The expression of miR‐125b in Nrf2‐silenced A549 cells exposed to hyperoxia and its relationship with apoptosis

Abstract Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects the quality of life of infants. At present, premature exposure to hyperoxia for extended periods of time is believed to affect the development of lung tissue and vascularity, resulting in BPD. The oxidative stress caused by hyperoxia exposure is an important risk factor for BPD in premature infants. Nuclear factor E2‐related factor 2 (Nrf2) is an important regulator of antioxidant mechanisms. As a microRNA, microRNA‐125b (miR‐125b) plays an important role in cell proliferation, differentiation and apoptosis. Although the Nrf2/ARE pathway has been extensively studied, little is known about the regulatory role of microRNAs in Nrf2 expression. In this study, the expression levels of Nrf2 and miR‐125b in the lung tissues of premature Sprague Dawley (SD) rats and A549 cells exposed to hyperoxia were detected by quantitative real‐time polymerase chain reaction (qRT‐PCR), and the apoptosis of A549 cells was detected by flow cytometry. The results showed that Nrf2 and miRNA‐125b in the lung tissues of premature rats increased significantly upon exposure to hyperoxia and played a protective role. Nrf2 was suppressed by small interfering RNA (siRNA) in A549 cells, miR‐125b was similarly inhibited, and apoptosis was significantly increased. These results suggest that miR‐125b helps protect against BPD as a downstream target of Nrf2.

to ROS-induced lung injury in pre-term concurs to the induction of certain number of antioxidant, anti-inflammatory and detoxification pathway. These elicited protective effects are able to counteract/ mitigate all multifaceted aspects of the disease and may support novel approaches for the management of BPD. 5

miR-125b belongs
to the has-miR-125b family and consists of miR-125b-1 (located on chromosome 11 q24) and miR-125b-2 (located on chromosome 22 q21), which protect endothelial cells from apoptosis under oxidative stress. 6 Lukiw and Pogue 7 isolated microRNAs from HN cells exposed to magnesium sulphate (control), aluminium sulphate or aluminium plus iron sulphate. microRNA arrays showed that miR-125b was up-regulated by metal sulphate-generated ROS, suggested that miR-125b to be modulated by oxidative stress. However, the regulatory role of microRNAs (miRNAs) in Nrf2 expression is not fully understood. Recent studies have shown that miRNAs regulate Nrf2 directly or indirectly, 8  Nrf2/ARE is a key pathway in the antioxidant response in the human body. As important post-transcriptional regulators, miR-125b and Nrf2 play irreplaceable roles in proliferation, differentiation and regulation. We hypothesized that the expression of miR-125b is significantly correlated with Nrf2 and has an effect on cell apoptosis.
We elucidated the cellular defence mechanism of Nrf2 in the development of BPD and provided new ideas for the prevention and treatment of BPD in premature infants.
We hypothesized that (1) oxidative stress induced by hyperoxia exposure can lead to BPD in premature infants and trigger a series of antioxidant mechanisms, which involve miR-125b as an important cell protective factor and that (2) miR-125b has an important role in the antioxidant and anti-apoptotic effects of the Nrf2 pathway.

| Ethics approval
The use and care of laboratory rodents was performed according to the Animal Laboratory Center of Pediatrics, Children's Hospital of Fudan University and approved by the Committee of Animal Laboratory Management and Ethics, Shanghai Children's Hospital.

| Animal experimental grouping
In total, 250-300 g healthy adult SPF Sprague Dawley (SD) rats, 25 females and 5 males, were raised in cages. The animal licence number SCXK (Shanghai) 2018-0006 was provided by Shanghai Xipuer-Bikai Laboratory Animal Co. Ltd. Eighty premature SD rats with a gestational age (GA) of 21 days (full-term GA 22 days) were randomly divided into two groups: the air group and hyperoxia group.
The premature rats in the air group were fed in a normal pressure room (21% O 2 ), and the premature rats in the hyperoxia group were fed in a 90 × 60 × 45 cm homemade plexiglass oxygen chamber. The oxygen flow rate in the oxygen chamber was 1-3 L/min. The oxygen concentration curve in the chamber was recorded by a medical digital oxygen monitor within 24 hours, and the oxygen volume fraction in the chamber was kept at 80 ± 5% O 2 . Female rats that gave birth naturally were used as surrogate suckling mice. At 1, 4, 7, 10 and 14 days after exposure, the lung tissues of premature rats were placed in 5-mL enzyme-free cryopreservation tube and cooled rapidly with liquid nitrogen. The specimens were transferred to a -80°C freezer for subsequent PCR experiments.

| Transfection and screening of A549 cells
On the day before transfection, 5 × 10 4 cells were inoculated in a 24-well plate, and 10% FBS and Dulbecco's modified Eagle's medium (DMEM) with antibiotics were added. After 24 hours, cell confluence up to 40%-70% was achieved. Next, 50, 100 and 200 nmol/L siRNA was added to the DMEM serum-free medium. Then, 1 µL A549 cells were randomly divided into four groups: non-interference air group, non-interference hyperoxia group, air group after interference and hyperoxia group after interference. The hyperoxia group was exposed to a highly pure gas mixture of 3 L/min 92% O 2 and 5% CO 2 and sealed after 10 minutes. Both groups were placed in an incubator (37°C, 5% CO 2 ). Forty-eight hours after incubation, total RNA was extracted from A549 cells.

| Expression of Nrf2, miR-125b and IL-1β detected by qRT-PCR
Nrf2, miR-125b and IL-1β were detected by qRT-PCR. The control genes were β-Actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The total RNA of A549 was extracted by the TRIzol method. qRT-PCR was carried out using SYBR Green technology.
The samples were pre-amplified at 95°C for 10 minutes, after which they were amplified for 40 cycles at 95°C for 2 seconds, 60°C for 20 seconds and 70°C for 10 seconds. qRT-PCR was performed on a PCR instrument (Bio-Rad CFX96). The relative expression of target gene mRNA in the samples was determined by 2 −△△Ct .

| Western blotting analysis
Western blotting analysis was performed to assess protein abundance as described in our previous study. The primary antibody was Nrf2, and the secondary antibody was horseradish peroxidase (HRP) goat-anti-rabbit. Bio-Rad Image Lab Software (Version 5) was used for the analysis. After transfection of the Nrf2 siRNA plasmid into A549 lung cancer cells, an appropriate amount of cell lysate was added, and proteins from the nucleus and cytoplasm were collected and extracted. The collected proteins were mixed with buffer solution and then cooled on ice after 5 minutes in a water bath at 100°C. A separation gel and concentration gel were prepared for electrophoresis for 90 minutes. Next, the polyvinylidene fluoride (PVDF) membrane was removed and placed in a culture dish, and an appropriated amount of buffer [PBS buffer containing 5% (w/v) skim milk powder] was added and incubated at room temperature for 1-2 hours. The culture dish was replaced, 10 mL of the abovementioned buffer solution was added, and the primary antibody diluted at 1:1000 was added. Then, the membranes were incubated overnight at 4°C, washed three times, transferred to another culture dish and incubated with the secondary antibody buffer containing 5% skim milk powder and with the secondary antibody (HRP goat-anti-rabbit IgG) at 1:5000. The membranes were incubated at room temperature for 1 hour. Then, the PVDF membrane was transferred to another culture dish and washed three times with an appropriate amount of secondary antibody buffer. Finally, the Electrochemiluminescence (ECL) substrate developer (Thermo) and the substrate developer were added for imaging.

| Flow cytometry
Flow cytometry was used to detect the effect of hyperoxia and siRNA targeting Nrf2 on apoptosis of A549 cells. Cells in the logarithmic growth phase were digested by trypsin and then made into a cell suspension. The cells were cultured at 37°C for 48 hours, the culture medium was removed, and the cells were washed twice with PBS. After trypsin digestion of the adherent cells, the cells were collected. The cells were washed twice with pre-cooled PBS at 4°C and then centrifuged for 10 min (1000 r/min, 40°C). Then, the supernatant was discarded, the cells were collected, and the procedure was repeated for twice. The cell suspension was suspended again in 200 μL binding buffer, and 10 μL Annexin V-FITC was added to the cell suspension to mix evenly for 15 minutes at room temperature in the dark. Then, 300 μL binding buffer was added to the cell suspension (total reaction volume 500 μL) for 15 minutes at room temperature; flow cytometry was used for detection.

| Statistics
The data were analysed by SPSS 20.0 statistical software, and the results represent the mean ± SD of independent experiments. Relative expression levels among the siRNA interference group (including siRNA-1, siRNA-2 and siRNA-3), the blank group, air group and hyperoxia group were tested by Student's t test. One-way ANOVA was used between the two groups on apoptosis of A549 cells. The results with P < .05 were considered significant.

| Expression of Nrf2 in the lung tissues of premature SD rats
Compared with that of the air group, the expression of Nrf2 in the hyperoxia group was significantly increased on the 7th day (P < .05), but no significant differences were observed on the 4th, 10th and 14th day (P > .05) ( Table 1 and Figure 1).

| Expression of miR-125b in the lung tissues of premature SD rats
Compared with that of the air group, the miR-125b increased significantly on the 1st, 4th and 7th days, and decreased significantly on the 10th and 14th days (P < .05) ( Table 2 and Figure 2).

| Expression of Nrf2 and IL-1β in A549 cells
The expression of IL-1β in the non-interference hyperoxia group after hyperoxia exposure at 48 hours was significantly different (t = 12.96, P = .028), and Nrf2 increased at the same time. When Nrf2 was suppressed by siRNA, the expression of IL-1β increased and shows no significant difference after hyperoxia exposure (t = −2.031, P = .114) ( Figure 5).

| Influences of hyperoxia and siRNA on the expression of Nrf2 and miR-125b in A549 cells
Compared with that of the non-interference air group, the expression of Nrf2 and miR-125b in the non-interference hyperoxia group increased significantly (P < .05). Compared with that of the air group after interference, the expression of Nrf2 showed no significant change in the hyperoxia group (t = 0.070, P = .804) after 48 hours of hyperoxia exposure, and the expression of miR-125b decreased significantly (t = 7.891, P < .05) ( Table 5 and Figure 6). qRT-PCT assays of lung Nrf2 levels in premature rats exposed to air or hyperoxia. Compared with the air group, Nrf2 increased gradually after birth and reached its peak on the 7th day and then decreased. There were significant differences on the 7th day. Data represent the mean ± SD *P < .05

| The influence of siRNA targeting Nrf2 and hyperoxia on A549 cell apoptosis
TA B L E 2 Changes in the expression of miR-125b in lung tissue of premature rats at different time-points in two groups F I G U R E 2 Changes in expression of microRNA-125b in lung tissue of premature rats at different time-points. Both air group and hyperoxia group, miR-125b increased gradually after birth. Compared with the air group, there were statistical differences at each time-point. miR-125b reached its peak on 10th, which is similar to Nrf2. Data represent the mean ± SD **P < 0.01, ***P ＜ .001 which is one of the pathogenic mechanisms of BPD. 13 In the immune system, there is a dynamic balance between pro-inflammatory cytokines and anti-inflammatory cytokines. Long-term exposure to high concentrations of oxygen activates and releases many inflammatory cytokines, resulting in endothelial peroxidation, increased vascular permeability and aggravated alveolar interstitial and airway oedema. 14 Free radicals such as ROS and RNS produced by oxidative stress directly or indirectly attack cellular DNA, leading to cell degeneration and apoptosis. 15 Therefore, enhancing the antioxidation ability and reducing ROS production are key to reducing the incidence of BPD and are important for improving the survival rate and quality of life of premature infants. 16 The periods of lung development include the embryonic, pseudoglandular, canalicular, saccular and alveolar periods. At present, the animal models of BPD are mainly generated by hyperoxia exposure, and rodents are the most widely used model. For example, the pregnancy cycles of SD rats are relatively shorter than those of other models, and rats commence alveolarization ex utero and are born at a similar stage of lung development to that of a very pre-term human infant (saccular stage). 17 Previous studies have shown that 95% O 2 exposure resulted in a pulmonary inflammatory response and fibrosis in neonatal rats. 18  F I G U R E 3 Nrf2 suppressed by siRNA. siRNA designed by GenePharma. The control group was that Nrf2 not suppressed by siRNA. siRNA1, siRNA2 and siRNA3 represent three different siRNAs transfected into A549 cells, respectively. Their primer sequences are shown in Table 4. Data represent the mean ± SD **P < .01 F I G U R E 4 Nrf2 suppressed by siRNA for 48 h and subsequently subjected to protein isolation. Nrf2 and GAPDH (as reference gene) were measured by Western blot analysis. Molecular weight of Nrf2 is 70 kD including tags and GAPDH is 146 kD F I G U R E 5 Nrf2 and IL-1β in A549 while Nrf2 suppressed by siRNA and hyperoxia exposed. While A549 exposed to hyperoxia, Nrf2 increased significantly but IL-1β decreased. After transfection of siRNA into A549 cells, the expression of Nrf2 was decreased. When Nrf2 was suppressed, then A549 cells were exposed to high concentration of oxygen, and there was no significant difference in the expression of Nrf2, but the expression of IL-1β was increased. Data represent the mean ± SD *P ＜ .05, **P ＜ .01, ***P ＜ .001 TA B L E 5 Influence of hyperoxia and siRNA silencing on the expression of Nrf2 and miR-125b in A549 cells

Group (n = 3) Nrf2 miR-125b
Non-interference air group an animal model of BPD through neonatal rats exposed to hyperoxia and suppressed Nrf2 by siRNA in A549 cells instead of AECII cells.
We explored the role of Nrf2 in BPD in animal and cell models and provided new ideas for the prevention and treatment of BPD.
Nrf2 is located on chromosome 2q31.2 and belongs to the CNC subfamily containing bZIP transcription factors. This group contains nuclear factor erythroid-derived 2 (NFE2) and the NFE2-related factors Nrf1, Nrf2 and Nrf3. 19 Nrf2 is regulated by Kelch-like ECHrelated protein 1 (Keap1), which controls the expression of key components of the glutathione (GSH) and thioredoxin (TXN) antioxidant system, as well as enzymes involved in NADPH regeneration, ROS and xenobiotic detoxification, and heme metabolism, thus playing a fundamental role in maintaining the redox homeostasis of the cell. 20 As shown by a complementary DNA (cDNA) microarray in lung tissues, Nrf2-mediated protection against hyperoxia-induced acute lung injury (ALI) may be mediated by transcriptional regulation of genes related to DNA replication, the cell cycle, various metabolic pathways and small molecules, cell-to-cell interactions and redox homeostasis in neonatal mouse lung tissues during the cystic phase.
Nrf2 deficiency in immature lung aggravated lung injury and alveolar arrest induced by hyperoxia in neonates. 21 Studies have shown that Nrf2 increased survival in hyperoxic conditions and attenuated hyperoxia-induced alveolar growth inhibition in newborn mice. 22 Therefore, as an antioxidant factor, Nrf2 has an important protective effect on oxidative stress induced by hyperoxia exposure in the respiratory tract.
miRNAs are non-coding single-stranded RNA molecules that post-transcriptionally regulate target genes by binding specifically to the 3' untranslated region of target gene mRNAs. Prolonged hyperoxia alters the expression of miRNAs during normal lung development. These data support the hypothesis that the dynamic regulation of miRNAs plays a prominent role in the pathophysiology of BPD. 23 The miR-125 family is involved in many cellular processes, including cell differentiation, proliferation, metastasis, F I G U R E 6 Nrf2 and miR-125b increased significantly in A549 cells exposed to hyperoxia, and miR-125b decreased while Nrf2 suppressed by siRNA after A549 cells exposed to hyperoxia, suggested that miR-125b may be regulated by Nrf2, and participates in protection mechanism. Data represent the mean ± SD *P ＜ .05, **P ＜ .01, ***P ＜ .001 F I G U R E 7 Apoptosis of A549 cells exposed to hyperoxia for 0, 24, 48 and 72 h after silencing Nrf2. Compared with the air group, the apoptotic fraction of A549 cells in the hyperoxia group after Nrf2 silencing was significantly higher at 24, 48 and 72 h (F = 886.878, 1205.854, 4470.786, P < .05), and these are 27.650 + 0.254% vs 11.000 ± 0.707%, 31.950 ± 0.212 vs 12.150 ± 0.778% and 38.450 ± 0.212% vs 12.150 ± 0.495%, respectively. One-way ANOVA was used between the two groups apoptosis and immune defence, and is composed of three homologs: hsa-miR-125a, hsa-miR-125b-1 and hsa-miR-125-2. 24 Compared with miR-125a, miR-125b has been more extensively studied, and several targets of miR-125b regulating cell survival, proliferation and differentiation have been suggested and experimentally confirmed. 25 Up-regulation of miR-125b expression maintained the body weight and survival of ALI mice and significantly reduced LPS-induced pulmonary inflammation. 26 Another study showed that increased miR-125b through toll-like  Nrf2-ARE binding regulates the expression of more than 200 genes involved in cellular antioxidant and anti-inflammatory defence. 33 In the in silico promoter analysis using rVISTA, a conserved putative ARE sequence was identified in the promoter regions of miR-125b1 or miR-125b2; consistently, treatment of NRK52E cells with sulforaphane (SFN, another activator of Nrf2) increased miR-125b, which indicates that Nrf2 regulates miR-125b through AREs. 34

| CON CLUS ION
Both Nrf2 and miR-125b are important factors in the antioxidant response. The increased expression of Nrf2 and miR-125b in premature rats and A549 cells induced by hyperoxia exposure is involved in mitigating oxidative stress-induced cell damage. miR-125b plays a role in cellular protective mechanisms as a downstream target of Nrf2, but the specific mechanism of Nrf2 regulating miR-125b has not yet been fully elucidated. Strategies to rationally regulate the expression of Nrf2 and miR-125b to prevent and treat BPD require further study.

ACK N OWLED G EM ENTS
The authors thank their neonatal rats for participating in this study.
This work was supported by the National Natural Science Foundation of China (81571467). We would like to thank the Animal Laboratory Center of Pediatrics, Children's Hospital of Fudan University.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R S' CO NTR I B UTI O N S
ZXY, CC and GXH: made substantial contributions to the conception and design, acquisition of data or analysis and interpretation of data.
ZXY, CXY, ZHL and CC: were involved in drafting the manuscript or revising it critically for important intellectual content. GXH and CC: revised the manuscript and gave the final approval of the version to be published. The authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.