miR-142 Targets TIM-1 in Human Endothelial Cells: Potential Implications for Stroke, COVID-19, Zika, Ebola, Dengue, and Other Viral Infections

T-cell immunoglobulin and mucin domain 1 (TIM-1) has been recently identified as one of the factors involved in the internalization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human cells, in addition to angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2), neuropilin-1, and others. We hypothesized that specific microRNAs could target TIM-1, with potential implications for the management of patients suffering from coronavirus disease 2019 (COVID-19). By combining bioinformatic analyses and functional assays, we identified miR-142 as a specific regulator of TIM-1 transcription. Since TIM-1 has been implicated in the regulation of endothelial function at the level of the blood-brain barrier (BBB) and its levels have been shown to be associated with stroke and cerebral ischemia-reperfusion injury, we validated miR-142 as a functional modulator of TIM-1 in human brain microvascular endothelial cells (hBMECs). Taken together, our results indicate that miR-142 targets TIM-1, representing a novel strategy against cerebrovascular disorders, as well as systemic complications of SARS-CoV-2 and other viral infections.

T-cell immunoglobulin and mucin domain 1 (TIM-1) is a type I cell-surface glycoprotein that includes four main domains: a carboxy-terminal cytoplasmic tail, a mucin domain, a single transmembrane domain, and an amino-terminal immunoglobulin (Ig)-like domain [21]. TIM-1 is expressed on endothelial cells, antigen-presenting cells, neurons and microglial cells, and proximal tubular cells in the kidney (it is also known as Kidney Injury Molecule-1; KIM-1).
Since TIM-1 has been implicated in the regulation of endothelial function at the level of the blood-brain barrier (BBB), and its levels have been shown to be associated with stroke and cerebral ischemia-reperfusion injury [21,40], the main aim of this study was to identify and validate miRNAs that specifically target TIM-1 in human brain microvascular endothelial cells (hBMECs).

miR-142 Targets TIM-1 in Different Species
By combining bioinformatic analyses and functional assays, we identified hsa-miR-142-3p (abbreviated as miR-142) as a specific and conserved regulator of TIM-1 transcription ( Figure 1). We also generated a mutant construct of TIM-1 3 -UTR (TIM-1 MUT), harboring nucleotide substitutions within the predicted miR-142 binding sites of TIM-1 3 -UTR ( Figure 1). and microglial cells, and proximal tubular cells in the kidney (it is also known as Kidney Injury Molecule-1; KIM-1).
Since TIM-1 has been implicated in the regulation of endothelial function at the level of the blood-brain barrier (BBB), and its levels have been shown to be associated with stroke and cerebral ischemia-reperfusion injury [21,40], the main aim of this study was to identify and validate miRNAs that specifically target TIM-1 in human brain microvascular endothelial cells (hBMECs).

Figure 1.
Identification of miR-142 as a specific modulator of TIM-1; the complementary nucleotides between the target region of TIM-1 3′-UTR and hsa-miR-142-3p are highly conserved across different species.

Validation in Human Endothelial Cells of the Transcriptional Regulation of TIM-1 by miR-142
Preclinical assays have shown that inhibiting TIM-1 protects against cerebral ischemia-reperfusion injury [21], while in a clinical study conducted on 4591 subjects, TIM-1 has been associated with a significantly increased incidence of stroke (both ischemic stroke and all-cause stroke) [40]. On these grounds, we validated, for the first time to the best of our knowledge, miR-142 as a functional modulator of TIM-1 in hBMECs, which are generally considered to be among the most suitable cell lines to recapitulate the human BBB in vitro [41,42].

Validation in Human Endothelial Cells of the Transcriptional Regulation of TIM-1 by miR-142
Preclinical assays have shown that inhibiting TIM-1 protects against cerebral ischemiareperfusion injury [21], while in a clinical study conducted on 4591 subjects, TIM-1 has been associated with a significantly increased incidence of stroke (both ischemic stroke and all-cause stroke) [40]. On these grounds, we validated, for the first time to the best of our knowledge, miR-142 as a functional modulator of TIM-1 in hBMECs, which are generally considered to be among the most suitable cell lines to recapitulate the human BBB in vitro [41,42].
We demonstrated via luciferase assays ( Figure 2) that TIM-1 is a specific molecular target of miR-142.
We demonstrated via luciferase assays ( Figure 2) that TIM-1 is a specific molecular target of miR-142. Figure 2. Validation of TIM-1 targeting by miR-142. Luciferase activity was measured in hBMECs 48 h after transfection, using the vector without TIM-1 3′-UTR (empty vector), the vector containing the wild-type TIM-1 3′-UTR, and the vector containing a mutated TIM-1 3′-UTR (TIM-1 MUT); a non-targeting miRNA (miR scramble) was employed as a further control. All experiments were performed at least in triplicate; the box-and-whiskers graph indicates the medians and the 5th-95th percentiles; * p < 0.01 vs. miR scramble.

TIM-1 Expression Levels Are Regulated by miR-142
We observed that miR-142 was able to reduce both the mRNA levels ( Figure 3; Table  1) and the protein levels ( Figure 4) of TIM-1. TIM-1 mRNA levels were measured using RT-qPCR in hBMECs transfected with miR-142 mimic, inhibitor, or scramble (negative control) for 48 h, normalizing to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All experiments were performed at least in triplicate; the box-and-whiskers graph shows the medians and the 5th-95th percentiles; * p < 0.01 vs. miR scramble. Sequences of oligonucleotide primers are reported in Table 1.

TIM-1 Expression Levels Are Regulated by miR-142
We observed that miR-142 was able to reduce both the mRNA levels ( Figure 3; Table 1) and the protein levels ( Figure 4) of TIM-1.
We demonstrated via luciferase assays ( Figure 2) that TIM-1 is a specific molecular target of miR-142. Figure 2. Validation of TIM-1 targeting by miR-142. Luciferase activity was measured in hBMECs 48 h after transfection, using the vector without TIM-1 3′-UTR (empty vector), the vector containing the wild-type TIM-1 3′-UTR, and the vector containing a mutated TIM-1 3′-UTR (TIM-1 MUT); a non-targeting miRNA (miR scramble) was employed as a further control. All experiments were performed at least in triplicate; the box-and-whiskers graph indicates the medians and the 5th-95th percentiles; * p < 0.01 vs. miR scramble.

TIM-1 Expression Levels Are Regulated by miR-142
We observed that miR-142 was able to reduce both the mRNA levels ( Figure 3; Table  1) and the protein levels ( Figure 4) of TIM-1. TIM-1 mRNA levels were measured using RT-qPCR in hBMECs transfected with miR-142 mimic, inhibitor, or scramble (negative control) for 48 h, normalizing to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All experiments were performed at least in triplicate; the box-and-whiskers graph shows the medians and the 5th-95th percentiles; * p < 0.01 vs. miR scramble. Sequences of oligonucleotide primers are reported in Table 1.  TIM-1 mRNA levels were measured using RT-qPCR in hBMECs transfected with miR-142 mimic, inhibitor, or scramble (negative control) for 48 h, normalizing to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All experiments were performed at least in triplicate; the box-and-whiskers graph shows the medians and the 5th-95th percentiles; * p < 0.01 vs. miR scramble. Sequences of oligonucleotide primers are reported in Table 1.  We also confirmed that TIM-1 was expressed in other endothelial cell types, namely adult human lung microvascular endothelial cells (HMVEC-Ls) and human umbilical vein endothelial cells (HUVECs), demonstrating that its expression was significantly reduced by miR-142 (Supplementary Figure S1).

Discussion
Our data indicate that miR-142 targets TIM-1, representing a novel potential strategy against cerebrovascular diseases, COVID-19, and other viral infections.
We established that TIM-1 is expressed in different types of endothelial cells and that miR-142 is able to significantly reduce its expression. One of the key findings of the We also confirmed that TIM-1 was expressed in other endothelial cell types, namely adult human lung microvascular endothelial cells (HMVEC-Ls) and human umbilical vein endothelial cells (HUVECs), demonstrating that its expression was significantly reduced by miR-142 (Supplementary Figure S1).
In our experimental setting, we demonstrated that miR-142 significantly attenuated endothelial permeability triggered by the main agonist of TIM-1, namely, TIMD4 [51,52] ( Figure 5).  We also confirmed that TIM-1 was expressed in other endothelial cell types, namely adult human lung microvascular endothelial cells (HMVEC-Ls) and human umbilical vein endothelial cells (HUVECs), demonstrating that its expression was significantly reduced by miR-142 (Supplementary Figure S1).

Discussion
Our data indicate that miR-142 targets TIM-1, representing a novel potential strategy against cerebrovascular diseases, COVID-19, and other viral infections.
We established that TIM-1 is expressed in different types of endothelial cells and that miR-142 is able to significantly reduce its expression. One of the key findings of the Our data on endothelial leakage were further supported by the regulation of a major tight-junction protein, namely occludin [53][54][55][56], by miR-142 (Supplementary Figure S2).

Discussion
Our data indicate that miR-142 targets TIM-1, representing a novel potential strategy against cerebrovascular diseases, COVID-19, and other viral infections.
We established that TIM-1 is expressed in different types of endothelial cells and that miR-142 is able to significantly reduce its expression. One of the key findings of the present work is the demonstration of the functional role of miR-142 in the regulation of endothelial leakage, which represents a fundamental step in several disorders caused by different viruses, which do not have to necessarily display a definite endotheliotropism [57][58][59][60][61][62][63][64]. Moreover, many viral infections, including COVID-19, have been shown to lead to the involvement of different tissues and organs, often culminating in a systemic inflammatory response, in which the endothelium plays key roles [50,[65][66][67][68]. Our results in terms of endothelial permeability are also corroborated by data showing that miR-142 can regulate the expression of Occludin, a tight-junction protein that is functionally involved in viral neuro-invasion [69].
Additional potential applications of our discovery include kidney disease, disturbances of iron metabolism, and the modulation of the immune response. Indeed, plasma levels of TIM-1 have been found to be associated with underlying tubulointerstitial and mesangial lesions and progression to kidney failure in two cohort studies of individuals with kidney diseases [104]. Preclinical investigations have demonstrated that TIM-2, the rodent homolog to TIM-1, is a binding partner to H-ferritin [105], a protein initially thought to be solely used for iron storage [106][107][108], which was later shown to serve as an iron delivery protein, secreted from the endothelial cells within the BBB as a source of iron for the brain [109]. TIM-1 can also mediate the tethering between T cells and endothelial cells in vivo and the rolling of lymphocytes on the vascular endothelium [110].
Our study is not exempt from limitations, including having performed the luciferase assays only in one cell type; nevertheless, we demonstrated that TIM-1 is indeed expressed (and regulated by miR-142) in three different types of endothelial cells, namely, hBMECs, HUVECs, and HMVEC-Ls. Further dedicated experiments are warranted to prove the effects of miR-142 in the pathophysiology of cerebrovascular events and on the actions of SARS-CoV-2 and other viruses.

Identification and Validation of miR-142 as a Regulator of TIM-1
To identify miRNAs targeting the 3 -UTR of TIM-1, we used TargetScanHuman 8.0, as we previously described [20,111,[114][115][116][117]. To assess the effects of miR-142 on TIM-1 gene transcription, we used a luciferase reporter containing the 3 -UTR of the predicted miRNA interaction site, both wild-type and mutated, in hBMECs cells. The mutant construct of TIM-1 3 -UTR (TIM-1 MUT, as shown in Figure 1), harboring a substitution of three nucleotides within the predicted miR-142 binding sites of TIM-1 3 -UTR was obtained using the NEBaseChanger and Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA, USA) as we previously described [111,114,116].

Immunoblotting
Immunoblotting assays were performed as previously described and validated by our group [111,116,119]; the intensity of the bands was quantified using FIJI ("Fiji Is Just ImageJ") software. The antibody for TIM-1 was purchased from Novus Biologicals (Centennial, CO, USA; catalog number; #NBP1-76701); the antibody for Occludin was purchased from Novus Biologicals (catalog number, #NBP1-87402); the antibody for β-Actin was purchased from abcam (Cambridge, MA, USA; catalog number, #ab8229); the antibody for GAPDH was purchased from Novus Biologicals (catalog number, #NB300-221).

Endothelial Permeability Assay
We performed the in vitro permeability assay in hBMECs as we previously described [53,111,113]. Briefly, hBMECs transfected with miR-142 mimic or miR scramble were grown on 0.4 mm fibronectin-coated transwell filters for 48 h; then, we replaced the medium in the upper well with FITC-dextran 70 kD (0.5 mg/mL in phosphate-buffered saline, PBS). Cells were stimulated in the lower well with PBS alone or PBS containing 50 ng/mL VEGF-A 165 (Bio-Techne Corporation, Minneapolis, MN, USA). Endothelial permeabilization was quantified by measuring at 520 nm the fluorescence of dextran that passed in the bottom chamber through the cell monolayer [111,113,120].

Statistical Analysis
All data were expressed as means ± standard errors of the means (SEMs). The statistical analyses were carried out using GraphPad 9 (Prism, San Diego, CA, USA). Statistical significance, set at p < 0.05, was tested using the two-way ANOVA followed by Tukey-Kramer multiple comparison test or the non-parametric Mann-Whitney U test, as appropriate.

Conclusions
Taken together, our results indicate that miR-142 targets TIM-1, representing a novel potential strategy against cerebrovascular disease, SARS-CoV-2, and other viral infections.

Conflicts of Interest:
The authors declare no conflict of interest.