miR-296-5p Inhibits the Secretion of Pulmonary Surfactants in Pulmonary Epithelial Cells via the Downregulation of Wnt7b/Œ≤-Catenin Signaling
Authors: Guang-Dong Fang
Ying-Zi Mei
Qin Zhou
Shan-Yu Jiang
Min Wang
Bei-Bei Jia
Ying-Hui Zhang
Dan-Ni Ye
Ren-Qiang Yu
Bing Zhang
Ai-Ling Chen
Journal: BioMed Research International (2021)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Neonatal respiratory distress syndrome (NRDS); microRNAs (miRNAs); miR-296-5p; preterm infants; lentiviral vectors
Affiliations: Department of Neonatology, The Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University, Wuxi, China., Translational Medicine Laboratory, Research Institute for Reproductive Health and Genetic Diseases, The Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University, Wuxi, China., Department of Gynaecology and Obstetrics, The Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University, Wuxi, China.

Abstract:  Neonatal respiratory distress syndrome (NRDS) is a common disease that occurs in premature infants. However, the mechanisms underlying the disease remain unclear. microRNAs (miRNAs) have been indicated to play a crucial role in the development of NRDS. In this study, we aimed to explore the regulatory mechanisms of miR-296-5p in NRDS. The expression levels of miR-296-5p in preterm infants with NRDS were determined using quantitative reverse-transcription polymerase chain reaction (RT-qPCR). A549 cells were transfected with lentiviral vectors encoding miR-296-5p, and the transfection efficiency was determined using RT-qPCR. Flow cytometry and CCK8 assay were performed to measure apoptosis and proliferation of A549 cells, respectively. The protein levels of pulmonary surfactant SP-A (SFTPA1), SP-B, Wnt7b, and Œ≤-catenin were measured using western blotting. We demonstrated an upregulation of miR-296-5p in NRDS. The miR-296-5p was successfully overexpressed in A549 cells via lentivirus transfection, and the upregulation of miR-296-5p inhibited cell proliferation and secretion of SP-A and SP-B and also induced downregulation of the Wnt7b/Œ≤-catenin in vitro. Therefore, miR-296-5p inhibits cell proliferation and secretion of pulmonary surfactants in A549 cells via downregulation of Wnt7b/Œ≤-catenin signaling. 

Retracted: No

PubPeer comments:
 According to a recent paper ( http: onlinelibrary.wiley.com doi 10.1002 anie.201700424 full ) showing that 6mA cannot be detected by highly sensitive and accurate mass-spec techniques applied on DNA harvested from mouse brain, even with a limit of detection of 0.4 6mA sites per million deoxyadenosines. Another study from 2006 did not detect any 6mA in the mouse brain, with a limit of detection of lt;1 6mA per million deoxyadenosines. It is important that the authors present the background level of 6mA in the blank digestion reactions, because many of the commercial enzymes used for digesting DNA into free nucleosides for mass spec contain some amount of DNA (which could contribute false positive 6mA signal). In addition, the authors of this paper did not present the level of bacterial DNA contamination in the different brain samples (which could also contribute to variable false positive 6mA signal). Even the brain is known to have its own microbiota, which can change under different pathologies and conditions . Thirdly, the authors do not present any loading controls in their 6mA dot blots to show that the amount of DNA cross-linked to the membrane is the same for all samples. Without this loading control, the differences in 6mA signal cannot be trusted. Finally, methylated DNA IP sequencing methodologies are prone to false positives (especially at repetitive DNA elements), so it is not surprising to falsely identify 6mA sites using 6mA IP . In conclusion, I am skeptical about the existence and epigenetic relevance of N6-mdA in mammals (and other metazoa). The presence of 6mA in these organisms should be re-evaluated using contaminant-free LC-MS reagents for DNA digestion, while carefully controlling for the presence of bacterial contamination that is common in bulk tissue samples. Can the authors provide evidence that any of the above artifacts could not explain their results? , 
 #1 I agree with peer #1. The identification of 6mA in mouse genomic DNA is not reproducible according to many papers, including http: onlinelibrary.wiley.com doi 10.1002 anie.201700424 full . Another paper with a highly sensitive limit of detection (LOD of 0.4 6mA sites per million deoxyadenosines) was also unable to detect 6mA in mouse genomic DNA from several tissues, including the brain . Also the DpnI sequencing method is most likely to yield false positive 6mA sites, since DpnI is supposedly specific to 6mA-methylated GATC (on both strands!). With a 6mA level of a few parts per million in the mouse brain (if it is present at all), the probability of finding even one 6mA-methylated GATC on both strands in the mouse genome is extremely low. Therefore, DpnI probably has off-target effects. The findings by Yao et al. should be re-evaluated while taking into consideration potential bacterial DNA contamination in the DNA digestion reaction enzymes and in the brain samples themselves. , 
 https: bmcgenomics.biomedcentral.com articles 10.1186 s12864-019-5754-6 , 
 We agree that improved sample preparation and sequencing sensitivity are necessary to accurately characterize genomic features of 6mA and reveal its biological functions. However, before concluding that 6mA is not present in eukaryotic genomes, several factors need to be considered. First, many published studies have conducted 6mA-IP-seq to identify differentially expressed 6mA regions between experimental conditions. Directly comparing control and genetic depletion or stressed samples could define 6mA in eukaryotic DNA with elimination of any bacterial contamination or IgG nonspecific binding. Analyses of differential 6mA signals between DMAD knockout and control fly neuronal cells revealed the alterations of 6mA at distinct genomic loci, and the cells were free of bacterial contamination as determined by bacterial 16S quantitative PCR. Therefore, it would be difficult to imagine that all these differential signals were due to bacterial contamination. In addition, 6mA reads from prokaryotic origin can be controlled for by sequencing alignment. Second, the HPLC method used by Ou2019Brown et al. as described in the paper might not be fully optimized. Offline Enrichment, specifically of 6mA, before LC-MS MS acquisition will reduce the likelihood of 6mA experiencing signal suppression due to isobaric interference. Third, 6mA abundance should differ among various cell types, tissues, developmental stages and environmental circumstances. Ou2019Brown looked at static tissues and cell types, such as human HEK293 and HeLa cells, which have very low levels of 6mA as reported previously. Detectable 6mA signal could be diluted using HPLC analysis of whole brain. Last but not the least, unlike what has been portrayed in public discussions, Ou2019Brown et al. found rare (0.9-3.7 ppm) 6mA signals above background in DNA samples from gnotobiotic mouse tissues, which are non-treated and non-stressed samples. Like 5fC, steady low levels of DNA modification could still play critical roles in specific circumstances. Low levels of 6mA in the adult brain of fly and mice are important for neuronal function. Targeting 6mA modification can significantly decrease cancer cell growth and extend survival of tumor-bearing mice. Again, these rare DNA modifications could have critical biological functions, such as marking genes or regulation elements, and recruiting specific u201creaderu201d protein to render their biological functions in a more dynamic manner that may be under-appreciated. , 
 #4 Seems like wishful thinking without substantiative experimental evidence.. Claims that 6mA is a real, regulated mark in higher eukaryotes are bogus and will likely not stand the test of time. , 
 Several labs are working on this and many unpublished works, some of which will be published soon, provide stronger experimental evidence on the presence of 6mA as well as the explanation why 6mA could not be detected in the previously published works. Failure of detecting something does not mean it does not exist. Calling claims are bogus is not helpful either. Stop behaving like Mitt Romney. If you are really interested in this topics and willing to invest time, funding and effort into this, we would be happy to collaborate and exchange the reagents to further test them. , 
 Yet another well executed study showing that previous reports of 6mA in mammals (including this one) are artifactual: No evidence for DNA N6-methyladenine in mammals https: advances.sciencemag.org content 6 12 eaay3335  Unfortunately, in the absence of a 6mdA detection technique robust to the multiple sources of error that we have outlined, we conclude that the evidence published to date is not sufficient to support the presence of 6mdA in mammals. ,