Carbonic Anhydrase IV Research Articles

Tiliroside as a CAXII inhibitor suppresses liver cancer development and modulates E2Fs/Caspase-3 axis

Liver cancer is the fatal cause of cancer deaths worldwide due to its aggressiveness and lack of effective therapies. Tiliroside (C30H26O13) is an active compound extracted from herb plant Tribulus terrestris L., which has been used as alternative therapy in clinic practice. However, its therapeutic use against liver cancer has not been previously reported. Here, we showed that Tiliroside exerted significantly higher anti-proliferation effect on liver cancer cell lines Hep3B and SNU-449 than on liver normal cell THLE-3 cells or NC group, respectively, by using MTS assay. Results from colony formation, immigration and invasion assays support the anticancer efficacy of Tiliroside and its low-toxic property while treating liver normal cell THLE-3. 3D spheroid formation and CD133 expression level also displays its anti-stemness effect. It has been showed that Tiliroside may function as Carbonic anhydrases XII (CAXII) inhibitor and affects apoptotic E2F1/E2F3/Caspase-3 axis by using CAXII esterase activity assay, Human carbonic anhydrase 12 (CA-12) ELISA Kit, quantitative reverse transcription PCR (RT-qPCR) as well as CaspACE Assay System, respectively. In summary, we demonstrate for the first time that Tiliroside suppresses liver cancer development possibly by acting as a novel CAXII inhibitor, which warrant further investigation on its therapeutic implications.

Large pH oscillations promote host defense against human airways infection.

The airway mucosal microenvironment is crucial for host defense against inhaled pathogens but remains poorly understood. We report here that the airway surface normally undergoes surprisingly large excursions in pH during breathing that can reach pH 9.0 during inhalation, making it the most alkaline fluid in the body. Transient alkalinization requires luminal bicarbonate and membrane-bound carbonic anhydrase 12 (CA12) and is antimicrobial. Luminal bicarbonate concentration and CA12 expression are both reduced in cystic fibrosis (CF), and mucus accumulation both buffers the pH and obstructs airflow, further suppressing the oscillations and bacterial-killing efficacy. Defective pH oscillations may compromise airway host defense in other respiratory diseases and explain CF-like airway infections in people with CA12 mutations.

Overexpressed CA12 has prognostic value in pancreatic cancer and promotes tumor cell apoptosis via NF-κB signaling.

Pancreatic adenocarcinoma (PAAD) is among the deadliest forms of cancer globally. Carbonic anhydrase 12 (CA12) is known to play central roles in regulating many cancers, but its function in the context of PAAD is rarely discussed. This study was, therefore, designed to assess the expression of CA12 in PAAD and to explore its underlying mechanistic role in this cancer type. Immunohistochemical staining was used to measure CA12 expression in PAAD samples. The functionality of pancreatic cancer cells expressing varying levels of CA12 was assessed through wound healing, Transwell, and CCK-8 assays. In addition, flow cytometry was used to measure apoptosis and cell cycle progression in these same cells, while Western blotting was used to analyze the expression of proteins associated with the NF-κB signaling pathway. PAAD tissue samples exhibited significant CA12 downregulation (P < 0.001), and lower CA12 expression was, in turn, associated with poorer overall survival (P < 0.001). CA12 overexpression significantly impaired the proliferation of PAAD cell lines, instead inducing their apoptotic death and G0/G1 phase cell cycle arrest (P < 0.05). We additionally found that CA12 may exert its tumor suppressive roles via modulating the NF-κB signaling pathway. These results indicate that CA12 functions as a tumor suppressor in PAAD and may thus be a novel therapeutic target that can be used to guide PAAD patient treatment.

Increased expression of fibroblast growth factor 23 is the signature of a deteriorated Ca/P balance in ageing laying hens

The present study concerned the effect of ageing in laying hens, from 23 to 90 weeks of age, on the regulation of Ca metabolism related to the requirement for eggshell mineralization. Samples were collected from parathyroid gland (PG), liver, jejunum, medullary bone (MB) and kidney for a quantitative study of candidate gene expression. Although parathyroid hormone (PTH) gene expression in the PG did not vary with age, a stronger challenge to Ca homeostasis was suggested in aged hens. Indeed gene expression of Ca transporters , Vitamin D Receptor (VDR) in the jejunum, and that of transient receptor potential channel subfamily V member 5 (TRPV5) in the kidney decreased. This could exacerbate bone resorption and impair bone accretion, as attested by a higher expression of the Carbonic Anhydrase 2 (CA2) gene and a lower expression of collagen type I alpha 1 chain (COL1A1) in the MB. The increased expression of Fibroblast Growth Factor 23 (FGF23) in the MB likely contributed to the decreased plasma levels of 1.25(OH)2D3 and the altered expression of target genes under its regulation. Our data highlights the molecular mechanisms underlying the osteoporotic syndrome previously documented in aged laying hens, thus providing new perspectives for future interventions.

Carbonic Anhydrase 12 Protects Endplate Cartilage From Degeneration Regulated by IGF-1/PI3K/CREB Signaling Pathway.

Lumbar intervertebral disc degeneration (IVDD) is the most common cause of low back pain (LBP). Among all the factors leading to IVDD, lumbar cartilage endplate (LCE) degeneration is considered a key factor. In the present study, we investigate the effect and regulation of carbonic anhydrase 12 (CA12) in LCE, which catalyzes hydration of CO2 and participates in a variety of biological processes, including acid–base balance and calcification. Our results show that CA12, downregulated in degenerated LCE, could maintain anabolism and prevent calcification in the endplate. Furthermore, CA12 is regulated by the IGF-1/IGF-1R/PI3K/CREB signaling pathway. When we overexpressed CA12 in LCE, the decreased anabolism induced by inflammatory cytokine could be rescued. In contrast, reducing CA12 expression, either with siRNA, PI3Kinhibitor, or CREB inhibitor, could downregulate anabolism and cause apoptosis and then calcification in LCE. The protective effects of IGF-1 are even diminished with low-expressed CA12. Similar results are also obtained in an ex vivo model. Consequently, our results reveal a novel pathway, IGF-1/IGF-1R/PI3K/CREB/CA12, that takes a protective role in LCE degeneration by maintaining anabolism and preventing calcification and apoptosis. This study proposes a novel molecular target, CA12, to delay LCE degeneration.

Class switching of carbonic anhydrase isoforms mediates remyelination in CA3 hippocampal neurons during chronic hypoxia

Chronic exposure to hypoxia results in cerebral white matter hyperintensities, increased P300 latency, delayed response and impairment in working memory. Despite burgeoning evidence on role of myelination in nerve conduction, the effect of chronic hypoxia on myelination of hippocampal neurons has been less studied. The present study provides novel evidence on alterations in myelination of hippocampal CA3 neurons following chronic hypoxic exposure. Sprague Dawley rats exposed to global hypobaric hypoxia simulating altitude of 25,000 ft showed progressive demyelination in CA3 hippocampal neurons on 14 days followed by remyelination on 21 and 28 days. The demyelination of CA3 neurons was associated with increased apoptosis of both oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (OLs), peroxidation of myelin lipids, and nitration induced reduced expression of Carbonic Anhydrase II (CAII). Prolonged hypoxic exposure of 21 and 28 days on the other hand resulted in peroxisome proliferator-activated receptor alpha (PPARα) induced upregulation of Carbonic Anhydrase IV (CAIV) expression in mature oligodendrocytes through iNOS mediated mechanisms along with reduction in lipid peroxidation and remyelination. Inhibition of carbonic anhydrase activity on the other hand prevented remyelination of CA3 neurons. Based on these findings we propose a novel iNOS mediated mechanism for regulation of myelination in hypoxic hippocampal neurons through class switching of carbonic anhydrases.

Abstract 6604: Drug responsive domain regulation of IL15-engineered T cells provides pharmacological control over antigen-independent cell expansion

Abstract Engineered T cell therapies have been remarkably successful in the treatment of B cell malignancies, yet lack of control over these “living drugs” can lead to significant toxicities or limited efficacy. One particular challenge is the achievement of durable anti-tumor responses because the reduction of tumor burden results in reduced antigen stimulation and therefore reduced antigen-dependent T cell expansion. Interleukin-15 (IL15) drives T and NK cell expansion and persistence in an antigen-independent manner, however unregulated expression of this cytokine may compromise the safety and efficacy of cellular immunotherapy. To address these issues, we engineered T cells with a pharmacologically controllable, membrane-bound IL15 that supports antigen-independent T cell expansion and has the potential to reduce the safety risks associated with continuous exposure to soluble IL15. Our approach utilizes Drug Responsive Domains (DRDs) which are fully human protein domains that are inherently unstable in the cell but are reversibly stabilized when bound to specific FDA-approved drugs. Fusion of a DRD to a protein of interest confers drug-dependent, reversible regulation of protein expression and function. We developed a DRD based on the carbonic anhydrase 2 (CA2) protein, which is stabilized in the presence of the FDA-approved drug acetazolamide (ACZ). Upon gene transfer of membrane-bound IL15 fused to a CA2 DRD, regulated IL15 expression on T cells was detected only in the presence of ACZ. In the absence of ACZ, the level of IL15 detected on the surface of gene-modified T cells is not substantially different from the level detected on untransduced T cells. ACZ treatment of gene-modified T cells increases surface IL15 expression in a dose-dependent manner. We observed prolonged survival and up to 15-fold expansion of IL15-CA2 DRD-transduced T cells in the absence of supplemental cytokines or antigen stimulation. In contrast, vehicle-treated IL15-CA2 DRD modified T cells and untransduced T cells did not survive or expand in vitro. Importantly, both IL15-CA2-transduced T cells and unengineered, co-infused NK cells survived and persisted significantly more in vivo in ACZ-treated but not vehicle-treated non-tumor-bearing NSG mice. Our results demonstrate that a novel membrane-bound IL15-CA2 fusion protein coupled with ACZ treatment induces antigen-independent T cell expansion and augments bystander NK cell persistence. Regulatable IL15 expression has significant implications for both T and NK cell therapies by providing more durable cell expansion and cytolytic activity in vivo, and thus the potential to significantly reduce cell dosing while maintaining clinical efficacy in patients. Citation Format: Steven Shamah, Kutlu Elpek, Tucker Ezell, Michelle Fleury, Michael Gallo, Jennifer Gori, Scott Heller, Mara Inniss, Meghan Langley, Grace Olinger, Celeste Richardson, Karen Tran, Dhruv Sethi, Dexue Sun, Vipin Suri. Drug responsive domain regulation of IL15-engineered T cells provides pharmacological control over antigen-independent cell expansion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6604.

Study on mechanisms of anti-Alzheimer's disease action of absorbed components of Gardeniae Fructus based on network pharmacology

Gardeniae Fructus has the traditional effects of promoting intelligence and inducing resuscitation, but its mechanism is unclear. In this study, the relationship between Gardeniae Fructus's traditional effect of promoting intelligence and inducing resuscitation and anti-Alzheimer's disease effect was taken as the starting point to investigate the anti-Alzheimer's disease mechanism of the major absorbed components in Gardeniae Fructus by the network pharmacology method. The network pharmacology research model of &quot;absorbed composition-target-pathway-disease&quot; was adopted. In this study, the active components screening and target prediction technology were used to determine the active components and targets of Gardeniae Fructus in treatment of Alzheimer's disease. The enrichment pathway and biological process of Gardeniae Fructus were studied by using the bioinformatics annotation database(DAVID), and the results of molecular docking validation network analysis were used to elaborate the mechanism of Gardeniae Fructus in treatment of Alzheimer's disease. It was found that 35 absorbed components of Gardeniae Fructus not only regulated 48 targets such as cholines-terase(BCHE) and carbonic anhydrase 2(CA2), but also affected 11 biological processes(e.g. transcription factor activity, nuclear receptor activity, steroid hormone receptor activity, amide binding and peptide binding) and 7 metabolic pathways(MAPK signaling pathway, Alzheimer disease and estrogen signaling pathway, etc.). Molecular docking results showed that more than 60% of the active components could be well docked with key targets, and the relevant literature also showed that the active components could inhibit the MAPK1 expression of key targets, indicating a high reliability of results. These results indicated that Gardeniae Fructus may play its anti-Alzheimer's disease action via a &quot;multi-ingredients-multi-targets and multi-pathways&quot; mode, providing a scientific basis for further drug research and development.

Expression of CA2 and CA9 carbonic anhydrases in ulcerative colitis and ulcerative colitis-associated colorectal cancer.

Ulcerative colitis (UC) is characterized by chronic inflammation in the colonic mucosa and submucosa with repeating relapse and remission, but the pathogenesis is unknown. Patients with long-standing UC are at high risk of neoplasm development. The aim of the present study was to identify molecules whose expression is associated with UC and UC-associated colorectal cancer (UCCA). Biopsy specimens from UC and normal colonic mucosae were analyzed using a proteomics approach, in which carbonic anhydrase 2 (CA2) was identified as a molecule downregulated in UC mucosae. Immunohistochemically, CA2 expression was detected in normal and diverticulitis mucosal epithelia, and its expression decreased as UC activity increased. CA2 expression was almost undetectable in UCCA. We also analyzed the expression of another carbonic anhydrase, CA9, and its upstream molecule, hypoxia-inducible factor-1α (HIF-1α), both of which are induced under hypoxic conditions. It was revealed that CA9 expression was relatively low in normal, diverticulitis and UC mucosae, and was upregulated in UCCA. HIF-1α expression was consistently low in all tissue types examined. In conclusion, these results suggest that CA2 and CA9 may be possible indicators of UC activity and UCCA development, respectively.

Carbonic anhydrases CA1 and CA4 function in atmospheric CO2-modulated disease resistance

Main conclusionCarbonic anhydrases CA1 and CA4 attenuate plant immunity and can contribute to altered disease resistance levels in response to changing atmospheric CO2 conditions.β-Carbonic anhydrases (CAs) play an important role in CO2 metabolism and plant development, but have also been implicated in plant immunity. Here we show that the bacterial pathogen Pseudomonas syringae and application of the microbe-associated molecular pattern (MAMP) flg22 repress CA1 and CA4 gene expression in Arabidopsis thaliana. Using the CA double-mutant ca1ca4, we provide evidence that CA1 and CA4 play an attenuating role in pathogen- and flg22-triggered immune responses. In line with this, ca1ca4 plants exhibited enhanced resistance against P. syringae, which was accompanied by an increased expression of the defense-related genes FRK1 and ICS1. Under low atmospheric CO2 conditions (150 ppm), when CA activity is typically low, the levels of CA1 transcription and resistance to P. syringae in wild-type Col-0 were similar to those observed in ca1ca4. However, under ambient (400 ppm) and elevated (800 ppm) atmospheric CO2 conditions, CA1 transcription was enhanced and resistance to P. syringae reduced. Together, these results suggest that CA1 and CA4 attenuate plant immunity and that differential CA gene expression in response to changing atmospheric CO2 conditions contribute to altered disease resistance levels.

Expression of protocadherin-20 in mouse taste buds

Taste information is detected by taste cells and then transmitted to the brain through the taste nerve fibers. According to our previous data, there may be specific coding of taste quality between taste cells and nerve fibers. However, the molecular mechanisms underlying this coding specificity remain unclear. The purpose of this study was to identify candidate molecules that may regulate the specific coding. GeneChip analysis of mRNA isolated from the mice taste papillae and taste ganglia revealed that 14 members of the cadherin superfamily, which are important regulators of synapse formation and plasticity, were expressed in both tissues. Among them, protocadherin-20 (Pcdh20) was highly expressed in a subset of taste bud cells, and co-expressed with taste receptor type 1 member 3 (T1R3, a marker of sweet- or umami-sensitive taste cells) but not gustducin or carbonic anhydrase-4 (markers of bitter/sweet- and sour-sensitive taste cells, respectively) in circumvallate papillae. Furthermore, Pcdh20 expression in taste cells occurred later than T1R3 expression during the morphogenesis of taste papillae. Thus, Pcdh20 may be involved in taste quality-specific connections between differentiated taste cells and their partner neurons, thereby acting as a molecular tag for the coding of sweet and/or umami taste.

Carbonic Anhydrase 4 serves as a Clinicopathological Biomarker for Outcomes and Immune Infiltration in Renal Cell Carcinoma, Lower Grade Glioma, Lung Adenocarcinoma and Uveal Melanoma.

Background: Carbonic anhydrase 4 (CA4) maintains homeostasis of carbon dioxide and bicarbonate. It is suggested to be a potential prognostic biomarker, while the correlations between CA4 and different cancers are indistinct.Methods: Differential mRNA expression of CA4 among different cancers and corresponding normal tissues was compared based on datasets on the Cancer Genome Atlas (TCGA) platforms. Then, survival analysis was performed using Tumor-immune system interactionsplatform and TCGA cohort on the basis of distinct comparison expression of CA4 in five kinds of tumors. In addition, molecular penal analysis and functional annotations of CA4-related genes was elaborated. The correlation between CA4 mRNA expression and tumor immune microenvironment were analyzed in detail.Results: Compared with adjacent normal tissues, CA4 mRNA expressions were found significantly lower in various tumors. Moreover, decreased expression of CA4 was significantly related to worse overall survival (OS) and progression-free survival (PFS) in kidney renal clear cell carcinoma (KIRC), brain lower grade glioma (LGG), lung adenocarcinoma (LUAD) and uveal melanoma (UVM), and worse OS of prostate adenocarcinoma (PRAD) (p<0.05). Cox regression analyses indicated that CA4 was a significant prognostic biomarker in KIRC, LGG, LUAD and UVM. Moreover, CA4 showed markedly relationship with tumor immune environment and diverse immune infiltration signatures in KIRC, LGG, LUAD and UVM.Conclusions: Our study revealed that CA4 was a potential biomarker for aggressive progression and poor prognosis in KIRC, LGG, LUAD, PRAD and UVM, correlated with immune infiltration in various tumor environments. These results suggested that CA4 possibly served as a promising prognostic and immune infiltration biomarker in many cancers.

Effects of hub genes on the clinicopathological and prognostic features of lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a common malignancy; however, the majority of its underlying molecular mechanisms remain unknown. In the present study, weighted gene co-expression network analysis was applied to construct gene co-expression networks for the GSE19804 dataset, in order to screen hub genes associated with the pathogenesis of LUAD. In addition, with the aid of the Database for Annotation, Visualization and Integrated Discovery, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes, pathway enrichment analyses were performed on the genes in the selected module. Using the GSE40791 dataset and The Cancer Genome Atlas database, the hub genes were identified. It was discovered that the turquoise module was the most significant module associated with the tumor stage of LUAD. After performing functional enrichment analyses, it was indicated that the turquoise module was mainly enriched in signal transduction. Additionally, at the transcriptional and translational level, nine hub genes were identified and validated: Carbonic anhydrase 4 (CA4), platelet and endothelial cell adhesion molecule 1 (PECAM1), DnaJ member B4 (DNAJB4), advanced glycosylation end-product specific receptor (AGER), GTPase, IMAP family member 6 (GIMAP6), chromosome 10 open reading frame 54 (C10orf54), dedicator of cytokinesis 4 (DOCK4), Golgi membrane protein 1 (GOLM1) and platelet activating factor acetylhydrolase 1b catalytic subunit 3 (PAFAH1B3). CA4, PECAM1, DNAJB4, AGER, GIMAP6, C10orf54 and DOCK4 were expressed at lower levels in the tumor samples, whereas GOLM1 and PAFAH1B3 were highly expressed in tumor samples. In addition, all hub genes were associated with prognosis. In conclusion, one module and nine genes were recognized to be associated with the tumor stage of LUAD. These findings may enhance the understanding of the progression and prognosis of LUAD.

Effects of insulin signaling on mouse taste cell proliferation.

Expression of insulin and its receptor (IR) in rodent taste cells has been proposed, but exactly which types of taste cells express IR and the function of insulin signaling in taste organ have yet to be determined. In this study, we analyzed expression of IR mRNA and protein in mouse taste bud cells in vivo and explored its function ex vivo in organoids, using RT-PCR, immunohistochemistry, and quantitative PCR. In mouse taste tissue, IR was expressed broadly in taste buds, including in type II and III taste cells. With using 3-D taste bud organoids, we found insulin in the culture medium significantly decreased the number of taste cell and mRNA expression levels of many taste cell genes, including nucleoside triphosphate diphosphohydrolase-2 (NTPDase2), Tas1R3 (T1R3), gustducin, carbonic anhydrase 4 (CA4), glucose transporter-8 (GLUT8), and sodium-glucose cotransporter-1 (SGLT1) in a concentration-dependent manner. Rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR) signaling, diminished insulin’s effects and increase taste cell generation. Altogether, circulating insulin might be an important regulator of taste cell growth and/or proliferation via activation of the mTOR pathway.

Study on Hypoglycemic Effect of the Drug Pair of Astragalus Radix and Dioscoreae Rhizoma in T2DM Rats by Network Pharmacology and Metabonomics.

Type 2 diabetes mellitus (T2DM) is a metabolic disease accompanied by a series of diseases such as diabetic nephropathy. The drug pair (HS) of Astragalus Radix (HQ) and Dioscoreae Rhizoma (SY) was designed by Dr. Shi Jinmo to improve the treatment of T2DM. However, the exact mechanism involved requires further clarification. In this work, 1H-NMR–based metabonomics and network pharmacology were adopted. Metabolic profiling indicated that the metabolic perturbation was reduced after HS treatment. The results found 21 biomarkers. According to the network pharmacology, we found that the regulation of T2DM was primarily associated with 18 active compounds in HS. These active compounds mainly had an effect on 135 targets. Subsequently, combining network pharmacology and metabonomics, we found four target proteins, which indicated that HS has potential hypoglycemic effects through regulating monoamine oxidases B (MAOB), acetyl-CoA carboxylase 1 (ACACA), carbonic anhydrase 2 (CA2), and catalase (CAT). In conclusion, the result showed that these four targets might be the most relevant targets for the treatment of T2DM with HS. This study clarified the mechanism of HS in the treatment of T2DM and also confirmed the feasibility of combining metabonomics and network pharmacology to study the mechanisms of traditional Chinese medicine (TCM). In the future, this approach may be a potentially powerful tool to discovery active components of traditional Chinese medicines and elucidate their mechanisms.

Identification of potential pathogenic candidates or diagnostic biomarkers in papillary thyroid carcinoma using expression and methylation profiles.

The mechanisms underlying the pathogenesis of papillary thyroid carcinoma (PTC) have not yet been elucidated. The aim of the current study was to identify potential pathogenic biomarkers in PTC by comprehensively analyzing gene expression and methylation profiles, and to increase the understanding of PTC pathogenesis. The gene expression profiles of the GSE97001 and GSE83520 datasets, the miRNA expression profiles of the GSE73182 dataset, and the DNA methylation profiles of the GSE86961 and GSE97466 datasets were downloaded from Gene Expression Omnibus database. The differentially expressed genes (DEGs) and the differentially expressed microRNAs (DEMs) were identified using the limma package in R, and the differentially methylated sites (DMSs) were identified using the β distribution and two-sample t-tests. The Database for Annotation, Visualization and Integrated Discovery, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome were subsequently used to perform functional and pathway enrichment analysis. The miRNA target genes were predicted using the online databases miRWalk. The protein-protein interactions (PPI) were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins. The regulatory network was constructed, and the gene expression and methylation levels of the key nodes were detected using reverse-transcription quantitative-polymerase chain reaction (PCR) and methylation-specific PCR. A total of 155 overlapping DEGs were identified between the GSE97001 and GSE83520 datasets, and 19 DEMs between PTC tissue and normal tissue samples were identified in the GSE73182 set. In the GSE86961 and GSE97466 datasets, 2,910 overlapping DMSs that were associated with 38 downregulated methylated genes were identified. The overlapping DEGs were enriched in 46 Gene Ontology terms and one KEGG pathway. A total of 60 PPI pairs were identified for the overlapping DEGs and 12 negative miRNA-gene pairs were identified for the DEMs. The expression levels of hsa-miR-199a-5p and decorin (DCN) were decreased in patients with PTC. C-X-C motif chemokine ligand 12 (CXCL12) was hypermethylated and had a decreased expression level in PTC tissues. LDL receptor related protein 4 (LRP4) and carbonic anhydrase 12 (CA12) were hypomethylated and had an increased expression level. The present study revealed that hsa-miR-199a-5p, DCN, CXCL12, LRP4 and CA12 may serve important roles in the pathogenesis of PTC.

SSTR5‑AS1 functions as a ceRNA to regulate CA2 by sponging miR‑15b‑5p for the development and prognosis of HBV‑related hepatocellular carcinoma.

Long non-coding RNAs (lncRNAs) have been implicated in the development and progression of cancer. However, the mechanisms of lncRNAs in hepatitis B virus (HBV) infection-induced hepatocellular carcinoma (HCC) remain unclear. The study aimed to reveal the roles of lncRNAs for HBV-HCC based on the hypothesis of competing endogenous RNA (ceRNA). The lncRNA (GSE27462), miRNA (GSE76903) and mRNA (GSE121248) expression profiles were collected from the Gene Expression Omnibus database. Differentially expressed lncRNAs (DELs), genes (DEGs) and miRNAs (DEMs) were identified using the LIMMA or EdgeR package, respectively. The ceRNA network was constructed based on interaction pairs between miRNAs and mRNAs/lncRNAs. The functions of DEGs in the ceRNA network were predicted using the DAVID database, which was overlapped with the known HCC pathways of Comparative Toxicogenomics Database (CTD) to construct the HCC-related ceRNA network. The prognosis values [overall survival, (OS); recurrence-free survival (RFS)] of genes were validated using the Cancer Genome Atlas (TCGA) data with Cox regression analysis. The present study screened 38 DELs, 127 DEMs and 721 DEGs. A ceRNA network was constructed among 17 DELs, 12 DEMs and 173 DEGs, including the FAM138B-hsa-miR-30c-CCNE2/RRM2 and SSTR5-AS1-hsa-miR-15b-5p-CA2 ceRNA axes. Function enrichment analysis revealed the genes in the ceRNA network that participated in the p53 signaling pathway [cyclin E2 (CCNE2), ribonucleotide reductase M2 subunit (RRM2)] and nitrogen metabolism [carbonic anhydrase 2 (CA2)], which were also included in the pathways of the CTD. Univariate Cox regression analysis revealed that six RNAs (2 DELs: FAM138B, SSTR5-AS1; 2 DEMs: hsa-miR-149, hsa-miR-7; 2 DEGs: CCNE2, RRM2) were significantly associated with OS; while seven RNAs (1 DEL: LINC00284; 3 DEMs: hsa-miR-7, hsa-miR-15b, hsa-miR-30c-2; and 3 DEGs: RRM2, CCNE2, CA2) were significantly associated with RFS. In conclusion, FAM138B-hsa-miR-30c-CCNE2/RRM2 and the SSTR5-AS1-hsa-miR-15b-5p-CA2 ceRNA axes may be important mechanisms for HBV-related HCC.

Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells.

We investigated the effects of a fibrin-hyaluronic acid hydrogel (FBG–HA) and fibroblast growth factor 18 (FGF-18) for nucleus pulposus (NP) regeneration. Healthy bovine (n = 4) and human degenerated NP cells (n = 4) were cultured for 14 days in FBG-HA hydrogel with FGF-18 (∆51-mutant or wild-type) in the culture medium. Gene expression, DNA content, and glycosaminoglycan (GAG) synthesis were evaluated on day 7 and 14. Additionally, histology was performed. Human NP cells cultured in FBG-HA hydrogel showed an increase in collagen type II (COL2) and carbonic anhydrase XII (CA12) gene expression after 14 or 7 days of culture, respectively. GAG release into the conditioned medium increased over 14 days. Healthy bovine NP cells showed increased gene expression of ACAN from day 7 to day 14. Wild type FGF-18 up-regulated CA12 gene expression of human NP cells. Histology revealed an increase of proteoglycan deposition upon FGF-18 stimulation in bovine but not in human NP cells. The FBG-HA hydrogel had a positive modulatory effect on human degenerated NP cells. Under the tested conditions, no significant effect of FGF-18 was observed on cell proliferation or GAG synthesis in human NP cells.

Molecular modelling and dynamics of CA2 missense mutations causative to carbonic anhydrase 2 deficiency syndrome

Carbonic anhydrase 2 (CA2) enzyme deficiency caused by CA2 gene mutations is an inherited disorder characterized by symptoms like osteopetrosis, renal tubular acidosis, and cerebral calcification. This study has collected the CA2 deficiency causal missense mutations and assessed their pathogenicity using diverse computational programs. The 3D protein models for all missense mutations were built, and analyzed for structural divergence, protein stability, and molecular dynamics properties. We found M-CAP as the most sensitive prediction method to measure the deleterious potential of CA2 missense mutations. Free energy dynamics of tertiary structure models of CA2 mutants with DUET, mCSM, and SDM based consensus methods predicted only 50% of the variants as destabilizing. Superimposition of native and mutant CA2 models revealed the minor structural fluctuations at the amino acid residue level but not at the whole protein structure level. Near native molecular dynamic simulation analysis indicated that CA2 causative missense variants result in residue level fluctuation pattern in the protein structure. This study expands the understanding of genotype-protein phenotype correlations underlying CA2 variant pathogenicity and presents a potential avenue for modifying the CA2 deficiency by targeting biophysical structural features of CA2 protein. Communicated by Ramaswamy H. Sarma

Humanized Monoclonal Antibody Blocking Carbonic Anhydrase 12 Enzymatic Activity Leads to Reduced Tumor Growth In Vitro.

Carbonic anhydrase 12 (CA12) is a membrane-associated enzyme that is highly expressed on many human cancers. It is a poor prognostic marker and hence an attractive target for cancer therapy. This study aimed to develop a humanized CA12-antibody with anti-cancer activity. Antibody libraries were constructed and screened by the Retrocyte display®. Antibody binding and blocking properties were determined by ELISA, flow cytometry and enzymatic activity assays. Spheroid viability was determined by Cell-Titer-Fluor assay. We developed a novel humanized CA12-specific antibody, 4AG4, which recognized CA12 as an antigen and blocked CA12 enzymatic activity. Our humanized CA12-antibody significantly inhibited spheroid growth of lung adenocarcinoma A549-cells in vitro by blocking CA12 enzymatic activity. Similar anti-tumor effects were recapitulated with CA12-gene knockout of A549-cells. Our newly identified humanized CA12-antibody with anti-cancer activity, represents a new tool for the treatment of CA12-positive tumors.