Introduction

The Qinghai-Tibet Plateau is known as the “roof of the world” and “water tower of Asia.” It is the highest plateau in the world1,2. It has a unique environment with low temperature, low oxygen, low pressure, and strong ultraviolet radiation. When people living at low altitude enter the plateau, some may develop a series of nonspecific symptoms and syndromes including headache, fatigue, vomiting, chest tightness, and nausea due to poor acclimatization, which is called acute altitude sickness3. Altitude sickness is the most obvious initial symptom of change in lung function. Alveolar hypoxia is the most significant feature of high-altitude environment4. Long-term exposure to high altitude and low-oxygen environment affects the cardiopulmonary system, which can eventually lead to pulmonary hypertension and pulmonary edema5. Tibetan pig is a unique local pig breed in the plateau areas of China. Tibetan pigs live in high-altitude areas with low temperature and low oxygen for a long time, and they have developed a unique mechanism to adapt to the extreme environment of the plateau6,7. Under hypoxia, investigation of the molecular mechanisms in the lungs of these pigs, as a representative model animal in high-altitude areas, can help in developing strategies for humans or other animals to adapt to hypoxic environment at high altitudes.

MicroRNAs (miRNAs), approximately 22 nucleotides in length, are non-coding RNAs that predominantly function through the pairing of their seed region—typically comprising the 2nd to the 7-8th nucleotides—with complementary sequences in the 3' untranslated regions (3'UTRs) of target mRNAs. This interaction leads to the formation of the RNA-induced silencing complex (RISC), resulting in gene expression suppression8,9. Studies demonstrate that miRNAs are indispensable components of various physiological processes, encompassing cell proliferation10, oxidative stress, and apoptosis11. They further engage in complex regulatory mechanisms, notably by hindering translation initiation through competition with cap-binding proteins via miRNA-loaded Ago proteins, influencing mRNA circularization12. Our investigation delves deeply into the role of miRNAs in lung injury precipitated by hypoxia, resonating with insights into the protective effect of the miR-144/GSK3β axis13, the modulation of HIF2α by miR-22314, and the exacerbation of lung damage by miR-14515. Preliminary miRNA sequencing in Tibetan pig lungs reveals a striking variation in ssc-miR-101-3p expression correlated with altitude, suggesting its engagement in the adaptation to high-altitude environments16 (Fig. 1G, Supplementary Table S1).

Fig. 1
figure 1

Morphological observation of ATII cells. (A) ATII cell morphology after 48 h treatment in the normal oxygen group (10 ×). (B) ATII cell morphology after 48 h treatment in the hypoxia group (10 ×). (C) The internal structure of ATII cells in the normoxic group 48 h after the treatment (5 μm). (D) Enlarged structure of ATII cells at specific locations in the normoxic group 48 h after the treatment (2 μm). (E) The internal structure of ATII cells in the hypoxic group 48 h after the treatment (5 μm). (F) Enlarged structure of ATII cells at specific locations in the hypoxic group 48 h after the treatment (2 μm). (G) The expression of ssc-miR-101-3p in the lungs of TGN and TJC as evaluated by RNA-seq. (H) qRT-PCR was used to evaluate the expression of ssc-miR-101-3p in the ATII cells in the normoxic and hypoxic groups. microvilli (Mv), nucleus (N), mitochondria (M), and lamellar bodies (LB) are indicated. *P < 0.05, **P < 0.01 (the same below).

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FOXO3 is a member of the Forkhead box O (FOXO) family of transcription factors. It regulates multiple biological processes including oxidative stress response, apoptosis, autophagy, proliferation, and immunity, thus playing an important role in cell fate determination17,18,19. In recent years, several studies have focused on the regulation of FOXO3 by miRNAs. Yin et al. reported that miRNA-96-5p may play a tumor-promoting role by negatively regulating FOXO3 to promote cell proliferation20. Cao et al. reported that miR-182-5p activates the Wnt signaling pathway by inhibiting the degradation of β-catenin and enhancing the interaction between β-catenin and TCF4; this is mediated by repressed FOXO3a so that miR-182-5p promotes the proliferation of hepatocellular carcinoma (HCC)21. Meng et al. reported that hypoxia can promote the upregulation of miR-155 in extracellular vesicles (EVs) inhibit the expression of FOXO3, and promote the proliferation of RCC cells, thereby promoting the progression of cell tumors22.

Alveolar type-II epithelial cells (ATII) are one of the important cells constituting the alveolar epithelium, and they play an important role in maintaining the structure and function of the alveoli23,24. However, although FOXO3 plays an important regulatory role in various types of apoptosis, its mechanism of action in ATII cells in Tibetan pigs under hypoxic conditions remains unclear. Therefore, in this study, we aimed to assess the effect of the miR-101/FOXO3 regulatory axis on the ATII cells in Tibetan pigs during hypoxic conditions. In addition, the target genes of ssc-miR-101-3p and their functions were predicted using three bioinformatic software to analyze the molecular mechanism of hypoxia-induced lung injury. This study would enhance the understanding of the regulation of miRNAs in lung injury under hypoxia.

Materials and methods

Ethics approval

The present study and all experimental techniques, including animal experiments, were approved by the Animal Ethics Committee of Gansu Agricultural University (GSAU-ETH-AST-2021-023), and comply with the relevant guidelines and regulations of ARRIVE.

Animal sample collection

Initially, 20 unrelated depopulated 6-month-old Tibetan pigs of similar weight were selected from Gannan Tibetan Autonomous Prefecture (Gannan, China). Among them, 10 Tibetan pigs were randomly selected for rearing in Gannan Tibetan Autonomous Prefecture. They were labeled as TGN, representing an altitude of 3000 m. The remaining 10 Tibetan pigs were moved from their native place in Gannan to Jingchuan County. They were labeled as TJC, representing an altitude of 1000 m, and reared under the same conditions as those for TGN for 6 months. Then, three pigs in each group were randomly selected for euthanasia by intramuscular injection of ketamine, and the left lower lung lobe was collected and rapidly frozen in liquid nitrogen for later RNA extraction and miRNA-seq16,25.

Cell culture

One newborn (7-day-old) Tibetan pig was selected, anesthetized, and aseptically sacrificed. The lung tissue was collected. Primary ATII cells were isolated from the lungs according to the method described by Yang et al16. The isolated ATII cells were randomly divided into two groups: the control group (21% O2, normoxia) and the experimental group (2% O2, hypoxia). ATII cells were cultured under normoxic (74% N2, 5% CO2, and 21% O2) or hypoxic (93% N2, 5% CO2, and 2% O2) conditions using a mixed three-gas incubator(ESCO, Singapore). For dual-luciferase reporter assay, HEK-293T cells were purchased from the cell bank of the Chinese Academy of Sciences. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, high glucose, HyClone, USA), supplemented with 12% (v/v) heat-inactivated GIBCO Fetal Bovine Serum (FBS, Thermo Fisher Scientific, USA), and 1% Penicillin–Streptomycin solution (Solarbio, Beijing).

Cell transfection

MiR-101 mimic (100 nM), mock negative control (NC; 100 nM), miR-101 inhibitor (50 nM), inhibitor NC (50 nM), small interfering RNA targeting FOXO3 (si-FOXO3), and control siRNA (150 nM) were synthesized by GEMA Pharmaceutical Technology (Shanghai, China). FOXO3 overexpression vector (pc-FOXO3; 1 µg) and pcDNA3.1 vector were purchased from GENEWIZ (Suzhou, China). When the ATII cells attained 80% confluency, they were transfected with miR-101-3p mimics, inhibitors, or their respective NCs using INVI DNA RNA Transfection Reagent™ (Invigentech, CA, USA) for 48 h. The details of miR-101 mimics, inhibitors, and si-FOXO3 with their respective NC sequences are given in Supplementary Table S2.

Cell proliferation assay

First, the cell viability was determined as follows. The transfected ATII cells were seeded in a 96-well plate at a density of 3 × 103 cells in 100 μL/well and incubated for 48 h. Further, 10 μL CCK-8 reagent (Solarbio, Beijing, China) was added to each well, and the plate was incubated at 37 °C for 2 h. The optical density (OD) was measured at 450 nm with an enzyme marker.

Cell proliferation was assessed using the BeyoClick EdU Cell Proliferation Kit (EdU, Beyotime, Shanghai, China) with Alexa Fluor 555. The transfected ATII cells were incubated with 10 μM EdU solution in growth medium for 3 h. The cells were stained with Hoechst 33,342 solution (blue) and observed under a fluorescent microscope (Olympus IX71, Tokyo, Japan) at 200 × magnification. EdU-positive cells were analyzed using ImageJ 1.6 software (National Institute of Health, USA).

Determination of cell cycle phase

The transfected ATII cells were seeded in six-well plates at the appropriate density for 48 h. The supernatant was discarded, and the cells were washed twice with PBS. Pre-chilled 70% alcohol was added to the cell suspension and incubated overnight at 4 °C. The cell cycle phase was determined as described previously26. The cell samples were centrifuged, and ethanol was removed. The cells were washed once with PBS, and 500 μL PBS was added to the cells. Further, the cells were resuspended in PBS and sent to Sevier Biotechnology Co., LTD (Wuhan, China) for apoptosis assay.

Cytotoxicity assay

The extent of cell damage was assessed by detecting the release of lactate dehydrogenase (LDH). ATII cells were inoculated in 24-well plates at a density of 1 × 105 cells/well and incubated for 24 h. When the cells attained approximately 70% confluency, miR-101 mimics, mimic NC, and miR-101 inhibitor or inhibitor NC were used to transfect the cells. The cells were incubated for 48 h, after which the medium was collected. The LDH activity in the supernatant was analyzed using LDH assay kit according to the manufacturer’s instructions (Nanjing Jincheng Institute of Biological Engineering, Nanjing, China). The absorbance was measured at 450 nm with an enzyme marker (Thermo Fisher Scientific).

To determine the levels of reactive oxygen species (ROS), the cells were washed with PBS. DCFH-DA probe diluted in 10 μmol/L serum-free cell culture medium (Gibco, USA) was added to each well and incubated for 20 min at 37 °C. The cells were washed three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. Further, the fluorescence was observed under a fluorescence microscope.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from ATII cells using AG RNAex Pro RNA Extraction Reagent (AG, Hunan, China) according to the manufacturer's instructions. Mir-X miRNA First Strand Synthesis Kit and Evo M-MLV RT Kit and gDNA clean cDNA (AG, Hunan, China) were used to reverse transcribe the isolated RNA into complementary DNA (cDNA) for miRNA and mRNA expression analysis. The cDNA was stored at − 20 °C. qRT-PCR was performed using 2 × RealStar Fast SYBR qPCR Mix (GeneStar, Beijing, China) and LightCycler 480 II instrument (Roche, Basel, Switzerland). β-actin and U6 were used as internal mRNA and miRNA references, respectively. Data were obtained from three independent experiments. Relative expression levels were determined using the 2−ΔΔCt method27. All primers were synthesized by Shanghai Prime Tech Biotechnology Co., Ltd (Shanghai, China), and the sequences are given in Table 1.

Table 1 Primers for qRT-PCR.
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Western blot analysis

ATII cells were seeded in 6-well plates and transfected for 48 h using RIPA buffer and protease inhibitor mixture (Beyotime, Shanghai, China). All procedures were performed according to the kit instructions. Protein concentrations were determined using the BCA protein concentration assay kit (Servicebio, Wuhan, China). For each sample, equal concentration of protein was denatured using 4 × SDS-PAGE loading buffer. The denatured protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking the PVDF membranes with 5% skim milk for 2 h at room temperature, the membranes were incubated with anti-FOXO3 (1:1000; GB11092-1, Servicebio, Wuhan, China) and anti-GAPDH (1:2000; GB15002, Servicebio) antibodies overnight at 4 °C. The next day, the PVDF membranes were washed 5 times with TBST and incubated with goat anti-rabbit horseradish peroxidase (HRP) IgG antibody (Servicebio) at room temperature for 1 h. After 3 washes with TBST for 5 min each, the protein bands on the PVDF membranes were visualized using enhanced chemiluminescence (ECL) and a gel imaging system (Bio-Rad, Foster City, CA, USA). The grayscale values were analyzed using Image J software (http://imagej.nih.gov/ij/).

Bioinformatic analysis

To predict the targets of ssc-miR-101-3p in Sus scrofa, we leveraged an integrated suite of online tools including RNAhybrid (v2.1.2) alongside svm_light (v6.01), TargetScan (v7.0), and Miranda (v3.3a). With a dedicated focus on the 3'-UTR regions to circumvent potential false positives arising from non-specific mRNA binding sites, we prioritized 8mer and 7mer-m8 targets—deemed by TargetScan as high-confidence candidates. Applying stringent criteria of low binding energy and superior sequence complementarity, we refined our predictions. Post-prediction, we characterized the biological functions of these targets through meticulous Gene Ontology (GO) analysis and insightful scatter plot visualizations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways28,29. Employing a one-tailed hypergeometric test, we precisely pinpointed significantly enriched terms among the ssc-miR-101-3p target genes. To ensure robust q-values, we subjected the initial p-values to a stringent Bonferroni correction, mitigating the risk of false positives stemming from multiple comparisons. We emphasized terms that maintained significance post-correction (P < 0.05), utilizing a background gene set specific to the cell lines under investigation to maintain relevance and specificity. Consequently, FOXO3 emerged as a preeminent candidate target gene of ssc-miR-101-3p, deserving of further in-depth inquiry.

Dual-luciferase reporter assay

HEK-293 T cells were inoculated in 24-well plates, and miR-101-3p or mock NC and FOXO3-WT or FOXO3-MUT were used to co-transfect the cells with confluency of 70%–80%. Luciferase activity was analyzed 48 h after transfection using a dual luciferase reporter kit (Promega, Madison, WI, USA) and normalized to Renilla luciferase activity values. Data were obtained from three independent experiments.

Statistical analysis

Graphs were plotted using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA), and all data were presented as mean ± SEM. Differences between groups were analyzed using two-tailed student’s t-test and one-way analysis of variance (ANOVA). *P < 0.05 and **P < 0.01 were considered significant and highly significant, respectively.

Ethics statement

The study and all experimental techniques were approved by the Ethics Committee for the Care and use of Laboratory Animals of Gansu Agricultural University (GSAU-ETH-AST-005). The experiments were performed in accordance with the relevant guidelines and regulations implemented by the Administration of Laboratory Animal Affairs approved by the National Science and Technology Commission on October 31, 1988.

Results

Morphological changes in the ATII cells under hypoxia

First, observe the morphological changes in ATII cells following hypoxia treatment. Compared to the normoxia group (Fig. 1A), the intercellular gaps in the hypoxia group widened, and more floating cells and cell debris were observed in the culture medium (Fig. 1B). Additionally, the ultrastructure of ATII cells was examined using transmission electron microscopy. After 48 h under normoxia, the intracellular and mitochondrial membranes in ATII cells were intact, and some concentric lamellar vesicles were visible (Fig. 1C, D). However, in the hypoxia group, the nuclei were irregular in shape. Most mitochondria were swollen; cristae were disrupted; lamellar vesicles were absent; autophagic vesicles formed, and signs of apoptosis were more pronounced (Fig. 1E, F).

Expression analysis of ssc-miR-101-3p

Ssc-miR-101-3p expression was determined in the lung tissue of Tibetan pigs at different altitudes. Pre-sequencing results of indicated that ssc-miR-101-3p expression was significantly higher in TGN than in TJC (P < 0.01; Fig. 1G; Supplementary Table S11). The results of qRT-PCR showed that in the hypoxia group, ssc-miR-101-3p expression was significantly elevated in ATII cells compared with the normoxia group (P < 0.05; Fig. 1H), which was consistent with the sequencing results. Therefore, ssc-miR-101-3p may play an important regulatory role in the lung tissue of Tibetan pigs under hypoxia.

Overexpression of ssc-miR-101-3p inhibits hypoxia-induced apoptosis of ATII cells in Tibetan pigs

Proliferation of ATII cells is crucial for the pulmonary adaptation to hypoxic environments30. To assess the regulatory role of ssc-miR-101-3p in the proliferation of ATII cells, methods such as CCK-8 assay, EdU staining, flow cytometry, and qPCR were employed. Following transfection with ssc-miR-101-3p mimics, inhibitors, and their respective NCs, there was a significant increase or decrease in the expression levels of ssc-miR-101-3p in ATII cells (Fig. 2A). Results from CCK-8 and EdU staining indicated that overexpression of ssc-miR-101-3p significantly enhanced cellular viability and proliferation of ATII cells, whereas knockdown of ssc-miR-101-3p exhibited the opposite effects (Fig. 2B,E,F). Flow cytometry results demonstrated that overexpression of ssc-miR-101-3p significantly increased the proportion of ATII cells in the G2 phase and decreased the proportion in the G1 phase (Fig. 2C,D). However, inhibition of ssc-miR-101-3p significantly reduced the proportion of cells in the S phase (Fig. 2C,D). Additionally, qRT-PCR analysis was used to examine the effects of ssc-miR-101-3p on the expression of apoptotic factors Bax and Bcl-2. After transfection with ssc-miR-101-3p mimics, Bcl-2 was upregulated and Bax was downregulated, with trends reversing following ssc-miR-101-3p silencing (Fig. 2G,H).

Fig. 2
figure 2

Ssc-miR-101-3p overexpression promoted the proliferation of ATII cells. (A–H) ATII cells were transfected with simulated NC (100 nM), miR-101 mimics (100 nM), inhibitor NC, and miR-101 inhibitor (50 nM) and incubated for 48 h. (A) Transfection efficiency of miR-101 compared with control group. (B) CCK-8 assay was performed to analyze cell viability. The absorbance was measured at 450 nm. (C) Analysis of cell cycle phases. (D) Flow cytometric analysis of cell cycle. (E) Number of cells testing positive for EdU, DNA-replicating cells stained with EdU (red), and nuclei stained with Hoechst (blue). (F) Proportion of EdU-positive cells. (G–H) qRT-PCR was used to detect the mRNA levels of apoptosis-related genes Bax and Bcl-2 after miR-101-3p transfection.

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Ssc-miR-101-3p attenuates hypoxia-induced inflammatory damage in the ATII cells

Living at high altitudes can lead to respiratory difficulty and pulmonary tissue infections in animals, primarily due to inflammatory damage in ATII cells. To explore the impact of ssc-miR-101-3p on the inflammatory processes in ATII cells, these cells were transfected with ssc-miR-101-3p mimics, inhibitors, or their respective NCs. Overexpression of ssc-miR-101-3p significantly inhibited LDH activity and reduced levels of ROS, while inhibition of ssc-miR-101-3p increased LDH activity and ROS levels (Fig. 3A,B). Furthermore, overexpression of ssc-miR-101-3p significantly lowered the transcription levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8); conversely, inhibition of ssc-miR-101-3p significantly increased the expression levels of these inflammatory factors. ssc-miR-101-3p may regulate the expression of inflammation-related genes and mitigate inflammatory damage in ATII cells.

Fig. 3
figure 3

Ssc-miR-101-3p overexpression attenuated the inflammatory damage in the ATII cells. (A) ROS levels were measured to assess apoptosis in the ATII cells transfected with miR-101-3p mimics, miR-101-3p inhibitors, or NC. (B) LDH activity was detected by measuring the absorbance at 450 nm. (C–F) qRT-PCR was used to evaluate the levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-8.

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Ssc-miR-101-3p targets the 3′ UTR of FOXO3 in ATII cells

To elucidate the regulatory mechanisms of ssc-miR-101-3p in ATII cells, three different miRNA online prediction tools—RNAhybrid(v2.1.2) + svm_light, Miranda, and TargetScan—were utilized to identify the target genes of ssc-miR-101-3p. An intersection analysis of the results from these tools revealed a total of 5037 common target genes (Supplementary Table S3). KEGG and GO functional analysis indicated significant enrichment of common target genes in the FOXO and PI3K-AKT signaling pathways (Fig. 4A,B; Supplementary Tables S4, S5). Based on pathway-based gene interaction scoring and binding site assessment principles, we identified the binding of ssc-miR-101-3p’s seed sequence (CA-GACAU) to the 3'-UTR region of FOXO3, suggesting that FOXO3 may be a target gene of ssc-miR-101-3p. Interestingly, we discovered a perfect match between the miRNA's nucleotides 13–18 (AUAACU) and the upstream region of the FOXO3 binding site (AGUUAU), which may indicate an additional compensatory mechanism (Fig. 4C). To accurately confirm the regulatory mechanism of ssc-miR-101-3p on FOXO3, we prioritized the conserved seed region for further work. We successfully constructed wild-type and mutant FOXO3 3'UTR dual-luciferase reporter vectors. As expected, the ssc-miR-101-3p mimic significantly inhibited the luciferase activity of the wild-type FOXO3 3'UTR, while the activity level of the mutant FOXO3 3'UTR remained unchanged (Fig. 4D). Additionally, overexpression of ssc-miR-101-3p significantly suppressed the mRNA and protein levels of FOXO3 in ATII cells, whereas the inhibitor of ssc-miR-101-3p significantly increased the mRNA and protein levels of FOXO3 (Fig. 4E,F). These results indicate that FOXO3 is a direct target gene of ssc-miR-101-3p.

Fig. 4
figure 4

Ssc-miR-101-3p directly binds to FOXO3 3`UTR FOXO3 (A, B) GO and KEGG pathway enrichment analyses of the target genes of miR-101-3p. (C) The sequence in the 3′-UTR of FOXO3 that binds to miR-101-3p, red boxes indicate additional compensation mechanisms. (D) Dual luciferase assay assessing the relationship between miR-101-3p and FOXO3. (E) mRNA level of FOXO3 after transfection with miR-101-3p. (F) Protein levels of FOXO3 after transfection with miR-101-3p.

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Effect of FOXO3 on the proliferation and apoptosis of ATII cells in Tibetan pigs under hypoxia

To investigate the regulatory effects of FOXO3 on ATII cell proliferation, these cells were transfected with FOXO3 overexpression and suppression vectors, yielding significant increases or decreases in FOXO3 protein and mRNA levels, respectively (Fig. 5A,B). CCK-8 and EdU staining results illustrated that overexpression of FOXO3 reduced cell viability and the number of EdU-positive cells. Conversely, treatment with FOXO3 inhibitors demonstrated opposite outcomes (Fig. 4C,F–G). Flow cytometry results indicated that overexpressing FOXO3 increased the proportion of ATII cells in the G1 phase and decreased the proportions in the S and G2 phases compared to cells transfected with pcDNA3.1 (P < 0.05). Furthermore, inhibition of FOXO3 led to an increase in the percentages of S and G2 phase cells relative to cells transfected with inhibitor NC, while maintaining a higher proportion of cells in the G1 phase (P < 0.05) (Fig. 5D,E). Additionally, overexpression of FOXO3 significantly upregulated Bax and downregulated Bcl-2, whereas inhibition of FOXO3 showed reverse effects (Fig. 5H,I). Therefore, overexpression of FOXO3 may promote hypoxia-induced apoptosis in ATII cells.

Fig. 5
figure 5

FOXO3 could regulate the survival rate and apoptosis of ATII cells. (A, B) The mRNA and protein levels of FOXO3 were evaluated using qPCR and western blot analysis, respectively, in ATII cells after FOXO3 overexpression or inhibition. (C) CCK-8 assay was used to detect the cell viability of ATII cells with FOXO3 overexpression or inhibition. (D, E) Determination of cell cycle phase of ATII cells using flow cytometry. (F) EdU staining. The ATII cells in S phase were stained red with EdU, whereas the nucleus was stained blue with Hoechst. (G) The proportion of proliferating cells was analyzed using Image J. (G, H) qRT-PCR was used to detect the mRNA levels of apoptosis-related genes Bax and Bcl-2 after FOXO3 overexpression and interference.

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FOXO3 increases hypoxia-induced toxicity and inflammatory damage in the ATII cells

To further explore the effect of FOXO3 in hypoxia-induced inflammation in ATII cells, we analyzed the effect of FOXO3 knockdown and overexpression on hypoxia-induced ATII cytotoxicity by assessing the ROS levels and LDH activity. LDH activity and ROS levels were significantly increased after FOXO3 overexpression and decreased after FOXO3 knockdown (Fig. 6A,B). In addition, qRT-PCR results indicated that the inflammatory factors TNF-α, IL-6, IL-8, and IL-1β were differentially upregulated after transfection with pc-FOXO3 (FOXO3 overexpression) and downregulated in varying degrees after transfection with si-FOXO3 (FOXO3 knockdown) (Fig. 6C,D). These results suggested that FOXO3 overexpression aggravates hypoxia-induced inflammatory damage in ATII cells.

Fig. 6
figure 6

FOXO3 overexpression exacerbates inflammatory damage in ATII cells. (A) ROS levels were measured to assess apoptosis in ATII cells transfected with vectors for FOXO3 overexpression or inhibition or NC. (B) LDH activity was assessed by measuring absorbance at 450 nm. (C, D) The transcript levels of TNF-α, IL-1β, IL-6, and IL-8 were detected using qPCR after FOXO3 overexpression or inhibition.

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ssc-miR-101-3p targets FOXO3 to regulate ATII cell damage induced by hypoxia

Based on the above results, we determined that a relationship and negative functional correlation exists between ssc-miR-101-3p and FOXO3. We further determined whether ssc-miR-101-3p alters the effect of FOXO3 on ATII cell proliferation and damage. ATII cells were co-transfected with ssc-miR-101-3p mimic and pc-FOXO3 vectors and with mimic NC and pc-FOXO3 as controls. CCK-8, EdU staining, and flow cytometry results indicated that ssc-miR-101-3p overexpression attenuated the inhibitory effects of FOXO3 on cell viability, proliferation, and cell cycle (Fig. 7A,E–H). The ssc-miR-101-3p mimic and pc-FOXO3 co-transfection inhibited the expression of the proapoptotic factor Bax and promoted the expression of the anti-apoptotic factor Bcl-2 (Fig. 7B,C). In addition, the co-transfection with ssc-miR-101-3p mimic and pc-FOXO3 significantly reduced the LDH activity and levels of ROS and inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) compared with the control transfection (Fig. 7D,I–J). Therefore, the above results suggested that ssc-miR-101-3p exhibits a protective effect on ATII cells by inhibiting the expression of FOXO3.

Fig. 7
figure 7

Ssc-miR-101-3p regulates apoptosis and inflammatory damage in ATII cells by targeting FOXO3. (A) CCK-8 assay for ATII cell viability 48 h after miR-101-mimics and FOXO3 overexpression plasmids were co-transfected. (B, C) Levels of apoptosis-related genes Bax and Bcl-2 mRNA after combined treatment. (D) LDH activity was assessed by measuring absorbance 450 nm. (E, F) Determination of cell cycle phase of ATII cells co-transfected with miR-101-3p mimic and FOXO3 overexpression plasmid using flow cytometry. (G) EdU staining was performed to detect the proliferation of ATII cells 48 h after co-transfection with miR-101-3p mimic and FOXO3 overexpression plasmid. (H) EdU-positive cells. (I) ROS levels were measured to assess apoptosis in ATII cells after co-transfection. (J) Expression levels of TNF-α, IL-1β, IL-6, and IL-8 after co-transfection.

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Discussion

High-altitude, low-oxygen environments affect mammalian physiology and pose a great challenge for mammalian survival31. One of the most important functions of the lung is to maintain adequate oxygenation in the body. When the lung is exposed to hypoxic environment, mitochondrial function in the lung cells is impaired, which in turn leads to increased oxidative damage in the lung32. Hoenderdos et al. reported that hypoxia induces a destructive phenotype in neutrophils with delayed apoptosis, impaired capacity of killing bacterial cells, and increased release of cytotoxic proteases, which in turn increases the likelihood of an inflammatory response in the organism33. ATII cells play an important role in maintaining alveolar integrity and function, and they may be critical for lung function and repair34. To determine whether hypoxic environment affects the morphology of ATII cells of Tibetan pigs, the changes in morphological structure of ATII cells were assessed using transmission electron microscopy after 48 h of hypoxia treatment in vitro. Compared with the normoxic group (21% O2), the apoptotic features of ATII cells were obvious in the hypoxic group (2% O2), with disappearance of lamellar vesicles, significant swelling of mitochondria, and reduction of cell membrane microvilli. This was consistent with the apoptotic morphology of rat ATII cells studied by Huang et al35. This indicated that long-term exposure of ATII cell to hypoxic environmental stimuli leads to apoptosis. Stimulus response is a cellular response in which the organism’s own defenses are activated when exposed to any stimulus (such as hypoxia); this occurs by the regulation of expression of various RNAs (miRNA) in the cell36. Several studies have reported that miRNAs play a key role in coordinating many fundamental biological processes, including proliferation, apoptosis, differentiation, and metabolism37,38. Our previous study reported that ssc-miR-101-3p was differentially expressed in the lung tissues of Tibetan pigs at different altitudes. Therefore, it is important to explore whether porcine ssc-miR-101-3p is involved in the regulation of adaptation to hypoxic conditions in Tibetan pigs at different altitudes.

MiRNAs are involved in post-transcriptional regulation of genes and play an important role in developmental processes, cell differentiation, and immune defense in organisms39. miRNA expression has been reported to trigger innate immune responses in hypoxic environments and enhance host defense. Shi et al. reported that hypoxia inhibits cell cycle progression and cell proliferation in brain microvascular endothelial cells through the miR-212-3p/MCM2 axis40. miR-181a and miR-150 target the JAK1-STAT1/c Fos pathway that regulates immune inflammatory response and cardiomyocyte apoptosis in dendritic cells41. miR-760 mediates hypoxia-induced proliferation and apoptosis in the smooth muscle cells of human pulmonary artery by targeting TLR442. These findings suggested that aberrant miRNA expression is a key factor in regulating cell proliferation and apoptosis under hypoxia. Our previous study reported that ssc-miR-101-3p was significantly expressed in the lung tissue of Tibetan pigs at high altitude, suggesting that ssc-miR-101-3p may play an important role in the adaptation of Tibetan pigs to hypoxic environment. In the present study, ssc-miR-101-3p expression was significantly elevated in the ATII cells after hypoxia treatment, suggesting that ssc-miR-101-3p may be involved in the regulation of ATII cells under hypoxia. Therefore, we further verified whether ssc-miR-101-3p affected hypoxia-induced apoptosis in the ATII cells. Notably, ssc-miR-101-3p overexpression promoted ATII cell proliferation and decreased LDH activity, ROS levels, and release of proinflammatory factors, whereas ssc-miR-101-3p inhibition exhibited the opposite effect. miR-101 overexpression is reported to significantly reduce apoptosis and promote proliferation of hair follicle stem cells (P < 0.01) and increase mRNA and protein levels of proliferation-related (PCNA) and antiapoptotic (Bcl-2) genes43. miR-101 overexpression was reported to inhibit increased autophagy and apoptosis in mouse and cardiac myocytes (P < 0.01)44, which is consistent with our findings. Therefore, it can be concluded that upregulation of ssc-miR-101-3p inhibits hypoxia-induced ATII cell injury.

The regulatory effects of miRNAs on cell proliferation and apoptosis are achieved by binding to target genes45. Xu et al. reported that prediction of target genes of miRNAs using bioinformatic softwares is a rapid and efficient method46. Therefore, in this study, three online software programs, namely, RNAhybrid + svm_light, TargetScan, and Miranda, were used to predict the target genes of ssc-miR-101-3p, and 5037 shared target genes were successfully predicted. GO and KEGG functional enrichment analyses indicated that the target genes were significantly enriched in FOXO and PI3K-AKT signaling pathways. The lipoylinositol 3-kinase (PI3K) and protein kinase B (AKT) signaling pathways play central roles in cell proliferation and survival. FOXO transcription factor is negatively regulated by the PI3K/AKT signaling pathway, and these pathways are directly involved in the regulation of cell proliferation and apoptosis under hypoxia47. In alignment with the principles of pathway-based gene interaction scoring and binding site evaluation, our study identified FOXO3 as a candidate target gene for miR-101-3p, based on its conserved region (CA-GACAU) that binds to this microRNA. However, the binding site was discontinuous. Notwithstanding this, we uncovered a perfect pairing between the upstream region of the FOXO3 binding site (AGUUAU) and nucleotides 13 to 18 (AUAACU) of the miRNA. This observation suggests a compensatory mechanism for less than ideal seed pairing. Research by Friedman, Jan et al. has highlighted that, beyond the seed region match, a shift of six nucleotides in either the 3' or 5' direction, offset by one nucleotide, can sometimes mediate detectable repression48,49. In parallel, Bartel et al. have noted that the complementarity of nucleotides 13–16 at the 3' end of the miRNA can augment the pairing within the seed region35. Yet, the impact of these 3'-terminal complementary pairing sites on affinity and effectiveness is negligible. Thus, we prioritized the conserved seed region for our subsequent investigations—emphasizing the significance of the core binding motif. Our conclusions were substantiated by qRT-PCR, Western blot, and dual-luciferase reporter assays, all of which collectively verified that ssc-miR-101-3p specifically targets and binds to FOXO3. Furthermore, FOXO3 overexpression further exacerbated hypoxia-induced ATII cell damage; however, ssc-miR-101-3p overexpression reversed this effect. FOXO3 is a widely studied transcription factor that plays an important role in a wide range of physiological and pathological processes by regulating a multigene regulatory network50. Essers et al. reported that under oxidative stress, including reduced oxygen levels, FOXO3 is activated through signaling pathways involving Ral and JNK. This suggested that FOXO3 activation is involved in oxidative-stress-induced apoptosis51. Clinical trials revealed that RG7112, an MDM2 antagonist, inhibited cancer cell growth in acute leukemia by inducing apoptosis through the reactivation of FOXO3 under hypoxic conditions52. As expected, our data indicated that FOXO3 overexpression exacerbated the hypoxia-induced inflammatory response and promoted apoptosis in ATII cells. We further explored whether ssc-miR-101-3p regulates hypoxia-induced ATII cell apoptosis by binding to FOXO3. The findings revealed that the roles of FOXO3 and ssc-miR-101-3p are completely opposite in hypoxia-induced apoptosis and inflammatory response. ssc-miR-101-3p may inhibit hypoxia-induced ATII cell apoptosis and inflammatory response by targeting FOXO3 (Fig. 8). Nevertheless, we are acutely aware of the limitations inherent in our study. While the interaction between ssc-miR-101-3p and FOXO3 in Tibetan pigs has revealed its significant role in mechanisms of hypoxic adaptation, we have yet to directly ascertain the specificity and ubiquity of this mechanism across other swine species. This realization underscores the necessity for future research to place a greater emphasis on interspecies comparative analyses, thereby enabling a comprehensive evaluation of the cross-species applicability and functional versatility of the ssc-miR-101-3p-FOXO3 interaction.

Fig. 8
figure 8

Schematic representation of ssc-miR-101-3p related regulatory mechanisms affecting ATII cells in Tibetan pigs under hypoxia. Ssc-miR-101-3p targeted FOXO3 alleviates hypoxia-induced apoptosis and inflammatory injury in ATII cells of Tibetan pigs.

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Conclusion

In conclusion, our study revealed that ssc-miR-101-3p targets 3′ UTR of FOXO3 to alleviate hypoxia-induced inflammation and to promote cell proliferation in ATII cells of Tibetan pigs. These results provided new insights into the function of miRNAs in the ATII cells of Tibetan pigs and would help in better understanding of the regulatory mechanisms by which lung tissues of Tibetan pigs adapt to hypoxic environment.