Screening for differentially expressed microRNAs in BALF and blood samples of infected COVID‐19 ARDS patients by small RNA deep sequencing

Abstract Background The pandemic COVID‐19 has caused a high mortality rate and poses a significant threat to the population of the entire world. Due to the novelty of this disease, the pathogenic mechanism of the disease and the host cell's response are not yet fully known, so lack of evidence prevents a definitive conclusion about treatment strategies. The current study employed a small RNA deep‐sequencing approach for screening differentially expressed microRNA (miRNA) in blood and bronchoalveolar fluid (BALF) samples of acute respiratory distress syndrome (ARDS) patients. Methods In this study, BALF and blood samples were taken from patients with ARDS (n = 5). Control samples were those with suspected lung cancer candidates for lung biopsy (n = 3). Illumina high‐throughput (HiSeq 2000) sequencing was performed to identify known and novel miRNAs differentially expressed in the blood and BALFs of ARDS patients compared with controls. Results Results showed 2234 and 8324 miRNAs were differentially expressed in blood and BALF samples, respectively. In BALF samples, miR‐282, miR‐15‐5p, miR‐4485‐3p, miR‐483‐3p, miR‐6891‐5p, miR‐200c, miR‐4463, miR‐483‐5p, and miR‐98‐5p were upregulated and miR‐15a‐5p, miR‐548c‐5p, miR‐548d‐3p, miR‐365a‐3p, miR‐3939, miR‐514‐b‐5p, miR‐513a‐3p, miR‐513a‐5p, miR‐664a‐3p, and miR‐766‐3p were downregulated. On the contrary, in blood samples miR‐15b‐5p, miR‐18a‐3p, miR‐486‐3p, miR‐486‐5p, miR‐146a‐5p, miR‐16‐2‐3p, miR‐6501‐5p, miR‐365‐3p, miR‐618, and miR‐623 were top upregulated miRNAs and miR‐21‐5p, miR‐142a‐3p, miR‐181‐a, miR‐31‐5p, miR‐99‐5p, miR‐342‐5p, miR‐183‐5p, miR‐627‐5p, and miR‐144‐3p were downregulated miRNAs. Network functional analysis for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), in ARDS patients' blood and BALF samples, showed that the target genes were more involved in activating inflammatory and apoptosis process. Conclusion Based on our results, the transcriptome profile of ARDS patients would be a valuable source for understanding molecular mechanisms of host response and developing clinical guidance on anti‐inflammatory medication.


| INTRODUC TI ON
Since December 2019, the virus that causes pneumonia has spread worldwide and became a pandemic. More detailed studies have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of respiratory distress. 1 Clinico-epidemiological studies have shown that the most common symptoms of this infection are as follows: fever, cough, fatigue, and shortness of breath. 2,3 Unfortunately, however, in some people, the disease progresses very quickly and causes acute respiratory distress syndrome (ARDS), which eventually leads to death. 4,5 Little is known about the pathogenesis of the disease. However, it is clear that when the innate immune system recognizes viral RNAs, three major classes of cytoplasmic recognition receptors are activated: Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs), which trigger expression of interferon (IFN) and activation of antiviral effectors such as natural killer cells, T CD8+ cells, and macrophages; [6][7][8][9] but uncontrolled host responses lead to aberrant immune cell activation and deregulated cytokine production. 10 However, the exact molecular mechanism of the pathogenesis of this infection is still unknown. In this study, we investigated the role of MicroRNAs (miRNAs) in the development of respiratory distress. MicroRNAs are small (18-25 nt) RNAs categorized as noncoding RNAs, and their function is to regulate gene expression. 11 They exhibit nearly 90% sequence homology among humans, mice, and rats. 12 MiRNAs control gene expression in two ways: by suppressing translation/transcription (RNAi) or by activating transcription (RNAa). Some critical features of miRNAs include tissue specificity, stability, and association with clinicopathological parameters. [13][14][15] Therefore, differential expression of miRNAs during the progression of COVID-19 may play an essential role in the host response, and these RNAs may function as biomarkers and even novel therapeutic targets for COVID-19. To the best of our knowledge, no data have been reported on the expression profile of miRNAs in the bronchoalveolar fluid (BALF) of the lungs of patients with COVID-19, and all information is based on bioinformatics and in silico studies. The analysis of miRNA profile of patients with COVID-19 infection would help us to understand the pathogenesis of the disease and find more effective strategies for diagnosis and treatment. Therefore, this study investigated the expression profile of miRNAs in BALF and blood samples from people with COVID-19 infectious disease.

| Sample collection
Blood and bronchoalveolar fluid samples and whole blood were obtained from five severe COVID-19 patients at Vellayat Hospital Qazvin, IRAN. The patients were informed about the sample collection process and had signed informed consent forms. The recruited patients were between 55 and 65 years old, with severe shortness of breath or breathlessness, rapid and labored breathing, extreme tiredness, and muscle fatigue, who were admitted to the ICU section. These patients had a positive result in the COVID-19 nasopharyngeal swab RT-PCR test (n = 5). Patients with obesity, diabetes, heart, kidney, and lung diseases were not included in the study due to many factors involved in the expression of miRNAs. Some characterization of the samples is mentioned in Table 1

| miRNA library construction and sequencing
In the first step, the Illumina Small RNA v1. 5

| Analysis of sequenced data
In the first step, Cutadapt software (version 2.10; https://cutad apt. readt hedocs.io/en/stabl e/) was used for adaptor trimming, lowquality reads, rRNA, and tRNA removal. The rRNA proportion less than 5% showed that the studied samples had good quality. Bowtie

| Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR)
To confirm miRNA sequencing results, the RNA samples of patients with ARDS (n = 25) and controls (n = 15) were used; moreover, the expression of miRNAs and their target genes, which had the highest score and significance, examined by the Real-Time PCR method.
We used TRIZOL reagent to isolate the total RNA from the samples  Table 2. Also, we used beta-actin as an internal control. Subsequently, real-time quantification was performed using Rotor gene-Q real-time PCR system (Qiagen). Each real-time PCR After the reactions, the mean Ct was determined from the triplicate PCRs. We used Ct values to evaluate the expression levels of the miRNAs and target genes. The expression value of miRNAs relative to internal controls was determined using the 2 -△Ct method.

| Bioinformatics analysis
For bioinformatics analysis of the sequenced miRNAs, we used miR-Walk (http://miRwa lk.uni-hd.de) and miRBase (http://www.miRba se.org) database. Gene Ontology (GO) was performed using multiple tools and algorithms for identifying miRNA-target genes and their corresponding pathways. GO pathway enrichment analysis of biological processes was applied for the predicted miRNA-target genes, and the human genome was a reference set. We used the TA B L E 2 Primer sequence of studied target genes Target Forward primer sequence Reverse primer sequence z test to determine GO terms or Kyoto Encyclopedia of Genes and The rate of degradation was very insignificant because the contribution of rRNA in the reads was <0.29%.

| Differential expression of miRNAs in BALFs and blood
Our results showed that differentially expressed miRNAs were higher in BALF samples than in blood samples. To select the differentially expressed miRNAs more accurately, we used fold change  Tables 3-6. As mentioned in Table 3,

| Functional enrichment analysis of the targeted genes of miRNAs
Since  Figure 3B).

| Comparison of detailed KEGG data analysis of infected BALF and blood
We used KEGG release 76.0 application for KEGG (https://www. genome.jp/kegg/) pathway analysis. In this regard, the same enrichment rule was applied as in GO, when the P-value and the Q-value were < 0.05. The KEGG pathway studied was considered enriched and included in the results for both infected BALF and blood in infected blood and BALF samples compared with control ( Figure 4).

| Results of the miRNA-mRNA correlation study
To better understand the function of the studied miRNAs, we an- In blood samples, we showed upregulation of miR-15b-5p with a high score. These miRNAs may target extracellular signal-regulated kinase 1 (ERK-1), inhibit cell proliferation, and induce apoptosis. 21 The second upregulated miRNA was miR-18a-3p, which may target the 3'UTR region of the HOXA1 gene, thus blocking its expression and triggering apoptosis. 22 The first downregulated miRNA was miR-21-5p; it is associated with elevated inflammatory immune responses by targeting smad-7. 23 The miR-142a-3p was the second downregulated miRNA that can target TGFBR1 and diminish TGFβ signaling. This may indicate a novel pathogenic pathway that simultaneously decreases the immunomodulatory effects of cytokine, and downregulation of this miRNA leads to an increased immune and cytokine response. 24

| Confirmation of RNA-seq results by QRT-PCR method
To confirm RNA-seq results, we selected 8 high-score (up and downregulated) miRRNA and their target genes in BALF and blood samples.
We also showed significant downregulation of miR-21-5p (p< 0.001) and miR-14a-3p (p < 0.0001) ( Figure 7A) and upregulation of CCL-20 (p < 0.001) and TGFβ (p < 0.0001) in infected blood samples ( Figure 7B). The results of the correlation study between miRNA and the expression of its target genes expression showed a significant negative correlation, which is listed in Table 7.

| DISCUSS ION
The COVID-19 pandemy started in 2019 and is still going on. In

general, infection with SARS-CoV-2 causes this disease and usually
begins with cold symptoms such as fever, sore throat, and headache. If left untreated, it enters the inflammatory phase, and lung involvement occurs. 25 The definitive treatment of this disease has not yet been known because the pathogenesis is not fully understood. Designing novel therapeutics approaches is difficult because our understanding of the host immune response to SARS-CoV-2 infection is limited. Studies have shown that viral infections alter transcriptome expression in host cells and ultimately provide the right conditions for the virus replication. [26][27][28] In this study, the expression profiles of the miRNAs in the lung BALF and blood samples of the  In BALF samples, miR-15a-5p with the highest score was selected; this miRNA is one of the five miRNAs that commonly bind to SARS-CoV, MERS-CoV, and SARS-CoV-2. 19 It was one of the most essential miRNAs in this study because it targets two critical genes in the path of inflammation and apoptosis (CRP, IL-6). C-reactive protein (CRP) is a protein made by the liver sent into the bloodstream in response to inflammation. It has been shown that CRP levels are correlated with levels of inflammation. CRP levels can promote phagocytosis and activate the complement system. 38 induction of GADD153 gene expression related to cell cycle arrest and DNA damage. 38 Another miRNA was miR-4284, which was significantly downregulated in the BALF samples. Typically, this miRNA can elevate MLL, BCL-10, and HDAC, which play a role in blocking apoptosis and increasing proliferation. In our study, we hypothesized that upregulation of miR-4284 promotes apoptosis with underexpression of MLL, BCL-10, and HDAC. 16 Based on the results of this study and the potential role of miRNAs in the pathogenesis of COVID-19 disease, it can be speculated that it is possible to use miRNAs as a target for the treatment. Since the expression of miRNAs is affected by various factors, we tried to eliminate interfering factors as much as possible. Patients were selected at approximately the same age range, and patients with a history of diabetes, heart, kidney, and lung disease were excluded from the study. Still, the rest of the interventions were out of control. The current study showed that differentially expressed microRNA (miRNA) in blood and bronchoalveolar fluid (BALF) samples of acute respiratory distress syndrome (ARDS) patients is involved in activating the inflammatory and apoptosis process; workflow of this research is shown in Figure 8.

ACK N OWLED G EM ENT
We thank all participants for their participation in the current study.
We also thank the Cellular and Molecular Research Center of the Qazvin University of Medical Science for providing laboratory facilities.

FU N D I N G I N FO R M ATI O N
This research was supported by the Cellular and molecular research center of the Qazvin University of Medical Science.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the results of this study are available from the corresponding author upon request [SM]. The data are not publicly available because they contain information that could compromise the privacy of the research participants.