POLR3A mutations by assessing the functional consequences of a missense and a splicing acceptor mutation.To expand the clinical phenotype of 1H]-magnetic resonance spectroscopy. Expression levels of Pol III target genes were measured by quantitative real-time (qRT)-PCR to study the pathogenicity of the biallelic mutations in patient fibroblasts.We performed whole-exome sequencing for identification of likely pathogenic mutations in a 9-year-old female patient with severe generalized dystonia, metabolic acidosis, leukocytosis, hypotonia, and dysphagia. Brain MRI showed basal ganglia atrophy and presence of lactate and lipid peaks by [POLR3A, the gene coding for the catalytic subunit of RNA polymerase III (Pol III). Aberrant splicing was observed for the c.1771-6C>G mutation. Decreased RNA expression levels of Pol III targets  were observed in patient fibroblasts with rescue to normal levels by overexpression of the wild-type protein but not by the p.Val1241Met variant.The patient is a compound heterozygous for a novel missense c.3721G>A  and the splicing region c.1771-6C>G mutation in POLR3A gene cause POLR3A-related hypomyelinating leukodystrophy with or without oligodontia or hypogonadotropic hypogonadism  and neonatal progeroid syndrome (OMIM: 264090), both with high phenotypic variability. We demonstrated the pathogenicity of c.1771-6C>G and c.3721G>A mutations causing an early-onset disorder. The phenotype of our patient expands the clinical presentation of POLR3A-related mutations and suggests a new classification that we propose designating as Neurodevelopmental Disorder with Regression, Abnormal Movements, and Increased Lactate.Mutations in the  POLR3A encodes the catalytic subunit A of Pol III, which is responsible for the constitutive transcription of transfer ribonucleic acids, mitochondrial RNA–processing RNA, 5S, H1, and noncoding RNAs (ncRNAs), and involved in translation of several mRNA.2 Because of its involvement in the transcription of so many different RNAs, it is not surprising that mutations in POLR3A can lead to a variety of phenotypes.POLR3A mutations: HLD7 (OMIM: 607694) and neonatal progeroid syndrome . HLD7 is an autosomal recessive early-onset leukodystrophy3 presented by hypomyelination, hypogonadotropic hypogonadism, hypodontia, spasticity, dystonia, and neurodevelopmental regression.4Two different conditions are associated with 5Like HLD7, NPS is also an autosomal recessive early-onset disorder presenting a wide range of phenotypes including macrocephaly, progeroid appearance, triangular face, micrognathia, nystagmus, hypodontia, muscle atrophy, hypertonia, and agenesis of the corpus callosum.POLR3A, with features not described in HLD7 nor NPS. We propose that these mutations extend the phenotype of POLR3A deficiency and create a new classification of patients with features similar to those presented here.Here, we present the c.3721G>A and c.1771-6C>G mutations in Informed consent was obtained according to the Ethical Standards Committee of UFPR (CAAE: 84773818.2.0000.0102).3 21.4 reference: 22–29 mEq/L). She could sit but neither crawl nor walk.The proband is a 9-year-old girl with a healthy mother who had no other pregnancies and a father diagnosed with depression. Gastroesophageal reflux and ineffective breast sucking were observed just after birth. When aged 5 months, she was apathetic and presented generalized dystonia. Four months later, she refused food with no swallowing disorder. Protein malnutrition was observed, and oral administration of hypercaloric diet was started with ensuing of metabolic acidosis  with total brain volume reduction and alterations in striatal bodies. Four years later, new MRI showed bilateral hyperintensity in T2  and FLAIal peaks .EEG showed slight diffuse disorganization in background activity, reflecting a diffuse cerebral dysfunction, with no association with specific pathologic features. No epileptiform activity was observed.Amino acid analysis in blood showed increased levels of threonine, glycine, alanine, alpha-aminobutyric acid, valine, cystine, isoleucine, leucine, tyrosine, phenylalanine, and ornithine. Urine amino acid analysis showed increased levels of 3-hydroxybutyric and reduced levels of methylsuccinic, 3-methylglutaconic, and 3-hydroxy-3-methylglutaric. In addition, she presents leukocytosis and persistent metabolic acidosis with increased lactate , decreased pH , and increased ammonia levels . Currently, she presents hypotonia, dysphagia, low weight, diffuse muscular hypotrophy, and severe generalized dystonia with worsening of the condition at night, but no triggers identified. Anticholinergic, benzodiazepine, antidopaminergic, gabaergic, and anticonvulsant medications were used to decrease dystonia, unsuccessfully. Physical neurologic examination shows orofacial dyskinesias, normal cranial circumference, and symmetry with neither abnormalities of cranial nerves nor facial dysmorphism. The patient is fully wheelchair dependent for ambulation.6 were used. Sanger sequencing was performed by Source BioScience, United Kingdom.DNA was extracted from blood . Whole-exome sequencing (WES) was prepared (Illumina® Nextera) and run (HiSeq 2000). Human GRCh37 reference genome and a GATK-based pipeline2). Cell immortalization and rescue experiments were performed by lentiviral transduction .Fibroblasts were derived from the patient's skin biopsy and grown  at 37°C (5% CO2 1.5 mM plus protease inhibitors), centrifuged, and supernatants mixed with loading buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Protein concentration was measured .For Western blot, cells were lysed . For POLR3A mRNA processing, cDNA was amplified by PCR, separated by agarose gel electrophoresis, gel extracted, and Sanger sequenced. qRT-PCR of target genes was performed in duplicate with Life Technologies TaqMan Assays (Applied Biosystems): ncRNA  and mRNAs: ubiquitin B (UBB), lactotransferrin (LTF), and HSP90AA1.p < 0.05) using the one-way analysis of variance and Tukey multiple comparison test. All data are presented as mean ± SD of the mean (SD) of 3 biological independent experiments in duplicates.The differences among groups were calculated with 95% confidence intervals , inherited from the mother, and a splicing region c.1771-6C>G (rs115020338), from the father (WES detected 2 mutations in e father . SequencThe c.3721G>A mutation has a frequency of <0.0001 (TOPMed), and MutationTaster2 classifies it as possibly pathogenic (score 0.99). The valine residue in the 1,241 position is highly conserved in metazoans with bilateral symmetry, except in some species, which present isoleucine instead .Electrophoresis from control and proband fibroblasts detected 2 transcripts of shorter length along with the canonical full-length mRNA . The lonPOLR3A_WT_1 and PPOLR3A_WT_2) or mutant , using the empty vector (PEV) as a negative control. Compared with the control (Ctrl), patient-derived cell lines (P and PEV) displayed low levels of POLR3A, whereas overexpressing cell lines presented higher levels of the protein, as also demonstrated by anti-HA antibody immunovisualization , and it has been reported in a family with HLD7, causing basal ganglia atrophy with no hypomyelination.3The c.1771-6C>G mutation is located 6 nucleotides upstream exon 14 of 3 Expression levels of HNRNPH2 were reduced in patient's fibroblasts, and we showed that overexpression of WT POLR3A, but not of the mutated p.Val1241Met variant, rescued the expression of HNRNPH2. Decreased expression of UBB, LTF, and HSP90AA1 has been previously found in other patients carrying the c.1771-6C>G mutation in POLR3A.3 All the mRNAs expression levels were decreased in the patient cells, with rescue of their expression levels when WT but not p.Val1241Met POLR3A was overexpressed, demonstrating the pathogenicity of the c.3721G>A mutation.Loss of exon 14 has been linked to reduced expression of the splicing factor HNRNPH2, responsible for the regulation of pre-mRNA splicing.9 This variant was recently classified as benign in ClinVar following the American College of Medical Genetics guidelines10 .No other VUS with correlation between the patient's phenotype and previously associated disease genes was identified in the nuclear genome. In mtDNA, the mutation m.14831G>A was found in a patient with Leber hereditary optic neuropathy, but no functional studies were performed.POLR3A mutations and even mutations in genes coding for POL III targets,8 highlighting the difficulty of a clinical diagnosis and reinforcing the importance of a detailed clinical report combined with molecular studies.The clinical phenotype of our patient resembles a combination of symptoms of 2 conditions associated with POLR3A, with neither leukodystrophy nor NPS features, which we propose designating as Neurodevelopmental Disorder with Regression, Abnormal Movements, and Increased Lactate.In conclusion, we suggest a new classification to patients carrying mutations in


High mobility group A (HMGA) proteins are oncofoetal chromatin architectural factors that are widely involved in regulating gene expression. These proteins are unique, because they are highly expressed in embryonic and cancer cells, where they play a relevant role in cell proliferation, stemness, and the acquisition of aggressive tumour traits, i.e., motility, invasiveness, and metastatic properties. The HMGA protein expression levels and activities are controlled by a connected set of events at the transcriptional, post-transcriptional, and post-translational levels. In fact, microRNA (miRNA)-mediated RNA stability is the most-studied mechanism of HMGA protein expression modulation. In this review, we contribute to a comprehensive overview of HMGA-targeting miRNAs; we provide detailed information regarding HMGA gene structural organization and a comprehensive evaluation and description of HMGA-targeting miRNAs, while focusing on those that are widely involved in HMGA regulation; and, we aim to offer insights into HMGA-miRNA mutual cross-talk from a functional and cancer-related perspective, highlighting possible clinical implications. HMGA1 gene, and HMGA2, which is derived from a different—although related—gene [The high mobility group A (HMGA) family is a family of architectural nuclear proteins that are involved in the modulation of chromatin structure and regulation of gene expression. The family comprises three main members: HMGA1a and HMGA1b, which are derived from the alternative splicing of the ted—gene . These pted—gene ,5. The cted—gene , thus pated—gene ,8,9. In ted—gene  and alteted—gene . HMGA prted—gene , differeted—gene , senesceted—gene , apoptosted—gene ,16, inflted—gene , metabolted—gene ,19, autoted—gene , DNA repted—gene , DNA repted—gene ,23, splited—gene , and virted—gene , given tHmga1 and Hmga2 [HMGA1 and HMGA2 genes has been extensively and recently reviewed: HMGA1 is an inducible gene that is mainly regulated by transcription factors at different promoter regions and enhancers, while the HMGA2 promoter seems to be constitutively active in different cell lines, and its activity can be modulated either positively or negatively by different DNA-binding factors [HMGA2 gene transcription modulation, providing an open chromatin conformation for HMGA2 transcriptional cis-regulatory sequences [HMGA proteins are highly and widely expressed during embryonic development ,27, whernd Hmga2 ,26,28,29nd Hmga2 ,31,32, and Hmga2 ,35,36,37 factors . In addiequences . Moreoveequences .HMGA proteins are subjected to many PTMs that regulate their ability to bind DNA and they interact with several other factors; therefore, post-translational regulation is a relevant step contributing to the regulation of their activity .Post-transcriptional regulation is a key process that regulates gene expression, and it is often altered in cancer cells ,43. AlthHMGA genes; (ii) offer a complete overview of HMGA-targeting miRNAs, focusing on those that have been more deeply investigated; (iii) discuss the hypothesis of the mutual influence of HMGA1/HMGA2; and, (iv) highlight the role of HMGA mRNAs as competing endogenous RNA (ceRNA) molecules in the context of cancer initiation and development.A comprehensive overview and critical evaluation of the role of miRNA in regulating HMGA1 and/or HMGA2 protein expression is still missing from the literature, despite this knowledge. In this review, we specifically focus on the HMGA-targeting miRNA network. We (i) provide insights into the structural organization of HMGA1 gene [Analysis of the organization of the GA1 gene  identifiGA1 gene ,180,181 GA1 gene . RemarkaHMGA1 gene. We used Ensembl Genome Browser 96 for this purpose, which collects open-access, integrated genome, gene, variation, gene regulation, and comparative genomic information [Currently, whole-genome sequencing projects, together with transcriptome analysis, can provide a more complete picture of transcripts that originate from a specific locus. Starting from this consideration, we investigated whether new coding transcripts can arise from the human ormation   that separates the final DNA-binding domain from the acidic C-terminal tail. Within the long intron, some additional exons have been described to be part of alternative splicing transcripts [HMGA2 locus  is involved in human haematopoietic stem cell development [The  tumours ,185. Indd intron ,185 and d intron . The HMGnscripts ,188. We A2 locus . In addielopment .More than one hundred miRNAs are involved in the regulation of HMGA mRNA . Among tHMGA2 gene leading to the loss of 3′UTR-mediated let-7 repression could activate HMGA2 expression and lead to cell transformation, anchorage-independent growth [The description of miRNA-mediated HMGA protein regulation begins with the finding that chromosomal abnormalities in the regions 12q15 and 6p21.3 were associated with the aberrant expression of either HMGA2 or HMGA1 in several benign mesenchymal tumours . Invariat growth , and prot growth . Followit growth , thus fuC. elegans, the human let-7 miRNA family includes 13 evolutionarily conserved members  [Unlike in miR-202) ,195 thatmiR-202) . In addimiR-202)  in severmiR-202) , gastricmiR-202)  and non-miR-202) , sarcomamiR-202) ,198, hepmiR-202)  and oesomiR-202) , uterinemiR-202) ,200, andmiR-202) , as a lomiR-202) . MoreovemiR-202) ,204,205.In addition to its role in cancer, the let-7/HMGA2 axis was shown to regulate physiological processes, such as adipose , osteogeThe factors controlling let-7 expression form a regulatory feedback loop with downstream targets of let-7 and are pivotal regulators of stemness or differentiation. The major molecules regulating let-7 are the two RNA-binding proteins LIN28A and LIN28B, , which rThe hsa-miR-26 family includes miR-26a and miR-26b. miR-26a is specifically located on chromosome 3p22, a region that is subjected to the loss of heterozygosity in cancer . In the miR33 gene family and it is located in a non-coding region of the human SREBP-1 gene at the 17p11.2 genomic locus  [The intronic human hsa-miR-33b-5p belongs to the e, HGNC) . miR-33be, HGNC) ,103,104.miR-33b is downregulated in breast tumour and melanoma tissue samples and it is inversely correlated with the clinical stage ,102.Ectopic expression of miR-33b, which affects the levels of HMGA2, SALL4 and Twist1, can decrease the stem cell-like properties of breast cancer cells. Moreover, miR-33b suppresses cell migration and invasion in vitro through decreases in HMGA2 and Twist1 expression . miR-33bmiR-33b modulates pathways controlling the levels of high-density lipoprotein (HDL) cholesterol, triglycerides, and insulin signalling, and negatively regulates adipogenesis ,226. miRhsa-miR-98-5p belongs to the let-7 gene family and it is located at the Xp11.22 genomic locus  [NC site) . SeveralNC site) ,111,112.NC site) . Other aNC site) ,110,111.NC site) ,108,111.NC site) .HMGA2 gene was demonstrated to increase HMGA2 protein expression via a ceRNA mechanism, in which it acts as a sponge for miR-16 and miR-15 [miR-16 is considered to be a critical tumour suppressor miRNA downregulated in many types of cancer, among the validated miRNAs targeting both HMGA1 and HMGA2 . Via a cd miR-15 .SOX2, which is a target gene of HMGA2 and a master regulator of stemness features, thus inducing the suppression of cancer cell pluripotency and tumour cell growth. Therefore, in GBM, miR-142-3p downregulation is required to maintain stem cell-like properties and cell proliferation through HMGA2 upregulation. Metformin, which is a first-line drug for type 2 diabetes mellitus, was proposed to disrupt the sponge effect of MALAT1 on miR-142-3p, enabling miR-142-3p to bind the HMGA2 3′UTR in cervical cancer and inhibit cervical cell invasion and migration [hsa-miR-142 shows pleiotropic functions in physiological processes that are connected with embryonic development and stem cell pluripotency, whereas the alteration of its expression supports the development of cardiovascular disease  and tumoigration . Moreoveigration .Among the 75 miRNAs experimentally explored for their ability to target HMGA mRNA, 26 targeted HMGA1, 64 targeted HMGA2, and 15 targeted both HMGA1 and HMGA2 . We usedA comparison between experimentally validated and bioinformatically predicted miRNAs revealed that of the experimentally validated miRNAs targeting HMGA1 (26), 54% (14) were also predicted by TargetScan and that almost the same percentage  of the 64 miRNAs targeting HMGA2 were also predicted by TargetScan , panel BFor each predicted miRNA, we report in HMGA genes might be linked through a ceRNA mechanism would not be surprising. A bioinformatic search for mRNAs, which can act as ceRNAs that target HMGA1 or HMGA2, showed that HMGA2 mRNA had the highest ceRNA score for HMGA1 (Competing Endogenous mRNA DataBase (ceRDB), https://www.oncomir.umn.edu/cefinder/index.php). [We believe that the shared regulation of HMGA1 and HMGA2 by a set of miRNAs implies a degree of interdependence between HMGA1 and HMGA2. Studies have already demonstrated that the expression of HMGA2 could be controlled by the expression levels of the HIF1A 3′UTR via a ceRNA mechanism . Therefoex.php). . Let-7 iHMGA genes is controlled by different pathways/stimuli [While considering that the transcription of the two /stimuli , the posThe power of miRNA regulation depends, in part, on the ability of these molecules to simultaneously target several mRNAs, thus affecting different pathways and inducing a fan-shaped effect. The types of targets and the pathways affected, however, occasionally make defining a miRNA as an oncogene or onco-suppressor difficult, as one miRNA sometimes performs opposing functions. The existence of self-regulating feedback loops or the effect of different cellular backgrounds might explain these dichotomies.http://mirtarbase.mbc.nctu.edu.tw) [An analysis of the miRTarBase database (.edu.tw)  allowed .edu.tw) .On the other hand, some let-7 targets function as molecular sponges that can remove let-7 molecules from other targets, hence functioning as oncogenes. In MYCN-amplified neuroblastoma cell lines, MYCN mRNA levels are exceptionally high and they are sufficient for sponge let-7 . Not onlMirTarBase screening for strongly validated targets shows that miR-26a also affects tumorigenesis, by often acting as a tumour suppressor and targeting genes that regulate fundamental cancer hallmarks, including the histone methyltransferase EZH2 ,247, theValidated, relevant miR-33b targets include PIM1  and XIAPThe strongly validated miR-98-5p targets include EZH2 , IL-6 2, PGRMC1 As miR-16 is considered to be a crucial onco-suppressor, many researchers have investigated the mechanism by which it affects cancer properties and have revealed that it is involved in targeting numerous genes that are crucial in mediating different cancer hallmarks . Via anamiR-142-3p has been validated to target a wide range of mRNAs . Among tThe abundance of miRNA response elements (MREs) in the HMGA1 and HMGA2 sequences not only makes them susceptible to negative regulation by miRNAs, but can also influence other transcripts or non-coding genes, such as lncRNAs or pseudogenes that share the MREs. Thus, the HMGA1 and HMGA2 mRNAs can reciprocally regulate each other by acting as ceRNAs.HMGA1P6 and HMGA1P7. The conserved seed sequences in miRNAs that target the HMGA1 and HMGA2 mRNAs that are also present in the HMGA1 pseudogenes transcripts can protect HMGA mRNAs from miRNA inhibition; therefore, pseudogene overexpression can sustain cancer cell proliferation and migration and inhibit cell death [HMGA1 pseudogenes upregulate several cancer-related genes [HMGA1P7 transgenic mouse embryonic fibroblasts (MEFs) revealed that HMGA1P7 mRNA induces overexpression of H19 and Igf2 by acting as ceRNA [One of the first lines of evidence indicating that HMGA mRNAs participate in the ceRNA network came from the identification and characterization of two HMGA1 non-coding processed pseudogenes, ll death . Consisted genes , and RNAas ceRNA . Interesas ceRNA .Accumulating lines of evidence reveal that lncRNAs are also critical regulators of HMGA-dependent tumour growth. HOXC13-AS, a lncRNA that is involved in the HMGA2 ceRNA network, was recently identified. It can function as a ceRNA in nasopharyngeal carcinoma to promote the expression of HMGA2 by sponging miR-383-3p, and its silencing induced cell cycle arrest and apoptosis, consistent with the inhibition of the pro-proliferative function of HMGA2 .HMGA2, among the most highly upregulated lncRNAs. RPSAP52 acts as an endogenous sponge, which protects HMGA1 and HMGA2 from miR-15a-, miR-15b-, and miR-16-mediated inhibition. RPSAP52 modulates HMGA expression and promotes cell proliferation by acting as a decoy for miRNAs and, most likely, redirecting them to other genes by interacting with RNA-binding proteins to alter the local DNA conformation [HMGA2 transcription [Moreover, a very recent analysis of lncRNA expression in gonadotroph adenomas revealed the RPSAP52 gene, which is the head-to-head natural antisense transcript of ormation . Intriguormation  and can cription . Table 2C. elegans [Hmga2 transcript was selected as a case study, and regulatory sequences in its 3′UTR were systematically identified [circNSUN2, by interacting with a short sequence within an ARE in the 3′UTR of HMGA2 mRNA, plays a critical role in promoting the formation of a ternary complex with the RNA-binding protein IGF2B2 that stabilizes HMGA2 mRNA to promote colorectal liver metastasis [As the discovery of miRNA-mediated post-transcriptional regulation of RNA in  elegans , this me elegans  and exte elegans  suggestsentified . A high-tastasis .In addition, the RNA-binding protein IMP3 has been found to form cytoplasmic granules that contain HMGA2 mRNA and other let-7 targets, including LIN28B, and to stabilize HMGA2 by opposing the action of let-7 on HMGA2 mRNA. This effect was not achieved by direct competition between IMP3 and let-7, but by providing HMGA2 with an IMP3-dependent, membrane-less cytoplasmic domain  that is devoid of RNA-induced silencing complex (RISC) pathway components .Accumulating evidence indicates not only the importance of miRNAs as molecular players in tumorigenesis, but also their valuable diagnostic and therapeutic applications. The term “exo-miRNAs” has been coined to describe miRNAs that are stored in cellular exosomes and released into biological fluids, where they can be easily detected due to their high stability to provide more precise diagnoses and more personalized therapeutic strategies .Among the HMGA-targeting miRNAs, the let-7 family members undoubtedly have the greatest clinical relevance, even though some of the data are contradictory and need further investigation . Let-7 eAn interesting application is in the monitoring of patients during therapy for cancers, such as lung cancer and acute promyelocytic leukaemia, since reduced let-7 plasma levels are considered to be an indicator of relapse .On the other hand, the HMGA-targeting miRNAs are also attractive for their potential applications in cancer therapy, acting as drug targets or as off-the-shelf drugs.Accordingly, some anticancer drugs have been shown to act through let-7 activation/induction, and their use has been proposed alone or in combination with other treatments. The pan-deacetylase inhibitor panobinostat, for example, affects liver cancer cell lines by inducing the let-7b-mediated downregulation of HMGA2 . SimilarEven more fascinating is the potential administration of let-7 mimics as a next-generation treatment for cancer . In thisIn addition to let-7, which appears to be a dominant miRNA that is involved in controlling HMGA proteins, particularly HMGA2, a knotty miRNA network clearly controls the regulation of HMGA protein expression. Obviously, each miRNA has been separately studied, but it is reasonable to assume that the final effect on the modulation of HMGA protein expression is achieved by the cooperation of multiple miRNAs and it is not necessarily a simple additive effect. In addition, the inverse mechanism is intriguing, i.e., the possibility that HMGA transcripts act as ceRNAs. This aspect, together with the observation that miRNAs have multiple targets, suggests that small changes in one factor could lead to connected and amplified biological outcomes, but the quantitative prediction of these effects is currently not possible.In conclusion, HMGA post-transcriptional regulation mechanisms appear to be much more complex than expected, and this field will certainly experience strong development in the future, with new miRNA and other ncRNA involved in HMGA regulation.


Skin Cutaneous Melanoma (SKCM) is a tumor of the epidermal melanocytes induced by gene activation or mutation. It is the result of the interaction between genetic, constitutional, and environmental factors. SKCM is highly aggressive and is the most threatening skin tumor. The incidence of the disease is increasing year by year, and it is the main cause of death in skin tumors around the world. CXC chemokines in the tumor microenvironment can regulate the transport of immune cells and the activity of tumor cells, thus playing an anti-tumor immunological role and affecting the prognosis of patients. However, the expression level of CXC chemokine in SKCM and its effect on prognosis are still unclear.Oncomine, UALCAN, GEPIA, STRING, GeneMANIA, cBioPortal, TIMER, TRRUST, DAVID 6.8, and Metascape were applied in our research.The transcription of CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL13 in SKCM tissues were significantly higher than those in normal tissues. The pathological stage of SKCM patients is closely related to the expression of CXCL4, CXCL9, CXCL10, CXCL11, CXCL12, and CXCL13. The prognosis of SKCM patients with low transcription levels of CXCL4, CXCL9, CXCL10, CXCL11, and CXCL13 is better. The differential expression of CXC chemokines is mainly associated with inflammatory response, immune response, and cytokine mediated signaling pathways. Our data indicate that the key transcription factors of CXC chemokines are RELA, NF-κB1 and SP1. The targets of CXC chemokines are mainly LCK, LYN, SYK, MAPK2, MAPK12, and ART. The relationship between CXC chemokine expression and immune cell infiltration in SKCM was closed.Our research provides a basis for screening SKCM biomarkers, predicting prognosis, and choosing immunotherapy. Skin Cutaneous Melanoma is a common skin tumor caused by abnormal hyperproliferation of melanocytes . Its incChemokines are a superfamily composed of small molecules of cytokine-like proteins . CXC cheIn SKCM there are many updated reviews on molecular factors important for biology, drug targeting, and prognosis  –13. HoweHence, the focus of this research is to find biological targets from CXC that can be used as the diagnosis and treatment of SKCM. In addition, we hope to discover the specific molecular mechanism of CXC affecting prognosis of SKCM through bioinformatics analysis.http://www.oncomine.org) is a great tumor gene chip database, which contains multiple functions include finding outliers, predicting co-expressed genes, and analyzing gene expression differences  can effectively analyze and mine cancer data on-line  is a newly developed interactive web server for analyzing the RNA sequencing expression data of 9,736 tumors and 8,587 normal samples from the TCGA and the GTEx projects, using a standard processing pipeline (GERIA (pipeline . It fillhttps://string-db.org/) is a database that searches for known interactions between proteins and predicts their interactions (STRING (ractions . The dathttp://www.genemania.org) is to predict the interactions between proteins, including Predicted, Physical Interactions, co-location, co-expression, Pathway, Shared protein domains, and Genetic Interactions  integrates data from 126 tumor genome studies  using a deconvolution algorithm from the gene expression profile (TIICs) concluded that tumor-infiltrating immune cells in abundance (TIMER (bundance . Gene exhttps://www.grnpedia.org/trrust/) is a record of transcription factors regulating the relationship database (TRRUST (database . TRRUST database . It inclhttps://david.ncifcrf.gov/summary.jsp)  to help users extract biological information from DAVID 6.8 (ary.jsp) . GO enrihttp://metascape.org) integrates more than 40 bioinformatics databases . The primer sequences of relative genes are provided in 10 SKCM tissues and 12 normal tissues were enrolled in our research to verify the CXC chemokines expression. We obtained consent of patients and ethical approval from the Xiangya hospital ethics committee. All procedures were executed by the ethics guidelines and regulations. We extracted total RNA of the tissue by using Trizol reagent . qRT-PCR was performed with SYBRThe expression differences of 16 CXC chemokines between SKCM patients and controls were analyzed in the Oncomine database Table 1.Relationship between 16 CXC chemokines and the pathological stage of SKCM was assessed by GEPIA. The results showed that CXCL4 (P = 0.0349), CXC9 (P = 7.74E-05), CXC10 (P = 0.000105), CXC11 (P = 0.000664), CXC12 (P =0.00989), and CXC13 (P =0.000815) were notably associated with the pathological stage of SKCM Figure 2P = 4.16E-02) and CXCL7 (P = 1.46E-02) level in primary SKCM patients was remarkably higher than metastasis SKCM patients (P = 4.16E-02). However, the expression level of CXCL9 (P = 1.37E-11), CXCL10 (P = 3.04E-03), CXCL12 (P = 5.05E-11), CXCL13 (P = 3.52E-06), and CXCL16 (P =1.18E-04) in patients with metastasis SKCM is notably higher than that of patients with primary SKCM. We concluded that CXCL1, CXCL 7, CXCL 9, CXCL 10, CXCL 12, CXCL 13, and CXCL 16 have obvious differences in expression in patients with primary SKCM and metastasis SKCM. No obvious difference has been seen in the expression levels of CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL8, CXCL11, CXCL14, and CXCL17 in patients with primary SKCM and metastasis SKCM. We also analyzed the expression levels of each gene of the CXC chemokine family in SKCM tissue through the GEPIA website , CXCL9 (P = 0.00024), CXCL10 (P = 2.8E-05), CXCL11 (P =1.9e-05), and CXCL13 (P = 2.7e-05) was remarkably related to longer overall survival in SKCM.We evaluated the worth of different CXC chemokines in SKCM clinical outcomes by using GEPIA. No obvious correlation between the level of CXC chemokine family transcription and disease-free survival time in SKCM patients was found Figure 5). The most common change in these samples was increased mRNA expression. Also, PPI network analysis was performed for CXC chemokines and Strings with different expressions to explore their possible interactions. Several 16 nodes and several 11 edges were acquired from the PPI network  were found to significantly regulated CXC chemokines in SKCM Table 2.P = 3.79E-02). The expression of CXCL2 was positively correlated with Neutrophil cell invasion in SKCM-primary patients . CXCL2 is positively correlated with CD8+ T cells  and Neutrophil cells  infiltrates in SKCM-metastasis patients. CXCL3 is mostly related to Neutrophil cell invasion  in SKCM-metastasis patients (P = 7.16E-03). In SKCM-metastasis, CXCL4 is negatively correlated with CD8+ T cells  and Neutrophil cells  and Dendritic cell  infiltrates. CXCL5 was definitely related to Neutrophil cell infiltration in SKCM-primary patients . In SKCM-metastasis, CXCL5 is positively correlated with invasion of B cells  and Neutrophil cells . The expression of CXCL7 in SKCM-primary was negatively correlated with Dendritic cells infiltration . In SKCM-metastasis patients, CXCL7 is negatively correlated with B cells  and Dendritic cells  infiltrate. CXCL8’s expression was positively correlated with Neutrophil cell infiltration in SKCM-primary patients . In SKCM-metastasis patients, CXCL8 is positively correlated with the infiltration of CD8 + T cells , Macrophage cells , and Neutrophil cells  and Dendritic cells . In SKCM-primary patients, down-regulation of CXCL9 enhanced infiltration of CD8 + T cells , CD4 + T cells , Neutrophil cells , and dendritic cells . In SKCM-metastasis patients, CXCL9 was positively correlated with infiltration of B cells , CD8+ T cells , CD4+ T cells , Macrophage cells , Neutrophil cells  and Dendritic cells . In SKCM-primary patients, CXCL10 was certainly related to infiltration of CD8 + T cells , CD4 + T cells , Neutrophil cells , and dendritic cells . In SKCM-metastasis, CXCL10 was positively correlated with infiltration of B cells , CD8+ T cells , CD4+ T cells , Macrophage cells , Neutrophil cells  and Dendritic cells .Clinical outcome is related to inflammatory response and immune cell infiltration in SKCM patients, in which CXC chemokines are also involved. Timer database was applied to comprehensively quest whether CXC chemokines expression is associated with immune infiltration in SKCM patients Table 4.Similar results were found in CXCL11, CXCL12, CXCL13, CXCL14, and CXCL16. The upregulation of these CXC chemokines enhanced infiltration of B cells, CD8+ T cells, CD4+ T cells, Macrophage cells, Neutrophils cells, and Dendritic cells  were identified as being expression difference between SKCM patient tissues and normal tissues. ONCOMINE database analysis results showed that the expression levels of these six CXC chemokines in SKCM tissues were significantly higher than that in normal tissues. Further validation by clinical samples, the mRNA level of CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL13 was considerably adjusted. One research has pointed out that CXCL1 down-regulation leads to reduced colorectal cell viability, invasion, and proliferation. ude mice . CXCL1 pude mice . Therefoude mice . The up-ude mice . CXCL9 pude mice . High exude mice . The CXCude mice . Overallude mice , 43.The expression differences of CXC chemokines between primary SKCM and metastatic SKCM were explored by using UALCAN database. The result showed that CXCL1 and CXCL7 were higher in the primary SKCM than in the metastatic SKCM. One research indicates that primary melanoma cells might down-regulate the invasion activity of metastatic melanoma cells through CXCL1 signaling . TherefoHelicobacter pylori infection was significantly associated with gastric cancer, EB virus infection can cause nasopharyngeal carcinoma, the human papillomavirus infection can cause cervical cancer or Burkitt lymphoma, etc. . Hunan province also provided financial support in the form of the Human Nature Science Foundation (No. 2018JJ2624).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Selenium is a vital trace element present as selenocysteine (Sec) in proteins that are, thus, known as selenoproteins. Humans have 25 selenoproteins, most of which are functionally characterized as oxidoreductases, where the Sec residue plays a catalytic role in redox regulation and antioxidant activity. Glutathione peroxidase plays a pivotal role in scavenging and inactivating hydrogen and lipid peroxides, whereas thioredoxin reductase reduces oxidized thioredoxins as well as non-disulfide substrates, such as lipid hydroperoxides and hydrogen peroxide. Selenoprotein R protects the cell against oxidative damage by reducing methionine-R-sulfoxide back to methionine. Selenoprotein O regulates redox homeostasis with catalytic activity of protein AMPylation. Moreover, endoplasmic reticulum (ER) membrane selenoproteins  are involved in ER membrane stress regulation. Selenoproteins containing the CXXU motif  are putative oxidoreductases that participate in various cellular processes depending on redox regulation. Herein, we review the recent studies on the role of selenoproteins in redox regulation and their physiological functions in humans, as well as their role in various diseases. Most reactive oxygen species (ROS) are generated as by-products of cellular redox processes, including mitochondrial respiration and are known to be harmful to human health when their cellular levels exceed the physiologically acceptable level. However, moderate ROS concentrations play a crucial role in regulating signal transduction and cellular functions, such as proliferation and differentiation, via protein oxidation . NeverthAntioxidant enzymes such as superoxide dismutase, catalase, and other redox enzymes, including selenoproteins, and low weight antioxidant molecules such as carotenoids, ascorbate, vitamin E, α-lipoic acid, and glutathione (GSH) are essential for maintaining the “steady state” concentration of ROS, which helps to regulate the redox balance and maintain cellular homeostasis. Most functionally characterized selenoproteins have catalytic activities owing to their selenocysteine (Sec) residue and act to neutralize and remove ROS. Therefore, they protect against oxidative stress. Selenium was considered a toxic element for humans and other mammals but is now considered an important trace element, as the benefits of dietary selenium supplementation have been identified . SeleniuSulfur and selenium have similar physicochemical properties as both are members of the chalcogen group and undergo thiol-disulfide exchange reactions in the form of cysteine (Cys) or Sec, respectively . However[Ser]Sec), a secondary stem-loop structure named selenocysteine insertion sequence (SECIS), SECIS Binding Protein 2 (SBP2), and other protein factors [[Ser]Sec synthesis, selenium can be intaken from dietary sources, including organic forms such as selenomethionine (Se-Met) and inorganic forms such as selenate and selenite Sec Sec . In euka factors ,26. This factors .Trsp deletion leads to oxidative stress, early-onset intraepithelial neoplasia [Trsp knockout (KO) mice showed that p53 and BRCA1 expression changed, resulting in enhancing susceptibility to cancer [Ka values of cysteines, configuring proteins for a particular redox function [Selenoproteins are essential for survival in many organisms, including humans. For example, prostate epithelium-specific selenocysteine tRNA gene eoplasia , and eareoplasia . Moreoveo cancer , which io cancer . SelP hao cancer . Over hao cancer ; its como cancer , becausefunction . Alterinfunction . The Trxfunction ,36,37,38Glutathione peroxidase (GPx) is an intracellular antioxidant enzyme mainly protects the organism against oxidative stress by catalyzing the reduction of hydrogen peroxide, lipid hydroperoxides, and organic hydroperoxides to water or corresponding alcohols, using GSH as an essential cofactor . It has 2O2 or other oxidants, which results in selenenic acid (GPx-SeOH) formation. Then, the GPx-SeOH is converted back to selenol via a two-step process. First, selenenyl sulfide (GPx-SeSG) is produced by the reaction between GPx-SeOH and GSH. Subsequently, the second GSH reduces GPx-SeSG back to selenol. Notably, owing to high levels of oxidative stress or low GSH concentrations, GPx-SeOH may be overoxidized to seleninic acid (GPx-SeO2H) (The selenol in the Sec residue of GPx is oxidized by Hx-SeO2H) .2O2 and lipid hydroperoxide reduction in conjunction with GSH with increased efficiency [2O2 and organic hydroperoxides, including tert-butyl hydroperoxide and cumene hydroperoxide. Its peroxidase activity and expression are affected by Se status [2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and linoleic acid hydroperoxide reduction [2O2, organic hydroperoxides, and lipid hydroperoxides to reduce systemic oxidative stress [Gpx6 gene is a modulator of mutant huntingtin toxicity, and its overexpression can dramatically alleviate both behavioral and molecular phenotypes associated with a mouse model of Huntington’s disease [Mammalian GPx has eight isoforms; of these, GPx1 , GPx2 , GPx3 (plasma), GPx4 (phospholipid hydroperoxide), and GPx6 (olfactory epithelium) contain a Sec residue in the active catalytic site and can, thus, catalyze Hficiency ,53. In cficiency . GPx1, te status . GPx1 loe status , lung [5e status , prostate status , and blae status  cancers.eduction . The maieduction . Florianeduction . Gpx3 ise stress . GPx3 hae stress . GPx3 pre stress ,64,65. Ge stress . GPx4 ise stress . A studye stress . GPx4 ace stress ,70. GPx6e stress , is highe stress . Synthet disease .2O2 [TrxR is an essential component of the Trx system, which comprises Trx, nicotinamide adenine dinucleotide phosphate (NADPH), and TrxR . The Trx2O2 , selenit2O2 , lipid h2O2 , ascorbi2O2 , α-lipoi2O2 ,85,86,872O2 , antibac2O2 , dehydro2O2 , and cyt2O2 . Similar2O2 ,92.TrxRs are ubiquitously expressed enzymes that regulate redox metabolism and play a critical role in protection against malignant transformation. There is increasing evidence that supports the idea that TrxRs inhibit multiple stages of tumor progression, from initiation to growth, invasion, and metastasis ,94,95. I497 and its neighboring cysteine residue Cys496. Moreover, this modified TrxR strongly induces NADPH oxidase activity, which leads to ROS production [The tumor suppressor PTEN negatively regulates the PI3K/AKT signaling pathway, which is pivotal for cell growth and survival. Numerous studies have demonstrated that PTEN catalytic activity is regulated via direct oxidation by ROS ,103. Theoduction ,121.SelR  is an antioxidant enzyme that uses Met to defend cellular macromolecules against oxidative stress. Met is a sulfur-containing amino acid that is readily oxidized to Met sulfoxide by ROS; subsequently, Met sulfoxide reductases (Msr) such as SelR reduce Met sulfoxide back to Met . Met sulSelR expression is regulated by dietary selenium; its mRNA expression level is low in a selenium-deficient diet, but this can be reversed by dietary selenium supplementation . SelR acSelR KO mice exhibit increased oxidative stress in the liver and kidney with exacerbated hepatotoxicity ,136. SelEscherichia coli is regulated by intramolecular disulfide bridge formation between a Cys residue in the activation loop (Cys272) and the Cys residue in the C-terminal region (Cys476), with the latter being replaced by a Sec residue in higher eukaryotes [SelO, the largest protein among the 25 mammalian selenoproteins, is expressed in a variety of organs, such as the brain, heart, liver, kidneys, lungs, and stomach ,143. Unlkaryotes . Using bkaryotes . Recentlkaryotes . SelO plkaryotes   is a single-pass transmembrane protein  that haseduction . An NF-κeduction . Moreoveeduction . This sueduction ,153.2+ mobilization and neuroendocrine secretion. Sec-containing SelT overexpression in PC12 cells was found to increase intracellular Ca2+ concentrations, whereas the Sec-to-Ala SelT mutant overexpression had no effect on Ca2+ release, suggesting that SelT regulates intracellular Ca2+ mobilization via the redox-active Sec residue [Human SelT is a 22 kDa protein localized to the Golgi apparatus and ER and present in the plasma membrane . SelT po residue . SelT ha residue .2+ homeostasis in human skeletal muscle cells [2O2-induced oxidative stress [2+ concentrations via the modulation of Ca2+ channels, followed by Ca2+ release or leaking [SelN is a 65 kDa transmembrane glycoprotein, which is localized to the ER that contains a transmembrane-addressing site in proximity to the EF-hand motif, which is a helix-loop-helix structural motif found in a large family of calcium-binding proteins. . Human Sle cells . Moreovee stress . Notably leaking .Organisms contain an array of defense systems, such as the thiol-dependent antioxidant system, which coordinate to remove ROS and reactive nitrogen species. This review focused on several mammalian selenoproteins, discussing their splicing forms, structures, and relationships with oxidative stress and disease. Although the functions of some selenoproteins still remain unclear, up-to-date research is advancing in the characterization of some of the less known selenoproteins, as well as their role in the development of various diseases as they may act as potential drug targets. Further studies should focus on revealing the detailed molecular mechanisms underlying the functions of selenoproteins, which can further help develop new guidelines for novel therapies in various diseases.


In response to the coronavirus disease 2019 (COVID-19), Saudi Arabia have imposed timely restrictions to minimize the infection spread, lower the risk for vulnerable groups, and reduce the pressure on healthcare services. The effectiveness of these measures has not been assessed comprehensively and, thereby, remains uncertain. Besides monitoring the number of COVID-19 cases diagnosed by molecular assays, the seroprevalence can serve as an indicator for the incidence rate among the general population. This study aimed to evaluate seroprevalence status of all healthy blood donors who attended one of the main largest hospital located in the western region of Saudi Arabia from 1 January to 31 May 2020. The study period covered two months prior to reporting the first COVID-19 case in the country on 2 March 2020. Importantly, it covered the period when “lock-down type” measures have been enforced. Samples were subjected to in-house enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and microneutralization (MN). The sero statuses of all samples were confirmed negative, demonstrating the lack of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among blood donors during COVID-19 lockdown period. This study supports the hypothesis that COVID-19 restrictions have potential for limiting the extent of the infection. The coronavirus disease 2019 (COVID-19) continues to pose a global threat to the human population. The first cases of COVID-19 were reported from Wuhan, China in late December 2019 [Officials in the affected countries have adopted varying strategies to deal with this crisis ,4,5,6,7.Molecular and serological tests are valuable tool for estimating virus circulation among populations ,13. SeroSeveral reports, from different countries, have investigated the seroprevalence status of their population ,20,21,22The Research Ethics Committee (REC) of the unit of biomedical ethics located at the Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia has approved this study (reference no. 487-20).Participants of this study were all healthy blood donors (n = 956) who attended the King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia from 1 January 2020 to 31 May 2020 and were eligible for donation. All blood donors were aware that their samples might be used for research purposes and written consent forms were obtained.® 2 HB, Bloomington, Minnesota, USA) were coated with 100 ng per well of SARS-CoV-2 spike recombinant protein . Following three washes with PBST (PBS containing 0.1% Tween 20), blocking was performed in PBST containing 5% skimmed milk (blocking buffer). Three washes with PBST were performed prior to addition of samples. Sera were diluted at 1:100 in blocking buffer, added, and incubated for an hour at 37 °C. The plates were washed three times with PBST. Secondary antibody  at a dilution of 1:64,000 in PBST was added and incubated for an hour at 37°. Three more washes were conducted. Then, 3,3′,5,5′-tetramethylbenzidine (TMB)  was added for 5 min before stopping the reaction using hydrochloric acid (HCL). The optical density was measured using an Elx 800 bioelisa Reader  at 450 nm (OD450) with OD450 values of ≥0.27 considered to be positive. Under these experimental conditions, the assay provides 100% sensitivity  and 98.4% specificity  [Sera of blood donors were screened for anti-SARS-CoV-2 IgG antibodies using our recently developed in-house ELISA . Microtiositive) . SamplesSamples that tested positive by ELISA were subjected to commercially available CLIA , according to manufacturer instructions.® CRL-1586™) were utilized to propagate and titrate the local SARS-CoV-2 clinical isolate (SARS-CoV-2/human/SAU/85791C/2020) (Genbank accession number MT630432.1). Inactivation of serum samples was conducted at 56 °C for 30 min. Serially diluted samples plus 100 TCID50 of SARS-CoV-2 were added on Vero E6 cells and incubated at 37 °C in 5% CO2 for 3 days. Positive sample had MN titer of ≥1:20.The MN assay, the gold standard for antibody detection, was conducted, as previously described . AfricanGraphPad Prism software version 9  was used for data curation.Despite the proactive response from Saudi Arabia to COVID-19, the MOH officially reported the first case on 2 March 2020. That is over three months from the first case discovered in Wuhan, China, and only a few days before declaring COVID-19 as a pandemic by the World Health Organization (WHO) . Since t24. Out of the 956 samples, 942 samples tested negative (OD450 < 0.27). The OD450 of the remaining 14 samples were ≥0.27 and considered to be positive , February (n = 78), March (n = 48), April (n = 175), and May (n = 101). Overall, these numbers are substantially lower than usually due to the control measures implemented in response to COVID-19 pandemic. Utilizing in-house ELISA, all sera were screened for anti-SARS-CoV-2 IgG antibodies. This assay offers 100% sensitivity and 98.4% specificity when previously evaluated against MN assay positive .Given that our in-house ELISA is a screening tool, those 14 samples tested positive were subjected to two more serological assays (CLIA and MN assay) to confirm their statuses. MN assay is the gold standard for determining the presence of SARS-CoV-2 antibody. All samples were tested “non-reactive, <1” by CLIA A. Their Many countries, including Saudi Arabia, implemented several control measures and restrictions in response to COVID-19 pandemic  3,4,5,6,65,6,8,9Initially, all samples were screened for anti-SARS-CoV-2 antibody by a recently optimized ELISA . This asTo our knowledge, this is the first investigation addressing the seroprevalence status of SARS-CoV-2 among general population. A recent national study conducted on healthcare workers demonstrated an overall seroprevalence rate of 2.37% . The ratIn summary, we have not identified any sero-positive cases among blood donors screened in this study. Our data suggest that COVID-19 restrictions and control measures played an effective role in limiting the initial spread of the infection. It is also indicative of the low herd immunity status during the study period. This observation is interesting as compared with reports from other countries  where high seroprevalence rates have been reported ,21,22. V


Subsequent research has pointed to a significant role of tetrapyrrole biosynthesis in retrograde signaling. Meanwhile, the molecular functions of GUN1, the proposed integrator of multiple retrograde signals, have not been identified yet. However, based on the interactions of GUN1, some working hypotheses have been proposed. Interestingly, GUN1 contributes to important biological processes, including plastid protein homeostasis, through transcription, translation, and protein import. Furthermore, the interactions of GUN1 with tetrapyrroles and their biosynthetic enzymes have been revealed. This review focuses on our current understanding of the function of tetrapyrrole retrograde signaling on chloroplast biogenesis.Chloroplast biogenesis requires the coordinated expression of the chloroplast and nuclear genomes, which is achieved by communication between the developing chloroplasts and the nucleus. Signals emitted from the plastids, so-called retrograde signals, control nuclear gene expression depending on plastid development and functionality. Genetic analysis of this pathway identified a set of mutants defective in retrograde signaling and designated  Meanwhile, fully conjugated (pigmented) porphyrin rings possess photodynamic properties: they can generate reactive oxygen species (ROS), primarily singlet oxygen under light excitation, which cause photooxidative damage and cell death [a, and Chl b. Although they are synthesized in plastids, these tetrapyrroles are widely distributed, with the exception of Chls. Especially, heme is found throughout the cell. In addition to prosthetic groups’ function, tetrapyrroles have been proposed as signaling molecules that control transcription and intracellular signaling. This review focuses on the signaling function of tetrapyrroles on chloroplast biogenesis in Arabidopsis thaliana. We inform interested readers of several comprehensive reviews on the signaling function of tetrapyrroles on other aspects of plant physiology [Tetrapyrroles are involved in various functions critical to whole organisms’ viability, including light absorption, electron transfer, and oxygen binding ,2. Thus,ysiology ,5,6.5 pathway [Glu to form glutamyl-tRNAGlu, a substrate for plastid protein biosynthesis. The following two enzymes, glutamyl-tRNA reductase (GluTR) and glutamate 1-semialdehyde aminotransferase (GSAT), synthesize ALA from glutamyl-tRNAGlu. In particular, the step of GluTR is the rate-limiting step of total tetrapyrrole biosynthesis, the activity of which is controlled by transcriptional and post-transcriptional regulations [Arabidopsis has two paralogous genes for GluTR, HEMA1 and HEMA2. HEMA1 is light-responsive, is actively expressed in green tissues, and contributes predominantly to Chl biosynthesis [In plant cells, tetrapyrrole biosynthesis takes place entirely in the plastid . The fir pathway . In thisulations ,9. Arabiynthesis ,11. ALA 2+ or Fe2+ by Mg-chelatase (MgCh) or ferrochelatase (FC), respectively, directing Proto into the Chl or heme biosynthetic pathways. MgCh consists of three subunits, CHLI, CHLD, and CHLH in plants. In Arabidopsis, CHLD and CHLH are encoded by a single gene, and CHLI is encoded by two isoforms, CHLI1 and CHLI2. CHLI1 is essential for photosynthesis [S-adenosylmethionine MgProto methyltransferase (encoded by CHLM) to form MgProto methyl ester (MgProtoME). MgProtoME cyclase catalyzes the formation of the fifth ring of the tetrapyrrole ring structure, which is further converted to 3,8-divinyl protochlorophyllide a (DV-Pchlide a). DV-Pchlide a is further converted to Pchlide a by DV-Pchlide a 8-vinyl reductase. Pchlide a accumulates in dark-grown angiosperms because the next enzyme, light-dependent NADPH:Pchlide oxidoreductase (POR), requires light to reduce Pchlide a to chlorophyllide a (Chlide a). Depending on the plant species, it is considered that the step of DV-Pchlide a 8-vinyl reductase occurs after POR reaction , which is the prosthetic group of b-type cytochromes and proteins, such as catalase and peroxidase. In these hemoproteins, the heme is noncovalently bound via coordination to the Fe atom by histidine and/or cysteine residues [FC1 and FC2) in Arabidopsis and cucumber, which show differential tissue-specific and development-dependent expression profiles: FC2 is light-dependent and mainly expressed in photosynthetic tissues, whereas FC1 is stress responsive and ubiquitously expressed in all tissues [a and heme c. Protoheme is also substrate for bilins. Heme oxygenase oxidatively cleaves protoheme to biliverdin IX. Then, phytochromobilin synthase converts biliverdin IX to 3Z-phytochromobilin. Finally, 3Z-phytochromobilin is isomerized to 3E-phytochromobilin, which functions as the chromophore for the phytochromes (PHYs) [In the heme branch, FC inserts Feresidues . There a tissues ,20. Somes (PHYs) .a and POR. Once the etiolated seedlings are exposed to light, most Pchlide a molecules in PLBs are immediately converted to Chlide a by POR, and then to Chl a via enzymatic processes [In plant cells, the chloroplast is one of the differentiated states in which plastids have a photosynthetic function . In the rocesses .Plastids originate from a free-living cyanobacterium in a process known as endosymbiosis . A primiThus, for efficient chloroplast biogenesis, communication between the nucleus and the plastids is paramount. The nucleus controls most aspects of chloroplast biogenesis  ,30, whilphotosynthesis-associated nuclear genes (PhANGs) is dependent on the presence of functional chloroplasts [PhANGs [genomes uncoupled (gun) mutants, which have a reduced ability to coordinate this nuclear response to the chloroplast function, were identified through the retention of PhANGs, such as Lhcb gene expression after treatment with norflurazon (NF) [Important insights into biogenic control of retrograde signals have come from the finding that the expression of many roplasts ,36. The  [PhANGs . Subsequzon (NF) . So far,zon (NF) ,37,38,39zon (NF) .gun mutant screening isolated five mutants (gun1 to gun5) that retained the expression of PhANGs after NF treatment [gun2, gun3, gun4, and gun5 are the four mutants of tetrapyrrole biosynthetic genes and encode heme oxygenase, phytochromobilin synthase, and the regulator and the CHLH subunit of MgCh, respectively [The original reatment . gun2, gectively   conditions, type and concentration of inhibitors used, and sensitivity of detection methods (RNA gel blot or quantitative reverse transcription-polymerase chain reaction (qRT-PCR)) in the gun phenotype evaluation (derepression of PhANG expression). In gun mutant screening, NF, an inhibitor of the carotenoid biosynthesis enzyme phytoene desaturase, is mainly used to block chloroplast functions that result in the intense repression of many PhANGs. Inhibition of carotenoid biosynthesis by NF may cause photooxidative stress during the conversion of proplastids to chloroplasts [Arabidopsis seedlings are grown on agar plates containing 1–5 µM NF for 4–10 days under illumination (~100 µmol m−2 s−1) for scoring of the gun phenotype. It is assumed that during growth on the NF-containing plates, free Chl or its precursors accumulate without the concomitant accumulation of carotenoids. In such a situation, ROS (singlet oxygen) are generated, which cause the photooxidative block of chloroplast biogenesis [PhANG expression [gun phenotype. It is sometimes difficult to distinguish whether tested lines are real gun mutants or not, and if the changes in PhANG expression are marginal or rare, but significant.As discussed below, researchers of the retrograde signaling field related to roplasts ,36. In gogenesis ,36. Howeogenesis . It is pogenesis ,43. Lighpression . Furthergun4 and gun5 mutations directly affect MgCh activity, and gun2 and gun3 mutants are unable to metabolize heme that may cause feedback inhibition of GluTR on ALA synthesis. These results led the hypothesis that the first intermediates of the Chl branch, MgProto and/or MgProtoME, function as mobile retrograde signals between the chloroplast and the nucleus. The signaling role of MgProto has been suggested in algae [Chlamydomonas reinhardtii, exogenous treatment of MgProto or MgProtoME induced the nuclear heat shock protein 70 (HSP70A) gene [Cyanidioschyzon merolae, MgProto is proposed to function as a coordinator of the cell cycle from plastid-to-nuclear DNA replication [The in algae . In the 0A) gene . Additiolication .Arabidopsis, the chld [chli1/chli2 double mutant [gun phenotype. Furthermore, higher accumulation of MgProto after NF treatment in the wild type than in the gun2 and gun5 mutants was detected [Lhcb1 expression in leaf protoplasts [Arabidopsis seedlings [PhANG expression [In the chld ,48 and cdetected . A treattoplasts . Subsequeedlings . These opression .Arabidopsis mutants deficient in the MgProto methyltransferase (chlm) [chl27) [gun phenotype. In barley, LHCB expression was greatly reduced in NF-treated seedlings, but no accumulation of MgProto was detected [Arabidopsis did not support any relationship between the levels of MgProto and gun phenotype in several of the gun mutants [Opponents have argued that e (chlm)  and MgPr [chl27)  accumuladetected . Further mutants ,53.Arabidopsis seedlings, the levels of MgProto and MgProtoME increased, peaking 72 h after NF treatment—the profile of which is opposite to that of Lhcb expression in the wild type [Arabidopsis after 3.5 h of treatment [CHLH/GUN5, CHLM, and CHL27 were repressed in 10-day-old Arabidopsis seedlings, which caused a transient increase in the levels of MgProto within 24 h [PhANGs, suggesting photooxidative damage is necessary to exhibit a gun phenotype. Concerning the cytosolic receptor of MgProto, the proteomic analysis identified heat shock proteins, especially in HEAT SHOCK PROTEIN 90 (HSP90), as MgProto-binding proteins [HSP90 genes in the gun5 background, significantly decreased levels of derepression of PhANG expression were observed when compared to gun5 in NF- or MV-treated seedlings [Meanwhile, a transient increase in MgProto that might regulate nuclear gene expression has been proposed ,55,56. Ireatment ,55. Conthin 24 h . Howeverproteins ,55. In Reedlings ,55.PhANGs is observed in the gun mutants after several days on NF-containing plates when the levels of MgProto are quite low, other mechanisms than MgProto signaling are likely to be involved.It is currently unknown how MgProto and/or MgProtoME can be exported from the dysfunctional chloroplast to the cytosol. Since MgProto and MgProtoME contain a fully conjugated ring structure, which has photodynamic properties, deregulated accumulation of these intermediates may cause phototoxicity to the cell . In addiCh. reinhardtii, heme is proposed as a signaling molecule that may substitute for light [Cy. merolae, abscisic acid (ABA) induced heme-scavenging tryptophan-rich sensory protein-related protein (TSPO), resulting in inhibition of the cell cycle G1/S transition [Arabidopsis showed heme-binding properties and was induced by ABA treatment [Arabidopsis TSPO localized to the secretory pathway [An alternative hypothesis is that another metabolite, protoheme (hereafter just “heme”), functions as a positive signal. Heme has been proposed to be a regulatory factor in controlling transcription and intercellular signaling in yeast, animals ,59, and or light , and theor light . In Cy. ansition . Since tansition . It is nreatment . However pathway .gun mutation (gun6-1D) resulting in the overexpression of FC1 has led to a model in which FC1-derived heme mediates plastid signaling [FC1-overexpressing line (gun6-1D) exhibited the gun phenotype, the FC2-overexpressing line did not exhibit gun phenotypes, suggesting increased flux of the FC1-derived heme may act as a signaling molecule that controls the PhANGs [FC1 resulted in the detection of FC1 protein in mitochondria with a concomitant increase in mitochondrial FC activity [fc1 and fc2 by the FC2 and FC1 genes, respectively, confirmed distinct functions of these FC isoforms [FC1 in plastids but not in mitochondria resulted in the gun phenotype, supporting the role of FC1-derived heme as a plastid-derived retrograde signal [gun phenotype. Since FC1 and FC2 are colocalized to the plastid, the importance of clarifying the precise localization of FC1 and intracellular heme trafficking mechanism is emphasized [Arabidopsis and Cy. merolae [A dominant ignaling . Althouge PhANGs . In plane PhANGs ,66, althe PhANGs . In tobaactivity . A functactivity . Partialisoforms . The ovee signal . Howevere signal ,42 and fe signal  heme didphasized . Proteom merolae . Furtherabi4 does not show a gun phenotype [ABSCISIC ACIC INSENSITIVE 4 (ABI4) is assumed to be the transcription factor that mediates retrograde signaling . Actuallhenotype ,77,78,79henotype . Howeverhenotype . From thhenotype ,82.gun mutant phenotype identified multiple alleles of the blue light photoreceptor cryptochrome 1 (CRY1). They also suggested a role for the red-light photoreceptor PhyB and the transcription factor ELONGATED HYPOCOTYL 5 (HY5) [Lhcb1 expression, because HY5 was converted from a positive to a negative regulator [gun1 cry1 and gun1 hy5 synergistically attenuated the plastid regulation of PhANG expression and chloroplast biogenesis, consistent with the integration of light and plastid-to-nucleus signaling [Contrastingly, it looks promising that biogenic retrograde signaling is closely linked to light signaling ,83. Scre 5 (HY5) . HY5 is  5 (HY5) . In the  5 (HY5) . When chegulator . Meanwhiignaling .GLK genes in pairs (GLK1 and GLK2). In Arabidopsis, GLK1 and GLK2 are functionally equivalent, and only the double knockout mutant (glk1 glk2) showed perturbed chloroplast development [PhANGs during chloroplast biogenesis [PhANG expression [Arabidopsis roots, it has been proposed that a combination of HY5 and GLK1/2 is crucial to the coordinated expression of PhANGs and key tetrapyrrole genes [gun phenotype [GOLDEN2-LIKE (GLK) also contributes to the retrograde signaling. Many plant species have elopment . GLK1/2 ogenesis ,87,88 anogenesis . GLK1 isogenesis , one of ogenesis . Currentpression . It is dpression ,92. Signpression . Based ole genes . As the henotype . When chhenotype .gun 2 to 6 related to the tetrapyrrole biosynthetic pathway, gun1 encodes a chloroplast protein containing a pentatricopeptide repeat (PPR) protein with a C-terminal small MutS-related (SMR) domain. Since gun1 can also prevent down-regulation of PhANG expression after treatment with lincomycin (Lin), an inhibitor of plastid translation [gun1 was shown to be hypersensitive to Lin or NF [Arabidopsis, suggesting the promiscuous nature of the GUN1 protein [Unlike mutant lines nslation , GUN1 hain or NF ,97,98. Sin or NF  and SMR in or NF  domains in or NF . Subsequin or NF ,102,103.in or NF  may corrin or NF . The bin protein . Althoug protein ,106, and protein , all of  protein ,109,110  protein ,107  exhibited granular fluorescence colocalizing with pTAC2, a component of transcriptionally-active complexes [Arabidopsis GUN1–GFP line [Arabidopsis lines [Concerning the localization of GUN1 in the plastid, it was first suggested that GUN1 localizes in nucleoids where plastid DNA is actively transcribed. Transiently expressed GUN1–GFP in tobacco , a member of the so-called plastid RNA editosome, and regulate plastid RNA editing ,115. Comreatment . Target reatment ,115.gun1 mutation genetically interacts with the mutations of these genes. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level [gun1 mutation aggravated the effects of decreased FUG1 levels on chloroplast protein translation [GUN1 interacts with several ribosomal subunits, such as the plastid-encoded ribosomal proteins S1 (PRPS1) and the nucleus-encoded plastid ribosomal protein L10 . The gunin level . Moreovein level . This suin level . The gunnslation . Based onslation .gun1, the reduced accumulation of NEP-dependent transcription of Tic214 [cphsc70-1 showed a gun phenotype in NF-treated, but not in Lin-treated, seedlings. The accumulation of precursor proteins in gun1 was confirmed by independent analysis, which accommodates the higher cytosolic HSP90 and HSP70 accumulation [PhANG expression and proposed to be directly involved in the development of the gun phenotype in the gun1 mutant [hsp90 mutants showed reduced derepression of the gun phenotype [GUN1 has been proposed to be involved in the regulation of protein import into the plastid ,97,107, henotype ,55, HSP9henotype  and GLK1henotype ,89.gun phenotype is not seen for other mutants with reduced NEP transcription, such as sca3 defective in RpoTp enzyme, [ppi1) encoding TOC33, toc75-III-3, and tic40-4 [ppi2) defective in Toc159 revealed the accumulation of several plastid pre-proteins in the cytosol with concomitant upregulation of HSP90.1 protein, but PhANG expression is largely repressed in this mutant [One critical point of this model is that a  enzyme,  or chlor tic40-4 . Proteoms mutant ,117. Thegun phenotype was observed in the gun1-1 gun4-1 and the gun1-1 gun5 double mutants relative to the single mutants [gun phenotype of a double mutant gun5 gun1-9, a nonsense allele of GUN1, was found to be indistinguishable from gun1-9, suggesting the tetrapyrrole signal acts upstream of GUN1 [gun1 sig2 relative to sig2 supported the interaction between these two plastid-to-nucleus signaling mechanisms [CHLD, the other three genes are not light responsive, but involved in the common pathway and the heme branch [At first, a synergistic enhancement of the  mutants , indicat of GUN1 . Transcr of GUN1 . The incchanisms . A direcchanisms . Yeast tchanisms  (Figure e branch . It shoue branch .Arabidopsis seedlings, gun1 accumulated more Pchlide a in darkness, while GUN1 overexpressors accumulated less Pchlide a when compared with the wild type [a accumulation in gun1 was also observed without ALA feeding [HEMA1, cannot be properly imported into chloroplasts of the gun1 mutant, which may cause impeded distribution of tetrapyrrole in the mutant [Arabidopsis leaves [It was found that when ALA is fed to etiolated ild type . Such hi feeding . Since t feeding . Further feeding . This mo feeding , or thro feeding . On the e mutant . On the e mutant . Currents leaves .FC1 is highly expressed in primordial tissues [PhANGs, including the key tetrapyrrole biosynthetic genes for massive Chl biosynthesis [It was shown that  tissues . Mutants tissues ,69. Thesynthesis  remainedgun1 seedling development is hypersensitive to sucrose and ABA signaling [gun1 seedlings. From these results, GUN1 is proposed to have roles for sucrose and ABA signaling during initial seedling development [It has been reported that ignaling ,121,122.elopment ,121. Meaelopment . GIP1 waelopment .Glu [As discussed in this article, GUN1 functions as a biogenic retrograde signaling hub by interacting with numerous proteins . AlthougGlu ,127 may PhANG derepression is only obvious when plastids become dysfunctional by NF or Lin treatment. One possibility is that the FC1-derived heme signal cannot reach the nucleus in the wild type, while it can be transferred to the nucleus in the gun1 mutant. Considering GUN1 enhances total tetrapyrrole flow and can bind Proto and metal porphyrins, including heme and MgProto [PhANG induction. In the wild type, Clp-dependent GUN1 degradation is blocked by inhibitors [gun1 mutant, certain upregulation of tetrapyrrole distribution occurs without droplet formation, which may trigger direct transfer of heme to the nucleus (PhANG induction in the wild type and gun1 mutant. Coupled with the results from gun2–6, this hypothesis explains most of the observed phenotypes of the gun mutants.The question is how GUN1-dependent droplet formation is related to the generation of the biogenic plastid-to-nucleus retrograde signal. The GUN1-deficient effect on  MgProto , we hypohibitors . Thus, p nucleus c. Meanwhtau glutathione transferases [Currently, FC1-specific heme is the most prominent candidate as chloroplast mobile biogenic signal. However, the possible involvement of other retrograde signaling pathways that interplay or antagonize the heme signaling cannot be excluded. Since hemoproteins are widely distributed within the cell, heme must be transported from the plastids to the target organelles . Howeversferases ,132, a nPhANG expression is activated. As shown in Arabidopsis [At present, it is unknown whether FC1-derived heme affects the chloroplast processes, such as transcription, editing, translation, and import . Previoubidopsis . TherefoPhANG expression. Live cell imaging using the fluorescent heme sensor [It is possible that such a GUN1-dependent droplet functions as a system, so it will be difficult to dissect the individual pathways. To clarify this hypothesis, in addition to biochemical and molecular biological methods, novel approaches to liquid–liquid phase separation would be beneficial. For instance, live imaging using GEMS  and labee sensor  would ai


Meningioma is a prevalent type of brain tumor. However, the initiation and progression mechanisms involved in the meningioma are mostly unknown. This study aimed at exploring the potential transcription factors/micro(mi)RNAs/genes and biological pathways associated with meningioma.  mRNA expressions from GSE88720, GSE43290, and GSE54934 datasets, containing data from 83 meningioma samples and eight control samples, along with miRNA expression dataset GSE88721, which had 14 meningioma samples and one control sample, were integrated analyzed. The bioinformatics approaches were used for identifying differentially expressed genes and miRNAs, as well as predicting transcription factor targets related to the differentially expressed genes. The approaches were also used for gene ontology term analysis and biological pathway enrichment analysis, construction, and analysis of protein-protein interaction network, and transcription factor-miRNA-gene coregulation network construction. κB/IL6, PTGS2, MYC/hsa-miR-574-5p, hsa-miR-26b-5p, hsa-miR-335-5p, and hsa-miR-98-5p, which are involved in the transcription factor-miRNA-mRNA coregulation network, were found to be associated with meningioma.  Fifty-six upregulated and 179 downregulated genes were identified. Thirty transcription factors able to target the differentially expressed genes were predicted and selected based on public databases. One hundred seventeen overlapping genes were identified from the differentially expressed genes and the miRNAs predicted by miRWalk. Furthermore, NF- The bioinformatics analysis identified several potential molecules and relevant pathways that may represent critical mechanisms involved in the progression and development of meningioma. This work provides new insights into meningioma pathogenesis and treatments. Meningioma accounts for 30% of primary brain tumors, with an incidence of 5 cases per 100,000 individuals, and commonly occurs between 60 and 70 years of age, and this type of cancer originates from the cap cells of the arachnoid layer of the meninges and is usually benign , 5. OccaTranscription factors (TFs) are DNA-binding proteins that inhibit tumors or act as oncogenes  and playMicroRNAs (miRNAs) are short noncoding RNAs composed of 18-25 nucleotides that regulate protein translation of mRNA . Mature Recently, bioinformatics analyses were applied to explore the underlying mechanisms of cancer to identify essential genes, noncoding RNAs, and TFs involved in initiation and progression for further experimental verification. Due to the urgent need for a better understanding of the mechanisms behind meningioma, this study aimed at exploring the potential transcription factors/microRNAs/genes and biological pathways associated with meningioma using bioinformatics approach.http://www.ncbi.nlm.nih.gov/geo). Four series (GSEs) were selected in this study, including the gene expression datasets GSE88720, GSE43290, and GSE54934, as well as the miRNA expression datasets GSE88721. GSE88721 contains 14 meningioma samples and one control sample, while GSE43290 contains 47 meningioma samples and four control samples. GSE54934 contains 22 meningioma samples and We searched several keywords separated or combined, including “meningioma,” “meningiomas,” “expression profiling by array,” and “Homo sapiens” in the Gene Expression Omnibus (GEO) database  were obtained from three datasets, including GSE88720, GSE43290, and GSE54934. The inclusion criteria included http://www.grnpedia.org/trrust/) database was used to predict TFs that regulated DEGs based on the existing literature [http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/miRpub.html) was used to predict the potential target genes of miRNA with validated information [The Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining , including 83 meningioma samples and eight normal tissues, were included in this study. The sva package was used in the following preprocessing to remove the batch effects . P < 0.0235 DEGs . The DEMIL6, CHRDL1, PTGS2, MTHFD2, SLC7A11, ADM, CRISPLD2, ROBO1, FHL2, SLC7A5, MYC, FOSL1, PLLP, HIF1A, and NAV2) , including leukocyte migration, chemotaxis, and taxis . In termThe PPIs with combined scores greater than 0.4 were selected to construct the PPI networks. The entire PPI network was analyzed using MCODE, and the top 5 modules were chosen . FurtherThe different colors represent different functional modules of the PPI network.Meningioma is typically a slow-growing tumor that is derived from the meninges, the membranous layers surrounding nerve tissue, such as the brain and spinal cord , 17. As κB), 4 miRNAs , and 15 key genes , which may play significant roles in meningioma. Among them, five key genes are coregulated by at least two key miRNAs or TFs, including IL6, PTGS2, SLC7A11, CRISPLD2, and MYC. Once the network was constructed, enrichment analysis and PPI analysis were performed to understand the underlying functions of these DEGs. Intriguingly, the key genes IL6, PTGS2, and MYC were also clustered into PPI module 2 or 3. Module 2 genes were enriched in pathways related to cancer, while module 3 genes were enriched in vascular smooth muscle contraction and cytokine-cytokine receptor interaction.In this study, 235 DEGs were identified from three mRNA datasets, including 179 downregulated and 56 upregulated genes. TRRUST databases were used to predict 86 TFs targeting DEGs, with the top 30 enriched TFs selected for analysis. Subsequently, a TF-miRNA-target gene coregulation network was constructed to study the potential molecular mechanisms, and the top 20 nodes ranked by degree were identified, including 1 TF  can inhibit hsa-miR-98-5p. After the inhibition of hsa-miR-98-5p, the efficacy of cisplatin on NSCLC cell lines was enhanced. The findings indicate that hsa-miR-98-5p could be a potential target in clinical cisplatin treatment of NSCLC .Our analysis also predicted MYC, PTGS2, and IL-6 in both the TF-miRNA-mRNA coregulation network and PPI network, with a high degree of interaction. This result indicates that they may act as independent factors associated with meningioma prognosis. Members of the MYC family of protooncogenes are the most commonly deregulated genes in all human cancers. MYC proteins drive an increased cellular proliferation and facilitate multiple aspects of tumor initiation and progression, thereby controlling all hallmarks of cancer . A previκB/hsa-miR-98-5p/IL6 coregulation pair is predicted to be most relevant to the pathogenesis of meningioma. These TFs, miRNAs, and key genes which are related to meningioma may serve as biomarkers for the detection, prognosis, monitoring, and prediction of therapeutic responses in meningioma and may provide a novel direction for further experiments.We constructed a TF-miRNA-mRNA coregulation network to analyze the potential molecular mechanisms of meningioma and identified 1 TF, 3 key genes, and 4 miRNAs. Though the indicated key genes are associated with meningioma, the functions of the miRNAs have not been previously evaluated. The results from the KEGG function analysis show that module genes mainly enrich in the chemokine and cytokine-signaling pathway. Among them, the NF-


Background: There has been no report regarding the clinicopathological features and genetic mutations regarding elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in gastric cancer (GC). Methods: The correlation among EMAST status, microsatellite instability (MSI) status, mutations of common GC-related genes and 16 DNA repair-associated genes, and the clinicopathological features were analyzed. Results: Among the 360 GC patients enrolled, there were 76 (21.1%) with EMAST+ tumors and 284 with EMAST− tumors, and 59 (16.4%) were MSI-high (MSI-H) tumors, and 301 were microsatellite stable (MSS) tumors. Patients with EMAST+ tumors exhibited an earlier pathological T category and had more genetic mutations in the PI3K/AKT pathway, ARID1A and DNA repair-associated genes than those with EMAST− tumors. Patients with MSI-H tumors have more genetic mutations in the PI3K/AKT pathway and DNA repair-associated genes than those with MSS tumors. In the subgroup analysis for MSI-H GC, EMAST+ tumors were associated with earlier pathological T and N categories, earlier TNM stages, higher frequency of DNA-repair-associated genetic mutations, and a better survival rate than EMAST− tumors. Conclusions:PI3K/AKT pathway mutations may play an important role in EMAST+ and/or MSI-H GC. EMAST+/MSI-H tumors seem to represent a different subtype of gastric cancer from EMAST−/MSI-H tumors. Gastric cancer (GC) ranks as the sixth most common cancer and the second leading cause of cancer-related deaths . AccordiIn colorectal cancer, the incidence of EMAST was similar to that of MSI and was approximately 20%–40%; EMAST+ tumors were associated with the MSI-H phenotype and more frequently located in the colon than in the rectum ,6. HowevIn our previous study , we desiAmong the 360 patients, 76 (21.1%) had EMAST+ tumors and 284 had EMAST− tumors; and 59 (16.4%) tumors were MSI-H, and 301 were MSS. According to the EMAST/MSI status, there were 35 EMAST+/MSI-H, 41 EMAST+/MSS, 24 EMAST−/MSS, and 260 EMAST−/MSS tumors.PI3K/AKT pathway and in ARID1A than those with EMAST− tumors. Low expression of MSH3 by IHC staining was not significantly different between patients with EMAST+ tumors and patients with EMAST− tumors (28.9% vs. 27.8%). Patients with MSI-H tended to be older, have a larger tumor size, have more EBV infections, have fewer HP infections, and have more genetic mutations in the PI3K/AKT pathway than those with MSS.As shown in As shown in EXO1, EPCAM, MSH2, TGFBR2, MLH1, MSH3, POLE, AXIN1, AXIN2, and BAX. MSI-H tumors showed a significantly higher frequency of genetic mutations than MSS tumors in EXO1, EPCAM, PMS1, TGFBR2, and BAX, while MSS tumors showed a significantly higher frequency of genetic mutations than MSI-H tumors in CTNNB1.Mutation profiling of DNA-repair-associated genes, using NGS analysis, was performed for 160 patients, according to their EMAST/MSI status. As shown in EXO1, EPCAM, MSH2, MSH6, TGFBR2, AXIN1, and BAX than the other three subtypes. EMAST+/MSS tumors showed a significantly higher frequency of genetic mutations in MSH3 and POLE than the other three subtypes.As shown in p = 0.034) than those with EMAST− tumors. There was no significant difference in the initial recurrence pattern between patients with MSI-H tumors and patients with MSS tumors.Among the 360 patients, 275 patients receiving curative surgery were enrolled in the analysis of initial recurrence patterns. As shown in p = 0.001). Among the initial recurrence patterns, patients with EMAST−/MSI-H tumors were associated with the highest distant metastasis rates compared to the other three GC subtypes, especially peritoneal recurrence.As shown in p = 0.689), or between patients with MSI-H and patients with MSS tumors .The five-year overall survival (OS) rates were not significantly different between patients with EMAST+ and patients with EMAST− tumors . There was no significant difference in five-year OS rates between other GC subtypes.As shown in As shown in For clinicopathological features of MSI-H GC, EMAST+ tumors showed fewer Borrmann type 3 and 4 tumors, less lymphovascular invasion, more lymphoid stroma, earlier pathological T and N categories, and earlier TNM stages than EMAST− tumors.MSH2, AXIN1, and POLD1 than EMAST− tumors.Regarding the mutational profiling in MSI-H GC, EMAST+ tumors showed a significantly higher frequency of genetic mutations in For the initial recurrence pattern and survival analysis for MSI-H GC patients receiving curative surgery, EMAST+ tumors showed less tumor recurrence and a better five-year OS rate than EMAST− tumors.PI3K/AKT pathway mutations were more frequent in EMAST+ and/or MSI-H tumors. Neither EMAST status nor MSI status was an independent prognostic factor. Subgroup analysis for MSI-H GC showed that EMAST+ tumors were associated with more favorable clinicopathological features and better survival than EMAST− tumors, demonstrating that EMAST+ and EMAST− tumors are different entities in MSI-H GC.To the best of our knowledge, this study is the first to investigate the clinical impact of EMAST status on genetic alterations and clinicopathological features in GC. Our results demonstrated that It was reported that EMAST was associated with the loss of MSH3 nuclear expression in colorectal cancer , while nEXO1, EPCAM, MSH2, MSH6, TGFBR2, AXIN1, and BAX than the other three GC subtypes. Our previous study [MLH1, MSH3, MSH6, PMS2, and EXO1 genetic mutations than the other three colorectal cancer subtypes. Comparing the results of the present study and our previous study [MSH6 and EXO1 genetic mutations were higher in EMAST+/MSI-H tumors than other subtypes, in both GC and colorectal cancer. It seems that MSH6 and EXO1 genetic mutations play an important role in gastrointestinal tract cancer with the EMAST+/MSI-H phenotype. Because there have been no reports investigating the differences in DNA-repair-associated genetic mutations among GC patients according to the EMAST/MSI status, our results might provide useful information for future studies in this field. More patients encompassing different races enrolled from different countries and further in vivo and in vitro studies are required to validate our results.In the present study, regarding the 16 DNA-repair-associated genes, EMAST+/MSI-H tumors had a higher frequency of us study  regardinus study  in colorMSH2, AXIN1, and POLD1 compared with EMAST− tumors. Consequently, the higher frequency of several DNA-repair-associated genetic mutations in EMAST+/MSI-H than EMAST−/MSI-H tumors demonstrated that combined EMAST/MSI status may be more promising than MSI status alone for the application of immunotherapy in GC treatment, which was similar to the findings of our previous study in colorectal cancer [Although immunotherapy was approved by the US Food and Drug Administration for MSI-H tumors, the response rate was approximately 30%–40% ,13. The l cancer . For valIn the present study, for MSS GC, the status of EMAST does not correlate with patient prognosis. There are two possible reasons. First, the patient number is limited and the difference is not easy to reach statistical significance. Second, in comparison with the major role of MSI status associated with a better prognosis, EMAST phenotype may play as an additional effect on improved prognosis. Only for MSI-H GC, EMAST+ tumors were associated with significantly more DNA-repair-associated genetic mutations than EMAST− tumors, which may cause immune response and improve patient survival.PI3K/AKT pathway compared with MSS GC, which was similar to our findings  in the EMAST+/MSI-H tumors than in other subtypes. It was reported that upregulation of PI3K/AKT pathway was observed in tumors with mismatch repair deficiency, including MSH2-mutant tumors [AXIN1 protected against tumors via inhibiting the PI3K/AKT pathway [MSH2 and AXIN1 may be involved in the PI3K/AKT pathway and play an important role in both EMAST+ and MSI-H tumors originating from gastrointestinal tract cancer. Further in vivo and in vitro studies are required to investigate the mechanism between EMAST status, MSI status, MSH2, AXIN1, and PI3K/AKT pathway in GC. Our findings might have clinical impact on the targeted therapy for EMAST+ and MSI-H GC.In previous studies ,15, Corsfindings . FurtherS tumors . Our pret tumors . In addi pathway . We specThere are some limitations in the present study. First, it is a retrospective study, and selection bias exists. Second, although significant survival difference was observed between the EMAST+/MSI-H group and EMAST−/MSI-H group, the patient number enrolled in the present study was limited, and more patients are required for the validation of our results. We hope our findings can have a clinical impact on immunotherapy and targeted therapy for GC treatment in the future.The normal and tissue samples of 360 GC patients who underwent curative surgery were obtained from the biobank of our hospital. After surgery, tumor and normal tissues were collected and immediately frozen and stored in liquid nitrogen. The study was approved by the Ethical Committee of Taipei Veterans General Hospital (Number: 2017-12-012CC). The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all study participants.The clinical data, including age, gender, tumor location, TNM stage, differentiation, pathological prognostic features, and follow-up conditions, were prospectively collected. After surgery, patients were followed up quarterly for the first 3 years and then semiannually thereafter. The follow-up examinations included panendoscopy, serum tumor markers , chest radiography, and sonography or computed tomography of the abdomen.DNA samples were extracted from freshly frozen tumors and normal tissues , using the QIACUBE  instrument and dedicated reagents and kits, according to the manufacturer’s instructions.According to international criteria, five reference microsatellite markers were used to determine MSI status: D5S345, D2S123, BAT25, BAT26, and D17S250. The MSI method was the same as that described in a previous report . SamplesAs described in a previous study , five teThe methods for identifying HP and EBV infection were the same as those described in a previous report . HP infep-value <0.05.As described in a previous study , the copTP53, ARID1A, PTEN, PIK3CA, AKT1, AKT2, AKT3, KRAS, and BRAF. Among them, mutations in the PI3K/AKT pathway were identified in at least any one of the following genes: PTEN, PIK3CA, AKT1, AKT2, and AKT3.As described in a previous study , a nine-AXIN1, AXIN2, BAX, CTNNB1, EPCAM, EXO1, MLH1, MSH2, MSH3, MSH6, PCNA, PMS1, PMS2, POLD1, POLE, and TGFBR2.As described in our previous study , we usedTissue sections of 5 µm thickness were deparaffinized, rehydrated, and pretreated with sodium citrate buffer , in a pressure cooker, at 121 °C, for 3 minutes. Immunohistochemical (IHC) staining was performed with the Novolink Poly Detection System , according to the manufacturer’s instructions. The tissue sections were incubated at 4 °C overnight, with MSH3 primary antibody . The samples were developed with DAB chromogen and then counterstained with hematoxylin. The slides were mounted, using DPX Mountant for histology . As defined in previous reports , low MSHp-value of <0.05 was considered to be statistically significant.Statistical analyses were performed by using IBM SPSS version 25.0. Categorical data were compared, using a χ2 test, with Yates correction or Fisher’s exact test. OS was measured from the operation date to the date of death or the final follow-up. DFS was defined as the length of time after surgery during which a patient survived without GC recurrence. The distributions of OS and DFS were estimated, using the Kaplan–Meier method. Multivariate analysis, using Cox proportional hazards models, was performed to explore the association of the clinical parameters with OS and DFS. A For MSI-H GC, EMAST+ tumors showed a more favorable prognosis and were associated with a higher frequency of several DNA-repair-associated genetic mutations than EMAST− tumors. EMAST+/MSI-H tumors are likely to be a different entity from EMAST−/MSI-H tumors. Combined EMAST/MSI status is recommended for the evaluation of immunotherapy for GC treatment. PI3K/AKT pathway mutations are more frequent in EMAST+ and/or MSI-H tumors than in EMAST−/MSS tumors. Further in vivo and in vitro studies are required to investigate of the correlation of EMAST/MSI status and genetic mutations in DNA-repair-associated genes and the PI3K/AKT pathway in GC. We hope our results can shed light on GC treatment, including immunotherapy and even targeted therapy.