Balkan Journal of Medical Genetics

DIFFERENTIALLY EXPRESSED CIRCULATING LONG-NONCODING RNAS IN PREMATURE INFANTS WITH RESPIRATORY DISTRESS SYNDROME
Bao ZD, Wan J, Zhu W, Shen JX, Yang Y, Zhou XY
*Corresponding Author: Dr. Yang Yang and Dr. Zhou Xiao‑Yu, E‑mail: yy860507@126.com (YY) and xyzhou161@163.com (XYZ), Tel:+ 86-25-83117362, Department of Neonatology, Children’s Hospital of Nanjing Medical University, Nanjing, Jiangsu 210008, P.R. China
page: 11

PATIENTS AND METHODS

Patients This prospective study enrolled 15 premature infants who were admitted to Jiangyin People’s Hospital of Nantong University between April 2019 and October 2019. Inclusion and Exclusion Criteria: Infants were eligible for enrollment in the study if they were (1) with a gestational age less than 36 weeks; (2) admitted within 4 hours after birth; (3) appropriate for gestational age. Patients were excluded for any of the following reasons: (1) severe cyanotic congenital heart diseases; (2) congenital chromosomal diseases or severe congenital malformations; (3) severe asphyxia at delivery (5 min Apgar score <5); (4) early symptoms of sepsis [10]. Severity grading: In the present study, the severity of RDS was determined clinically using a combination of PS treatment coupled with a degree of aeration of the lungs on chest X-ray [11]. The degree of aeration of the lungs on chest X-ray was graded as follows: (1) slightly reduced radiolucency with still sharp cardiac and diaphragmatic margins; (2) markedly reduced radiolucency with retained cardiac and diaphragmatic margins; (3) severely reduced radiolucency with air bronchogram and blurred cardiac and diaphragmatic margins; and (4) almost completely white lung fields with or without air bronchogram and barely visible cardiac and diaphragmatic margin [12]. Grouping: Of the 15 included infants, 5 were neonates without RDS and 10 were newborns diagnosed with RDS (presenting as cyanosis, groan, intercostal retractions, polypnea, and nasal flaring combined with changed aeration of the lungs on chest X-ray [11]). Babies who were worsening when FiO2 >0.30 or positive end-expiratory pressure (PEEP) > 6 cm H2O were given PS replacement [11]. Infants were given PS only once with lung X-ray grade 1or 2 were further defined as mild RDS, while infants who needed PS re-dosing with X-ray grade 3 or 4 were defined as severe RDS. Accordingly, based on the patients’ clinical features and chest X-ray results, 10 RDS infants were further divided into mild RDS group (n=5) and severe RDS group (n=5). Data collecting: Data provided by all patients were collected in detail using a standard data collection form, including age, gender, gestational age, weight, 5 min Apgar score, maternal gestational diabetes mellitus, antenatal glucocorticoid use, etc. The collection was completed by two individuals independently and verified by a third person. Patient information has been processed anonymously before statistical analysis. Sample preparation and RNA-sequencing Peripheral blood samples (2ml for each person) were collected from all infants between 1 and 6 hours after birth. Among them, it should be noted that, for RDS patients, samples were drawn before PS replacement. All blood samples were frozen in the −80°C refrigerator following a specific process which includes centrifugation at 3,000 × g for 10 min at 4°C and then separation of clear upper liquid into an RNase-free tube. Total RNA was then extracted from the blood samples using the TRIzol reagent according to the manufacturer’s instructions and a previous study [13]. After quality control of RNA, the RNA library of each sample was prepared using NEB Next Ultra RNA Library Prep Kit for the Illumina platform (BioLabs Inc., USA). The RNA sequencing analysis was performed by Genminix Informatics Co., Ltd (Shanghai, China) with the GeneChip® Human Transcriptome Array 2.0 (Affymetrix Inc., US) served as a gene expression profiling tool. Identification of differentially expressed genes The expression profile of the lncRNAs were analyzed by Deseq package (Affymetrix Inc., US). Samples were hybridized on the Human Clariom D (Thermo Fisher Scientific) gene chip. Background-adjustment, normalization, and log-transformation of signals intensity were performed with the Signal Space Transformation-Robust Multi-Array Average algorithm (RMA). Raw data were analyzed by the transcriptome analysis console (TAC) 4.0 software (Applied Biosystems, Foster City, CA, USA) awaiting further analysis [14]. The differentially expressed lncRNAs and mRNAs were screened according to the criteria of gene differential expression with |log2-fold change| (FC) more than 2 times and adjusted P<0.05. The differentially expressed lncRNAs were afterward clustered by a heatmap drawn to show the results. Real-time quantitative PCR validation For lncRNA expression analysis, total RNA was transcripted to cDNA using a Reverse Transcription Kit (PrimeScript RT Master Mix, Takara Bio Inc., Otsu, Japan), then real-time quantitative PCR (qRT-PCR) validation was performed using the SYBR method (SYBR® Premix Ex Taq™, Takara Bio Inc., Otsu, Japan) according to the product instructions. An aliquot of 1 μg total RNA was added to each reaction mixture. qRT-PCR was performed on an ABI 7500 thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc. US) with SYBR Green (Roche Diagnostics Co., Ltd. GER). The thermocycling conditions were as follows: 95˚C for 5 min, followed by 40 cycles of 95˚C for 20 sec and 55˚C for 20 sec. At the end of each run, a melting curve analysis was performed at 72˚C to monitor primer dimers and formation of non‑specific products. For data analysis, the comparative Ct method (2ΔΔCt) was used. Results were expressed as fold changes of gene expression adjusted to housekeeping gene GAPDH [15]. All primers used in the present study were shown in table 1. LncRNA‐mRNA co-expression network Multivariate statistical analysis was used to calculate the Pearson correlation coefficient between differentially expressed lncRNAs and mRNAs. The greater the cor relation coefficient, the greater possibility that there was a regulatory relationship between certain lncRNAs and mRNAs. The co-expression network was constructed with the Pearson correlation coefficient r> 0.99 and P< 0.05 as the filtering standard in this study. GO and KEGG pathway analysis GO and KEGG pathway analysis were applied to predict functions of the differentially expressed genes. The GO project offers a controlled vocabulary to label gene and gene product attributes in any organism (geneontology. org). GO results were mainly classified into three subgroups namely biological process, cellular component, and molecular function. GO analysis provides an interpretation of the relevance of genes differentially expressed between the groups. Fisher’s exact test and the χ2 test were performed to calculate the P‑value and false discovery rate of each GO term function. KEGG (kegg.jp/kegg/ pathway.html) pathway analysis is a functional analysis tool, mapping a set of genes that may be associated with a certain lncRNA to potential pathways. The enrichment probability of a differentially expressed gene set in a term entry was represented by an enrichment score (EC), with a higher EC indicating a higher significance of the entry. The EC was calculated as the negative base 10 log of the P value. The input used in the bioinformatics analysis was the differential mRNA genes co‑expressed with lncRNA that were screened in the lncRNA expression profile. Statistical analysis For clinical results (clinical characteristics), data were analyzed using SPSS 17.0 software. Quantitative data are expressed as mean ± standard deviation (SD). One-way variance analysis was applied to detect differences among the three groups. In terms of qualitative data, the Pearson Chi-square test was performed. Significant differences were considered as P < 0.05.

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