miR‐20a‐5p regulates pulmonary surfactant gene expression in alveolar type II cells

Abstract MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR‐20a‐5p had such regulatory functions on alveolar type II (AT‐II) cells. To accomplish this, miR‐20a‐5p–overexpressed and miR‐20a‐5p–inhibited adenoviral vectors were constructed and transfected into cultured AT‐II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR‐20a‐5p levels were verified by real‐time PCR. The CCK‐8 assay was used to compare the proliferation ability of AT‐II cells that had over‐ or underexpressed miR‐20a‐5p. The expression of surfactant‐associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real‐time PCR and Western blotting. In AT‐II cells, transfection resulted in over‐ or under‐regulation of miR‐20a‐5p. While overexpression of miR‐20a‐5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR‐20a‐5p regulation of PTEN. As a result, when miR‐20a‐5p was rendered overexpressed, PTEN was down‐regulated. By contrast, when miR‐20a‐5p was underexpressed, PTEN was up‐regulated. Neither overexpression nor underexpression of miR‐20a‐5p altered the cell proliferation. miR‐20a‐5p plays no role in proliferation of foetal AT‐II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.


| INTRODUC TI ON
The alveolar type II (AT-II) epithelial cells at the alveolar horn have highly specialized functions for the synthesis, secretion and reutilization of pulmonary surfactant. 1  Thus, in this present study, we used miRNA microarrays to first detect the differential expression of miRNAs in peripheral blood of premature infants with versus without RDS. We found that miR-20a-5p was down-regulated in RDS premature infants who lacked pulmonary surfactant (PS). The result is consistent with our previous work in rats that showed that the expression of miR-20a gradually decreased in the late stage of foetal lung development. 12 Given these results, which were further validated by real-time qPCR, and in view of the role of PS in RDS, the association between miR-20a and RDS and the role of miR-20a in lung development, we further explored the role of miR-20a-5p in pulmonary surfactant gene expression.
Bioinformatic analysis has revealed that phosphatase and tensin homolog (PTEN) is one of the target genes for miR-20a-5p, which is in keeping with what was reported by Wang et al. 13,14 PTEN is abundant in lung epithelia where it plays an important role in pulmonary development and pulmonary function. 15 PTEN inhibition improves wound healing in lung epithelia. 16,17 In addition, it is well known that PTEN/PI3K/Akt signalling promotes proliferation of lung alveolar progenitor type II cells. 18 We found that the expression of PTEN increased gradually in the late stage of foetal rat lung development, which was negatively correlated with the expression of miR-20a-5p. Furthermore, it was found that miR-20a-5p regulated PS and PTEN gene expression in foetal AT-II cells in a reverse manner. While overexpression of miR-20a-5p promoted PS, it inhibited PTEN. By contrast, while underexpression of miR-20a-5p inhibited PS, it promoted PTEN. Thus, for the first time we have shown that miR-20a-5p plays an important role in regulation of synthesis of pulmonary surfactant. Based upon these findings, we postulate that miR-20a-5p might be important in controlling essential developmental and physiological events in the lung, including the synthesis and metabolism of pulmonary surfactant in AT-II, and may also be involved in the occurrence and development of RDS.

| Clinical samples
The study was approved by the Ethics Committee of Children's Hospital of Nanjing Medical University, and all the guardians of participants signed an informed consent for participation in this study (approval number: 201701011). Blood samples were obtained from premature infants with RDS <37 weeks' gestational age in 2014. As control, a group of age/sex-matched premature infants without RDS who had been admitted to the same hospital was also recruited. The diagnosis of RDS was based on the European consensus guidelines updated in 2013. 19

| miRNA array
The miRNA microarray assay was completed by LC Bio in Hangzhou.
The main testing steps included sample extraction and quality control, miRNA labelling, miRNA array hybridization, image acquisition and data analysis. The fluorescence intensity of the array was scanned using an GenePix 4000B array scanner (Molecular Devices Co.), and the original image intensity was read using software (Axon).
The data were analysed using unsupervised hierarchical clustering (Cluster 3.0) and Tree View analysis (Stanford University, Stanford, CA, USA).

| Animals
Sprague Dawley rats were used in this study. These include ten female rats, weight 250-300 g, and five male rats, weight 300-400 g in total. All rats were maintained in a specific pathogen-free animal facility at Animal Center of the Nanjing Medical University. FBS on 24-well or 6-well plates and cultured at 37°C and 5% CO2.

| Isolation, purification and primary culture of foetal AT-II
The nature of the cultures was identified by electron micrograph.
Cell viability was >98% as determined by the trypan blue exclusion assay. The purity was >90% as determined by immunohistochemistry with vimentin and fluorescent immunocytochemistry with SP-C ( Figure S1).

| Proliferation and cell survival assays
Cell viability was assessed using the Cell Counting Kit-8 assay
The 293T cells were divided into four groups, and the two types of recombinant plasmids were cotransfected into 293T cells with miR-20a-5p mimic or miR-20a-5p negative control, respectively, by Lipofectamine™ 3000 (Invitrogen). After 48 hours, firefly and Renilla luciferase activities were detected by dual-luciferase reporter assay (Promega Corporation).

| Protein extraction and Western blot analysis
Proteins were extracted from AT-II cells in RIPA buffer (Beyotime) supplemented with protease inhibitor. The mixed protein from the

| Statistical analysis
Data were presented as mean ± SE. from three independent experiments. Statistical analysis was carried out with Student's t test (between two means) or one-way ANOVA test (among more than two means). P < .05 was considered as statistically significant difference.

| Differential expression of miR-20a and PTEN
We performed miRNA profiling analysis in peripheral blood from premature infants with RDS and without RDS (controls). We observed that miR-20a was significantly down-regulated in peripheral blood from infants with RDS relative to infants without RDS.
Previously, our group have found that the expression of miR-20a in rat foetal lungs gradually decreases with lung development. 12 To validate these results, we measured miR-20a-5p expression in the foetal lung at three time-points of rat lung development [embryonic (E) day 16 (E16), E19, E21] using a conventional real-time qPCR assay. In keeping with the miRNA array finding, miR-20a-5p was down-regulated during the lung development. In addition, we examined the expression levels of PTEN at these three time-points in rat lung development and found that its expression gradually increased with lung development. This is in contrast to the expression trend of miR-20a-5p ( Figure 1).

| Up-regulation/down-regulation of miR-20a-5p in AT-II cells
To investigate whether miR-20a-5p plays any role in surfactant synthesis, the adenovirus-expressing miR-20a was transfected into AT-II cells that were isolated from rat foetal lungs of 19 days' gestation and cultured. In these experiments, the transfection efficiency was determined by observing the expression of green fluorescent protein (GFP) carried by adenoviral vector. After 48 hours of infection, more than 90% of the cells had positively expressed GFP, showing that the transduction of the adenovirus into AT-II cells reached over 90% ( Figure S2A). To examine whether miR-20a was overexpressed in AT-II cells, total RNAs were isolated and mature miR-20a level was measured by real-time PCR. As shown in Figure 2A, adenoviral vector for up-regulation of miR-20a resulted in a significant higher expression level of miR-20a compared to negative control. To obtain more evidence that the gene expression of pulmonary surfactant is regulated by miR-20a-5p, the adenoviral vector expressing miR-20a-5p inhibitor was also used to downgrade miR-20a-5p expression.
Again, GFP+ cells were more than 90%, and compared with the control group, the expression level of miR-20a-5p decreased by 60.6% ( Figure 2B; Figure S2B).

| miR-20a has no effect on cell proliferation
The CCK-8 assay was used to detect proliferation ability of AT-II cells that had overexpressed or underexpressed miR-20a-5p.
There was no significant difference between the virus control group and the miR-20a overexpressed or inhibited group in cell proliferation ( Figure 3). In addition, we also assessed protein expressions of these SP genes. The proteins were found to be significantly increased as well ( Figure 4E).
In another set of experiments, we measured SP gene and protein expression after inhibition of miR-20a-5p in order to further assess the role that miR-20a-5p may play in the regulation of SP.
The level of SP-A, SP-B and SP-C, but not SP-D, decreased when miR-20a-5p was inhibited ( Figure 5). Thus, it appeared that the effect of miR-20a silencing was not as obvious as that of overexpression. Alternatively, we failed to detect changes in SP-D expression after miR-20a silencing probably because the expression of SP-D was low.

| miR-20a-5p targeted expression of PTEN
We predicted that miR-20a targets the PTEN 3′-UTR. Using TargetScan, we found a perfect base pairing between the seed sequence of miR-20a and the 3′-UTR of PTEN mRNA and that these seed sequences were conserved across species, such as humans, rat, and mouse. Dual-luciferase reporter assay system revealed that the relative luciferase activity of WT-3′UTR vector and miR-20a-5p mimic cotransfected group was decreased by 31.39% comparing with miRNA controls, the differences were statistically significant, while the luciferase activity of MUT-3′UTR vector was not affected obviously ( Figure 6A,B), which is consistent with the results reported in the related literature. 13,14 Western blot and qRT-PCR analysis indicated PTEN activity had an inverse correlation with miR-20a-5p.
Overexpression of miR-20a-5p reduced the expression of PTEN by an average of 68% in comparison with the negative control virus-transfected cells ( Figure 6C). And the level of PTEN increased by 86% following miR-20a-5p inhibition in AT-II cells ( Figure 6D). Cells were harvested for Western blot analysis to confirm this effect at the protein level. Densitometric analysis of these protein blots revealed a similar regulatory pattern at the protein level ( Figure 6E,F). miR-20a is a member of the miR-17~92 cluster, which is the most extensively studied cluster that has an oncogenic function.

| D ISCUSS I ON
In addition to its roles in tumorigenesis, miR-20a has been shown to contribute to hepatic glycogen synthesis by targeting p63, 22 inhibit TCR-mediated signalling and cytokine production, 23 promote the proliferation and migration of human pulmonary artery smooth muscle cells (PASMC) and inhibit their differentiation, 24 and regulate autophagy in C2C12 cells via targeting ULK1. 25 Our study shows  Figure 6C,E) and that inhibition of miR-20a-5p increased its expression ( Figure 6D,F). We also utilized the luciferase reporter gene technique and also concluded that PTEN is the target gene of miR-20a-5p. Similar findings were reported by two other research groups, Wang and Zhang. 13,14 Phosphatase and tensin homolog is relatively abundant in lung epithelia. As a major suppressor of PI3K/Akt signalling pathway and a vital survival pathway in lung parenchymal cells, 32 PTEN has been implicated in pulmonary development and pulmonary function. [15][16][17] PI3K/Akt signalling pathway has been widely studied both in vitro and in vivo. The essential biochemical role of PTEN is to remove the D3 phosphate from the inositol ring of phosphoinositides, 33  Consistent with this notion, in the present study we observed that F I G U R E 6 Effects of miR-20a-5p on PTEN expression. A, Target position of miR-20a-5p at 3′-UTR of human, rat and mouse PTEN. B, The relative fluorescence activity of PTEN-WT-3′UTR and miR-20a-5p mimic cotransfected group was decreased significantly comparing with others. mRNA and protein levels were examined using real-time PCR and Western blot. β-Actin was used as an internal control. Overexpression of miR-20a-5p decreases the PTEN mRNA (C) and protein (E) expression. Inhibition of miR-20a-5p increased the PTEN expression (D, F). ****P < .0001, **P < .01 overexpression of miRNA-20a-5p in AT-II improved the synthesis of SP-A, SP-B, SP-C and SP-D, and concurrently suppressed the expression of PTEN both at mRNA level and at protein level. On the contrary, inhibition of miRNA-20a-5p inhibited the expression of SP-A, SP-B and SP-C, and concurrently increased the expression of PTEN. These data support the postulation that miR-20a-5p exerts its action via PTEN in AT-II.
As an oncogene, miR-20a has been reported to play an important role in cell proliferation, differentiation and apoptosis.
Overexpression of miR-20a promoted gastric cancer cell cycle progression and inhibited cell apoptosis, whereas knockdown of miR-20a resulted in cell cycle arrest and increased apoptosis. 35 Studies in mouse suggested that mir-17-92 normally promotes the high proliferation and undifferentiated phenotype of lung epithelial progenitor cells. 11 However, others reported that miR-17/20a miR-NAs function as tumour suppressors by reprogramming tumour cells for NK cell-mediated cytotoxicity. 36 In this study, we found that the In conclusion, our current studies show that in AT-II cells, overexpressing miR-20a-5p promotes pulmonary surfactant synthesis, whereas silencing miR-20a-5p inhibits pulmonary surfactant synthesis. We further show that overexpression and underexpression of miR-20a-5p cause up-and down-regulation of PTEN. Our findings suggest that miR-20a is a regulatory molecule capable of modulating the metabolic processes of pulmonary surfactant, and, as such, may have potential role in the treatment of lung-related diseases such as RDS.

ACK N OWLED G M ENTS
We thank doctors and nurses of Children's Hospital of Nanjing Medical University for patient management, sample collection and analysis in this work. We would also like to thank Professor Zhan-Jun Jia for his excellent theoretical assistance.

CO N FLI C T O F I NTE R E S T
The authors indicate no potential conflicts of interest in this work.