Evaluation of the Expression of Infection-Related Long Noncoding RNAs among COVID-19 Patients: A Case-Control Study

Background and Aims Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a worldwide pandemic, activates signaling cascades and leads to innate immune responses and secretion of multiple chemokines and cytokines. Long noncoding RNAs (lncRNAs) have a crucial role in inflammatory pathways. Through our search on the PubMed database, we discovered that existing research has primarily focused on examining the regulatory impacts of five lncRNAs in the context of viral infections. However, their role in regulating other conditions, including SARS-CoV-2, has not been explored. Therefore, this study aimed to investigate the expression pattern of lncRNAs in the peripheral blood mononuclear cells (PBMC) and their potential roles in SARS-CoV-2 infection. Potentially significant competing endogenous RNA (ceRNA) networks of these five lncRNAs were found using online in-silico techniques. Methods Ethylenediaminetetraacetic acid (EDTA) blood samples of the control group consisted of 45 healthy people, and a total of 53 COVID-19-infected patients in case group, with a written informed consent, was collected. PBMCs were extracted, and then, the RNA extraction and complementary DNA (cDNA) synthesis was performed. The expression of five lncRNAs (lnc ISR, lnc ATV, lnc PAAN, lnc SG20, and lnc HEAL) was assessed by real-time PCR. In order to evaluate the biomarker roles of genes, receiver operating characteristic (ROC) curve was drawn. Results Twenty-four (53.3%) and 29 (54.7%) of healthy and COVID-19-infected participants were male, respectively. The most prevalent symptoms were as follows: cough, general weakness, contusion, headache, and sore throat. The results showed that three lncRNAs, including lnc ISR, lnc ATV, and lnc HEAL, were expressed dramatically higher in the case group compared to healthy controls. According to ROC curve analysis, lnc ATV has a higher AUC and is a better biomarker to differentiate COVID-19 patients from the healthy controls. Then, using bioinformatics methods, the ceRNA network of these lncRNAs enabled the identification of mRNAs and miRNAs with crucial functions in COVID-19. Conclusion The considerable higher expression of ISR, ATV, and HEAL lncRNAs and the significant area under curve (AUC) in ROC curve demonstrate that these RNAs probably have a potential role in controlling the host innate immune responses and regulate the viral replication of SARS-CoV-2. However, these assumptions need further in vitro and in vivo investigations to be confirmed.

Background and Aims.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a worldwide pandemic, activates signaling cascades and leads to innate immune responses and secretion of multiple chemokines and cytokines.Long noncoding RNAs (lncRNAs) have a crucial role in infammatory pathways.Trough our search on the PubMed database, we discovered that existing research has primarily focused on examining the regulatory impacts of fve lncRNAs in the context of viral infections.However, their role in regulating other conditions, including SARS-CoV-2, has not been explored.Terefore, this study aimed to investigate the expression pattern of lncRNAs in the peripheral blood mononuclear cells (PBMC) and their potential roles in SARS-CoV-2 infection.Potentially signifcant competing endogenous RNA (ceRNA) networks of these fve lncRNAs were found using online in-silico techniques.Methods.Ethylenediaminetetraacetic acid (EDTA) blood samples of the control group consisted of 45 healthy people, and a total of 53 COVID-19-infected patients in case group, with a written informed consent, was collected.PBMCs were extracted, and then, the RNA extraction and complementary DNA (cDNA) synthesis was performed.Te expression of fve lncRNAs (lnc ISR, lnc ATV, lnc PAAN, lnc SG20, and lnc HEAL) was assessed by real-time PCR.In order to evaluate the biomarker roles of genes, receiver operating characteristic (ROC) curve was drawn.Results.Twenty-four (53.3%) and 29 (54.7%) of healthy and COVID-19-infected participants were male, respectively.Te most prevalent symptoms were as follows: cough, general weakness, contusion, headache, and sore throat.Te results showed that three lncRNAs, including lnc ISR, lnc ATV, and lnc HEAL, were expressed dramatically higher in the case group compared to healthy controls.According to ROC curve analysis, lnc ATV has a higher AUC and is a better biomarker to diferentiate COVID-19 patients from the healthy controls.Ten, using bioinformatics methods, the ceRNA network of these lncRNAs enabled the identifcation of mRNAs and miRNAs with crucial functions in COVID-19.Conclusion.Te considerable higher expression of ISR, ATV, and HEAL lncRNAs and the signifcant area under curve (AUC) in ROC curve demonstrate that these RNAs probably have a potential role in controlling the host innate immune responses and regulate the viral replication of SARS-CoV-2.However, these assumptions need further in vitro and in vivo investigations to be confrmed.

Introduction
SARS-CoV-2 was frst discovered in Wuhan, China, in December 2019 and rapidly became a pandemic all around the world [1][2][3].COVID-19 clinical manifestations vary widely among patients from asymptomatic to high severe states [4].Te most frequent signs and symptoms of COVID-19 are fever, shortness of breath, nonproductive cough, sore throat, and diarrhea [5].
Almost 80% of eucaryotic genome is composed of noncoding sequences that are consequently transcribed to noncoding RNAs (ncRNAs) [12].Tese RNAs are categorized into two groups as follows: short ncRNAs with a length <200 nucleotides and long noncoding RNAs (lncRNAs) with a length over 200 nucleotides [13].Although most of the lncRNAs are not protein-coding, they are involved in modulation of transcription patterns and regulation of diferent protein activities [13][14][15][16].LncRNAs have a crucial role in infammatory pathways and infammasomes [17].LncRNAs are involved in cellular and molecular procedures and have shown to act as epigenetic regulators in several key molecular processes [18].By exposing a sponge efect on miRNAs, lncRNAs can control the mRNAs that target them [19].Recently, it has been showed that interactions of noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), and lncRNAs are related to SARS-CoV-2 infection, so, in this research, we have also focused on the interactions of ncRNAs.
Angiotensin-converting enzyme (ACE) which is the receptor of SARS-CoV-2 is highly expressed in lungs, testis, and kidneys [20].Te lncRNAs and miRNAs are able to afect ACE2 expression [21].ACE2 is expressed in humans and is also altered in many viral infections, such as the hepatitis C virus (HCV), the hepatitis B virus (HBV), the infuenza virus, the human immunodefciency virus (HIV), the herpes simplex virus (HSV), and the SARS-CoV-2 [22][23][24].
We scanned the PubMed database for a list of recently discovered lncRNAs that have been validated by other research.Tus, interferon-stimulated lncRNA (lncRNA ISR), lncRNA ATV, PA-associated noncoding RNA (lncRNA PAAN), interferon-stimulated gene 20 (lncRNA ISG20), and HIV-1-enhanced lncRNA (lncRNA HEAL) were the fve lncRNAs that were chosen.We found that while current research has concentrated on the regulatory efects of fve lncRNAs in viral infection, their involvement in regulating other disorders, such as SARS-CoV-2, has not been investigated by searching the PubMed database.
We searched for ceRNA networks on which in-silico studies had been performed in viral infections.Te roles of lnc ISR and lnc PAAN have been proven with bioinformatics studies [25].For example, lnc ISR is a lncRNA that is expressed concurrently with infuenza A virus (IAV) infection as a result of interferon (IFN) signaling [26,27].IFN signaling is implicated in both the protective and damaging aspects of SARS-CoV-2 [28].According to the fndings of Wang et al., lnc PAAN facilitates IAV replication [29].In line with a study by Chai et al., the expression of lnc ISG20 is suppressed by IFN-β stimulation, which inhibits the replication of IAV [30].Lnc ATV is another lncRNA that is substantially expressed when IFN types I and III are present.It is also elevated in viral infections such as Zika and hepatitis C [31].Additionally, throughout the active phase of HIV infection, the expression of lnc HEAL increases in Tcell lymphocytes, microglia, and monocyte-derived macrophages [32].Some studies have been performed on the status of lncRNAs during COVID-19 infection.Previous studies showed that the expression of some lncRNAs is dysregulated in SARS-CoV-2-infected cell lines and these RNAs might be involved in immune response against COVID- 19 [25, 33, 34].Tus, this study aimed to investigate the potential roles of lncRNAs in SARS-CoV-2 infection and the lncRNA profles among COVID-19 patients in comparison to the healthy control group.

Sample Collection.
A case-control study was performed from January to March 2021.Tere were 45 healthy people in the control group and 53 COVID-19-infected patients in the case group.Te healthy group consists of individuals who had no infectious or immune-related diseases, such as autoimmunity, allergy, cancer, and liver diseases, and these subjects are negative in RT-PCR test results.RT-PCR, the gold-standard method for COVID-19 diagnosis, was also performed to detect SARS-CoV-2 infection.Tis study was performed in Valiasr Hospital, Fasa, Fars Province.EDTA blood samples and written informed consent were collected from all participants.Te ethical approval was obtained from the ethic committee of Fasa University of Medical Sciences (IR.FUMS.RES.1399.040).

Clinical Signs and Symptoms. COVID-19 patients
showed variable clinical manifestations.Te most prevalent symptoms were as follows: cough, general weakness, contusion, headache, and sore throat, which were observed in over 50% of patients.On the other hand, nausea, vomiting, diarrhea, conjunctivitis, and stomachache were rarely reported.Six (11.6%) of patients had underlying diseases (Table 2).

Expression Pattern of lncRNAs.
Real-time PCR was used to evaluate the expression of fve noncoding RNAs including lnc ISR, lnc ATV, lnc PAAN, lnc SG20, and lnc HEAL.
According to the fndings, the expression levels of several lncRNAs were signifcantly diferent in COVID-19-infected patients compared to healthy controls.Two lncRNAs, including lncRNA ATV (P ≤ 0.001) and lncRNA HEAL (P ≤ 0.001), were expressed dramatically higher in the case group compared to healthy controls (Figures 1(a) and 1(b)).Moreover, the expression of lncRNA ISR was signifcantly higher in COVID-19-infected patients (P ≤ 0.05), see Figure 1(c).No signifcant diferent expression of lncRNA PAAN and lncRNA ISG20 was observed between two groups (P > 0.05) (Figures 2(a) and 2(b)).Te detailed statistical analyses are shown in Table 3.
According to our ROC curve analysis, lnc ATV has a higher AUC (AUC � 0.762) which represented it might be a great biomarker to diferentiate COVID-19 patients from the healthy controls (Figure 3).

Discussion
One of the most contagious viruses, SARS-CoV-2, is a member of the Coronaviridae (CoV) family, which has become a pandemic and is linked to high rates of morbidity and mortality [35].Te increasing amount of evidence suggests that the host transcriptome changes following the viral infections and several signaling pathways are stimulated.For instance, Merkel cell carcinoma (MCC) is caused by Merkel cell polyomavirus (MCPyV), which is a small DNA tumor virus and oncogenic virus [36].In recent years, a long type of noncoding RNAs known as lncRNAs has attracted incredible attention.LncRNAs are one of the major regulators of antiviral immune responses [26].
Numerous lncRNAs have been shown to be diferently expressed in COVID-19 patients, and important lncRNAs for virus-host interactions have also been identifed, along with improvements in research instruments and methodologies.Furthermore, targeting gene transcription and protein translation are the main targeted therapies for SARS-

Genetics Research
CoV-2-infected cells, based on the properties of poorly conserved lncRNAs that are extensively involved in cell proliferation, diferentiation, apoptosis, and other biological processes.Moreover, a number of bioanalysis-based investigations have discovered a variety of dysregulated lncRNAs connected to SARS-CoV-2 replication [33].
According to some research, the pathogenesis of SARS-CoV-2 is facilitated by epigenetic changes in both individual genomes and the virus.Nongenomic alterations in gene expression and function are heritable known as epigenetic modifcations.Deoxyribonucleic acid (DNA) methylation, histone changes, and noncoding RNA-associated gene silencing are three key epigenetic processes [37].
Te lncRNAs listed below are examples of those with the mentioned previously functions.In a study, Di Qu et al. introduced the lncRNA GM26917 in HIV-1 by sponging the miR-124-3p.Te lncRNA NORAD is implicated in cytokine storms because it has the ability to target fve of the ten cytokines that are engaged in them.Additionally, new research on HIV-1 has shown that MALAT1 regulates promoter-enhancer interactions to enhance viral transcription and infection [38].It is interesting to note that lncRNAs can also control the SARS-CoV-2 innate immune response by connecting to the IFN pathway.IFN-1 response was shown to be considerably downregulated in the ncRNA regulatory network in a recent study employing total transcriptome RNA sequencing in COVID-19 patients [39].In the context of viral infections, particularly COVID-19, the regulation and function of lncRNAs are intricate and can be changed based on a number of variables, including the infection's stage, the viral strain, and individual characteristics.In line with our analysis, previous research has indicated that upregulated lncRNA-NEAT1 and lncRNA-TUG1 could infuence cytokine storms in both moderate and severe forms of COVID-19 infection [40].
IFNs are a family of secreted proteins that are able to hinder viral infection and replication.Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling cascade leads to transcription of several IFNstimulated genes.In the context of SARS-CoV-2, IFN signaling is involved in both protective and detrimental aspects of this infection [41].Tus, assessment of IFN-associated genes might provide a new insight into the pathogenesis of SARS-CoV-2.Te majority of studied lncRNAs in the present study were IFN-stimulated genes including lnc ISR, lnc ATV, and lnc ISG20 [27,30,31].
Lnc ISR is a lncRNA which is synergically expressed along with IAV infection, by IFN signaling.On the other hand, hosts with type I IFN receptor (IFNAR1) defciency, lnc ISR is not induced.It has been observed that silencing of lnc ISR results in replication of IAV.Tus, lnc ISR and IFN signaling are involved in antiviral immune responses.In other words, lnc ISR suppresses the growth and replication of IAV [26,27].Next, expression of lncRNA ISR was assessed in mouse tissues and cell lines infected with or without IAV, and their results showed that lncRNA ISR expression was signifcantly increased after the IAV infection [27].Our experimental results indicate that lnc ISR is  Lnc ATV is another lncRNA that is highly expressed under the infuence of IFN type I and III.In a study by Jingjing Fan et al., microarray was used to determine the changes in host lncRNA expression in Huh7 cells stimulated by type I or type III IFNs, and their results demonstrated that 272 lncRNAs were upregulated while 631 were downregulated.In the next step, lnc ATV was selected and its expression was assessed by qRT-PCR.IFNα2b and IFNλ1 signifcantly upregulated the expression of endogenous lnc ATV in Huh7 cells.Furthermore, silencing of this lncRNA inhibits the viral replication in several RNA viruses such as Zika virus, hepatitis C virus, Sendai virus, and Newcastle disease virus.Reciprocally, the knockdown of lnc ATV leads to enhanced activity of IFN and RIG-I antiviral signaling pathways.Tus, this human-specifc lncRNA has a considerable role in suppression of host innate immunity during viral infection [31].In our study, this lncRNA was also signifcantly expressed higher in COVID-19 patients, like Huh7 cells.Moreover, it is stated that the IFN I/III response changes in animal models of COVID-19-and SARS-CoV-1-infected patients [25].Hence, this study confrms our observation regarding the higher expression of lnc ATV in COVID-19 patients that might be resulted from IFN I/III response.
Te nomenclature of lnc HEAL refers to its role in HIV-1 infection.Te expression of lnc HEAL increases in T cell lymphocytes, microglia, and monocyte-derived macrophages during active phase of HIV infection, while its expression is downregulated in HIV-infected latent CD4+T lymphocytes [32].Te complex of lnc HEAL and FUS RNA-binding protein accelerates HIV replication.It has been reported that knockdown of lnc HEAL or disturbing the HEAL-FUS complex can be employed as a cure for AIDS and helps eradication of HIV reservoir, but the strategies are still unidentifed [42].Our experimental results indicate that lnc HEAL is upregulated in COVID-19 patients compared to the control group, similar to its expression in diferent cells during active phase of HIV infection.Also, this lncRNA with signifcant AUC in ROC curve could have a biomarker role.
Although no signifcant relation was observed between the expression of lnc PAAN and lnc ISG20 and SARS-CoV-2 infection, Wang J et was not altered during infection with enterovirus 71, VSV-Gpseudo-type of HIV-1, and Zika virus [29].It seems that lnc PAAN is a specifc prognostic marker for IAV infection.Lnc ISG20 hinders the replication of HBV via exonuclease activity through binding to the epsilon stem loop of HBV RNA.In concordance with these fndings, the IFNgamma (IFN-c) is used for treatment of HBV by blocking the viral replication [41].On the other hand, a study conducted by Chai et al. revealed that silencing of lnc ISG20 leads to higher viral replication.Indeed, lnc ISG20 is a negative regulator of IAV replication [30].Although no signifcant association was found between the expression of lnc ISG20 and SARS-CoV-2, the expression of this lncRNA was lower in COVID-19 patients in comparison to healthy patients, which seems that there might be a reverse association between lnc ISG20 and replication of SARS-CoV-2.
Furthermore, in order to better understand the signifcance of these lncRNAs in COVID-19 progression, we designed the ceRNA regulatory network of these lncRNAs using online databases.Also, bioinformatics construction of the lncRNA/miRNA/mRNA network suggests that the RNF24, F2RL3, ACVR2B, and hsa-miR-23b-3p, hsa-miR-629-5p, hsa-miR-30d-3p, hsa-miR-1307-3p, hsa-miR-342-5p, and hsa-miR-221-5p had the most interaction among the mRNAs and miRNA, respectively [43][44][45][46][47][48][49][50].Furthermore, a few of these genes have signifcant involvement in the development and prognosis of COVID-19, and their interactions with lncRNAs highlight their signifcance.LncRNAs have a wide range of roles in pathogenesis, and current research on COVID-19 has focused on interferons and cytokine storms [34].With the aid of the potential gene network created by bioinformatics, these investigations may serve as a precursor to future discoveries about the functions of these noncoding genes in COVID-19.According to our knowledge, this is the frst investigation to develop a novel perspective on the functions of these lncRNAs in regulating Genetics Research 7 the immune response to SARS-CoV-2.Nonetheless, additional research is necessary to investigate the clinical ramifcations of these fndings.

Conclusion
Te considerable higher expression of ISR, ATV, and HEAL lncRNAs demonstrates that these RNAs probably have a remarkable role in the host innate immune responses and the viral replication of SARS-CoV-2.Tese three lncRNAs need to be knocked down to observe their efect on innate immune response and signaling pathways.Hence, these assumptions need further in vitro and in vivo investigations to be confrmed.

Figure 2 :
Figure 2: Box and whisker plot of expression of lncRNAs in COVID-19-infected patients and control group.(a) lncRNA PAAN and (b) lncRNA ISG20.Te grey columns show the expression of lncRNAs in healthy participants, and the dotted columns show the expression of lncRNAs in COVID-19-infected patients (ns: P > 0.05).

Figure 3 :
Figure3: ROC curve analysis.Lnc ATV has a higher AUC (AUC � 0.762) and is a better biomarker to diferentiate COVID-19 patients from the healthy controls.

Figure 4 :
Figure4: Based on lncRNA-miRNA pairings, miRNA-mRNA pairs, and PPI, the lncRNA-miRNA-mRNA triple regulatory network was constructed.mRNAs are shown as blue, miRNAs as green, and lncRNAs as pink.
2.4.Data Analysis.Te expression of each noncoding RNA was interpreted based on the 2 −ΔΔCT (fold change) method.Before using this method, amplifcation efciency was assessed by standard curve.Te statistical data were analyzed using SPSS V.22 software.Te nonparametric Mann-Whitney test was used to compare the expression between the normal control group and the group infected with COVID-19.We used the pROC package in R software to draw the ROC curves for diferentially expressed genes and calculate the AUC.Larger AUC value means the gene can well distinguish COVID-19 patients from the healthy controls, and this gene is a better biomarker.Comparison of groups in terms of age and gender was carried out with Chisquare statistical test.P value less than 0.05 was considered signifcant.

Table 1 :
List of primers used in this study.

Table 3 :
Comparison of lncRNAs expression between healthy people and COVID-19 patients.
al. reported that lnc PAAN enhances the replication of IAV.But similar to our results, this lncRNA