Study group
This retrospective cohort study included 210 patients (50 ICU and 160 non-ICU patients) diagnosed with COVID-19 at the Department of Infectious, Tropical Diseases and Acquired Immunodeficiency at the Pomeranian Medical University in Szczecin, Poland. Nasopharyngeal swabs analyzed by real-time polymerase chain reaction (RT-PCR) were used to confirm SARS-CoV-2 virus infection. The control population comprised 80 healthy individuals from the hospital staff whose RT–PCR results for SARS-CoV-2 from nasopharyngeal swab were negative, and their ELISA results for SARS-CoV-2-specific IgG, IgM, and IgA antibodies were also negative. In the study design, ICU patients constituted the research group, while non-ICU individuals constituted the control group. The control population was included to validate the laboratory kits and ensure that SARS-CoV-2 patients had truly positive results. In study participants, blood was collected to assess the expression of predetermined genes, as well as the levels of specific chemokines and complement components. In addition, participants were required to complete comprehensive questionnaires regarding their overall health. The study was approved by the Ethics Committee of the Pomeranian Medical University in Szczecin (KB-0012/83/2020) and was in accordance with the principles of the Declaration of Helsinki. Before starting the study, each participant signed an informed consent form. Patients were enrolled to the study from July 2020 to April 2021. More details can be found in our previous articles10,11.
General health questionnaire
All patients were interviewed and examined to document symptoms such as fever, shortness of breath, cough, cold, sore throat, fatigue, chest pain, changes in smell/taste, headache, body aches, and diarrhea, along with details of severity and duration of symptoms. Medical records were reviewed to collect data on laboratory test results, need for oxygen or respiratory support, presence of pneumonia on chest computed tomography, need for hemodialysis, and patient outcomes, including mortality. Additionally, patient demographics, family medical history, and pre-existing health conditions such as hypertension, hyperlipidemia, smoking, diabetes, cardiovascular problems, liver disease, respiratory disorders, rheumatic diseases, and previous cerebrovascular events were documented. The severity of COVID-19 cases was assessed retrospectively by categorizing COVID-19-infected patients into two groups according to the severity of the disease. Group 1 consisted of intensive care unit patients (ICU patients) requiring intensive care due to respiratory failure, hospital stays longer than 14 days due to COVID-19 or with a fatal outcome. Group 2 encompassed non-ICU patients, including asymptomatic or mildly symptomatic cases with an oxygen saturation level of at least 95%, not requiring hospitalization, as well as symptomatic patients, with an oxygen saturation below 95%, requiring hospitalization for up to fourteen days. More details can be found in our previous articles10,11.
Material
Plasma collection
Peripheral blood samples were collected upon the patient’s admission to hospital on day 1, and subsequently during hospitalization/isolation on days 7, 14 and 28 following the COVID-19 diagnosis. Approximately 7.5 mL of peripheral blood was collected into EDTA tubes and centrifuged at 2000 rpm for 10 min. The resulting plasma was then transferred to a new tube and subjected to a second centrifugation cycle under the same parameters. Blood samples were collected only at the hospital, no samples were collected prior to admission.
Viral RNA isolation
Viral RNA isolation was performed using the MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit (Thermo Fisher Scientific, ON, CA) according to the manufacturer’s protocol. MagMAX Viral/Pathogen nucleic acid isolation was processed using an automated KingFisher Flex instrument (Thermo Fisher Scientific, ON, CA). The isolation procedure has been described in detail in our previous articles10,11.
qRT–PCR assays for detecting SARS-CoV-2 RNA
For the detection of SARS-CoV-2 RNA, qRT-PCR assays were conducted utilizing a QuantStudio 5 PCR system and a TaqPath COVID-19 CE IVD RT-PCR Kit (Thermo Fisher Scientific, Markham, ON, Canada) following the manufacturer’s guidelines. After completion of RT-PCR, results were assessed using Applied Biosystems COVID-19 Interpretive Software v1.5.1 (Thermo Fisher Scientific, Markham, ON, Canada). Positive test results were determined if a minimum of 2 out of the 3 analyzed SARS-CoV-2 genes (ORF1ab, N, S) exhibited Ct values of ≤ 37. For further information, please refer to our prior publications10,11.
miRNA isolation
miRNA was extracted from 900 µL of plasma utilizing the NucleoSpin miRNA Plasma Mini kit designed for circulating miRNA (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. Subsequently, the concentration of isolated miRNA from plasma was assessed using Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) in conjunction with the Qubit microRNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
Affymetrix GeneChip miRNA microarray and data analysis
miRNA was extracted from the plasma of all patients, although miRNA microarrays were solely conducted on selected samples (four representative samples at each time points and control group) and pooled to generate one sample for subsequent experimental procedures. The study utilized Affymetrix miRNA 4.1 Array Stripes (Affymetrix, Santa Clara, CA, USA) with n = 3 technical replicates for each miRNA probe. The procedure commenced with a poly(A) tailing reaction followed by ligation of the biotinylated signal molecule to the target RNA. The sample was then hybridized on an Affymetrix miRNA 4.1 Array Strip (Affymetrix, Santa Clara, CA, USA). The final step involved the addition of streptavidin-PE followed by array scanning using the Affymetrix GeneAtlas system (Affymetrix, Santa Clara, CA, USA). Microarray data analysis was performed using Bioconductor12. The normalized data were integrated into the description file “pd.mirna.4.1”, including miRNA names, types and sequences. Differential expression was determined by applying linear models to microarray data, facilitated by the “limma” library13. Differentially up- and down-regulated miRNAs were graphically presented in volcano plots based on predefined cut-off criteria (fold change > abs. 2). Experimentally validated miRNA target genes were obtained from the miRTarBase database, focusing solely on targets associated with differentially expressed miRNAs. The target gene lists from each comparison were functionally annotated and clustered using DAVID (Database for Annotation, Visualization and Integrated Discovery)14. Differentially expressed miRNA target symbols were entered into DAVID using the Bioconductor library “RDAVIDWebService”15, matching the targets to the corresponding gene ontology (GO) terms.
qRT-PCR for validation of selected miRNA expression
The results of miRNA microarray studies facilitated the identification of specific miRNAs with altered expression levels in the plasma of severely and moderately ill COVID-19 patients compared to healthy controls. Then, the expression of selected miRNAs (miR-106a-5p, miR-17-5p, miR-181a-5p, miR-191-5p, miR-20a-5p, miR-423-5p, miR-451a) was assessed through qRT-PCR on samples from all patients.
Initially, miRNA reverse transcription was conducted using the qScript microRNA cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA, USA), which contains all the necessary components for qRT-PCR. Then, primers for miRNA were custom designed by miRPrimer and purchased at the Laboratory of DNA Sequencing and Oligonucleotide Synthesis at the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences in Warsaw, Poland. The qRT-PCR protocol included an initial 10-minute denaturation at 95 °C, denaturation at 95 °C for 15 s, annealing at 54–57 °C (depending on the primer) for 30 s, and extension at 30 °C for 60 s. The relative gene expression was quantified using the comparative Ct method (2ΔCt), where ΔCt = (Ct of miRNA) – (Ct of endogenous control). All PCR products were characterized by high specificity by determining the melting point at a transition rate of 0.1 °C/s. The qRT-PCR reaction mixture comprised 5 µL of PerfeCTa SYBR Green SuperMix (Bio-Rad Inc., Hercules, CA, USA), 1 µL of microRNA cDNA, 0.2 µL of microRNA-specific primer, 0.2 µL of PerfeCTa Universal PCR Primer, and 4.6 µL of nuclease-free water. Reactions were performed on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad Inc., Hercules, CA, USA), with all miRNA expressions performed in two technical replicates.
Statistical analysis
Quantitative data were expressed as mean and standard deviation. Medical history and medications taken the study were expressed as percentages. The Mann–Whitney test was used in our analyses to compare quantitative parameters between groups. Fisher’s exact test was implemented to assess the differences between categorical variables. A p value of < 0.05 was considered statistically significant. All calculations were performed in RStudio version 1.2.1335. ROC analysis was performed in PQ Stat software.
