Application of CRISPR/Cas12a in miRNA-155 detection: A novel homogeneous electrochemiluminescence biosensor.

BACKGROUND: MicroRNAs (miRNAs) are important non-coding RNA entities that affect gene expression and function by binding to target mRNAs, leading to degradation of the mRNAs or inhibiting their translation. MiRNAs are widely involved in a variety of biological processes, such as cell differentiation, development, metabolism, and apoptosis. In addition, miRNAs are associated with many diseases, including cancer. However, conventional detection techniques often suffer from shortcomings such as low sensitivity, so we need to develop a rapid and efficient detection strategy for accurate detection of miRNAs. RESULTS: We have developed an innovative homogeneous electrochemiluminescence (ECL) biosensor. This biosensor employs CRISPR/Cas12a gene editing technology for accurate and efficient detection of microRNA (miRNA). Compared to conventional technologies, this biosensor employs a unique homogeneous detection format that eliminates laborious probe fixation steps and greatly simplifies the detection process. By using two amplification techniques - isothermal amplification and T7 RNA polymerase amplification - the biosensor improves the sensitivity and specificity of the assay, providing excellent detection performance in the assay. This makes it possible to evaluate miRNA directly from a variety of biological samples such as cell lysates and diluted human serum. Experimental results convincingly demonstrate the extraordinary performance of this biosensor, including its extremely low detection limit of 1.27 aM, high sensitivity, reproducibility and stability. SIGNIFICANCE: The application of our constructed sensor in distinguishing between cancerous and non-cancerous cell lines highlights its potential for early cancer detection and monitoring. This innovative approach represents a major advancement in the field of miRNA detection, providing a user-friendly, cost-effective, and sensitive solution with broad implications for clinical diagnosis and patient care, especially in point-of-care settings.

CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.

A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.

Detection of MicroRNA-155 based on lambda exonuclease selective digestion and CRISPR/cas12a-assisted amplification.

In numerous malignancies, miRNA-155 is overexpressed and has oncogenic activity because it is one of the most efficient microRNAs for inhibiting apoptosis in human cancer cells. As a result, the highest sensitive detection of the miRNA-155 gene is a technological instrument that can enable early cancer screening. In this study, a miRNA-155 biosensor was created to create a hairpin probe that can bind to the miRNA-155 gene using lambda nucleic acid exonuclease, which can cut the 5' phosphorylated double strand, and by the DNA probe is recognized by the Cas12a enzyme, which then activates Cas12a to catalyze trans-cutting produces strong fluorescence. Research finding, the target concentration's logarithm and corresponding fluorescence intensity have a strong linear connection, and the limit of detection (LOD) of the sensing system was determined to be 8.3 pM. In addition, the biosensor displayed exceptional specificity, low false-positive signal, and high sensitivity in detecting the miRNA-155 gene in serum samples. This study's creation of a biosensor that has high sensitivity, good selectivity, and is simple to operate provides promising opportunities for research into biosensor design and early cancer detection.

Near-infrared light-induced homogeneous photoelectrochemical biosensor based on 3D walking nanomotor-assisted CRISPR/Cas12a for ultrasensitive microRNA-155 detection.

The dysregulation of microRNA (miRNA) expression levels is intricately linked to a myriad of human diseases, and the precise and delicate detection thereof holds paramount significance in the realm of clinical diagnosis and therapy. Herein, a near-infrared (NIR) light-mediated homogeneous photoelectrochemical (PEC) biosensor was constructed for miRNA-155 detection based on NaYF4: Yb, Tm@ZnIn2S4 (NYF@ ZIS) coupled with a three-dimensional (3D) walking nanomotor-assisted CRISPR/Cas12a strategy. The upconverted light emitted by the NYF in the visible and UV region upon NIR light excitation could be utilized to excite ZIS to produce a photocurrent response. The presence of target miRNA-155 initiated an amplification reaction within the 3D walking nanomotor, resulting in the production of multiple nucleic acid fragments. These fragments could activate the collateral cleavage capability of CRISPR/Cas12a, leading to the indiscriminate cleavage of single-stranded DNA (ssDNA) on ALP-ssDNA-modified magnetic beads and the subsequent liberation of alkaline phosphatase (ALP). The released ALP facilitated the catalysis of ascorbic acid 2-phosphate to generate ascorbic acid as the electron donor to capture the photogenerated holes on the NYF@ZIS surface, resulting in a positively correlated alteration in the photocurrent response. Under optimal conditions, the NIR light-initiated homogeneous PEC biosensor had the merits of good linear range (0.1 fM to 100 pM), an acceptable limit of detection (65.77 aM) for miRNA-155 detection. Considering the pronounced sensitivity, light stability, and low photodamage, this strategy presents a promising platform for detecting various other miRNA biomarkers in molecular diagnostic practice.

Kaempferol Ameliorated Alcoholic Hepatitis through Improving Intestinal Barrier Function by Targeting miRNA-155 Signaling.

INTRODUCTION: The aim of this study was to investigate the effect and mechanism of kaempferol on alcoholic steatohepatitis. METHODS: C57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. Kaempferol was given as the interventional drug to chronic alcohol-fed mice for 6 weeks to assess its effects. In vitro, intestinal epithelial Caco-2 cells were stimulated by alcohol, and miRNA-155 mimics were used to further study the effect of kaempferol to miRNA-155 signaling in intestinal epithelial cells. HE staining and oil red O staining were used to observe the liver and intestinal tissue damage in each group of mice, and ALT, AST, IL-1ß, and TNF-α were detected by kits; lipopolysaccharide (LPS) expression was detected by ELISA kit, and the expression of IL-1ß and TNF-α was assessed by qRT-PCR; Western blot was utilized to assess the excessive inflammatory response of liver and colon tissue and the related signaling pathway activation. RESULTS: Kaempferol treatment significantly improved pathological changes such as steatosis and vacuolated lesions in liver tissue of the alcohol diet model group, and reduced serum ALT and AST enzyme activities and liver tissue interleukin-1ß and tumor necrosis factor-α mRNA expression levels. Kaempferol significantly reduced the expression of miRNA-155 in the intestinal tissue of alcohol-fed mice, significantly increased their cytokine suppressor signaling 1 (SOCS1) protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and zonula occludens-1. More importantly, kaempferol significantly reduced serum LPS levels in alcoholic steatohepatitis mice. In vitro experiments showed that compared with the control group, kaempferol significantly inhibited the expression level of miRNA-155 in Caco-2 cells under ethanol exposure, decreased the activation of nuclear factor kappa-B, led to an increase in the expression of SOCS1 protein, and increased the production level of occludin protein in Caco-2 cells under the effect of alcohol. In contrast, overexpression of miRNA-155 significantly decreased occludin and SOCS1 protein production and increased nuclear factor kappa-B activation levels in Caco-2 cells, and the administration of kaempferol significantly inhibited this effect. CONCLUSION: Kaempferol improved the stability of gut barrier function to ameliorate hepatic injury induced by alcohol intake through enhancing occludin protein expression, by targeting miR-155 to inhibit the excessive inflammatory response in the intestine.

Chitosan nanoparticles-based in vivo delivery of miR-155 modulates the Viral haemorrhagic septicaemia virus-induced antiviral immune responses in zebrafish (Danio rerio).

Viral haemorrhagic septicaemia virus (VHSV) is one of the highly pathogenic virus, which causes viral haemorrhagic septicaemia disease in both marine and freshwater fish. Micro RNA-155 (miRNA-155) is a multifunctional small non-coding RNA and it involves regulation of immune responses during viral infection. In this study, dre-miR-155 mimics were encapsulated into chitosan nanoparticles (CNPs). Resulted encapsulated product (miR-155-CNPs) was investigated for its immunomodulation role in zebrafish during experimentally challenged VHSV infection. Successful encapsulation of dre-miR-155 mimics into CNPs was confirmed through average nanoparticle (NPs) size (341.45 ± 10.00 nm), increased encapsulation efficiency percentage (98.80%), bound dre-miR-155 with chitosan, sustained release in vitro (up to 40%), and the integrity of RNA. Overexpressed miR-155 was observed in gills, muscle, and kidney tissues (5.42, 19.62, and 140.72-folds, respectively) after intraperitoneal delivery of miR-155-CNPs into zebrafish upon VHSV infection (miR-155-CNPs + VHSV). The miR-155-CNPs + VHSV infected fish had the highest cumulative survival (85%), which was associated with low viral copy numbers. The miR-155-overexpressing fish showed significantly decreased expression of ifnγ, irf2bpl, irf9, socs1a, il10, and caspase3, compared to that of the miR-155 inhibitor + VHSV infected fish group. In contrast, il1ß, tnfα, il6, cd8a, and p53 expressions were upregulated in miR-155-overexpressed zebrafish compared to that of the control. The overall findings indicate the successful delivery of dre-miR-155 through miR-155-CNPs that enabled restriction of VHSV infection in zebrafish presumably by modulating immune gene expression.

Relation between microRNA-155 and inflammatory mediators in multiple sclerosis.

Multiple sclerosis (MS) is a complex autoimmune condition affecting the central nervous system characterized by axonal damage, demyelination, and chronic inflammation. Multiple molecular and cellular components mediate neuroinflammation in MS. In human macrophages and microglia, miRNA-155 is an essential proinflammatory noncoding RNA that regulates phenotypic and functional polarization properties. This study was conducted to detect the plasma level of miRNA-155 in RRMS and assess its relationship with inflammatory and anti-inflammatory mediators. The study included 60 MS patients and 30 healthy controls. Real-time quantitative polymerase chain reaction was utilized to detect miRNA-155, iNOS, and SMAD2, whereas ELISA was used to determine TNF-α, IFN-É£, TGF-ß, and IL-10 levels. There was no significant difference in miRNA-155, SMAD2, and iNOS expression in MS patients compared to control subjects. In addition, there was a statistically significant increase in TNF-α, INF-É£, and TGF-ß levels. IL-10 levels did not differ significantly between MS patients and healthy controls. There was a positive correlation between miRNA-155 and TNF-α (p < 0.000, r = 0.922), INF-É£ (p < 0.000, r = 0.81), and iNOS (p < 0.000, r = 0.916) and inverse correlation between miRNA-155 and IL-10 (p < 0.000, r = -0.928), TGF-ß (p < 0.000, r = -0.904) and SMAD2 (p < 0.000, r = -0.848). We conclude that expression of miRNA-155 in MS may modulate macrophage/microglia polarization by increasing the secretion of TNF-α, IFN-É£ & iNOS and decreasing anti-inflammatory mediators IL10 and TGF-ß.

Epigenetic alterations of miR-155 and global DNA methylation as potential mediators of ochratoxin A cytotoxicity and carcinogenicity in human lung fibroblasts.

Ochratoxin A (OTA) is a well-known mycotoxin that adversely affects different human cells. Inhalational exposure to OTA and subsequent pulmonary diseases have been previously reported, yet its potential carcinogenicity and underlying molecular mechanisms have not been fully elucidated. This study aimed to evaluate the OTA-induced cytotoxicity and the epigenetic changes underlying its potential carcinogenicity in fetal lung fibroblast (WI-38) cells. OTA cytotoxicity was assessed by MTT assay; RT-qPCR was used to determine the expression of BAX, BCL-2, TP53, and miR-155, while ELISA was used for measuring 5-methyl cytosine percentage to assess global DNA methylation in OTA-treated versus control cells. WI-38 cells demonstrated sensitivity to OTA with IC50 at 22.38 µM. Though BAX and Bcl-2 were downregulated, with low BAX/BCL-2 ratio, and TP53 was upregulated, their fold changes showed decline trend with increasing OTA concentration. A significant dose-dependent miR-155 upregulation was observed, with dynamic time-related decline. Using subtoxic OTA concentrations, a significant global DNA hypermethylation with significant dose-dependent and dynamic alterations was identified. Global DNA hypermethylation and miR-155 upregulation are epigenetic mechanisms that mediate OTA toxicity on WI-38 cells. BAX downregulation, reduced BAX/BCL-2 ratio together with miR-155 upregulation indicated either the inhibition of TP53-dependent apoptosis or a tissue specific response to OTA exposure. The aforementioned OTA-induced variations present a new molecular evidence of OTA cytotoxicity and possible carcinogenicity in lung fibroblast cells.

Regulation of miRNA-155-5p ameliorates NETosis in pulmonary fibrosis rat model via inhibiting its target cytokines IL-1ß, TNF-α and TGF-ß1.

Idiopathic pulmonary fibrosis (IPF) is an age-related inflammatory disease with no cure up till now.It is accompanied by neutrophils infiltration as the main responders to inflammation and fibrosis. Importantly, neutrophils release neutrophil extracellular traps (NETs) through NETosis process. The function of microRNAs during inflammation became of great biological attention. Owing to microRNAs' central role in immune system, microRNA-155-5p (miR-155-5p) is intensely involved in the inflammatory response. Capsaicin (Cap) is a bioactive compound that exhibits antioxidative and anti-inflammatory functions. Recent studies have shown its role in regulation of certain microRNAs' expressions. Accordingly, the present study aims to investigate the effect of miR-155-5p regulation in suppressing NETs production via ameliorating its target inflammatory cytokines, IL-1ß, TNF-α and TGF-ß1, in bleomycin (BLM)-induced pulmonary fibrosis rat model treated by Cap. The obtained results demonstrated that miR-155-5p downregulation was associated with significant decrease in IL-1ß, TNF-α, TGF-ß1, which consequently, reduced hydroxyproline (HYP), NETs activity markers as NE and PAD-4, and alleviated CTGF levels in lung tissues of animals treated by Cap. Furthermore, NETosis ultrastructure examination by transmission electron microscope (TEM), MPO immunohistochemical staining and histopathological studies confirmed an abolishment in NETs formation and an improvement in lung tissue architecture in Cap-treated rats. This study concluded that Cap quenched the inflammatory response through interrupting IL-1ß, TNF-α and TGF-ß1 pathway via modulating miR-155-5p expression. In addition, Cap was able to alleviate pulmonary NETosis markers by restraining NETs activity markers. These findings provide novel insight into the application of Cap-based treatment in ameliorating pulmonary damage in IPF.

Comparison of Plasma Levels of MicroRNA-155-5p, MicroRNA-210-3p, and MicroRNA-16-5p in Rheumatoid Arthritis Patients with Healthy Controls in a Case-control Study.

Rheumatoid arthritis is a chronic autoimmune disease that causes inflammation and destruction of the joints. The objective of the current study was to evaluate the expression of microRNA (miR)-155-5p, miR-210-3p, and miR-16-5p in the plasma of patients with rheumatoid arthritis in comparison with a healthy control group to attain an expression profile for earlier diagnosis and treatment. To carry out this study, 100 individuals were chosen as two equally sized groups of rheumatoid arthritis patients and healthy controls. Five milliliters of blood were drawn from each individual, and plasma RNA was extracted using Trisol solution. Complementary DNAs were synthesized using the Moloney leukemia virus (MMLV) and deoxynucleoside triphosphate (dNTP). Finally, real-time polymerase chain reaction (PCR) was implemented using the SYBR Green kit. The mean expression of miR-155-5p, miR-210-3p, and miR-16-5p were 2.46±2.79, 1.97±1.90, and 69.62±88.44 in the rheumatoid arthritis group, and 0.34±0.33, 9.82±9.34, and 7.94±7.09 in the healthy group, respectively. Additionally, significant differences were revealed in the relative  expression of the selected microRNAs in 4 subgroups of rheumatoid arthritis patients with different disease activities based on the disease activity score 28 (DAS28). ROC curve analysis showed that miR-155-5p (area under the curve, AUC=0.90, sensitivity=80%, specificity=81%), miR-210-3p (AUC=0.75, sensitivity=66%, specificity=71%), and miR-16-5p (AUC=0.96, sensitivity=89%, specificity=82%) could be potential biomarkers for rheumatoid arthritis diagnosis. Increased expressions of miR-16-5p and miR-155-5p and decreased expression of miR-210-3p in rheumatoid arthritis patients compared with healthy individuals demonstrate the effectiveness of these microRNAs in disease incidence and progression. Thus, the expression levels of these microRNAs can be used for diagnostic and therapeutic purposes.

MicroRNA-155 mediates endogenous angiotensin II type 1 receptor regulation: implications for innovative type 2 diabetes mellitus management.

Type 2 diabetes mellitus (T2DM) is a lifelong condition and a threat to human health. Thorough understanding of its pathogenesis is acutely needed in order to devise innovative, preventative, and potentially curative pharmacological interventions. MicroRNAs (miRNA), are small, non-coding, one-stranded RNA molecules, that can target and silence around 60% of all human genes through translational repression. MiR-155 is an ancient, evolutionarily well-conserved miRNA, with distinct expression profiles and multifunctionality, and a target repertoire of over 241 genes involved in numerous physiological and pathological processes including hematopoietic lineage differentiation, immunity, inflammation, viral infections, cancer, cardiovascular conditions, and particularly diabetes mellitus. MiR-155 Levels are progressively reduced in aging, obesity, sarcopenia, and T2DM. Thus, the loss of coordinated repression of multiple miR-155 targets acting as negative regulators, such as C/EBPß, HDAC4, and SOCS1 impacts insulin signaling, deteriorating glucose homeostasis, and causing insulin resistance (IR). Moreover, deranged regulation of the renin angiotensin aldo-sterone system (RAAS) through loss of Angiotensin II Type 1 receptor downregulation, and negated repression of ETS-1, results in unopposed detrimental Angiotensin II effects, further promoting IR. Finally, loss of BACH1 and SOCS1 repression abolishes cytoprotective, anti-oxidant, anti-apoptotic, and anti-inflammatory cellular pathways, and promotes ß-cell loss. In contrast to RAAS inhibitor treatments that further decrease already reduced miR-155 Levels, strategies to increase an ailing miR-155 production in T2DM, e.g., the use of metformin, mineralocorticoid receptor blockers (spironolactone, eplerenone, finerenone), and verapamil, alone or in various combinations, represent current treatment options. In the future, direct tissue delivery of miRNA analogs is likely.

Construction of electrochemical biosensors based on MoSe2@1T-MoS2 heterojunction for the sensitive and rapid detection of miRNA-155 biomarker in breast cancer.

MiRNA-155 is a typical biomarker for breast cancer. Since its low concentration in the physiological environment and the limitations of conventional miRNA detection methods like Northern imprinting and RT-qPCR, convenient, real-time, and rapid detection methods are urgently needed. In this work, an electrochemical biosensor was constructed based on the flower-like MoSe2@1T-MoS2 heterojunction electrode material and specific RNA recognition probes, which can realize the rapid determination of miRNA-155 content with a wide detection range from 1 fM to 1 nM and a limit of detection (LOD) as low as 0.34 fM. Furthermore, the contents of miRNA-155 in blood samples of tumor-bearing mice and normal mice were measured as 724.93 pM and 21.42 pM, respectively by this biosensor, demonstrating its strong identification ability and miRNA-155 can be regarded as an ideal diagnostic marker. On this basis, a portable sensor platform was designed for on-site detection simulation and showed good recovery efficiency from 95.80% to 98.69%. Meanwhile, compared with the standard detection method RT-qPCR, the accuracy and reliability of the biosensor were verified, indicating that the biosensor has the potential to provide point-of-care testing (POCT) for the early diagnosis of breast cancer.

Platelet miRNAs: differential expression in coronary artery disease and associations with course of left ventricular systolic function.

BACKGROUND: MicroRNAs are paramount in post transcriptional gene regulation. We investigated platelet miRNAs in patients with CAD and examined potential associations with course of left ventricular ejection fraction (LVEF%). MATERIALS AND METHODS: In a first cohort, 62 MiRNAs were measured in platelets of 100 patients suffering from CAD. Expression profiles of individuals with chronic coronary syndrome (CCS) and MI were compared (CCS n = 67, MI n = 33). Also, associations between miRNA profiles and change in left ventricular ejection fraction (LVEF%) were investigated. In a second cohort of patients suffering from CCS (n = 10), MI (n = 11) or no CAD (n = 13), we measured miRNA expression in platelets, platelet supernatant and serum. This was carried out before and after in vitro platelet activation with CRP. RESULTS: Platelet miRNAs 103a-3p and 155-5p demonstrated higher expression in patients with CCS then in individuals with MI. Furthermore, multiple miRNAs were significantly higher expressed in matched controls compared to MI patients. 8 miRNAs showed higher expression in patients with improving LVEF% after a 1-year follow-up. In our second cohort, we found higher concentrations of 6 miRNAs in the platelet supernatant of patients with CCS, MI and no CAD after in vitro platelet activation. Most of these miRNAs showed a higher abundance in serum of MI patients as compared to CCS. CONCLUSION: Several miRNAs show higher expression in platelets of CCS compared to MI. After in vitro platelet activation, a release of multiple miRNAs out of the thrombocyte was observed. Furthermore, upregulation of serum miRNAs was found in MI patients when compared to CCS patients and individuals without CAD. Hence, platelets could present a source of upregulated circulating miRNAs in MI and additionally affect course of LVEF%.

Fever-range whole body hyperthermia leads to changes in immune-related genes and miRNA machinery in Wistar rats.

OBJECTIVE: Fever is defined as a rise in body temperature upon disease. Fever-range hyperthermia (FRH) is a simplified model of fever and a well-established medical procedure. Despite its beneficial effects, the molecular changes induced by FRH remain poorly characterized. The aim of this study was to investigate the influence of FRH on regulatory molecules such as cytokines and miRNAs involved in inflammatory processes. METHODS: We developed a novel, fast rat model of infrared-induced FRH. The body temperature of animals was monitored using biotelemetry. FRH was induced by the infrared lamp and heating pad. White blood cell counts were monitored using Auto Hematology Analyzer. In peripheral blood mononuclear cells, spleen and liver expression of immune-related genes (IL-10, MIF and G-CSF, IFN-γ) and miRNA machinery (DICER1, TARBP2) was analyzed with RT-qPCR. Furthermore, RT-qPCR was used to explore miRNA-155 levels in the plasma of rats. RESULTS: We observed a decrease in the total number of leukocytes due to lower number of lymphocytes, and an increase in the number of granulocytes. Furthermore, we observed elevated expressions of DICER1, TARBP2 and granulocyte colony-stimulating factor (G-CSF) in the spleen, liver and PBMCs immediately following FRH. FRH treatment also had anti-inflammatory effects, evidenced by the downregulation of pro-inflammatory macrophage migration inhibitor factor (MIF) and miR-155, and the increased expression of anti-inflammatory IL-10. CONCLUSION: FRH affects the expression of molecules involved in inflammatory processes leading to alleviated inflammation. We suppose these effects may be miRNAs-dependent and FRH can be involved in therapies where anti-inflammatory action is needed.

CTLA4 mRNA is downregulated by miR-155 in regulatory T cells, and reduced blood CTLA4 levels are associated with poor prognosis in metastatic melanoma patients.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies.

CRISPR/Cas13a-triggered Cas12a biosensing method for ultrasensitive and specific miRNA detection.

Constructing an ultrasensitive CRISPR/Cas-based biosensing strategy is highly significant for the detection of trace targets. Here we presented a dual-amplified biosensing method based on CRISPR/Cas13a-triggered Cas12a, namely, Cas13a-12a amplification. As proof-of-principle, the developed strategy was used for miRNA-155 detection. The target bound to the Cas13a-crRNA complex and activated the cleavage activity of Cas13a for cleaving uracil ribonucleotides (rU) in the bulge structure of blocker strand (BS), resulting in the release of primer strand (PS) from the BS modified on magnetic beads. Then, the released PS activated the cleavage activity of Cas12a to cleave single-strand DNA reporter probes, producing a significantly increased fluorescent signal. The detection limit of the Cas13a-12a amplification using synthetic miRNA-155 was as low as 0.35 fM, which was much lower than that of the only Cas13a-based assay. The applied performance of this amplification strategy was verified by accurately quantifying miRNA-155 expression levels in different cancer patients. Therefore, the developed strategy offers a supersensitive and highly specific miRNAs sensing platform for clinical application.

Mechanisms underlying the improvement of preeclampsia through salvianolic acid B-regulated miRNA-155/CXCR4.

Introduction: The aim of this study was to evaluate the effects and mechanisms of salvianolic acid B (Sal B) in preeclampsia treatment by in vivo and in vitro study. Material and methods: Rats were randomly divided into 5 groups. In order to establish the model of preeclampsia, endotoxin was administered to the rats in the Sal B intervention and model groups. The systolic blood pressure (SBP) of the tail artery and urine protein concentration were observed at different points, the miRNA-155 and CXCR4 gene expression levels by RT-PCR and the CXCR4 and p-AKT protein expression by WB assay. Using HTR8/SVneo to explain the mechanisms; evaluating the miRNA-155 and CXCR4 mRNA expression by RT-PCR assay, measuring the cell invasion and migration by transwell and wound healing assay in different groups; evaluating the CXCR4 and p-AKT protein expression by WB assay and p-AKT nucleation volume by cellular immunofluorescence were evaluated. Results: Compared with the normal group, the systolic blood pressure and urine protein were significantly increased in the model group (p < 0.05), serum NO concentration was significantly down-regulated (all p < 0.05), CXCR4 and miRNA-155 mRNA expression was significantly different and CXCR4 and p-AKT protein expression was significantly suppressed (all p < 0.05). With Sal B supplement, the SBP, urine protein and NO concentration were significantly improved with dose-dependent (all p < 0.05). In the cell experiment, the cell invasion and migration ability were significantly improved with Sal B supplement (both p < 0.05). However, with miRNA-155 transfection, the cell invasion and migration ability were suppressed with Sal B treatment (both p < 0.05). Conclusions: Sal B improved preeclampsia via regulation of miRNA-155/CXCR4 in the in vitro and vivo study.

Ochratoxin A induces endoplasmic reticulum stress and fibrosis in the kidney via the HIF-1α/miR-155-5p link.

Ochratoxin A (OTA) is a ubiquitous fungal toxin found in agricultural products and foods that is toxic to both humans and animals. OTA mainly affects kidney, but the mechanisms underlying OTA-induced nephrotoxicity remain not fully understood. MicroRNA (miRNA) is involved in key cellular processes. The toxic mechanism and regulatory effects of miRNAs on OTA toxicity in kidney, and particularly the role of HIFα-1/miR-155-5p on OTA-caused ER stress and fibrosis, were investigated in this study. OTA induced hypoxia-like conditions such as ER stress and fibrosis in HK-2 cells and renal tissues via modulating HIF-1α, which was followed by regulation of ER stress-related proteins (GRP78 and ATF-4), as well as fibrosis-related markers (fibronectin, α-SMA, and E-cadherin). Notably, a total of 62 miRNAs showed significant differential expression in kidney of OTA-treated mice. Under OTA exposure, HIF-1α enhanced miR-155-5p expression, causing ER stress and fibrosis in HK-2 cells. HIF-1α knockdown decreased OTA-induced miR-155-5p expression as well as ER stress and fibrotic responses, whereas miR-155-5p overexpression restored this. Our data suggest that OTA enhances ER stress and fibrosis in the kidney through upregulating the HIF-1α/miR-155-5p link.

MicroRNA 155, Factor XIII and Type 2 Diabetes Mellitus and Coronary Heart Disease.

There is a rise in the number of individuals diagnosed with type 2 diabetes mellitus (T2DM) in South Africa. Cardiovascular disease is among the macrovascular complication of type 2 diabetes mellitus and accounts for the high mortality rate in patients with T2DM. The disease is characterized by insulin resistance, hyperglycaemia, oxidative stress, inflammation, hypofibrinolysis and hypercoagulation. The impairment of fibrinolysis, hyperactivation of coagulation and the inflammatory pathways result in an increased risk of developing coronary heart disease. Factor XIII-A is one of the key coagulation factors that play a crucial role in the last stage of the coagulation cascade, and it has been shown to play a critical role in the development of thrombotic diseases. In addition, several studies show the influence of FXIII-A polymorphisms on thrombotic diseases. The influence of genetic variations such as single nucleotide variants and gene expression regulators (micro-RNAs) are important factors involved in the hyperactivation of coagulation and hypofibrinolysis. Thus, this review aims to summarise key aspects of coagulation, FXIII-A expression, potential FXIII-A genetic variations and epigenetic mediators (micro-RNA-155) in T2DM and patients with coronary artery disease.

Vitamin D and miRNA-155 in Behçet's Disease: Possible Association with the Disease and Disease Activity.

Background: Vitamin D (vit D) controls inflammation and immunity. In Behçet's disease (BD), microRNA-155 is recognized as a significant immune response regulator. We aimed to investigate the role of vit D on immunomodulation and downregulation of inflammatory pathways associated with BD and detect the role of miRNA-155 in BD. Methods: miRNA-155 expression by Real Time -Polymerase Chain Reaction (RT-PCR), and vit D, nuclear factor Kappa-light-chain-enhancer of activated B cells (NF-κB), and Tumor necrosis fact of TNF-α) expression by Enzyme Linked Immunosorbent Assay (ELISA) were assessed. Results: BD patients had a significantly higher relative expression of microRNA-155 (P< 0.001), it was significantly related to vascular manifestations (P< 0.001). Vit D relative expression was significantly low in BD (P< 0.001). There was a significant rise in miRNA-155 in the active group compared to the inactive group (P< 0.001). A significant decrease in vit D levels (IU) was found in inactive and active individuals suffering from BD when compared to controls (P< 0.001). A significant rise was found in vit D levels in inactive BD cases (P< 0.001). A significant positive correlations were found between miRNA-155, NF-κB, TNF-α, and negative correlations with vit D relative expression in BD patients. Conclusions: miRNA-155 relative expression is higher in BD is significantly related to vascular manifestations. It may have a relationship to disease activity. Vitamin D relative expression is significantly low in BD patients, which can significantly influence immunomodulatory BD therapy. Vitamin D deficiency linked to active BD.