Perfluorooctanoic acid (PFOA) exposure induces renal filtration and reabsorption disorders via down-regulation of aquaporins.

Perfluorooctanoic acid (PFOA) exposure is associated with kidney dysfunction, however the exact mechanisms by which PFOA induces nephrotoxicity and the specific involvement of aquaporins (AQPs) in kidney tissue remains unclear. In this study, adult male Sprague-Dawley (SD) rats were exposed to PFOA by oral gavage for 28 days and compared with controls. Body weight, water intake and urine volume were recorded daily. At the end of the experiment, blood and kidney samples were collected, and serum urea, creatine and uric acid levels were assessed. The renal expression levels of water channel proteins AQP1, AQP3, AQP2 and p-AQP2 (Ser256) were observed by immunohistochemical staining, and the corresponding transcription levels were detected by Western blot and qRT-PCR. The results showed that PFOA exposure inhibited weight gain and increased water intake, urine volume, kidney weight and renal visceral index. PASM staining and transmission electron microscopy revealed pathological thickening of the glomerular capsule and basement membrane. Serum urea levels were increased, while serum creatine levels were decreased compared to controls. Additionally, the expression levels of AQP1, AQP3, AQP2 and p-AQP2 in kidney tissues were decreased, and the phosphorylation of AQP2 at Ser256 was inhibited. In conclusion, we demonstrate that PFOA exposure can damage the renal filtration barrier and reduce the expression level of AQPs in renal tissues, leading to renal filtration and reabsorption disorders.

PFOA exposure induces aberrant glucose and lipid metabolism in the rat liver through the AMPK/mTOR pathway.

Perfluorooctanoic acid (PFOA) is the most prominent member of a widely utilized family of compounds named Perfluoroalkyl substances (PFASs). Initially produced for use in both industrial and consumer applications, it has since been recognized that PFASs are extremely persistent in the environment where they have been characterized as persistent organic pollutants (POPs). While previous studies have demonstrated that PFOA may induce disorders of lipid and carbohydrate metabolism, the precise mechanisms by which PFOA produces this phenotype and the involvement of downstream AMPK/mTOR pathways remains unclear. In this study, male rats were exposed to 1.25, 5 and 20 mg PFOA/kg body weight/day for 28 days by oral gavage. After 28 days, blood was collected and tested for serum biochemical indicators and livers were removed and weighed. To investigate aberrant metabolism in rats exposed to PFOA, livers were analyzed by performing LC-MS/MS untargeted metabolomics, quantitative real-time PCR, western blotting, immunohistochemical staining was also performed on exposed tissues. Our results showed that exposure to PFOA induced liver damage, increased the expression of glucose and lipid related biochemical indexes in liver and serum, and altered the expression levels of AMPK/mTOR pathway related genes and proteins. In summary, this study clarifies the mechanisms responsible for PFOA toxicity in the liver of exposed animals.

Exposure to PFOA and its novel analogs disrupts lipid metabolism in zebrafish.

Perfluorooctanoic acid (PFOA), a typical perfluoroalkyl group compound, has received worldwide attention due to its significant environmental toxicity. Following regulatory bans on the production and emission of PFOA, concerns have been raised about the potential health risks and the safety of novel perfluoroalkyl analogues. HFPO-DA (trade name Gen-X) and HFPO-TA are two perfluoroalkyl analogues known to be bioaccumulative, whose level of toxicity and whether they are safe alternatives to PFOA remain unclear. In the following study, the physiological and metabolic effects of exposure to PFOA and its novel analogues were explored in zebrafish using 1/3 LC50 (PFOA 100 µM, Gen-X 200 µM, HFPO-TA 30 µM). At the same LC50 toxicological effect, exposure to PFOA and HFPO-TA resulted in abnormal phenotypes such as spinal curvature, pericardial edema and aberrant body length, while Gen-X was little changed. Metabolically, PFOA, HFPO-TA and Gen-X all significantly increased total cholesterol in exposed zebrafish with PFOA and HFPO-TA also increasing total triglyceride levels. Transcriptome analysis showed that the number of differentially expressed genes in PFOA, Gen-X, and HFPO-TA treated conditions compared to control groups were 527, 572, and 3, 933, respectively. KEGG and GO analysis of differentially expressed genes revealed pathways and functions related to lipid metabolism as well as significant activation of the peroxisome proliferators-activated receptor (PPARs) pathway. Furthermore, RT-qPCR analysis identified significant dysregulation in the downstream target genes of PPARα, which is responsible for lipid oxidative catabolism, and the SREBP pathway, which is responsible for lipid synthesis. In conclusion, both perfluoroalkyl analogues HFPO-TA and Gen-X exhibit significant physiological and metabolic toxicity to aquatic organisms and their environmental accumulation should be closely regulated.

Rutin ameliorates perfluorooctanoic acid-induced testicular injury in mice by reducing oxidative stress and improving lipid metabolism.

This study investigated the protective effect of rutin on reproductive and blood-testis barrier (BTB) damage induced by perfluorooctanoic acid (PFOA) exposure. In this study, male ICR mice were randomly divided into three groups, Ctrl group (ddH2O, 5 mL/kg), PFOA group (PFOA, 20 mg/kg/d, 5 mL/kg), PFOA + rutin group (PFOA, 20 mg/kg/d, 5 mL/kg; rutin, 20 mg/kg/d, 5 mL/kg). Mice were exposed to PFOA for 28 days by gavage once daily in the presence or absence of rutin. Histopathological observations demonstrated that rutin treatment during PFOA exposure can reduce structural damage to testis and epididymis such as atrophy of spermatogenic epithelium and stenosis of epididymal lumen, while increase in the number and layers of spermatogenic cells. Biochemical detection demonstrated that rutin can reduce 8-hydroxy-2'-desoxyguanosine (8-OHdG) concentration in the serum and testis tissues. Rutin can also ameliorate glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) content, and reduce malondialdehyde (MDA) and total cholesterol (TC) content in testis tissues. Biotin tracking immunofluorescence and transmission electron microscopy demonstrated that rutin can ameliorate BTB structural damage during PFOA exposure. Rutin ameliorated the stress expression of tight junction proteins occludin and claudin-11. In conclusion, our findings suggested that rutin has a degree of protection in reproductive and BTB damage, which could put forward a new perspective on the application of rutin to prevent reproductive damage.

Neuronal-epithelial cross-talk drives acinar specification via NRG1-ERBB3-mTORC2 signaling.

Acinar cells are the principal secretory units of multiple exocrine organs. A single-cell, layered, lumenized acinus forms from a large cohort of epithelial progenitors that must initiate and coordinate three cellular programs of acinar specification, namely, lineage progression, secretion, and polarization. Despite this well-known outcome, the mechanism(s) that regulate these complex programs are unknown. Here, we demonstrate that neuronal-epithelial cross-talk drives acinar specification through neuregulin (NRG1)-ERBB3-mTORC2 signaling. Using single-cell and global RNA sequencing of developing murine salivary glands, we identified NRG1-ERBB3 to precisely overlap with acinar specification during gland development. Genetic deletion of Erbb3 prevented cell lineage progression and the establishment of lumenized, secretory acini. Conversely, NRG1 treatment of isolated epithelia was sufficient to recapitulate the development of secretory acini. Mechanistically, we found that NRG1-ERBB3 regulates each developmental program through an mTORC2 signaling pathway. Thus, we reveal that a neuronal-epithelial (NRG1/ERBB3/mTORC2) mechanism orchestrates the creation of functional acini.

iTRAQ-based quantitative proteomics analysis of Sprague-Dawley rats liver reveals perfluorooctanoic acid-induced lipid metabolism and urea cycle dysfunction.

Perfluorooctanoic acid (PFOA) is a typical C8 representative compound of perfluoroalkyl and polyfluoroalkyl substances (PFAS) widely used in industrial and domestic products. It is a persistent organic pollutant found in the environment as well as in the tissues of humans and wildlife. Despite emerging scientific and public interest, the precise mechanisms of PFOA toxicity remain unclear. In this study, male rats were exposed to 1.25, 5, and 20 mg PFOA/kg body weight/day for 14 days by gavage; food intake and bodyweight changes were recorded every day. After 14 days, blood was collected for sera biochemistry, livers were quickly stripped and weighed after execution. Part of the liver tissue was frozen by liquid nitrogen for iTRAQ-Based Quantitative Proteomics Analysis; and some was fixed in 4% paraformaldehyde (PFA) for histological section and hematoxylin-eosin (HE) staining. Urine samples were also collected and monitored by raising rats in metabolic cages. Real-time quantitative PCR and western blot was used to validate the proteomics assay after bioinformatics analysis. The results demonstrate that 20 mg/kg/d PFOA exposure cause body weight loss and significant liver swelling and reduced urea metabolism. The sera biochemistry assay shows that ALT, GGT, BILD and UREA levels have significant changes compared with normal control group and reference range of rat sera. The subsequent iTRAQ-based quantitative proteomics analysis of rat livers identified 3,327 non-redundant proteins of which 112 proteins were significantly upregulated and 80 proteins were downregulated. Gene ontology analysis revealed proteins are primarily involved in cellular, metabolic and single-organism processes. Among them, eight proteins (ACOX1, ACOX2, ACOX3, ACSL1, EHHADH, GOT2, MTOR and ACAA1) were related to oxidation of fatty acids and two proteins (ASS1 and CPS1) were found to be associated with urea cycle disorder. The downregulation of urea synthesis proteins ASS1 and CPS1 after exposure to PFOA was then confirmed through qPCR and western blot analysis. Together, these data demonstrate that PFOA exposure directly influences urea metabolism and provides insight into specific mechanisms of hepatotoxicity as a result of PFOA exposure.

Interactions of perfluorooctanoic acid with acyl-CoA thioesterase 1 (Acot1).

Perfluorooctanoic acid (PFOA), a typical representative of per- and polyfluoroalkyl substances (PFASs), is a widely utilized persistent organic pollutant (POP) known to induce liver toxicity in laboratory animals and wildlife. Evidence suggests that PFOA interacts with Acyl-CoA thioesterase 1 (Acot1) to modulate levels of ß-oxidation. Specifically, PFOA accelerates ß-oxidation, while Acot1 is inhibitory. Few studies have investigated the specific relationship between PFOA and Acot1 and the mechanism of their interaction remains unclear. In the following study, purified rat Acot1 protein was synthesized via bacterial recombination and the structural features that facilitate its binding to PFOA were assessed via molecular docking technology. Additionally, through use of circular dichroism spectroscopy (CD) and isothermal titration calorimetry (ITC) we demonstrate that PFOA binds to WT-Acot1 through electrostatic attraction and low strength non-covalent hydrogen bonding at a molar ratio of 1:1. Furthermore, we identify N326 and H373 amino acid residues as key regulators of the binding process. Together, these findings clarify the interaction pattern of PFOA and Acot1 proteins and provide insight into the specific molecular mechanisms that induce PFOA toxicity in humans and animals.

Tumor innervation and clinical outcome in pancreatic cancer.

Pancreatic cancer is a highly aggressive malignancy characterized by poor survival, recurrence after surgery and resistance to therapy. Nerves infiltrate the microenvironment of pancreatic cancers and contribute to tumor progression, however the clinicopathological significance of tumor innervation is unclear. In this study, the presence of nerves and their cross-sectional size were quantified by immunohistochemistry for the neuronal markers S-100, PGP9.5 and GAP-43 in a series of 99 pancreatic cancer cases versus 71 normal adjacent pancreatic tissues. A trend was observed between the presence of nerves in the tumor microenvironment of pancreatic cancer and worse overall patient survival (HR = 1.8, 95% CI 0.77-4.28, p = 0.08). The size of nerves, as measured by cross-sectional area, were significantly higher in pancreatic cancer than in the normal adjacent tissue (p = 0.002) and larger nerves were directly associated with worse patient survival (HR = 0.41, 95% CI 0.19-0.87, p = 0.04). In conclusion, this study suggests that the presence and size of nerves within the pancreatic cancer microenvironment are associated with tumor aggressiveness.

The neurotrophic tyrosine kinase receptor 1 (TrkA) is overexpressed in oesophageal squamous cell carcinoma.

Nerve growth factor (NGF) and its receptors, the neurotrophic receptor tyrosine kinase 1 (NTRK1/TrkA) and the common neurotrophin receptor (NGFR/p75NTR), are increasingly implicated in cancer progression, but their clinicopathological significance in oesophageal cancer is unclear. In this study, the expression of NGF, NTRK1 and NGFR were analysed by immunohistochemistry in a cohort of 303 oesophageal cancers versus 137 normal adjacent oesophageal tissues. Immunostaining was digitally quantified and compared to clinicopathological parameters. NGF and NGFR staining were found in epithelial cells and at similar levels between oesophageal cancers and normal oesophageal tissue. NGFR staining was slightly increased with grade (p=0.0389). Interestingly, NTRK1 staining was markedly higher in oesophageal squamous cell carcinoma (OR 2.31, 95%CI 1.13-4.38, p<0.0001) and significantly lower in adenocarcinoma (OR 0.50, 95%CI 0.44-0.63, p<0.0001) compared to normal oesophageal tissue. In addition, NTRK1 staining was decreased in grade 2 and grade 3 (OR 0.51, 95%CI 0.21-1.40, p<0.0001) compared to grade 1, suggesting a preferential involvement of this receptor in the more differentiated forms of oesophageal carcinomas. Together, these data point to NTRK1 as a biomarker and a candidate therapeutic target in oesophageal squamous cell carcinoma.

The Receptor Tyrosine Kinase TrkA Is Increased and Targetable in HER2-Positive Breast Cancer.

The tyrosine kinase receptor A (NTRK1/TrkA) is increasingly regarded as a therapeutic target in oncology. In breast cancer, TrkA contributes to metastasis but the clinicopathological significance remains unclear. In this study, TrkA expression was assessed via immunohistochemistry of 158 invasive ductal carcinomas (IDC), 158 invasive lobular carcinomas (ILC) and 50 ductal carcinomas in situ (DCIS). TrkA was expressed in cancer epithelial and myoepithelial cells, with higher levels of TrkA positively associated with IDC (39% of cases) (p < 0.0001). Interestingly, TrkA was significantly increased in tumours expressing the human epidermal growth factor receptor-2 (HER2), with expression in 49% of HER2-positive compared to 25% of HER2-negative tumours (p = 0.0027). A panel of breast cancer cells were used to confirm TrkA protein expression, demonstrating higher levels of TrkA (total and phosphorylated) in HER2-positive cell lines. Functional investigations using four different HER2-positive breast cancer cell lines indicated that the Trk tyrosine kinase inhibitor GNF-5837 reduced cell viability, through decreased phospho-TrkA (Tyr490) and downstream AKT (Ser473) activation, but did not display synergy with Herceptin. Overall, these data highlight a relationship between the tyrosine kinase receptors TrkA and HER2 and suggest the potential of TrkA as a novel or adjunct target for HER2-positive breast tumours.

Schwann Cell Stimulation of Pancreatic Cancer Cells: A Proteomic Analysis.

Schwann cells (SCs), the glial component of peripheral nerves, have been identified as promoters of pancreatic cancer (PC) progression, but the molecular mechanisms are unclear. In the present study, we aimed to identify proteins released by SCs that could stimulate PC growth and invasion. Proteomic analysis of human primary SC secretome was performed using liquid chromatography-tandem mass spectrometry, and a total of 13,796 unique peptides corresponding to 1,470 individual proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment were conducted using the Database for Annotation, Visualization, and Integrated Discovery. Metabolic and cell-cell adhesion pathways showed the highest levels of enrichment, a finding in line with the supportive role of SCs in peripheral nerves. We identified seven SC-secreted proteins that were validated by western blot. The involvement of these SC-secreted proteins was further demonstrated by using blocking antibodies. PC cell proliferation and invasion induced by SC-conditioned media were decreased using blocking antibodies against the matrix metalloproteinase-2, cathepsin D, plasminogen activator inhibitor-1, and galectin-1. Blocking antibodies against the proteoglycan biglycan, galectin-3 binding protein, and tissue inhibitor of metalloproteinases-2 decreased only the proliferation but not the invasion of PC cells. Together, this study delineates the secretome of human SCs and identifies proteins that can stimulate PC cell growth and invasion and therefore constitute potential therapeutic targets.

Nerve growth factor and its receptor tyrosine kinase TrkA are overexpressed in cervical squamous cell carcinoma.

Nerve growth factor (NGF) and its receptors are increasingly implicated in cancer progression, but their expression in cervical cancer is unclear. The objective of this study was to define the protein expression of NGF, its precursor (proNGF), as well as their receptors, the tyrosine kinase receptor TrkA, the common neurotrophin receptor p75NTR and the pro-neurotrophin receptor sortilin in cervical cancer. Immunohistochemistry was performed in a cohort of cervical cancers (n = 287), including the two major subtypes of the disease: squamous cell carcinomas (SCC) and adenocarcinomas (AC). Normal cervical tissues (n = 28) were also analyzed. Protein expression was determined by computer-based digital quantification of staining intensity and comparative statistical analyses were made with clinicopathological parameters including histological subtype, age, grade, tumor size, lymph node invasion, and stage. The expression of NGF, proNGF, TrkA, p75NTR, and sortilin was higher in cervical cancer compared to normal cervical tissues. NGF and TrkA were found overexpressed in SCC compared to AC (P = .0006 and P < .0001, respectively). The expression of NGF (P = .0053), proNGF (P = .0022), and p75NTR (P = .0002), but not that of TrkA or sortilin, was associated with increasing grade in SCC. In addition, nerve infiltration into the tumor microenvironment was assessed using the pan-neuronal marker PGP9.5. Infiltrating nerves were detected in 27% of cervical tumors and expressed TrkA. Functional investigations using the HELA cervical cancer cell line indicated that the Trk tyrosine kinase inhibitor GNF-5837 reduced cell viability through decreased ERK1/2 activation. Together, these data reveal the overexpression of NGF and TrkA in cervical SCC, suggesting a potential therapeutic value of targeting the NGF-TrkA signaling pathway in this subtype of cervical cancer.

The Membrane Protein Sortilin Can Be Targeted to Inhibit Pancreatic Cancer Cell Invasion.

Pancreatic cancer has a dismal prognosis, and there is no targeted therapy against this malignancy. The neuronal membrane protein sortilin is emerging as a regulator of cancer cell development, but its expression and impact in pancreatic cancer are unknown. This study found that sortilin expression was higher in pancreatic cell lines versus normal pancreatic ductal epithelial cells, as shown by Western blot analysis and mass spectrometry. The increased sortilin level in pancreatic cancer cells was confirmed by immunohistochemistry in a series of 99 human pancreatic adenocarcinomas versus 48 normal pancreatic tissues (P = 0.0014). Sortilin inhibition by siRNA and the pharmacologic inhibitor AF38469 strongly reduced the adhesion and invasion of pancreatic cancer cells without affecting cell survival and viability. Sortilin inhibition also decreased the phosphorylation of the focal adhesion kinase in Tyr925. Together, these data show that sortilin contributes to pancreatic cancer invasion and could eventually be targeted in therapy.

Clinicopathological Significance of Nerves in Esophageal Cancer.

Nerves are emerging promoters of cancer progression, but the innervation of esophageal cancer and its clinicopathologic significance remain unclear. In this study, nerves were analyzed by immunohistochemistry in a cohort of 260 esophageal cancers, including 40 matched lymph node metastases and 137 normal adjacent esophageal tissues. Nerves were detected in 38% of esophageal cancers and were more associated with squamous cell carcinomas (P = 0.04). The surrounding or invasion of nerves by cancer cells (perineural invasion) was detected in 12% of esophageal cancers and was associated with reduced survival (P = 0.04). Nerves were found to express the following receptors for nerve growth factor (NGF): neurotrophic receptor tyrosine kinase 1 and nerve growth factor receptor. An association was suggested between high production of NGF by cancer cells and the presence of nerves (P = 0.02). In vitro, NGF production in esophageal cancer cells was shown by Western blot, and esophageal cancer cells were able to induce neurite outgrowth in the PC12 neuronal cells. The neurotrophic activity of esophageal cancer cells was inhibited by anti-NGF blocking antibodies. Together, these data suggest that innervation is a feature in esophageal cancers that may be driven by cancer cell-released NGF.

Innervation of papillary thyroid cancer and its association with extra-thyroidal invasion.

Nerves are emerging regulators of cancer progression and in several malignancies innervation of the tumour microenvironment is associated with tumour aggressiveness. However, the innervation of thyroid cancer is unclear. Here, we investigated the presence of nerves in thyroid cancers and the potential associations with clinicopathological parameters. Nerves were detected by immunohistochemistry using the pan-neuronal marker PGP9.5 in whole-slide sections of papillary thyroid cancer (PTC) (n = 75), compared to follicular thyroid cancer (FTC) (n = 13), and benign thyroid tissues (n = 26). Nerves were detected in most normal thyroid tissues and thyroid cancers, but nerve density was increased in PTC (12 nerves/cm2 [IQR 7-21]) compared to benign thyroid (6 nerves/cm2 [IQR: 3-10]) (p = 0.001). In contrast, no increase in nerve density was observed in FTC. In multivariate analysis, nerve density correlated positively with extrathyroidal invasion (p < 0.001), and inversely with tumour size (p < 0.001). The majority of nerves were adrenergic, although cholinergic and peptidergic innervation was detected. Perineural invasion was present in 35% of PTC, and was independently associated with extrathyroidal invasion (p = 0.008). This is the first report of infiltration of nerves into the tumour microenvironment of thyroid cancer and its association with tumour aggressiveness. The role of nerves in thyroid cancer pathogenesis should be further investigated.

Targeting neurotrophin signaling in cancer: The renaissance.

Nerve outgrowth in the tumor microenvironment (tumor neurogenesis) has recently been shown to be essential for cancer progression and the concept of nerve dependence is emerging in oncology. Neurotrophins such as nerve growth factor (NGF) have long been identified as drivers of neurogenesis during development and regeneration, but intriguingly they were also known to be expressed in human tumors where they can stimulate cancer cell growth. Recent findings have unraveled that NGF released by cancer cells is also a driver of tumor neurogenesis, via the stimulation of NGF receptors on nerve endings. In return, nerves infiltrated in the tumor microenvironment secrete neurotransmitters, which can stimulate both the growth of tumor cells and angiogenesis. This neurotrophic role of NGF in cancer is likely to be relevant to a large variety of human malignancies, as well as other neurotrophins, and may have ramifications in cancer pain. Therefore, pharmacological interventions against neurotrophin signaling have the potential not only to target cancer cells directly, but also to inhibit neurogenesis and its stimulatory impact on cancer progression and pain.

The neurotrophic tyrosine kinase receptor TrkA and its ligand NGF are increased in squamous cell carcinomas of the lung.

The neurotrophic tyrosine kinase receptor TrkA (NTRK1) and its ligand nerve growth factor (NGF) are emerging promoters of tumor progression. In lung cancer, drugs targeting TrkA are in clinical trials, but the clinicopathological significance of TrkA and NGF, as well as that of the precursor proNGF, the neurotrophin co-receptor p75NTR and the proneurotrophin co-receptor sortilin, remains unclear. In the present study, analysis of these proteins was conducted by immunohistochemistry and digital quantification in a series of 204 lung cancers of different histological subtypes versus 121 normal lung tissues. TrkA immunoreactivity was increased in squamous cell carcinoma compared with benign and other malignant lung cancer histological subtypes (p < 0.0001). NGF and proNGF were also increased in squamous cell carcinoma, as well as in adenocarcinoma (p < 0.0001). In contrast, p75NTR was increased across all lung cancer histological subtypes compared to normal lung (p < 0.0001). Sortilin was higher in adenocarcinoma and small cell carcinoma (p < 0.0001). Nerves in the tumor microenvironment were negative for TrkA, NGF, proNGF, p75NTR and sortilin. In conclusion, these data suggest a preferential therapeutic value of targeting the NGF-TrkA axis in squamous cell carcinomas of the lung.

Next-generation sequencing reveals broad down-regulation of microRNAs in secondary progressive multiple sclerosis CD4+ T cells.

BACKGROUND: Immunoactivation is less evident in secondary progressive MS (SPMS) compared to relapsing-remitting disease. MicroRNA (miRNA) expression is integral to the regulation of gene expression; determining their impact on immune-related cell functions, especially CD4+ T cells, during disease progression will advance our understanding of MS pathophysiology. This study aimed to compare miRNA profiles of CD4+ T cells from SPMS patients to healthy controls (HC) using whole miRNA transcriptome next-generation sequencing (NGS). Total RNA was extracted from CD4+ T cells and miRNA expression patterns analyzed using Illumina-based small-RNA NGS in 12 SPMS and 12 HC samples. Results were validated in a further cohort of 12 SPMS and 10 HC by reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: The ten most dysregulated miRNAs identified by NGS were selected for qPCR confirmation; five (miR-21-5p, miR-26b-5p, miR-29b-3p, miR-142-3p, and miR-155-5p) were confirmed to be down-regulated in SPMS (p < 0.05). SOCS6 is targeted by eight of these ten miRNAs. Consistent with this, SOCS6 expression is up-regulated in SPMS CD4+ T cells (p < 0.05). This is of particular interest as SOCS6 has previously been shown to act as a negative regulator of T cell activation. CONCLUSIONS: Ninety-seven percent of miRNA candidates identified by NGS were down-regulated in SPMS. The down-regulation of miRNAs and increased expression of SOCS6 in SPMS CD4+ T cells may contribute to reduced immune system activity in progressive MS.