Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite.

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.

Chloroquine corrects enlarged lysosomes in FIG4 null cells and reduces neurodegeneration in Fig4 null mice.

Loss-of-function mutations of FIG4 impair the biosynthesis of PI(3,5)P2 and are responsible for rare genetic disorders including Yunis-Varón Syndrome and Charcot-Marie-Tooth Disease Type 4 J. Cultured cells deficient in FIG4 accumulate enlarged lysosomes with hyperacidic pH, due in part to impaired regulation of lysosomal ion channels and elevated intra-lysosomal osmotic pressure. We evaluated the effects of the FDA approved drug chloroquine, which is known to reduce lysosome acidity, on FIG4 deficient cell culture and on a mouse model. Chloroquine corrected the enlarged lysosomes in FIG4 null cells. In null mice, addition of chloroquine to the drinking water slowed progression of the disorder. Growth and mobility were dramatically improved during the first month of life, and spongiform degeneration of the nervous system was reduced. The median survival of Fig4 null mice was increased from 4 weeks for untreated mutants to 8 weeks with chloroquine treatment (p < 0.009). Chloroquine thus corrects the lysosomal swelling in cultured cells and ameliorates Fig4 deficiency in vivo. The improved phenotype of mice with complete loss of Fig4 suggests that chloroquine could be beneficial FIG2 in partial loss-of-function disorders such as Charcot-Marie-Tooth Type 4 J.

G1-Cyclin2 (Cln2) promotes chromosome hypercondensation in eco1/ctf7 rad61 null cells during hyperthermic stress in Saccharomyces cerevisiae.

Eco1/Ctf7 is a highly conserved acetyltransferase that activates cohesin complexes and is critical for sister chromatid cohesion, chromosome condensation, DNA damage repair, nucleolar integrity, and gene transcription. Mutations in the human homolog of ECO1 (ESCO2/EFO2), or in genes that encode cohesin subunits, result in severe developmental abnormalities and intellectual disabilities referred to as Roberts syndrome and Cornelia de Lange syndrome, respectively. In yeast, deletion of ECO1 results in cell inviability. Codeletion of RAD61 (WAPL in humans), however, produces viable yeast cells. These eco1 rad61 double mutants, however, exhibit a severe temperature-sensitive growth defect, suggesting that Eco1 or cohesins respond to hyperthermic stress through a mechanism that occurs independent of Rad61. Here, we report that deletion of the G1 cyclin CLN2 rescues the temperature-sensitive lethality otherwise exhibited by eco1 rad61 mutant cells, such that the triple mutant cells exhibit robust growth over a broad range of temperatures. While Cln1, Cln2, and Cln3 are functionally redundant G1 cyclins, neither CLN1 nor CLN3 deletions rescue the temperature-sensitive growth defects otherwise exhibited by eco1 rad61 double mutants. We further provide evidence that CLN2 deletion rescues hyperthermic growth defects independent of START and impacts the state of chromosome condensation. These findings reveal novel roles for Cln2 that are unique among the G1 cyclin family and appear critical for cohesin regulation during hyperthermic stress.

Increased migration and motility in XIAP-null cells mediated by the C-RAF protein kinase.

The product encoded by the X-linked inhibitor of apoptosis (XIAP) gene is a multi-functional protein which not only controls caspase-dependent cell death, but also participates in inflammatory signalling, copper homeostasis, response to hypoxia and control of cell migration. Deregulation of XIAP, either by elevated expression or inherited genetic deletion, is associated with several human disease states. Reconciling XIAP-dependent signalling pathways with its role in disease progression is essential to understand how XIAP promotes the progression of human pathologies. In this study we have created a panel of genetically modified XIAP-null cell lines using TALENs and CRISPR/Cas9 to investigate the functional outcome of XIAP deletion. Surprisingly, in our genetically modified cells XIAP deletion had no effect on programmed cell death, but instead the primary phenotype we observed was a profound increase in cell migration rates. Furthermore, we found that XIAP-dependent suppression of cell migration was dependent on XIAPdependent control of C-RAF levels, a protein kinase which controls cell signalling pathways that regulate the cytoskeleton. These results suggest that XIAP is not necessary for control of the apoptotic signalling cascade, however it does have a critical role in controlling cell migration and motility that cannot be compensated for in XIAP-knockout cells.

An enhanced triple fluorescence flow-cytometry-based assay shows differential activation of the Notch signaling pathway by human papillomavirus E6 proteins.

Human papillomaviruses are DNA tumor viruses. A persistent infection with high-risk HPV types is the necessary risk factor for the development of anogenital carcinoma. The E6 protein is a viral oncoprotein that directly interacts with different cellular regulatory proteins mainly affecting the cell cycle, cellular differentiation and polarization of epithelial cells. In dependency of the phylogenetic classification of HPV different interaction partners of E6 have been described. The Notch pathway seems to be one common target of HPV, which can be up or down regulated by different E6 proteins. Our novel triple fluorescence flow-cytometry-based assay allows a semi-quantitative comparison of the E6 proteins´ effect on the Notch pathway using a Notch-responsive reporter plasmid. As a result, all E6 proteins of beta-HPV repressed the Notch reporter expression, of which HPV38 E6 showed the greatest repression potential. In contrast, alpha-HPV E6 of HPV16, activates the reporter expression most significantly, whereas E6 of HPV31 and low-risk HPV6b showed significant activation only in a p53-null cell line. Interestingly, HPV18 E6, with the second highest carcinogenic risk, shows no effect. This high divergence within different genus of HPV is important for targeting the Notch pathway regarding a potential HPV therapy.

Loss of PRC2 subunits primes lineage choice during exit of pluripotency.

Polycomb Repressive Complex 2 (PRC2) is crucial for the coordinated expression of genes during early embryonic development, catalyzing histone H3 lysine 27 trimethylation. Two distinct PRC2 complexes, PRC2.1 and PRC2.2, contain respectively MTF2 and JARID2 in embryonic stem cells (ESCs). In this study, we explored their roles in lineage specification and commitment, using single-cell transcriptomics and mouse embryoid bodies derived from Mtf2 and Jarid2 null ESCs. We observe that the loss of Mtf2 results in enhanced and faster differentiation towards cell fates from all germ layers, while the Jarid2 null cells are predominantly directed towards early differentiating precursors, with reduced efficiency towards mesendodermal lineages. These effects are caused by derepression of developmental regulators that are poised for activation in pluripotent cells and gain H3K4me3 at their promoters in the absence of PRC2 repression. Upon lineage commitment, the differentiation trajectories are relatively similar to those of wild-type cells. Together, our results uncover a major role for MTF2-containing PRC2.1 in balancing poised lineage-specific gene activation, whereas the contribution of JARID2-containing PRC2 is more selective in nature compared to MTF2. These data explain how PRC2 imposes thresholds for lineage choice during the exit of pluripotency.

Upregulation of mNEIL3 in Ogg1-null cells is a potential backup mechanism for 8-oxoG repair.

Reactive oxygen species formation and resultant oxidative damage to DNA are ubiquitous events in cells, the homeostasis of which can be dysregulated in a range of pathological conditions. Base excision repair (BER) is the primary repair mechanism for oxidative genomic DNA damage. One prevalent oxidised base modification, 8-oxoguanine (8-oxoG), is recognised by 8-oxoguanine glycosylase-1 (OGG1) initiating removal and repair via BER. Surprisingly, Ogg1 null mouse embryonic fibroblasts (mOgg1-/- MEFs) do not accumulate 8-oxoG in the genome to the extent expected. This suggests that there are backup repair mechanisms capable of repairing 8-oxoG in the absence of OGG1. In the current study, we identified components of NER (Ercc1, Ercc4, Ercc5), BER (Lig1, Tdg, Nthl1, Mpg, Mgmt, NEIL3), MMR (Mlh1, Msh2, Msh6) and DSB (Brip1, Rad51d, Prkdc) pathways that are transcriptionally elevated in mOgg1-/- MEFs. Interestingly, all three nucleotide excision repair genes identified: Ercc1 (2.5 ± 0.2-fold), Ercc4 (1.5 ± 0.1-fold) and Ercc5 (1.7 ± 0.2-fold) have incision activity. There was also a significant functional increase in NER activity (42.0 ± 7.9%) compared to WT MEFs. We also observed upregulation of both Neil3 mRNA (37.9 ± 1.6-fold) and protein in mOgg1-/- MEFs. This was associated with a 3.4 ± 0.4-fold increase in NEIL3 substrate sites in genomic DNA of cells treated with BSO, consistent with the ability of NEIL3 to remove 8-oxoG oxidation products from genomic DNA. In conclusion, we suggest that in Ogg1-null cells, upregulation of multiple DNA repair proteins including incision components of the NER pathway and Neil3 are important compensatory responses to prevent the accumulation of genomic 8-oxoG.

METTL3-dependent m6A modification programs T follicular helper cell differentiation.

T follicular helper (TFH) cells are specialized effector CD4+ T cells critical to humoral immunity. Whether post-transcriptional regulation has a function in TFH cells is unknown. Here, we show conditional deletion of METTL3 (a methyltransferase catalyzing mRNA N6-methyladenosine (m6A) modification) in CD4+ T cells impairs TFH differentiation and germinal center responses in a cell-intrinsic manner in mice. METTL3 is necessary for expression of important TFH signature genes, including Tcf7, Bcl6, Icos and Cxcr5 and these effects depend on intact methyltransferase activity. m6A-miCLIP-seq shows the 3' UTR of Tcf7 mRNA is subjected to METTL3-dependent m6A modification. Loss of METTL3 or mutation of the Tcf7 3' UTR m6A site results in accelerated decay of Tcf7 transcripts. Importantly, ectopic expression of TCF-1 (encoded by Tcf7) rectifies TFH defects owing to METTL3 deficiency. Our findings indicate that METTL3 stabilizes Tcf7 transcripts via m6A modification to ensure activation of a TFH transcriptional program, indicating a pivotal function of post-transcriptional regulation in promoting TFH cell differentiation.

Clinical characteristics and outcomes of null-cell versus silent gonadotroph adenomas in a series of 1166 pituitary adenomas from a single institution.

OBJECTIVE: Nonfunctioning pituitary adenomas present without biochemical or clinical signs of hormone excess and are the second most common type of pituitary adenomas. The 2017 WHO classification scheme of pituitary adenomas differentiates null-cell adenomas (NCAs) and silent gonadotroph adenomas (SGAs). The present study sought to highlight the differences in patient characteristics and clinical outcomes between NCAs and SGAs. METHODS: The records of 1166 patients who underwent transsphenoidal surgery for pituitary adenoma between 2012 and 2019 at a single institution were retrospectively reviewed. Patient demographics and clinical outcomes were collected. RESULTS: Of the overall pituitary adenoma cohort, 12.8% (n = 149) were SGAs and 9.2% (n = 107) NCAs. NCAs were significantly more common in female patients than SGAs (61.7% vs 26.8%, p < 0.001). There were no differences in patient demographics, initial tumor size, or perioperative and short-term clinical outcomes. There was no significant difference in the amount of follow-up between patients with NCAs and those with SGAs (33.8 months vs 29.1 months, p = 0.237). Patients with NCAs had significantly higher recurrence (p = 0.021), adjuvant radiation therapy usage (p = 0.002), and postoperative diabetes insipidus (p = 0.028). NCA pathology was independently associated with tumor recurrence (HR 3.64, 95% CI 1.07-12.30; p = 0.038), as were cavernous sinus invasion (HR 3.97, 95% CI 1.04-15.14; p = 0.043) and anteroposterior dimension of the tumor (HR 2.23, 95% CI 1.09-4.59; p = 0.030). CONCLUSIONS: This study supports the definition of NCAs and SGAs as separate subgroups of nonfunctioning pituitary adenomas, and it highlights significant differences in long-term clinical outcomes, including tumor recurrence and the associated need for adjuvant radiation therapy, as well as postoperative diabetes insipidus. The authors also provide insight into independent risk factors for these outcomes in the adenoma population studied, providing clinicians with additional predictors of patient outcomes. Follow-up studies will hopefully uncover mechanisms of biological aggressiveness in NCAs and associated molecular targets.

USP22 positively modulates ERα action via its deubiquitinase activity in breast cancer.

Estrogen receptor α (ERα) is the crucial factor in ERα-positive breast cancer progression. Endocrine therapies targeting ERα signaling is one of the widely used therapeutic strategies for breast cancer. However, a large number of the patients become refractory to therapy. Abnormal expression of ERα co-regulator facilitates breast cancer development and tendency of endocrine resistance. Thus, it is necessary to discover the novel co-regulators modulating ERα action. Here, we demonstrate that histone deubiquitinase USP22 is highly expressed in breast cancer samples compared with that in the benign tissue, and high expression of USP22 was significantly associated with poorer overall survival in BCa samples. Moreover, USP22 associates with ERα to be involved in maintenance of ERα stability. USP22 enhances ERα-induced transactivation. We further provide the evidence that USP22 is recruited together with ERα to cis-regulatory elements of ERα target gene. USP22 promotes cell growth even under hypoxia condition and with the treatment of ERα antagonist in breast cancer cells. Importantly, the deubiquitination activity of USP22 is required for its functions on maintenance of ERα stability, thereby enhancing ERα action and conferring endocrine resistance in breast cancer.

Preserved specific anti-viral T-cell response but associated with decreased lupus activity in SLE patients with cytomegalovirus infection.

OBJECTIVES: SLE is an autoimmune disease characterized by aberrant autoantibody production and immune dysfunctions. Whether the anti-CMV immunity is impaired in SLE patients is poorly understood. We investigated the specific anti-viral T-cell response in SLE patients with CMV infection and its possible impacts on clinical manifestations in lupus. METHODS: CD28 null T-cell percentages were measured by flow cytometry in 89 SLE patients and 58 healthy controls. A specific anti-CMV CD8 T-cell response was assessed ex vivo by the production of intracellular cytokines in response to CMV phosphoprotein 65 (pp65) by flow cytometry. Clinical manifestations and immune parameters were analysed in SLE patients according to their CMV serostatus. RESULTS: CD28 null T cells were significantly expanded in SLE patients. When the anti-CMV pp65 CD8 polyfunctional T cell response was analysed, as defined by production of at least three of four functional cytokines or effectors (intracellular IFN-γ, IL-2, TNF-α and surface CD107a), the results demonstrated that it was not impaired in SLE patients. In contrast, when comparing clinical manifestations, there were lower anti-ds-DNA levels and decreased SLEDAI in SLE patients with CMV infection. Furthermore, the expansion of CD4+CD28 null T cells was negatively associated with anti-ds-DNA levels and SLEDAI in these lupus patients. CONCLUSION: In SLE patients with CMV infection, the specific anti-CMV CD8 T-cell response is preserved but is associated with decreased disease activity and lower anti-DNA levels among these patients, suggesting CMV infection may mitigate lupus activity.

Clinical, pathologic, and imaging characteristics of pituitary null cell adenomas as defined according to the 2017 World Health Organization criteria: a case series from two pituitary centers.

PURPOSE: The 2017 World Health Organization classification of pituitary tumors redefined pituitary null cell adenomas (NCAs) by restricting this diagnostic category to pituitary tumors that are negative for pituitary transcription factors and adenohypophyseal hormones. The clinical behavior of this redefined entity has not been widely studied, and this is a major shortcoming of the classification. This study evaluated the imaging and clinical features of NCAs from two pituitary centers and compared them with those of gonadotroph adenomas (GAs). METHODS: Imaging, pathologic, and clinical characteristics of NCAs and GAs were retrospectively reviewed. Tumor immunohistochemistry was performed to confirm absence of adenohypophyseal hormones and pituitary transcription factor expression. RESULTS: Thirty-one NCAs were compared with 38 GAs. NCAs were more likely to invade the cavernous sinus (15/31 [48%] vs. 5/38 [13%], P = .003) and had a higher proliferative index (i.e., MIB-1 > 3%, 11/31 [35%] vs. 5/38 [13%], P = .04). Gross total resection was less likely in the NCA group (19/31 [61%] vs. 33/38 [87], P = .02). Progression-free survival was worse in the NCA cohort (5-year progression-free survival, 0.70 vs. 1.00; P = .011, by log-rank test). CONCLUSIONS: Compared with GAs, NCAs are more invasive at the time of presentation and have a more aggressive clinical course. This study provides evidence that NCAs represent a distinct clinicopathologic entity with behavior that differs adversely from that of GAs. This may inform clinical decision-making, including frequency of postoperative tumor surveillance and timing of adjunctive treatments.

Endothelial cell-specific molecule-1 as an invasiveness marker for pituitary null cell adenoma.

BACKGROUND: Endothelial cell-specific molecule-1 (ESM-1) is a biomarker associated with tumor progression in pituitary adenoma. We specifically focused on one type of pituitary adenoma, namely null cell adenoma (NCA) and evaluated the relationship between invasion and ESM-1 expression in both vascular endothelial and adenoma tissues. METHODS: Tissue samples from 94 patients with pituitary NCA were obtained through microscopic transsphenoidal resection. Tumor size and invasion were determined through preoperative magnetic resonance imaging. Immunohistochemical staining was performed to detect ESM-1 expression. ESM-1 index of ≥3 was defined as high expression. RESULTS: Signs of invasion were observed in 46 (47.9%) of the 94 patients. Significant differences were observed in the invasion state and maximum tumor diameter between high and low expression of ESM-1 in vascular endothelial tissues (both P < 0.05). Significant positive associations were noted between ESM-1 expression in vascular endothelial tissues and tumor invasion (P = 0.002) and tumor size (P = 0.020). However, only tumor size was associated with ESM-1 expression in adenoma tissues (P = 0.016). CONCLUSION: In NCA, a significant positive association between tumor invasion and ESM-1 expression was observed only in vascular endothelial tissues, suggesting that tumor progression occurs mainly through ESM-1-associated mechanism.

The E3 ligase Hrd1 stabilizes Tregs by antagonizing inflammatory cytokine-induced ER stress response.

Treg differentiation, maintenance, and function are controlled by the transcription factor FoxP3, which can be destabilized under inflammatory or other pathological conditions. Tregs can be destabilized under inflammatory or other pathological conditions, but the underlying mechanisms are not fully defined. Herein, we show that inflammatory cytokines induce ER stress response, which destabilizes Tregs by suppressing FoxP3 expression, suggesting a critical role of the ER stress response in maintaining Treg stability. Indeed, genetic deletion of Hrd1, an E3 ligase critical in suppressing the ER stress response, leads to elevated expression of ER stress-responsive genes in Treg and largely diminishes Treg suppressive functions under inflammatory condition. Mice with Treg-specific ablation of Hrd1 displayed massive multiorgan lymphocyte infiltration, body weight loss, and the development of severe small intestine inflammation with aging. At the molecular level, the deletion of Hrd1 led to the activation of both the ER stress sensor IRE1α and its downstream MAPK p38. Pharmacological suppression of IRE1α kinase, but not its endoribonuclease activity, diminished the elevated p38 activation and fully rescued the stability of Hrd1-null Tregs. Taken together, our studies reveal ER stress response as a previously unappreciated mechanism underlying Treg instability and that Hrd1 is crucial for maintaining Treg stability and functions through suppressing the IRE1α-mediated ER stress response.

Expanded peripheral CD4+CD28null T cells and its association with atherosclerotic changes in patients with end stage renal disease on hemodialysis.

End-stage renal disease (ESRD) patients, including those on hemodialysis, possess a high risk for cardiovascular diseases, as the first leading cause of death among them. Traditional risk factors do not utterly elucidate this. Throughout the last two decades, CD4+CD28null T cells; an unusual subset of T lymphocytes, was detected high with excess cardiovascular (CV) mortality. We aimed to investigate the circulating CD4+CD28null T cells frequency in ESRD patients on hemodialysis and to evaluate their relationship with atherosclerotic changes. High-resolution carotid ultrasonography was done to assess the common carotid artery intima media thickness in a number of ESRD patients, accordingly patients were selected and subdivided into two groups; 30 with atherosclerosis (mean [SD] age, 51.6 [6.3] years) and 30 without (mean [SD] age, 48.9 [5.5] years). Another 30 healthy individuals (mean [SD] age, 48.5 [6.8] years) were enrolled. Analysis of CD4+CD28null T-cells frequency by flow-cytometry was performed in all studied subjects. CD4+CD28null T cell percentage was significantly higher in ESRD patients, (mean [SD], 7.3 [2.7] %) compared to healthy individuals (mean [SD], 3.0 [0.8] %), (p < 0.001). Additionally, the expansion of these unusual T lymphocytes was significantly higher in ESRD patients with atherosclerotic changes (mean [SD], 9.47 [0.75] %) compared to those without atherosclerosis (mean [SD], 5.22 [2.14] %), (p < 0.001). In conclusion circulating CD4+CD28null T lymphocyte population showed expansion in ESRD patients, and of interest in correlation to preclinical atherosclerotic changes.

Clinically non-functioning pituitary adenomas: Pathogenic, diagnostic and therapeutic aspects / Adenomas hipofisarios clínicamente no funcionantes: aspectos patogénicos, diagnósticos y terapéuticos

Clinically non-functioning pituitary adenomas (NFPAs) are among the most common tumors in the sellar region. These lesions do not cause a hormonal hypersecretion syndrome, and are therefore found incidentally (particularly microadenomas) or diagnosed based on compressive symptoms such as headache and visual field defects, as well as clinical signs of pituitary hormone deficiencies. Immunohistochemically, more than 45% of these adenomas stain for gonadotropins or their subunits and are therefore called gonadotropinomas, while 30% of them show no immunostaining for any hormone and are known as null cell adenomas. The diagnostic approach to NFPAs should include visual field examination, an assessment of the integrity of all anterior pituitary hormone systems, and magnetic resonance imaging of the sellar region to define tumor size and extension. The treatment of choice is transsphenoidal resection of the adenoma, which in many instances cannot be completely accomplished. The recurrence rate after surgery may be up to 30%. Persistent or recurrent adenomas are usually treated with radiation therapy. In a small proportion of these cases, drug treatment with dopamine agonists and, to a lesser extent, somatostatin analogs may achieve reduction or at least stabilization of the tumor (AU)

Los adenomas hipofisarios clínicamente no funcionantes son los tumores más frecuentes de la región selar. Dado que estas lesiones no resultan en un síndrome de hipersecreción hormonal, se manifiestan por síntomas compresivos como cefalea y alteraciones campimétricas, así como por manifestaciones clínicas de hipopituitarismo, o bien son descubiertos de forma incidental (en particular los microadenomas). Inmunohistoquímicamente, más del 45% de estos adenomas inmunotiñen para gonadotropinas o sus subunidades, por lo que se los conoce como gonadotropinomas; mientras que el 30% de los casos no inmunotiñe para ninguna hormona y se los denomina adenomas de células nulas. El abordaje diagnóstico de los adenomas hipofisarios clínicamente no funcionantes debe incluir la evaluación de los campos visuales y la medición de las hormonas de la hipófisis anterior, así como una resonancia magnética nuclear para establecer el tamaño y la extensión del tumor. El tratamiento de elección es la resección transesfenoidal del adenoma, que en ocasiones no se logra completamente. La tasa de recurrencia después de la cirugía puede ser de hasta el 30%. Los adenomas persistentes o recurrentes suelen ser tratados con radioterapia. Una proproción pequeña de estos pacientes puede responder de forma favorable a agonistas dopaminérgicos y, en menor medida, a análogos de la somatostatina (AU)

Long non-coding RNA C5orf66-AS1 is downregulated in pituitary null cell adenomas and is associated with their invasiveness.

Pituitary null cell adenoma is a challenging clinical condition, and its pathogenesis remains to be elucidated. We performed this study to determine the roles of C5orf66-AS1, NORAD, and TINCR in the pathogenesis and invasion of pituitary null cell adenomas. Expression of the three long non-coding RNAs in pituitary null cell adenoma tissues of 11 patients and normal pituitary tissues from four donors was examined by performing quantitative reverse transcription-polymerase chain reaction. We found that C5orf66-AS1 expression was lower in pituitary null cell adenoma tissues than in normal pituitary tissues. Moreover, C5orf66-AS1 expression level was significantly lower in invasive pituitary null cell adenomas than in non-invasive ones. After transfection of C5orf66-AS1 into pituitary adenoma cells, assessment of cell viability and invasion suggested that overexpressed C5orf66-AS1 inhibited cell viability and cell invasion. In silico algorithms predicted several cis- and trans-acting target genes of C5orf66-AS1, including PITX1 and SCGB3A1. In addition, expression of some of the predicted target genes was determined using microarray data of another cohort with pituitary null cell adenomas. It showed that some of these target genes were differentially expressed between pituitary null cell adenoma tissues and normal pituitary tissues as well as between invasive and non-invasive tumors. Co-expression analysis in RNA sequencing data showed that PAQR7 was the most correlated gene of C5orf66-AS1 and that several predicted trans-acting target genes, including SCGB3A1, were highly correlated with C5orf66-AS1. NORAD and TINCR expression was not statistically significant in the complete cohort; however, a negative correlation was observed between NORAD expression and maximum tumor diameter in some subgroups. These results indicate that C5orf66-AS1 suppresses the development and invasion of pituitary null cell adenomas. However, our results do not provide enough statistical evidence to support the roles of NORAD and TINCR in the development and invasion of pituitary null cell adenomas.