Nischarin inhibits the epithelial-mesenchymal transition process and angiogenesis in breast cancer cells by inactivating FAK/ERK signaling pathway via EGF like repeats and discoidin domains 3.

BACKGROUND: Our previous study has demonstrated that Nischarin (NISCH) exerts its antitumor effects in breast cancer (BC) by suppressing cell migration and invasion. This study aims to explore the underlying mechanism through which NISCH functions in BC. METHODS AND RESULTS: The relevance between EGF Like Repeats and Discoidin Domains 3 (EDIL3) mRNA expression and the overall survival of tumor patients was depicted by the Kaplan-Meier curve. The findings revealed that overexpressed NISCH attenuated cell motility and colony-forming capacities of Hs578T cells, yet silenced NISCH in MDA-MB-231 cells led to contrasting results. Western blot (WB) analysis indicated that overexpression of NISCH significantly down-regulated the Vimentin and Slug expression, and inactivated the FAK/ERK signaling pathway. RNA sequencing (RNA-seq) was performed in NISCH-overexpressed Hs578T cells and the control cells to analyze differentially expressed genes (DeGs), and the results showed a significant down-regulation of EDIL3 mRNA level upon overexpression of NISCH. Subsequent functional analyses demonstrated that overexpression of EDIL3 attenuated the inhibitory effect of NISCH on cell migration, invasion, colony formation, and tube formation. CONCLUSION: In summary, our finding preliminarily revealed that NISCH inhibits the epithelial-mesenchymal transition (EMT) process and angiogenesis in BC cells by down-regulating EDIL3 to inactivate the FAK/ERK signaling pathway, thereby suppressing the progression of BC. Our results hold promise for contributing to the deep understanding of BC pathogenesis and identifying new therapeutic strategies for clinical application.

Identification of a psychiatric risk gene NISCH at 3p21.1 GWAS locus mediating dendritic spine morphogenesis and cognitive function.

BACKGROUND: Schizophrenia and bipolar disorder (BD) are believed to share clinical symptoms, genetic risk, etiological factors, and pathogenic mechanisms. We previously reported that single nucleotide polymorphisms spanning chromosome 3p21.1 showed significant associations with both schizophrenia and BD, and a risk SNP rs2251219 was in linkage disequilibrium with a human specific Alu polymorphism rs71052682, which showed enhancer effects on transcriptional activities using luciferase reporter assays in U251 and U87MG cells. METHODS: CRISPR/Cas9-directed genome editing, real-time quantitative PCR, and public Hi-C data were utilized to investigate the correlation between the Alu polymorphism rs71052682 and NISCH. Primary neuronal culture, immunofluorescence staining, co-immunoprecipitation, lentiviral vector production, intracranial stereotaxic injection, behavioral assessment, and drug treatment were used to examine the physiological impacts of Nischarin (encoded by NISCH). RESULTS: Deleting the Alu sequence in U251 and U87MG cells reduced mRNA expression of NISCH, the gene locates 180 kb from rs71052682, and Hi-C data in brain tissues confirmed the extensive chromatin contacts. These data suggested that the genetic risk of schizophrenia and BD predicted elevated NISCH expression, which was also consistent with the observed higher NISCH mRNA levels in the brain tissues from psychiatric patients compared with controls. We then found that overexpression of NISCH resulted in a significantly decreased density of mushroom dendritic spines with a simultaneously increased density of thin dendritic spines in primary cultured neurons. Intriguingly, elevated expression of this gene in mice also led to impaired spatial working memory in the Y-maze. Given that Nischarin is the target of anti-hypertensive agents clonidine and tizanidine, which have shown therapeutic effects in patients with schizophrenia and patients with BD in preliminary clinical trials, we demonstrated that treatment with those antihypertensive drugs could reduce NISCH mRNA expression and rescue the impaired working memory in mice. CONCLUSIONS: We identify a psychiatric risk gene NISCH at 3p21.1 GWAS locus influencing dendritic spine morphogenesis and cognitive function, and Nischarin may have potentials for future therapeutic development.

Evaluation of neurodevelopmental symptoms in 10 cases of neonatal ichthyosis and sclerosing cholangitis syndrome.

Neonatal ichthyosis and sclerosing cholangitis (NISCH) syndrome is an extremely rare entity with only 19 patients described in the literature. We report an extended family with the disorder and investigate the association of neurodevelopmental symptoms. Patients with CLDN1 mutations, and specifically « the Moroccan¼ c.200_201delTT deletion, may be an increased risk for neurodevelopmental symptoms such as learning disabilities, mental retardation, and language delay.

The Fate of Epidermal Tight Junctions in the stratum corneum: Their Involvement in the Regulation of Desquamation and Phenotypic Expression of Certain Skin Conditions.

We evaluated the presence of tight junction (TJ) remnants in the stratum corneum (SC) of in vitro reconstructed human epidermis and human skin explants subjected or not to an aggressive topical treatment with beta-lipohydroxy salicylic acid (LSA) for 24 h. LSA-treated samples showed an increased presence of TJ remnants in the two lowermost layers of the SC, as quantified with standard electron microscopy. The topical aggression-induced overexpression of TJ-like cell-cell envelope fusions may influence SC functions: (1) directly, through an enhanced cohesion, and (2) indirectly, by impeding accessibility of peripheral corneodesmosomes to extracellular hydrolytic enzymes and, thus, slowing down desquamation. Observations of ichthyotic epidermis in peeling skin disease (PSD; corneodesmosin deficiency; two cases) and ichthyosis hypotrichosis sclerosing cholangitis syndrome (IHSC/NISCH; absence of claudin-1; two cases) also demonstrated increased persistence of TJ-like intercellular fusions in pathological SC and contributed to the interpretation of the diseases' pathological mechanisms.

Tight Junction Proteins and the Biology of Hepatobiliary Disease.

Tight junctions (TJ) are intercellular adhesion complexes on epithelial cells and composed of integral membrane proteins as well as cytosolic adaptor proteins. Tight junction proteins have been recognized to play a key role in health and disease. In the liver, TJ proteins have several functions: they contribute as gatekeepers for paracellular diffusion between adherent hepatocytes or cholangiocytes to shape the blood-biliary barrier (BBIB) and maintain tissue homeostasis. At non-junctional localizations, TJ proteins are involved in key regulatory cell functions such as differentiation, proliferation, and migration by recruiting signaling proteins in response to extracellular stimuli. Moreover, TJ proteins are hepatocyte entry factors for the hepatitis C virus (HCV)-a major cause of liver disease and cancer worldwide. Perturbation of TJ protein expression has been reported in chronic HCV infection, cholestatic liver diseases as well as hepatobiliary carcinoma. Here we review the physiological function of TJ proteins in the liver and their implications in hepatobiliary diseases.

Evaluating NISCH and CDH1 Promoter Hypermethylation in Nonsmokers, Cancer Free Smokers and Lung Cancer Patients: A Case Control Study.

Lung cancer has very high mortality due to late stage diagnosis not amenable to curative resection. Cancer specific methylation patterns of tumor suppressor genes may precede precursor lesions of lung cancer. Our aim was to evaluate the promoter hypermethylation of tumor suppressor gene NISCH and CDH1 in cfDNA from plasma of lung cancer patients and its possible correlation with smoking status and various clinicopathological parameters. Forty histopathologically confirmed lung cancer cases, thirty smoker and thirty nonsmoker controls were enrolled. Plasma cfDNA was extracted and subjected to bisulfite treatment followed by MS-PCR. Serum nischarin levels were estimated by ELISA. The frequency of promoter hypermethylation of NISCH and CDH1 was significantly higher in lung cancer patients as compared to lifelong non-smoker controls (p < 0.05). It did not vary with smoking status among cancer cases. No significant association was found with staging or histological grading. NISCH methylation was found to be significantly higher among smoker controls. Pack years and packs per day were significantly higher in the methylated group. Serum nischarin levels showed no significant association with NISCH methylation or clinicopathological variables. NISCH is highly methylated in both high risk smoker controls as well as cancerousnon-smokers and may mark the convergence of varied etiologies of lung cancer. Hence NISCH and CDH1 are highly methylated in plasma cfDNA of lung cancer patients.

Investigation of morin-induced insulin secretion in cultured pancreatic cells.

Morin is a flavonoid contained in guava that is known to reduce hyperglycemia in diabetes. Insulin secretion has been demonstrated to increase following the administration of morin. The present study is designed to investigate the potential mechanism(s) of morin-induced insulin secretion in the MIN6 cell line. First, we identified that morin induced a dose-dependent increase in insulin secretion and intracellular calcium content in MIN6 cells. Morin potentiated glucose-stimulated insulin secretion (GSIS). Additionally, we used siRNA for the ablation of imidazoline receptor protein (NISCH) expression in MIN6 cells. Interestingly, the effects of increased insulin secretion by morin and canavanine were markedly reduced in Si-NISCH cells. Moreover, we used KU14R to block imidazoline I3 receptor (I-3R) that is known to enhance insulin release from the pancreatic ß-cells. Without influence on the basal insulin secretion, KU14R dose-dependently inhibited the increased insulin secretion induced by morin or efaroxan in MIN6 cells. Additionally, effects of increased insulin secretion by morin or efaroxan were reduced by diazoxide at the dose sufficient to open KATP channels and attenuated by nifedipine at the dose used to inhibit L-type calcium channels. Otherwise, phospholipase C (PLC) is introduced to couple with imidazoline receptor (I-R). The PLC inhibitor dose-dependently inhibited the effects of morin in MIN6 cells. Similar blockade was also observed in protein kinase C (PKC) inhibitor-treated cells. Taken together, we found that morin increases insulin secretion via the activation of I-R in pancreatic cells. Therefore, morin would be useful to develop in the research and treatment of diabetic disorders.

Canavanine induces insulin release via activation of imidazoline I3 receptors.

The aim of the present study was to identify the effect of canavanine on the imidazoline receptor because canavanine is a guanidinium derivative that has a similar structure to imidazoline receptor ligands. Transfected Chinese hamster ovary-K1 cells expressing imidazoline receptors (nischarin (NISCH)-CHO-K1 cells) were used to elucidate the direct effects of canavanine on imidazoline receptors. In addition, the imidazoline I3 receptor has been implicated in stimulation of insulin secretion from pancreatic ß-cells. Wistar rats were used to investigate the effects of canavanine (0.1, 1 and 2.5 mg/kg, i.v.) on insulin secretion. In addition the a specific I3 receptor antagonist KU14R (4 or 8 mg/kg, i.v.) was used to block I3 receptors. Canavanine decreased blood glucose by increasing plasma insulin in rats. In addition, canavanine increased calcium influx into NISCH-CHO-K1 cells in a manner similar to agmatine, the endogenous ligand of imidazoline receptors. Moreover, KU12R dose-dependently attenuated canavanine-induced insulin secretion in HIT-T15 pancreatic ß-cells and in the plasma of rats. The data suggest that canavanine is an agonist of I3 receptors both in vivo and in vitro. Thus, canavanine would be a useful tool in imidazoline receptor research.

UBTF mediates activation of L3MBTL2 to suppress NISCH expression through histone H2AK119 monoubiquitination modification in breast cancer.

Lethal(3)malignant brain tumor-like protein 2 (L3MBTL2) has been related to transcriptional inhibition and chromatin compaction. Nevertheless, the biological functions and mechanisms of L3MBTL2 are undefined in breast cancer (BRCA). Here, we revealed that L3MBTL2 is responsible for the decline of Nischarin (NISCH), a well-known tumor suppressor, in BRCA, and explored the detailed mechanism. Knockdown of L3MBTL2 reduced monoubiquitination of histone H2A at lysine-119 (H2AK119ub), leading to reduced binding to the NISCH promoter and increased expression of NISCH. Meanwhile, the knockdown of L3MBTL2 decreased proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of BRCA cells, and increased apoptosis, which were abated by NISCH knockdown. Nucleolar transcription factor 1 (UBTF) induced the transcription of L3MBTL2 in BRCA, and the suppressing effects of UBTF silencing on EMT in BRCA cells were also reversed by NISCH knockdown. Knockdown of UBTF slowed tumor progression and attenuated lung tumor infiltration, whereas simultaneous knockdown of NISCH accelerated EMT and increased tumor lung metastasis. Taken together, our results show that L3MBTL2, transcriptionally activated by UBTF, exerts oncogenic functions in BRCA, by catalyzing H2AK119Ub and reducing expression of NISCH.

Promoter Methylation of Two HOXA9 and NISCH Genes in Opium Users.

Background: Opiate abuse has been critically increased in the world, especially in Iran. Owing to the association of opiate use with multiple human cancers and neurological disorders, seeking for genetic and epigenetic effects of opium can pave the way for early diagnosis of major health defects in addicted users. Accordingly, the present study aimed to determine the methylation status of the promoter of two genes, which are actively involved in neurodevelopment and cancer evolution. Methods: DNA was isolated from peripheral blood of 28 opium abusers and 19 healthy controls and then subjected to sonication. Sonicated DNAs undergone methylated DNA immunoprecipitation-real time polymerase chain reaction (MeDIP-Real Time PCR) using specific primer pairs designed for HOXA9 and NISCH genes. Obtained data were analyzed using SPSS software. Findings: HOXA9 and NISCH genes were found to be significantly methylated in addicted users compared to controls (P<0.001) which was significantly associated with the mean of the age regarding HOXA9 gene (P=0.002). Neither opium amount nor duration or route of using was associated with the methylation status of HOXA9 or NISCH genes. Conclusion: Hypermethylation of HOXA9 and NISCH genes as tumor suppressor in opium-addicted individuals can be considered as confirmatory evidence for carcinogenesis of opium. Further studies are required to figure out the role of epigenetic alterations in cancer evolution among opium users.

LncRNA RP11-214F16.8 drives breast cancer tumorigenesis via a post-translational repression on NISCH expression.

Increasing research interests have been aroused in exploring the function of long non-coding RNA (lncRNA) in breast cancer and developing lncRNA-targeted diagnosis, treatment and prognosis. In GEPIA2 database, we compared the expression pattern of the lncRNA RP11-214F16.8 in normal mammary tissues and breast cancer tumors and its correlation with the overall death rate of breast cancer patients. Gain- and loss-of function assays were employed to study function of the lncRNA in breast cancer cell lines in vitro while xenograft tumor growth assay was performed to investigate its function in tumorigenesis in vivo. We also used RNA pull-down coupled with mass spectrometry to identify the lncRNA binding partner, and RIP, EMSA, ChIP and Co-IP assays as well to testify these physical interactions.We identified that up-regulation of the lncRNA RP11-214F16.8 is subtype-independently associated with a higher overall death rate in breast cancer patients. Increased RP11-214F16.8 expression endows breast cancer cells enhanced capabilities in the aspects of proliferation, invasion, migration and tumor-initiation, while loss of the lncRNA exerts the opposite effects. Mechanistically, the oncogenic property of RP11-214F16.8 lies to its post-translational repression on the tumor suppressor NISCH via recruiting SENP3-mediated de-SUMOylation and ubiquitin-proteasome-mediated protein degradation. NISCH in turn inhibits the transcription of RP11-214F16.8 through restraining the expression of the transcription factors located downstream of RAC1, PAK1 and ERK1/2 signaling transduction pathways. In all, dysregulation of RP11-214F16.8 not only stimulates activation of the proliferation- and migration-promoting signaling cascades, but also facilitates the removal of restrictions on self-transcription, which ensures the progression of tumorigenesis.

Transcription factor KLF15 inhibits the proliferation and migration of gastric cancer cells via regulating the TFAP2A-AS1/NISCH axis.

BACKGROUND: Recently, overwhelming evidence supports that long noncoding RNAs (lncRNAs) play crucial roles in the occurrence and progression of tumors. However, the role and mechanism of lncRNA TFAP2A-AS1 in human gastric cancer (GC) remains unclear. Thus, the biological role and regulatory mechanisms of TFAP2A-AS1 in GC were explored. METHODS: Quantitative real-time PCR (qPCR) was applied to detect gene expression. Western blot was used to measure protein expression. Cell proliferation and migration were determined by functional assays. Fluorescence in situ hybridization (FISH) assays were performed to determine the subcellular distribution of TFAP2A-AS1 in GC. Mechanism investigations were conducted to explore the downstream genes of TFAP2A-AS1 and the upstream transcription factor of TFAP2A-AS1 in GC cells. RESULTS: TFAP2A-AS1 inhibits the proliferation and migration of GC cells. In the downstream regulation mechanism, miR-3657 was verified as the downstream gene of TFAP2A-AS1 and NISCH as the target of miR-3657. NISCH also suppresses cell proliferation and migration in GC. In the upstream regulation mechanism, transcription factor KLF15 positively mediates TFAP2A-AS1 to suppress GC cell proliferation and migration. CONCLUSION: KLF15-mediated TFAP2A-AS1 hampers cell proliferation and migration in GC via miR-3657/NISCH axis.

Identification of morin as an agonist of imidazoline I-3 receptor for insulin secretion in diabetic rats.

Morin is a flavonoid contained in guava that is known to reduce hyperglycemia in diabetics. Morin has been demonstrated to increase plasma insulin. However, the mechanism(s) remains unknown. The present study is designed to investigate the effect of morin on the imidazoline receptor (I-R) that regulates insulin secretion. We used Chinese hamster ovary (CHO) cells transfected with an I-R expression construct (NISCH-CHO-K1 cells) to identify the direct effect of morin on the I-R. Moreover, the imidazoline I3 receptor (I-3R) is known to be present in pancreatic ß cells and involved in insulin secretion. Therefore, we applied a specific antagonist (KU14R) to block I-3R in diabetic rats. Additionally, the effect of morin on insulin secretion was characterized in isolated pancreatic islets. Morin decreased blood glucose levels by increasing plasma insulin levels in diabetic rats. In CHO cells expressing an I-R, morin increased calcium influx in a dose-dependent manner. Additionally, KU14R dose-dependently inhibited the morin-induced effects, including hypoglycemia and the increase in insulin secretion and plasma C-peptide levels, in diabetic rats. Furthermore, morin enhanced insulin secretion from isolated pancreatic islets, and this effect was also dose-dependently inhibited by KU14R. Phospholipase C (PLC) is known to couple with the I-R, and a PLC inhibitor dose-dependently attenuated the insulin secretion induced by morin in isolated pancreatic islets. Taken together, these data suggest that morin can activate I-3R to enhance insulin secretion. Therefore, it would be useful to develop morin into a treatment for diabetic disorders.

Cigarette smoke induces methylation of the tumor suppressor gene NISCH.

We have previously identified a putative tumor suppressor gene, NISCH, whose promoter is methylated in lung tumor tissue as well as in plasma obtained from lung cancer patients. NISCH was observed to be more frequently methylated in smoker lung cancer patients than in non-smoker lung cancer patients. Here, we investigated the effect of tobacco smoke exposure on methylation of the NISCH gene. We tested methylation of NISCH after oral keratinocytes were exposed to mainstream and side stream cigarette smoke extract in culture. Methylation of the promoter region of the NISCH gene was also evaluated in plasma obtained from lifetime non-smokers and light smokers (<20 pack/year), with and without lung tumors, and heavy smokers (20+ pack/year) without disease. Promoter methylation of NISCH was tested by quantitative fluorogenic real-time PCR in all samples. Promoter methylation of NISCH occurred after exposure to mainstream tobacco smoke as well as to side stream tobacco smoke in normal oral keratinocyte cell lines. NISCH methylation was also detected in 68% of high-risk, heavy smokers without detectable tumors. Interestingly, in light smokers, NISCH methylation was present in 69% of patients with lung cancer and absent in those without disease. Our pilot study indicates that tobacco smoke induces methylation changes in the NISCH gene promoter before any detectable cancer. Methylation of the NISCH gene was also found in lung cancer patients' plasma samples. After confirming these findings in longitudinally collected plasma samples from high-risk populations (such as heavy smokers), examining patients for hypermethylation of the NISCH gene may aid in identifying those who should undergo additional screening for lung cancer.