A total of 32 Sprague-Dawley neonatal rats (weight 6.2±0.3 g) were purchased from the Animal Center of Jiangsu University (Zhenjiang, China). The rats were randomly assigned to 4 groups with 8 rats in each group. Each group of neonatal rats was fed by a 3-month-old female rat. Rats were kept on a 12-h light/dark cycle at a room temperature of 23±2°C and supplied with sufficient food and water. Neonatal rats were sacrificed via cervical dislocation, followed by a sternotomy. Phosphate buffered saline (PBS) was gently perfused into the trachea with an injector to clear the lung tissue. Lungs were collected and frozen immediately for further study at postnatal day 1, 4, 7 and 14 (P1, P4, P7 and P14). All animal research was approved by the Animal Center of Jiangsu University.

Tissues were fixed with 4% paraformaldehyde for 24 h at 4°C and washed with PBS. Subsequently, samples were dehydrated using an alcohol gradient (75% alcohol, 1.5 h; 95% alcohol, 1.5 h; 100% alcohol, 1.5 h; 100% alcohol, 1 h; two xylene washes, 0.5 h each) and embedded in paraffin. Sections were sliced at 3 µm, followed by conventional dewaxing in water. Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) and boiled for 20 min. The tissue sections were stained with hematoxylin and eosin (Solarbio Science & Technology Co., Ltd., Beijing, China) 2-3 min each for histological analysis. All steps were performed at a room temperature. The sections were acquired by confocal light microscopy (Olympus Corporation, Tokyo, Japan) at magnification of ×400.

RT-qPCR was used to detect the mRNA expression of SUMO1 and SENP1 at each time-point. Total RNA was isolated from the tissue samples using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). For RT, cDNA was synthesized using the Prime Script RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturerֺ’s instructions. qPCR was performed using the SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd., Dalian, China). The reaction conditions were as follows: 95°C for 30 sec, and 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Target sequences of SUMO1, SENP1 and β-actin were as follows: SUMO1, 5′-AAG TTA TTG GAC AGG ACA GCA-3′ and 5′-CAT TCC CAG TTC TTT TGG AG-3′; SENP1, 5′-CGC CAG ATT GAA GAG CAG A-3′ and 5′-AGA GGA ACA CGA AGG TGG AG-3′; β-actin, 5′-TGT CAC CAA CTG GGA CGA TA-3′ and 5′-GGG GTG TTG AAG GTC TCA AA-3′. The specificity of the PCR product was ensured by melting curve analysis using the LightCycler^® 96 (Roche Diagnostics, Basel, Switzerland). All calculations of the relative expression of the target gene were performed using the 2^−ΔΔCq method (22).

For the experiments, human primary type II alveolar epithelial cells (AT2; cat. no. HUM-iCELL-a002; purchased from iCell Bioscience, Inc., Shanghai, China) were cultured in Dulbeccoֺ’s modified Eagleֺ’s medium/F12 (DME/F-12; HyClone; GE Healthcare Life Sciences, Logan, UT, USA), supplemented with 10% fetal bovine serum (Wisent, Inc., St. Bruno, QC, Canada), in a 5% CO₂ at 37°C. For AT2 differentiation, retinoic acid (RA) was added to medium at 1 µM (23). RA powder was purchased from Target Molecule Corp. (Boston, MA, USA) and dissolved in dimethyl sulfoxide. To minimize the effect of AT2 cells transforming into AT1 in vitro and ensure stable growth, the primary AT2 cells were passaged for three generations prior used for differentiation. After reaching 80-90% confluency, the cells were divided into normal control group (NC group), RA group (with 1 µM RA in the medium), si-NS group, si-SENP1 group, RA + si-NS group, and RA + si-SENP1 group. The si-SENP1 cells and si-NS cells were transfected with SENP1 small interfering RNA (siRNA) or non-specific (NS) siRNA.

All protein extraction handling was performed on ice. Free SUMO1 and SUMOylated proteins were analyzed. As described by Sharma et al (24), individual lung tissue protein lysates were prepared either using 4% sodium dodecyl sulfate (SDS) or 1% Nonident P40 (NP40). SDS denatures the action of SENPs and preserves conjugated SUMO. Therefore, the measured free SUMO1 is the naturally existing free unconjugated SUMO1 protein. NP40 separates SUMO1 from the target. Thus, the measured free SUMO1 represents total SUMO1 including unconjugated and separated SUMO1 in lung tissue. Free SUMO1 and SUMOylated proteins were extracted by 4% SDS, unless otherwise indicated. Protein extraction for SENP1 detection was performed as described. AT2 cells were harvested using the radioimmunoprecipitation assay buffer containing protease inhibitor phenylmethanesulfonyl fluoride (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for cell lysis. The extract was centrifuged at 12,000 × g, 4°C for 15 min and the supernatant was collected. The protein concentration was detected using a bicinchoninic acid kit (Beyotime Institute of Biotechnology, Haimen, China). Protein extracts (10 µl) were fractionated by 8-16% SDS-PAGE (GenScript, Piscataway, NJ, USA) and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% milk-Tris-buffered saline (TBS) buffer containing 0.1% Tween-20 at 37°C for 1 h, then incubated overnight at 4°C with primary antibodies. Primary antibodies were applied at dilutions of 1:200 for SENP1 (cat. no. HPA011765; Sigma-Aldrich; Merck KGaA), 1:1,000 for surfactant protein C (SP-C; cat. no. sc-13979; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), 1:1,000 for aquaporin-5 (AQP5; cat. no. sc-514022; Santa Cruz Biotechnology, Inc.), 1:1,000 for SUMO1 and 1:1,000 for β-actin (cat. no. 4903 and cat. no. 3700 from Cell Signaling Technology, Inc., Danvers, MA, USA). Following washing with TBS buffer-0.1% Tween-20, horseradish peroxidase-coupled secondary antibodies (1:2,000; cat. nos. FMS-MS01, FMS-Rb01 and FMS-Gt01) from Fcmacs Biotech Co., Ltd. (Nanjing, China) were added for 1 h at 37°C. The signals were visualized using FluorChem FC3 chemiluminescence (ProteinSimple, San Jose, CA, USA). Due to the significant difference in the protein expression quantity of free SUMO1 and conjugated SUMO1, the exposure time of conjugated SUMO1 (20 sec) was shorter than that of free SUMO1 (50 sec). Thus, they are typically shown separately in immunoblot bands (24,25). Semi-quantitative analysis was performed using LANE 1D software (Beijing Sage Creation Science Co., Ltd., Beijing, China). The target protein and β-actin were analyzed, and the expression of the target protein at each time-point was compared with with the corresponding internal reference. Statistical analysis was conducted to estimate the relative expression of the target proteins.

Retinoic acid (RA) is a major bioactive metabolite of vitamin A. It regulates normal lung development and maturation, and preserves alveolar formation in a caloric restriction model (26,27). To investigate the effect of RA on AT2 differentiation, 6×10⁵ cells were seeded in 2 ml medium. At a density of 1×10⁷ cells per 6-cm culture dish, AT2 cells were randomly divided into negative control (NC) group and RA group (with 1 µM RA added to the medium). Cells were harvested at 24, 48 and 72 h. Double immunofluorescent staining was used to observe the expression of SP-C and AQP5. AT2 cells were seeded in 24-well plates at a density of 1×10⁴ cells/well. The cells were separated into NC group and RA group. Cells were allowed to grow for 24, 48 and 72 h, then fixed with 4% paraformalde-hyde at room temperature for 20 min and blocked in 3% fetal calf serum at 37°C for 1 h. Primary antibodies against SP-C (1:200; cat. no. sc-13979) and AQP5 (1:200, cat. no. sc-514022; Santa Cruz Biotechnology, Inc.) were incubated overnight at 4°C. Anti-SP-C was detected using a secondary fluorescein isothiocyanate (FITC)-conjugated antibody (goat anti-rabbit IgG; cat. no. A22120; 1:200; Abbkine Scientific Co., Ltd., Wuhan, China) and anti-AQP5 was detected using a secondary Dylight 594-conjugated antibody (goat anti-mouse IgG; cat. no. ab96873; 1:200; Abcam, Cambridge, MA, USA) with incubated for 1 h at 37°C. Cell nuclei were stained with DAPI (1:1,000) for 3 min at a room temperature of 23±2°C. Fluorescent images were obtained using a fluorescent microscope (Olympus Corporation). Quantification of expression levels of SP-C and AQP5 in fluorescent images was performed using Image-Pro Plus III (Media Cybernetics, Inc., Rockville, MD, USA). The percentage of SP-C or AQP5 is the expression level of SP-C or AQP5/(SP-C+AQP5 expression level).

Transfections were performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), in accordance with the manufacturerֺ’s instructions, when the cells reached 33% confluency. To verify the transfection efficiency, AT2 cells were divided into si-NS group and si-SENP1 group with no RA added in medium. To investigate the suppression impact of SENP1 on differentiation, cells were transfected with a SENP1 siRNA or NS siRNA with RA added in the medium. The concentration of each siRNA used for transfection was 50 nM. Cells were harvested after transfection for 24, 48 and 72 h. The transfection efficiency of SENP1 was linked to the resulting mRNA and protein expression levels of conjugated SUMO1. The suppressive influence of SENP1 with RA added was detected by RT-qPCR and western blot analysis. Protein and gene expression levels of SP-C and AQP5 were measured to analyze the effects on differentiation. The siRNA sequences were as follows: SENP1 siRNA, 5′-GCC UGA CCA UUA CAC GCA ATT-3′ and 5′-UUG CGU GUA AUG GUC AGG CTT-3′; NS siRNA: 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and 5′-ACG UGA CAC GUU CGG AGA ATT-3′. Primer sequences for detecting SP-C, AQP5 and β-actin were as follows: SP-C, 5′-TTA CCA CTG CCA CCT TCT CC-3′ and 5′-TCA AGA CTG GGG ATG CTC TC-3′; AQP5, 5′-ACT GGG TTT TCT GGG TAG GG-3′ and 5′-GTG GTC AGC TCC ATG GTC TT-3′; β-actin, 5′-UGA CCU CAA CUA CAU GGU UTT-3′ and 5′-AAC CAU GUA GUU GAG GUC ATT-3′. The RT-qPCR processes were performed as described.

Cell counting kit-8 (CCK-8; Biosharp, Hefei, China) was used to analyze cell proliferation. AT2 cells were randomly into si-NS group and si-SENP1 group. They were then cultured in DME/F-12 for 24, 48 and 72 h. The cells were then seeded in a 96-well plate at a density of 5,000 cells/well in 100 µl medium and 10 µl CCK-8; four duplicate wells were set for each sample. After 2 h in the cell incubator, and ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to measure absorbance at 450 nm.

An Annexin V/propidium iodide (PI)-FITC apoptosis detection kit I (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze cell apoptosis by flow cytometry. AT2 cells were plated at a density of 6×10⁵ cells per 6-cm culture dish, and divided into si-NS and si-SENP1 groups. Cells were harvested after transfection for 24, 48 and 72 h. Cells and medium were collected in a 15 ml centrifuge tube. Trypsinization without EDTA was used to suspend cells. Samples were handled according to the manufacturerֺ’s instructions of the apoptosis detection kit. The apoptosis rate of cells in each group was analyzed using an Accuri C6 flow cytometer (CANTO 10C; BD Bioscience).

Data are expressed as the mean ± standard deviation and analyzed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used to analyze data differences among multiple groups, and then comparisons between groups were assessed using a Student-Newman-Keuls test. Student’s t-test was used to analyze the difference between groups. P<0.05 was considered to indicate a statistically significant difference.

As presented in Fig. 1, irregularity in the structures could be observed in the primary alveoli at P1. At P4, the late canalicular stage, an increasing number of alveolar septa were formed with some ridges protruding into the alveolar space. At P7, the size of air sacs tended to be homogeneous and the interstitial tissues thinned out. By P14, mature alveoli were the basic unit in the lung, appeared uniformly sized and separated by a thin septum.

To investigate the role of SUMOylation in lung development, whether the process involves changes in the levels of SUMO1 conjugation was determined. Neonatal rats 14 days after birth were used. At this age, their lungs are in the saccular and alveolar period, an important phase of lung maturation. Initially, the protein levels of naturally existing free SUMO1 were detected. As shown in Fig. 2A, free SUMO1 increased at day 4 compared with day 1, visibly decreased at day 7 compared with day 4, and then expressed at a similar level until day 14. Day 4 and day 14 were selected as time-points for detecting the degree of SUMO conjugation. In western blot analysis, free SUMO1 and bands of higher molecular mass (>70 kDa) were detected indicating SUMOylated proteins (Fig. 2B). Free SUMO1 prepared using 4% SDS (which represented naturally existing free SUMO1) was compared with free SUMO1 prepared using 1% NP40 (which represented total SUMO1). Naturally existing free SUMO1 at day 14 exhibited a sharp decrease compared with day 4, while no significant changes were observed in total SUMO1 (Fig. 2B). The results indicate that the total SUMO1 was steadily expressed and the SUMOylation and deSUMOylation were maintained in a dynamic balance.

Gene expression analysis was performed to further analyze the role of SUMO1 in lung development. RT-qPCR results indicated that SUMO1 mRNA was not significantly changed during lung development from P1 to P4 (Fig. 3A). This indicates that mRNA expression of SUMO1 was maintained in at a stable level. Subsequently, gene expression analysis of SENP1 was performed (Fig. 3B). The results demonstrated an increase in SENP1 mRNA at P4 compared with P1, which decreased at P7 and was maintained a steady level from P7 to P14. To confirm if varied gene expression is consistent with protein expression, SENP1 was also detected using western blot analysis. As presented in Fig. 4, the gene and protein levels of SENP1 exhibited a similar trend during lung development. However, compared with the induction of SENP1 mRNA at P4, the change in protein expression level was marginal. This difference may be associated with the post-translational modifications, and that protein translation is slightly delayed compared with mRNA transcription. This confirmed that SENP1 may impact lung development.

In a previous study, tritium [³H] was injected into pregnant rats and the fetal rats were examined (28). This experiment revealed that AT2 cells could convert to AT1 cells during lung development. According to the previous study in rats, during the 14 days after birth, the expression of SP-C was gradually reduced, whereas the expression of AQP5 increased (17,29). SP-C and AQP5 are specific markers of AT2 and AT1 cells, respectively (30,31). This indicated that AT2 differentiation to AT1 may occur during this period. To determine whether SENP1 affects lung development by participating in the differentiation of AT2, AT2 cells were cultured in vitro in the current study and RA was used to promote differentiation. Initially, the differentiation efficiency of RA was examined. AT2 cells were exposed to 1 µM RA and the expression levels of SP-C and AQP5 were then detected using western blot analysis. As shown in Fig. 5A-C, the expression of SP-C was decreased at 24 and 72 h following treatment with RA. However, it increased was 48 h, which may have been caused by cell proliferation. RA enhanced the expression of AQP5 at 24, 48 and 72 h compared with untreated cells (Fig. 5C). As shown in Fig. 5A and D, SENP1 protein was increased over time in the control and RA groups. Additionally, the expression of SENP1 in the RA group was increased compared with the control group at 24, 48 and 72 h. To validate the changes in SP-C and AQP5, the expression were also measured by immunofluorescence (Fig. 6A and B). These results also demonstrated that the decrease in SP-C was accompanied by an increase in AQP5. The experiments demonstrated that RA promoted the differentiation of AT2. Taken together, these results suggest that SENP1 may participate in the differentiation of AT2.

siRNA was used to inhibit SENP1 expression in AT2. Cells were transfected with si-SENP1 and non-specific RNA (si-NS). Transfection efficiency was demonstrated by RT-qPCR and western blot analysis. The results indicated that the gene expression of SENP1 was decreased at 24, 48 and 72 h after transfection compared with si-NS. SENP1 was decreased 8.8-fold at 48 h compared with the corresponding control group, indicating efficient inhibition (Fig. 7A). As SENP1 is a SUMO-specific protease, mediating protein deSUMOylation is its main role. Changes in SUMOylated proteins were detected when SENP1 was depleted using siRNA. The level of conjugated SUMO1 increased sharply, while free SUMO1 was decreased by SENP1 knockdown compared with si-NS (Fig. 7B). This result indicates that the inhibition of SENP1 expression interfered with deSUMOlation. The effect of SENP1 inhibition on cell differentiation was investigated further. AT2 cells were cultured in medium containing RA, then transfected with si-NS (RA + si-NS group) or si-SENP1 (RA + si-SENP1 group). Western blot analysis demonstrated that the expression of SENP1 protein decreased significantly in the RA + si-SENP1 group compared to the RA + si-NS group (Fig. 8A and B), with a 1.5-fold decrease in protein levels at 24 h, 3.2-fold at 48 h, and 1.6-fold at 72 h. The inhibition efficiency was the most pronounced at 48 h, in accordance with the changes in mRNA expression. The protein expression levels of SP-C and AQP5 were also assessed using western blot analysis. As shown in Fig. 8C and D, compared to the control group, the inhibition of SENP1 decreased the protein level of AQP5 expression at 24, 48 and 72 h. The protein expression of SP-C was increased at 24 and 48 h, but decreased at 72 h. The decrease of SP-C may have been caused by growth inhibition. The change in SUMOylated proteins was also detected. As shown in Fig. 8E, the conjugated SUMO1 was increased by si-SENP1 and free SUMO1 was decreased, which was similar to the result with no RA was added (Fig. 7B). The expression level of free and total SUMO1 under these two conditions revealed no statistically significant difference. These results indicate that the inhibition efficiency of SENP1 on conjugated and free SUMO1 has not been affected by the effect of RA on the differentiation of AT2 cells. To determine the effect of SENP1 on cell differentiation, SP-C and AQP5 levels were also analyzed by RT-qPCR. SP-C and AQP5 mRNA expression levels exhibited a similar expression pattern to the relevant protein levels following SENP1 knockdown (Fig. 9). These results indicate that inhibition of SENP1 hinders the differentiation of AT2, and that SENP1 is an important regulator of the differentiation process.

SENP1 has an important role in regulating the proliferation and apoptosis of cells. CCK-8 was used to evaluate the AT2 proliferation in cells transfected with SENP1 siRNA for 24, 48 and 72 h. As shown in Fig. 10A, cell proliferation was reduced in the si-SENP1 group compared with si-NS (P<0.05). To examine whether SENP1 knockdown also affects apoptosis, flow cytometry analysis and an Annexin V-FITC/PI apoptosis detection kit was used. Early apoptosis rate (Q3) and late apoptosis rate (Q2) were combined as the total apoptosis rate. SENP1 inhibition promoted cell apoptosis when AT2 cells were transfected with si-SENP1 for 24, 48 and 72 h (Fig. 10B). Following transfection with si-SENP1 for 24 h, the percentage of cells in the early apoptosis subgroup was significantly higher than in the si-NS group (7.230±0.816 vs. 4.573±0.398%); however, SENP1 inhibition had no influence on the late apoptosis. The percentage of cells in both the early and late apoptosis subgroups were higher compared with the si-NS group (12.833±1.266 vs. 7.317±0.831%; and 7.453±0.479 vs. 5.183±0.629%, respectively) when SENP1 inhibition was increased to 48 h. Furthermore, the percentage of cells in the early and late apoptosis subgroups were higher compared with the si-NS group (17.500±1.080 vs. 13.467±1.034%; and 8.637±0.585 vs. 6.860±0.483%, respectively) at 72 h after transfection. The significant decrease in cell apoptosis confirmed that the suppression of SENP1 caused AT2 cell death. Overall, the data indicate that SENP1 inhibition can reduce the proliferation of AT2 cells and promote apoptosis.