2-treated H9c2 cells (23).
It is well known that miRNAs exert their biological functions by targeting the 3′-untranslated regions of gene transcripts, thereby repressing gene expression, and Xuan et al. have reported that FOXO1 is one of the target genes of miR-184 (24). In line with this, our work shows that when miR-184 expression increases, FOXO1 expression decreases at both the mRNA and protein levels in HBE cells infected with C. psittaci. Furthermore, after transfection with miR-184 inhibitor, FOXO1 expression increases, while the opposite change was observed after transfection with miR-184 mimic, with or without C. psittaci infection. These results confirm the regulatory relationship between miR-184 and its target, FOXO1.
We also show here that, by targeting FOXO1, miR-184 can regulate the oxidative stress experienced by HBE cells following infection with C. psittaci. In other words, inhibiting FOXO1 expression via miR-184 can promote the oxidative stress caused by C. psittaci. It is widely reported that the action of FOXO1 can reduce the effect of oxidative stress on cells. For example, FOXO1 can restore osteoblast differentiation and function by reducing oxidative stress levels and osteoblast apoptosis (32). Ji et al. demonstrated that FOXO1 overexpression ameliorates oxidative damage by attenuating cellular ROS production, thereby attenuating high glucose-induced renal proximal tubular cell damage (33).
The Wnt/β-catenin signaling pathway also plays a key role in the repair of oxidative damage in cells and tissues (27). Our study shows that the mRNA levels of c-myc and cyclin D1 and the protein levels of Wnt3a, β-catenin, c-myc, and cyclin D1 increased in C. psittaci-infected HBE cells, indicating that the Wnt/β-catenin pathway had been activated. The levels of ROS and MDA decreased, while SOD activity and GSH levels increased, after cells were treated with a specific inhibitor of the Wnt/β-catenin signaling pathway, ICG-001. The results suggest that the Wnt/β-catenin signaling pathway can regulate the oxidative stress induced by C. psittaci.
Multiple studies have shown that FOXO1 interacts with β-catenin and plays a role in various diseases (34, 35). For example, Zou et al. reported that the coupling of FOXO1 to β-catenin in the cytoplasm can prevent accumulation of β-catenin in the nucleus, thereby downregulating Wnt signaling activity; this then stimulates the activation of key regulators of cancer stemness and promotes breast cancer progression (36). Guan et al. also found that FOXO1 reduces the survival of osteosarcoma cells by inhibiting Wnt/β-catenin signaling, thereby blocking osteosarcoma oncogenesis (37). In this study, we found that, after ICG-001 treatment, the increased levels of oxidative stress due to either miR-184 overexpression or FOXO1 knockdown were attenuated in HBE cells infected with C. psittaci, indicating that inhibition of the Wnt/β-catenin signaling pathway can reverse the stimulatory effect of miR-184 mimic or FOXO1 siRNA on oxidative stress in C. psittaci-infected HBE cells. These results further explain the changes in Fig. 4B, downregulation of miR-184 allows its target FOXO1 to bind β-catenin and thereby block the Wnt/β-catenin signaling pathway, resulting in amelioration of the oxidative stress induced in HBE cells by C. psittaci. After adding FOXO1 siRNA, the expression of FOXO1 was inhibited, reducing the effect of miR-184 inhibitor and enhancing the oxidative stress induced by C. psittaci in HBE cells.
In conclusion, we demonstrate that inhibition of miR-184 plays a protective role against C. psittaci-induced oxidative damage in HBE cells, by negatively regulating FOXO1 and blocking Wnt/β-catenin signaling pathway (Fig. 7). And next, we will further study whether C. psittaci may interact with the Wnt/β-catenin signaling pathway via other mechanisms, the other consequences of C. psittaci-induced ROS, or which C. psittaci factors induce ROS and elevate miR-184.
Fig 7.
Schematic representation of miR-184 inhibition as a means of counteracting oxidative stress in HBE cells infected with C. psittaci. C. psittaci can upregulate miR-184, resulting in downregulation of FOXO1, and promotion of oxidative stress in HBE cells via the Wnt/β-catenin signaling pathway. Downregulation of miR-184 allows its target FOXO1 to bind β-catenin and thereby block the Wnt/β-catenin signaling pathway, resulting in amelioration of the oxidative stress induced in HBE cells by C. psittaci.
MATERIALS AND METHODS
Cell culture
Human bronchial epithelial cells (HBE; ATCC 135-E6E7) were cultured in cell flasks and 6- or 24-well plates (Corning, NY, USA) to a suitable density and maintained in Dulbecco’s modified Eagle medium (DMEM) (Hyclone, Logan, USA) containing 10% (vol/vol) fetal bovine serum (Gibco BRL, Gaithersburg, USA) at 37°C in an incubator supplied with 5% CO2.
Cell treatment
For the RNA interference experiment, the cells were transfected according to the instructions of Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA). First, 5 µL Lipofectamine 2000 and 195 µL serum-free medium were mixed and incubated for 5 min; in another tube, 2.5 µL miR-184 mimic, miR-184 inhibitor, FOXO1 siRNA, or negative control sequence (Table 1) (GenePharma, China; 97% purity) with a mother liquor concentration of 20 mM was added to 797.5 µL serum-free medium; both solutions were mixed and incubated for 20 min with a final RNA concentration of 50 nM, and then, the mixture was added to the culture wells (38). After incubating for 6 h, the medium was replaced with fresh DMEM. Then, the cells were infected with C. psittaci 6BC at a multiplicity of infection (MOI) of 3 for 44 h.
TABLE 1.
siRNA sequences
| siRNA | Sequence |
|---|---|
| miR-184 inhibitor | 5′-ACCCUUAUCAGUUCUCCGUCCA-3′ |
| NC inhibitor | 5′-CAGUACUUUUGUGUAGUACAA-3′ |
| miR-184 mimic | 5′-UGGACGGAGAACUGAUAAGGGU-3′ (sense) |
| 5′-CCUUAUCAGUUCUCCGUCCAUU-3′ (antisense) | |
| NC mimic | 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) |
| 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense) | |
| FOXO1 siRNA | 5′-GCUCAAAUGCUAGUACUAUTT-3′ (sense) |
| 5′-AUAGUACUAGCAUUUGAGCTT-3′ (antisense) | |
| NC siRNA | 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) |
| 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense) |
For the pathway inhibition experiment, prior to C. psittaci 6BC infection, the cells were treated with or without 30 µM Wnt/β-catenin inhibitor ICG-001 (Selleck, Houston, USA) for 2 h. The outcome of both gene silencing and pathway inhibition experiments was measured by western blotting or real-time fluorescence quantitative PCR (RT-qPCR).
Oxidative stress index
Quantification of ROS, MDA, SOD, and GSH was carried out strictly according to the manufacturer’s protocol using a corresponding assay kit (ROS/MDA/SOD/GSH Assay Kit, Nanjing Jiancheng Bioengineering Institute, China).
Real-time fluorescence quantitative PCR (RT-qPCR)
Total RNAs from cells were extracted using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The RNA concentration and the absorbance at 260 and 280 nm were measured. If the A260/A280 was between 1.8 and 2.1, the RNA quality was considered of an acceptable standard and was used in subsequent experiments. Total RNA was converted into cDNA using a miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, Nanjing, China) or HiScriptIII 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China). The expression level of miRNA or mRNA was determined using a fluorescent quantitative PCR system (Applied Biosystems, Foster City, CA, USA) and a miRNA Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) or ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). MiR-184 expression was normalized to that of U6 small nuclear RNA (U6), and mRNA expression was standardized against that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative fold changes were calculated using the 2−ΔΔCt method. The primer sequences for the PCR assays are listed in Table 2.
TABLE 2.
Primer sequences
| Gene | Sequence |
|---|---|
| U6 (Reverse transcription) | 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATA-3′ |
| miR-184 (Reverse transcription) | 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACCCTT-3′ |
| U6 | 5′-AGAGAAGATTAGCATGGCCCCTG-3′(Forward) |
| 5′-AGTGCAGGGTCCGAGGTATT-3′(Reverse) | |
| miR-184 | 5′-CGCGTGGACGGAGAACTGAT-3′(Forward) |
| 5′-AGTGCAGGGTCCGAGGTATT-3′(Reverse) | |
| GAPDH | 5′-TGGCACCGTCAAGGCTGAGAAC-3′(Forward) |
| 5′-CTCGCTCCTGGAAGATGGTGATGG-3′(Reverse) | |
| FOXO1 | 5′-AAACACCAGTTTGAATTCACCC-3′(Forward) |
| 5′-TCGACTTATTGTCCTGAAGTGT-3′(Reverse) | |
| c-myc | 5′-CGACGAGACCTTCATCAAAAAC-3′(Forward) |
| 5′-CTTCTCTGAGACGAGCTTGG-3′(Reverse) | |
| cyclin D1 | 5′-GTCCTACTTCAAATGTGTGCAG-3′(Forward) |
| 5′-GGGATGGTCTCCTTCATCTTAG-3′(Reverse) |
Western blot analysis
Total protein from cultured HBE cells was isolated using Strong RIPA lysis buffer (Beijing Solarbio Science & Tecnology Co., Ltd., China) supplemented with cocktail protease inhibitor (Beijing ComWin Biotech Co., Ltd., China). The supernatant was obtained by centrifugation at 4°C using a high-speed centrifuge. Protein concentrations were determined using a bicinchoninic acid (BCA) kit (Beijing ComWin Biotech Co., Ltd., China). An equal amount of total protein was separated on 10%–15% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, USA). After blocking with 5% skimmed milk dissolved in TBST (0.02% Tween-20 and 1 M Tris buffer, pH 8.0) for 2 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4°C. Primary antibodies used: anti-c-myc, anti-beta catenin (Proteintech, Wuhan, China, Rabbit, 1:1,000); anti-FOXO1, anti-cyclin D1 (Cell Signaling Technology, Danvers, MA, USA, Rabbit, 1:1,000); anti-Wnt3a (Abcam, Cambridge, MA, USA, Rabbit, 1:1,000); anti-GAPDH (Proteintech, Wuhan, China, Rabbit, 1:5,000) was used as internal reference antibody. After washing three times with TBST, the membrane was incubated with HRP-secondary antibody (Proteintech, Wuhan, China, Rabbit, 1:10,000) for 1 h at room temperature and then analyzed using an Azure C300 (Azure Biosystems, California, USC) imaging system. Image J software was used for band density analysis.
Statistical analysis
Data are shown as the mean ± standard deviation (SD) of three independent experiments, and statistical analysis of the data was performed using SPSS version 23.0 software (SPSS, Chicago, IL, USA). One-way analysis of variance (ANOVA) was used for the comparison of multiple groups, and an independent sample t-test was used to analyze the difference between two groups. Presentation items were produced using GraphPad Prism 8.0 software. A difference was considered to be statistically significant at P < 0.05.
ACKNOWLEDGMENTS
This work was supported by the Natural Science Foundation of Hunan Province (2021JJ70098, 2020JJ4527), the Scientific Research Fund of Hunan Provincial Education Department (20A438), the Graduate Research Innovation Project of Hunan Province (CX20221027).
Q.H. designed and analyzed experimental data and drafted the manuscript. Y.L., Y.Z., T.T., and Y.C. validated the proposed method with practical experiments. Q.B., Z.L., and C.L. analyzed the measured data. L.C. and Z.L. critically revised the manuscript.
All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.
Informed consent was obtained from all individual participants included in the study.
The authors affirm that human research participants provided informed consent for publication of the final manuscript.
All authors declare that they have no conflict of interest.
Contributor Information
Lili Chen, Email: chlili720612@163.com.
Zhongyu Li, Email: lzhy1023@hotmail.com.
Guy H. Palmer, Washington State University, Pullman, Washington, USA
DATA AVAILABILITY
The data sets generated during and analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data sets generated during and analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.