LINC01087 is a recently described long non-coding RNA. Its potential implication within oncology is being increasingly acknowledged. Here, we explored the clinical interest of LINC01087 in the diagnosis and prognostication of multiple cancer types. A series of in-depth in silico analyses revealed LINC01087 as a potential diagnostic indicator of some hormone-related tumors, including breast cancer (BC), ovarian carcinoma, and testicular germ cell tumors, as well as other cancer histotypes, such as esophageal and stomach cancers. Moreover, LINC01087 appears as a prognostic indicator in BC and renal papillary cell carcinoma. Therefore, the quantitation of LINC01087 in tissue specimens might have a clinical utility to improve the management of these pathologies in the future.(1) Background: Long non-coding RNAs may constitute epigenetic biomarkers for the diagnosis, prognosis, and therapeutic response of a variety of tumors. In this context, we aimed at assessing the diagnostic and prognostic value of the recently described long intergenic non-coding RNA 01087 (LINC01087) in human cancers. (2) Methods: We studied the expression of LINC01087 across 30 oncological indications by interrogating public resources. Data extracted from the TCGA and GTEx databases were exploited to plot receiver operating characteristic curves (ROC) and determine the diagnostic performance of LINC01087. Survival data from TCGA and KM-Plotter directories allowed us to graph Kaplan–Meier curves and evaluate the prognostic value of LINC01087. To investigate the function of LINC01087, gene ontology (GO) annotation and Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analyses were performed. Furthermore, interactions between LINC01087 and both miRNA and mRNA were studied by means of bioinformatics tools. (3) Results: LINC01087 was significantly deregulated in 7 out of 30 cancers, showing a predominant upregulation. Notably, it was overexpressed in breast (BC), esophageal (ESCA), and ovarian (OV) cancers, as well as lung squamous cell carcinoma (LUSC), stomach adenocarcinoma (STAD), and uterine carcinosarcoma (UCS). By contrast, LINC01087 displayed downregulation in testicular germ cell tumors (TGCT). ROC curve analyses identified LINC01087 as a potential diagnostic indicator in BC, ESCA, OV, STAD, and TGCT. Moreover, high and low expression of LINC01087 predicted a favorable prognosis in BC and papillary cell carcinoma, respectively. In silico analyses indicated that deregulation of LINC01087 in cancer was associated with a modulation of genes related to ion channel, transporter, and peptide receptor activity. (4) Conclusions: the quantification of an altered abundance of LINC01087 in tissue specimens might be clinically useful for the diagnosis and prognosis of some hormone-related tumors, including BC, OV, and TGCT, as well as other cancer types such as ESCA and STAD. Moreover, our study revealed the potential of LINC01087 (and perhaps other lncRNAs) to regulate neuroactive molecules in cancer. Carcinogenesis is a dynamic process that inexorably and time-dependently perturbs the normal functions of an organism ,2. BesidMGMT) gene is recognized as a potential prognostic/predictive marker in glioblastoma [BCAT1) in plasma may distinguish between lung disease and healthy individuals [MEG3) lncRNA predicts poor survival in breast cancer (BC) patients [Progress in high-throughput technologies contributed to the identification of new molecules, referred to as biomarkers, such as gene sequences, proteins, and metabolites, which are quantifiable in a variety of human samples  ,4,5,6. Tblastoma . Moreoveividuals . Recentlividuals . Non-codividuals ,19,20,21ividuals ,23,24. Tividuals ,26. Moreividuals ,27. For ividuals ,29,30. Ipatients . Additiopatients ,33. Notapatients . Furtherpatients .+/Mn2+ dependent 1E (PPM1E) [The long intergenic non-coding RNA 01087 (LINC01087) is a recently described lncRNA whose function remains poorly investigated. The contribution of LINC01087 to the pathogenesis of cancer, as well as its pivotal role as a biomarker, has been described only in a few oncological indications  ,37,38,39 (PPM1E) . Indeed, (PPM1E) .Here, we shed light on the value of LINC01087 as an indicator for the diagnosis and prognostication of multiple cancer types. Using bioinformatics methods, we first assessed the level of expression of LINC01087 across several tumors. Then, we validated the diagnostic value of LINC01087 using receiver operating characteristics (ROC) curves analysis. We correlated its altered expression with patient survival and clinicopathological features. Finally, the potential functions of LINC01087 were identified by building co-expression networks and gene ontology (GO) enrichment analyses. Our results revealed that LINC01087 is mainly upregulated in multiple cancers, particularly in BC, esophageal, stomach, and ovarian carcinomas, as well as in testicular germ cell tumors. Additionally, LINC01087 could serve as a prognostic indicator in BC and papillary renal cell carcinoma. Interestingly, in silico analyses revealed a potential role for LINC01087 in carcinogenesis through the regulation of gene products that sense or exchange extracellular molecules.http://www.gtexportal.org/home/datasets, 1 August 2022). To perform the differential expression analysis of LINC01087, we compared the transcriptomic data of primary tumors extracted from TCGA to paired normal tissues  combined with healthy samples of the tissue of origin (extracted from GTEx). The TCGA and GTEx datasets originally comprised 32 and 35 tissues, respectively. However, we excluded several breast cancer and liver subtypes  from both datasets, as well as some organs/tissues with no data (n = 3) from GTEx, resulting in 24 (TCGA) and 27 (GTEx) tissues, reflecting a total of 30 distinct tissues. Of note, only datasets with information about LINC01087 expression were included. Samples with some unassigned values  were removed. Analysis of the differential expression of LINC01087 between normal and malignant tissues was performed using DeSeq2 R/Bioconductor in the 30 types of organs/tissues. LINC01087 was considered as significantly deregulated in cancer when log2 fold change (FC) absolute value ≥ 1.5 and p-value ≤ 0.05 (Wilcoxon test). For each dataset, the differential expression of LINC01087 was calculated based on the dataset median value used as a threshold. The association between the expression of LINC01087 in different tumor types and the clinicopathological parameters  was calculated using Fisher’s exact test. For each dataset, a high versus low discrimination of LINC01087 expression was achieved, using the dataset median value as a threshold.RNA-sequencing (RNA-seq) and clinical data of 24 primary tumors and paired normal tissues were extracted from The Cancer Genome Atlas (TCGA) pan-cancer 2018 online repository using TCGAbiolinks package in R ,24,40. Rhttps://portal.gdc.cancer.gov 15 September 2022) and the Kaplan–Meier Plotter (KM-Plotter) datasets  [p-values. For each TCGA dataset, depending on the number of available samples, patients were subdivided into two groups according to the median expression level of LINC01087: high or low. A p-value ≤ 0.05 was considered statistically significant.To evaluate the effect of LINC01087 on the overall survival (OS) across patients affected with various cancer histotypes, we used the pan-cancer RNA-seq data from TCGA (ly 2022) ,42. In Kp-value ≤ 0.05 were applied as a cut-off criterion to consider LINC01087 as a good diagnostic biomarker. For each dataset, a high versus low discrimination of LINC01087 expression was considered based on the dataset best cut-off value.ROC curves were generated and the area under the curve (AUC) was calculated using the R packages MASS and pROC ,44. All p-value < 0.05. Volcano plots and Venn diagrams were drawn using EnhancedVolcano and VennDiagram R packages, respectively. In the second step, we used the web tool DIANA-LncBASE v3  to identify candidate miRNA targets of LINC01087 [https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/index.php, 20 October 2022) to predict mRNA targets of the miRNAs derived from both analyses [For the identification of interactions between LINC01087 and mRNAs and miRNAs, we employed a dual strategy. In the first step, we performed a Spearman correlation test to identify mRNAs and miRNAs that correlated with LINC01087 expression in BC, ESCA, OV, STAD, and TGCT by extracting data from TCGA datasets. We defined significant associations as having a correlation coefficient (R) > 0.4 and a INC01087 . Then, wanalyses . Finallyp < 0.05) in BC, ESCA, STAD, and TGCT tumor tissues derived from TCGA .To investigate the biological pathways and processes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) term enrichment analyses were performed using the clusterProfiler R package on genes significantly associated with LINC01087 expression  in 6 out of the 30 cancers as compared to their normal  tissue counterparts , esophageal carcinoma , lung squamous cell carcinoma , ovarian cancer , stomach adenocarcinoma , and in uterine carcinosarcoma  (p ≤ 0.05) altered expression of LINC01087 was detected in clear cell  and papillary  renal cell carcinomas, hepatocellular carcinoma , lung adenocarcinoma , rectum adenocarcinoma , and skin cutaneous melanoma   . In addi.99e-23) .p = 6.59e-73) (p ≤ 0.05) expressed in acute myeloid leukemia , brain lower grade glioma , pancreatic adenocarcinoma , pheochromocytoma and paraganglioma , and thyroid carcinoma   . Moreove.13e-10) .p = 1.16e-35), as well as esophageal carcinoma , ovarian  and stomach  cancers, and in testicular germ cell tumors  (p< 0.05), AUC values of LINC01087 in clear cell  and papillary renal cell carcinomas , acute myeloid leukemia , lower grade glioma , hepatocellular carcinoma , lung adenocarcinoma , lung squamous cell carcinoma , pheochromocytoma and paraganglioma , skin cutaneous melanoma , and uterine carcinosarcoma   . Further.69e-05) .p < 0.05) and diagnostic performance , when such information was available. These criteria restricted the analysis to BC, ESCA, OV, STAD, and TGCT (p = 0.04) and tumor size (p = 0.01) in BC (p = 0.05) . To address this point, we focused on cancers in which LINC01087 showed both up- or down-regulation  in BC . In TGCT = 0.05) .Altogether, these results imply that the perturbation of LINC01087 expression might play a critical role in carcinogenesis. Moreover, LINC01087 shows great potential as an indicator for the diagnosis of BC, as well as esophageal carcinoma and stomach adenocarcinomas, OV and TGCT. These findings require further experimentation to validate such interest.Subsequently, we examined the prognostic value of LINC01087 by evaluating the relationship between the expression of LINC01087 and patient overall survival (OS). For this purpose, cohorts gathered in the TCGA directory were used for derivation, while inputs of the KM-Plotter database initially served as validation cohorts.p = 2.86E-04) (p = 0.01). Such prognostic interest was validated using KM-Plotter datasets  . Contrardatasets .Further interrogation of the KM-Plotter database revealed that a low abundance of LINC01087 matched an extended OS in HCC, as well as in uterine corpus endometrial carcinoma .p ≤ 0.05) with LINC01087 expression in each of the tissue types where LINC01087 demonstrated a strong diagnostic potential , namely BC, ESCA, OV, STAD, and TGCT  correlations between the expression of LINC01087 and 574 transcripts in BC, 691 in ESCA, 141 in OV, 773 in STAD, and 636 in TGCT . Similarly, in ESCA, among the top 10 genes associated with LINC01087 were pseudogenes and lncRNAs, as well as the neurofilament heavy polypeptide (NEFH) gene, whose deregulation had been associated with the development of several malignancies, such as BC [p ≤ 0.05) correlated with LINC01087; therefore, they were not considered for further analysis. To refine our analysis, we compared the correlating gene sets of every cancer type  for overlapping genes. We identified 2 transcripts that were positively associated with LINC01087 expression in all 5 cancers: the pseudogene POTE ankyrin domain family member K (POTEKP) and the uncharacterized lncRNA AC093838.1 , ESCA (R = 0.95), STAD (R = 0.93), and TGCT (R = 0.98), with the exception of OV, where it showed a moderate yet significant correlation (R = 0.61) (AC093838.1 demonstrated the strongest association with LINC01087 (R = 0.91). The latter transcript also displayed a strong correlation with LINC01087 in the 4 other types of cancer (MED15P4) showed a strong association with LINC01087 in 3 out of the 5 tumor types , and a moderate correlation in ESCA ( = 0.61) . In OV, f cancer . In addi in ESCA .p ≤ 0.05). We identified a total of 35 GO terms subcategorized as follows: 1 “biological process” (BP), 13 “cellular component” (CC) and 21 “molecular function” (MF)  and glutamate ionotropic receptors   . Among td GRIN1) . Of noteand GLRB . GABRG2 ly in OV . Concordly in OV .In summary, this orthogonal investigation revealed that LINC01087 might impact cancer progression by modulating the ability of cells to interact with their environment.LncRNAs participate in carcinogenesis by modulating gene expression via either direct binding to mRNAs, or indirectly by functioning as a competing endogenous RNA (ceRNA) in the cytoplasm. This latter mechanism is characterized by the binding of lncRNAs to miRNAs, thus preventing their interaction with target mRNAs . LINC010p ≤ 0.05) , ephrin A5 (EFNA5), hook microtubule-tethering protein 3 (HOOK3), myozenin 3 (MYOZ3), pleomorphic adenoma gene 1 (PLAG1), DNA Polymerase Iota (POLI), solute carrier family 2member 3 (SLC2A3), small nuclear ribonucleoprotein polypeptide A’ (SNRPA1), as well as two isoleucine/glutamine (IQ) motif genes, namely Purkinje cell protein 4 like 1 (PCP4L1) and IQ motif containing G (IQCG)  .Ultimately, this study led to the construction of a core network of 10 mRNA targets and 28 miRNAs related to LINC01087 .C8orf37 (namely has-miR-644a and has-miR-7-5p), EFNA5 , HOOK3 , MYOZ3 , and PCP4L1 , with 3 miRNAs targeting the mRNAs of SLC2A3  and SNRPA1 , and 4 miRNAs interfering with the transcripts of IQCG , PLAG1 , and POLI . Interestingly, most of these mRNAs and miRNAs have been connected to tumor suppressive and/or oncogenic functions in multiple cancers [In more detail, LINC01087 seemed to interfere with 2 miRNAs targeting the messenger of  cancers ,66,67.Altogether, these results documented a constellation of both protein-coding and translation-interfering transcripts that are directly or indirectly regulated by LINC01087 and that might contribute to the initiation and progression of neoplasms.clinicaltrials.gov (accessed on 1 August 2022)) aim at evaluating their clinical relevance in a variety of malignancies, including bladder (NCT05270174), colorectal (NCT04729855), gastric , head and neck (NCT04946968), kidney (NCT04946266), liver (NCT05088811), and prostate (NCT05141383) cancers. In the present report, bioinformatics analyses uncovered the role of LINC01087 in the diagnosis and prognostication of different human cancers.LncRNAs represent promising epigenetic diagnostic, prognostic, and therapeutic biomarkers . No lessTo examine the clinical interest of LINC01087, we first analyzed its expression across a large array of tumor types using TCGA and GTEx databases, then calculated the ROC scores to assess its diagnostic performance. Among the 30 cancers evaluated, LINC01087 displayed a significant modulation of its expression in 7 of them, mainly hormone-dependent. More precisely, LINC01087 showed an increased expression in BC, OC, and UCS, and an inversely reduced level in TGCT as compared to non-tumor/healthy control samples. Additionally, an elevation of LINC01087 expression was witnessed in LUSC, as well as in two digestive cancers, namely ESCA and STAD.Our results were consistent with the literature regarding the abundance of LINC01087 in BC and ESCA ,37,38,68Out of the 7 cancers harboring an aberrant expression of LINC01087, a diagnostic test revealed promising performances in 5 of them . In detail, the measurement of LINC01087 levels was an excellent (AUC = 1) indicator of malignancy in TGCT, and a fair (0.7 < AUC ≤ 0.8) diagnostic indicator in BC, ESCA, OV, and STAD. Of note, we reported that the measurement of the level of LINC01087 in BC also allowed us to further distinguish between the luminal and triple-negative subtypes .Next, we evaluated the prognostic value of LINC01087 by comparing cancer patient OS according to its level of expression using the cohorts of TCGA and KM-plotter databases for derivation and validation, respectively. LINC01087 deregulation predicted patient outcome in two oncological indications. First, a downregulation of LINC01087 correlated with favorable survival in patients suffering from pRCC. Second, a high expression level of LINC01087 indicated a better prognosis in subjects affected with BC.We proceeded by evaluating the potential functional significance of LINC01087 in the 5 cancer types in which it was most upregulated, namely, BC, ESCA, OV, STAD, and TGCT. At first sight, we screened for genes correlated with LINC01087 expression in the 5 tumor types. Such analysis identified 2 transcripts overlapping in the 5 cancer types, namely POTEKP and AC093838.1. These genes are both located on chromosome 2 but their role in human malignancies has not been elucidated.GABRG1, GABRG2, GABBR2, GABRA2, GABRA6) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) . Based on this information, we speculate that the involvement of LINC010187 in tumorigenesis might be associated with the “neuro-cancer axis” [GABRA1 by binding to miR-181a-5p [Interestingly, GO annotation and KEGG enrichment analyses performed on the genes positively correlated with LINC01087 expression in BC, ESCA, OV, STAD, and TGCT, found a spectrum of genes and pathways associated with the regulation of ion channel activities and neurotransmission. This cluster consisted mainly of genes related to the receptors for GABA  exhibits oncogenic activity, whereas miR-34a-5p and hsa-miR-644a  possess tumor-suppressive properties [GABRA3 [GABRA2 and GABRA6. Furthermore, some miRNAs of the LINC01087 network like miR-34a-5p, miR-197-3p, miR-423-5p, and hsa-miR-3174, were reported as targets of lncRNAs. For instance, miR-34a-5p can be trapped by LINC00665, HOTAIR, or else nuclear paraspeckle assembly transcript 1 (NEAT1) [NEAT1 is also able to target miR-98-5p to promote glioma cancer progression [Some mRNAs and miRNAs of the established LINC01087 network are related to malignant disease ,52,53. Foperties ,77,78,79operties ,86,87,88 [GABRA3 . In an a (NEAT1) ,91,92,93gression . Out of gression ,96,97. Fgression . Moreovegression .PLAG1 was reported as an immunohistochemical marker in pleomorphic adenoma [PLAG1 was found to be upregulated in OV, in which its silencing promoted chemosensitivity [HOOK3 as a predictor of poor prognosis in prostate cancer [HOOK3 as a target of miR-181-5p [POLI predicted poor prognosis in esophageal squamous cell carcinoma (ESCC) patients [Concerning the protein-encoding genes identified in the LINC01087 network, several of them were described as potential cancer-relevant biomarkers. Indeed,  adenoma . Moreovesitivity . Anothere cancer . FurtherR-181-5p . Similarpatients ,69,70. Fpatients . The afoSLC2A3 in ESCA. SLC2A3, also known as glucose transporter 3 (GLUT3), is a member of the SLC2 family, mediating glucose transport across the plasma membrane. The oncogenic potential of SLC2A3 has been extensively investigated. An overexpression or copy number gain of SLC2A3 has been witnessed in liquid and solid tumors [SLC2A3 were associated with unfavorable outcomes in CRC, gastric (GC), and head and neck squamous cell carcinoma (HNSCC) [SLC2A3 seems directly targeted by miRNAs along tumorigenesis [SLC2A3 [SLC2A3, thereby contributing to progression and poor prognosis in glioblastoma patients [SLC28A1, SLC9A3, SLC4A9, and SLC17A8) in luminal and TNBC BC subtypes [Here, we found a high expression of d tumors ,107,108. (HNSCC) ,106,107.igenesis ,110,111.patients . In GC, subtypes . These sEFNA5, a member of the ephrinA ligand family, is deregulated (up or down) in a variety of cancers [EFNA5 mRNA was found to be overexpressed in most tumors compared to normal tissues. Moreover, an association between the mRNA level of EFNA5 and DNA methylation of its promoter were documented for ccRCC, chRCC, pRCC, mesothelioma, prostate cancer, thymoma, and diffuse large B-cell lymphoma. At the protein level, EFNA5 was downregulated in glioblastoma multiforme and ccRCC, but highly expressed in endometrial carcinoma. In addition, a high abundance of EFNA5 in both TCGA and gene expression omnibus (GEO) databases correlated with poor OS in patients with advanced LUAD [ cancers ,115,116. cancers . EFNA5 mced LUAD .SNRPA1, a spliceosome component responsible for processing pre-mRNA into mRNA, showed strong prognostic potential in prostate cancer, HCC, and ccRCC [SNRPA1 in STAD, as well as a positive correlation between LINC01087 and SNRPA1. Interestingly, Chen and colleagues reported the interaction between the lncRNA RMRP  and the protein SNRPA1 as a mediator of TP53 inactivation and as a promoter of cell proliferation in CRC [The gene nd ccRCC ,117,118.nd ccRCC . Here, wn in CRC .IQCG is a protein-encoding gene involved in sperm axoneme assembly. Here, we reported an association between LINC01087, miR-181a-5p, and IQCG mRNAs in cancer. The link between miR-181a-5p and IQCG has previously been observed in endometrial carcinoma using an in silico approach [approach .C8orf37 and PCP4L1 in malignancies. While C8orf37 has been only associated with genetic disorders, the possible involvement of PCP4L1 in CRC and prostate cancer progression requires further confirmation [Conversely, there is limited knowledge on irmation ,123,124.Based on our results and previous observations, we speculate that the deregulation of LINC01087 might be related to the carcinogenesis of BC, ESCA, OV, STAD, and TGCT. However, the mechanisms behind LINC01087 involvement in the development/progression of these malignant diseases remain to be explored experimentally.Since its discovery, interest in and functional knowledge of LINC01087 in the field of oncology have rapidly expanded. Further bench-to-bedside investigations are required to demonstrate the clinical relevance of LINC01087 as a diagnostic and prognostic biomarker.In summary, the deregulation of LINC01087 plays a potential role in the diagnosis and prognosis of different tumor types. Notably, a high expression of LINC01087 exhibits a strong diagnostic value in breast, esophageal, stomach, and ovarian carcinomas. Moreover, a decreased expression of LINC01087 could be used for the diagnosis of malignant testicular germ tumors. LINC01087 also appears to predict the survival of patients suffering from breast and papillary kidney malignancies. Furthermore, the involvement of LINC01087 in cancer development might be related to its direct and indirect (miRNA-mediated) role in modulating biochemical exchanges between the intracellular milieu and the surrounding microenvironment. Future studies must shed light on the biological activity of LINC01087 and confirm its clinical utility as a diagnostic and/or prognostic biomarker. Ultimately, the quantitation of LINC01087 may ease clinical decision-making and improve the management of these oncological pathologies.


High expression of HMGA1 leads to a low survival rate, which is associated with HMGA1, proliferation, apoptosis, angiogenesis, epithelial-mesenchymal transition, and tyrosine kinase.The prognosis of advanced gastric cancer remains poor. Overexpression of high mobility group A 1 (HMGA1) in breast cancer and neuroblastoma indicates a poor prognosis. However, the relationship between HMGA1 expression and gastric cancer development remains unclear. Treatment strategies can be developed by identifying potential markers associated with gastric cancer. We used a constructed tissue array and performed hematoxylin and eosin and immunohistochemical staining. We quantified the staining results and performed statistical analysis to evaluate the relationship between HMGA1 expression and prognosis. HMGA1 expression was related to the expression of Ki-67, caspase3, CD31, N-cadherin, fibronectin, pAkt, and pErk. In the Kaplan–Meier graph, higher HMGA1 expression levels were associated with a relatively poor survival rate ( Helicobacter pylori infection also causes gastric cancer. These patients show reduced expression of the microRNA Let7, which directly regulates high mobility group A (HMGA) 2 [Gastric cancer is a serious disease associated with a poor prognosis. Globally, more than one million people are diagnosed with gastric cancer annually. Moreover, this cancer ranks among the top ten causes of cancer-related deaths ,2,3. Wit(HMGA) 2 . HoweverHMGA protein family consists of four members, divided into the major categories of HMGA1 and HMGA2. HMGA1 can be divided into HMGA1a, HMGA1b, and HMGA1c, whereas HMGA2 has only one encoding gene . MembersAlthough the role of HMGA2 in the metastasis and growth of malignant tumors in the esophagus, stomach, and colorectum has been examined , the rolThis retrospective study of medical records was approved by the Institutional Review Board (IRB) of Cardinal Tien Hospital (IRB number: CTH-101-3-5-054) and the requirement for patient consent was waived. To construct the tissue microarray, we selected 181 patients for whom both tumor and adjacent non-tumor samples were available. Briefly, a tissue microarray was constructed as previously described. All tissue samples were immersed in formalin and embedded in paraffin. Selected tissue samples were sectioned on glass slides and stained with hematoxylin and eosin. After selecting the tumor and non-tumor samples, the area of interest in the paraffin block was perforated using a 2.0 mm cylindrical specimen, and the specimen was inserted into the recipient paraffin block to form a complete tissue array. Finally, 5 mm slices were cut from the complete array blocks and attached to glass slides, followed by histological and immunohistochemical staining.Briefly, the tissue sections were heated at 75 °C for 1 h, and then xylene and alcohol were added at different concentrations for deparaffinization and rehydration, respectively. The tissue sections were processed in citrate buffer (pH 6.0) for heat-induced antigen retrieval at 95 °C for 15 min. The prepared sections were blocked with normal goat serum and incubated with the primary antibody for 1 h at room temperature. After washing with phosphate-buffered saline, the corresponding secondary antibody was added and incubated at room temperature for 1 h. The slides were then washed with phosphate-buffered saline to remove the secondary antibody and horseradish peroxidase-conjugated secondary antibody was added, followed by incubation at room temperature for 30 min. Simultaneously, 0.01% 3,3-diaminobenzidine tetrahydrochloride  was added to develop the immunostaining signal. The time was adjusted according to the color status and maintained under the same conditions for each tissue section. All immunohistochemical-stained slides were reviewed, and the immunostaining intensity was marked as 0 for no staining, 1 for weak staining, 2 for medium staining, and 3 for strong staining . The perThe following primary antibodies were used: HMGA1 , Ki-67 , cleaved caspase-3 , CD31 , E-cadherin , N-cadherin , fibronectin , protein kinase B (AKT)-phosphorylated , extracellular signal-regulated kinase (ERK)-phosphorylated , signal transducer and activator of transcription 3 (STAT3)-phosphorylated , and activated protein kinase (AMPK)-phosphorylated .p < 0.05.All statistical analyses were performed using SPSS software . To compare high and low HMGA1 expression levels, the chi-square test was used to analyze categorical variables. Bivariate correlation was used to determine the relationship between relevant tumorigenesis markers and HMGA1. For overall survival, the hazard ratio probability was calculated based on high or low HMGA1 expression. Finally, a Kaplan–Meier survival plot with the log-rank test was applied. All statistical tests were two-sided, and the results were considered statistically significant at p = 0.015). Sex, degree of differentiation, and cancer stage did not affect HMGA1 expression  with an average age of 71.1 years. We further divided the patients by age as those over and under 65 years. Younger subjects exhibited higher HMGA1 expression , apoptosis (cleaved-caspase 3), angiogenesis (CD31), epithelial-mesenchymal transition , and tyrosine kinase . Pearson correlation analysis revealed that the  results . Represep = 0.04)  . Moreove = 0.04) .We observed that high HMGA1 expression was associated with poor overall survival. Moreover, higher HMGA1expression was associated with a risk of harm, which was 1.78-fold greater than that observed in the low expression group. A previous in vitro study reported that HMGA1 regulates EMT and accelerates the development of gastric cancer . Our tisKi-67 is a common protein and indicator of proliferation . PreviouEpithelial cells differentiate into active mesenchymal cells via a process known as EMT, which promotes tissue fibrosis and tumor progression ,33,34. Ep-value of 0.048. Further studies are needed to confirm the relationship between pErk and HMGA1 expression.Activation of Akt and Erk is often abnormally regulated in cancer; these proteins play important roles in the growth and survival of tumor cells ,41. In sOur study has several limitations. We identified several proteins related to the expression of HMGA1; however, an in-depth assessment of the relationship between these proteins and HMGA1 was not performed. Our findings are similar to those of observational studies and not mechanistic studies. However, analysis of a larger number of human specimens may provide further evidence for the role of HMGA1 in gastric cancer.High HMGA1 expression indicates a poor prognosis for gastric cancer. Moreover, the expression of HMGA1 can affect tumor proliferation, apoptosis, angiogenesis, and N-cadherin and fibronectin expression, possibly through pAkt or pERK. However, additional studies of the underlying mechanisms are needed.


Ceriporia lacerata is an endophytic white-rot fungus that has lignocellulolytic and terpenoid-biosynthetic abilities. However, little is known about the genomic architecture of this fungus, even at the genus level. In this study, we present the first de novo genome assembly of C. lacerata (CGMCC No. 10485), based on PacBio long-read and Illumina short-read sequencing. The size of the C. lacerata genome is approximately 36 Mb . It encodes a total of 13,243 genes, with further functional analysis revealing that these genes are primarily involved in primary metabolism and host interactions in this strain’s saprophytic lifestyle. Phylogenetic analysis based on ITS demonstrated a primary evolutionary position for C. lacerata, while the phylogenetic analysis based on orthogroup inference and average nucleotide identity revealed high-resolution phylogenetic details in which Ceriporia, Phlebia, Phlebiopsis, and Phanerochaete belong to the same evolutionary clade within the order Polyporales. Annotation of carbohydrate-active enzymes across the genome yielded a total of 806 genes encoding enzymes that decompose lignocellulose, particularly ligninolytic enzymes, lytic polysaccharides monooxygenases, and enzymes involved in the biodegradation of aromatic components. These findings illustrate the strain’s adaptation to woody habitats, which requires the degradation of lignin and various polycyclic aromatic hydrocarbons. The terpenoid-production potential of C. lacerata was evaluated by comparing the genes of terpenoid biosynthetic pathways across nine Polyporales species. The shared genes highlight the major part of terpenoid synthesis pathways, especially the mevalonic acid pathway, as well as the main pathways of sesquiterpenoid, monoterpenoid, diterpenoid, and triterpenoid synthesis, while the strain-specific genes illustrate the distinct genetic factors determining the synthesis of structurally diverse terpenoids. This is the first genomic analysis of a species from this genus that we are aware of, and it will help advance functional genome research and resource development of this important fungus for applications in renewable energy, pharmaceuticals, and agriculture. Ceriporia lacerata was identified as a new species that causes white rot in both angiosperm and gymnosperm wood in 2003  present in soil and sediments  and filter paper cellulase activities. Notably, Ceriporia can also synthesize a series of secondary metabolites and has gained attention as a microbial source of biocatalysts for the biotransformation of natural terpenoid products  with other 14 genera based on morphological, physiological, and biochemical characteristics  (rpb1)  , the nuc) (rpb1) , but duehylogeny .C. lacerata strain isolated from the Changbai Mountain in Northeast China and deposited as CGMCC No. 10485, was able to grow rapidly on non-sterilized lignocellulosic substrates , then cultivated for 3.5 days in a rotary shaker at 150 rpm and 25°C.The mycelium of 2PO4 5 g/L, sterilized at 115°C for 30 min) and grown in a rotary shaker at 150 rpm and 25°C for sequential sampling on the 2nd day , day 2 , day 5 day , and day 8   according to the manufacturer’s instruction. PacBio sequencing was performed by Novogene  on a PacBio RS platform with one SMART cell, which generated long-read data. In addition, another run of Illumina sequencing was performed on a NovaSeq 6000 platform (standard 2 × 150 paired-end libraries), which generated short-read data. All the raw data in FASTQ format were processed to get clean reads by removing adapters, N bases, and low-quality bases.™ FastSelect−5S/16S/23S Kit (QIAGEN) to remove rRNA, and cDNA libraries were prepared using the QIAseq™ Stranded Total RNA Lib Kit according to the manufacturer’s instructions. Illumina sequencing was performed on a NovaSeq 6000 platform by Novogene . Raw data were filtered to obtain the high-quality RNA-seq data using the same procedure described above for genomic short-read data. The raw sequencing data (both genomic and transcriptomic) have been submitted to the NCBI Sequence Read Archive under BioProject PRJNA804482 (link).1Total RNA collected at CL1-CL2-CL3-CL4 was extracted using the RNAprep Pure Cell/Bacteria Kit . The integrity of the total RNA was assessed using 1.0% agarose gel electrophoresis. The concentration and purity were determined using a NanoDrop 2000 spectrophotometer. Total RNA (1 μg) was treated with the QIAseqde novo assembled using CANU 2.1 as described previously (Long-read data generated by the PacBio platform were eviously . Brieflyeviously . The geneviously .2 was used for masking repetitive elements in nucleotide sequences of contigs. The genomic structure annotation was performed using BRAKER2 (version 2.1.6) (The RepeatMasker program (version 4.1.1)n 2.1.6) , which an 2.1.6)  and AUGUn 2.1.6)  using RNn 2.1.6)  was usedn 2.1.6)  and RNAmn 2.1.6) , respectn 2.1.6) .3 NR4 and eukaryotic orthologous groups (KOG)5 databases using Diamond 2.0.9    . All proion 4.1) . Similarion 4.1) ,6 which (CAZyme)  were ann(CAZyme) . The pro7 were used to compute orthogroups and construct genome-level phylogenetic trees with single-copy orthologous genes based on Maximum Likelihood (ML) algorithm. FastANI , 20 Gb of Illumina short-read data (∼500 × coverage), and 12 Gb of Illumina cDNA data for the hybrid assembly and genome annotation. The genome was initially assembled using Canu via reads correction, trimming and assembly based on PacBio long-fragment sequences, then polished using Pilon based on Illumina short reads. The final C. lacerata genome assembly contained 58 scaffolds with a total consensus genome size of 36,361,585 bp (∼36 Mb) and GC content of 49.33%, which was comparable to the genome sizes of previously sequenced fungi from the order Polyporales (28∼60 Mb). The maximal scaffold size was 4,415,373 bp, and the N50 scaffold size was 3,409.20 kb. The BUSCO evaluation showed 98.4% completeness of this genome, indicating a high-quality genome assembly  provide excellent information for assessing genomic variation and are thus frequently used as molecular markers for distinguishing even closely related strains. A total of 5,475 repetitive sequences  were identified, accounting for 0.76% of the whole genome. More than 81% of these were simple sequence repeats, followed by transposable elements (TEs), including LTRs, LINEs, SINEs, and transposons . MoreoveC. lacerata genome, 4,215 genes were successfully annotated with 3,170 KEGG Orthologous (KO) terms. These terms were grouped into 42 KEGG BRITE functional hierarchies, including enzymes, membrane trafficking, chromosome and associated proteins, ribosome biogenesis, mitochondrial biogenesis, messenger RNA biogenesis, exosome, DNA repair and recombination proteins, ubiquitin system, and spliceosome  were annotated with GO terms classified as biological process , molecular function , and cellular component , respectively. The top GO terms were protein binding, oxidation-reduction process, ATP binding, nucleic acid binding, zinc ion binding, integral component of the membrane, protein phosphorylation, membrane, regulation of transcription, DNA-templated, carbohydrate metabolic process, nucleus, and host cell nucleus . In the iceosome . Both GOThe KOG database is a eukaryote-specific version of the COG (clusters of orthologous groups of proteins) database. Proteins orthologs typically occupy the same functional niche in different species and refer to proteins evolved from vertical families (speciation) from different species. In this study, a total of 2,721 genes  were annotated with KOG terms and grouped into 23 classes, generally covering the essential functions of metabolism, genetic information processing, environmental responses, and cellular processes. The top KOG terms were R: General function prediction only, O: Posttranslational modification, protein turnover, chaperones, J: Translation, ribosomal structure and biogenesis, C: Energy production and conversion, as well as A: RNA processing and modification .C. lacerata, 1,163 genes were found to encode secretory proteins that contain N-terminal signal peptides and no transmembrane domains, accounting for 8.8% of all proteins across the whole genome. Among them, 527 were annotated with GO terms , aspartic-type endopeptidase activity (GO:0004190), serine-type carboxypeptidase activity (GO:0004185), and proteolysis, serine-type endopeptidase activity (GO:0006508), and metalloendopeptidase activity (GO:0004222). Additionally, a few secretory proteins were involved in a host of diverse and vital biological processes, including calcium ion binding (GO:0005509), zinc ion binding (GO:0008270), signal transduction (GO:0007165), cell surface receptor signaling pathway (GO:0007166), mycotoxin biosynthetic process (GO:0043386), and regulation of cell cycle (GO:0051726), which are related to signal transduction, proliferation, survival, defense, and virulence factors.The “secretome” constitutes the entire set of secreted proteins of a microorganism , includiGO terms . SignifiC. lacerata, most of them were performed using ITS or 18S rDNA sequence alignment  and average nucleotide identity (ANI), provide a more robust and reliable taxonomy and phylogeny due to their greater resolution of evolutionary relatedness between different species . As shower genes . General8Carbohydrate-active enzymes (CAZymes) that can act synergistically on a wide range of glycosidic monomers, oligomers, or polymers in a regio- or stereo-specific manner, are required for the breakdown of carbohydrates, along with lignin and hemicellulose. In general, CAZymes include glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), auxiliary activity proteins (AAs), and carbohydrate-binding modules (CBMs).C. lacerata, encompassing 41.8% GHs, 21.8% GTs, 1.74% PLs, 7.32% CEs, 13.5% AAs, and 13.8% CBMs  and GH9 were more abundant compared with the other species used in this study ] and AA2  ] . Lignin 1.1.16)] . In the  species . AA4 proous PACs .via the mevalonic acid (MVA) pathway and the 2-C-methyl-d-erythritol 4-phosphate/1-deoxy-d-xylulose 5-phosphate (MEP/DOXP) pathway , as well as the typical terpenoid-producing species Ganoderma sinense, Trametes versicolor, T. pubescens, T. coccinea, and T. cinnabarina -ipsdienol, (6E)-8-hydroxylinalool, trans-8-oxolinalool, and (-)-trans-carveol, respectively.9 In pathway 00904 .10 In pathway 00909  and germacrene A (triterpenoid).11According to the KO annotations shown in ay 00904 , 20∼ 26 ay 00909 , 22 ∼ 27In conclusion, the characteristic and fingerprint KOs provide an effective identifier to distinguish the main or branching KEGG pathways and are of great importance for understanding strain-specific terpenoid synthesis enzymes.Ganoderma lucidum, have been employed as natural remedies in traditional Chinese medicine. Purified cell wall polysaccharides and triterpenoids from these fungi offer a wide spectrum of medicinal effects , gelatoporia , core polyporoids , and phlebioids   . In the C. lacerata based on lignocellulosic biomass. Several genes encoding endoglucanases, exoglucanases and β-glucosidases were found in the genome of C. lacerata and assigned to GH families, supporting the potential of this species to degrade cellulose. During lignin degradation, white-rot fungi generally secrete extracellular lignin-modifying enzymes, the best characterized of which are laccases, lignin peroxidases and manganese peroxidases. The characterized AA1 enzymes (laccases) are multi-copper oxidases that use diphenols and related substances as donors with oxygen as the acceptor, while AA2 family enzymes are class II lignin-modifying peroxidases. It was reported that C. lacerata could secrete such enzymes to degrade the softwood of Pinus densiflora , as well as most soft-rot fungi (13∼115), brown-rot fungi (21∼53), and Trametes versicolor (89)  , demonstGanoderma and Trametes .The data presented in the study are deposited in the NCBI SRA repository, accession number XL and WJ conceived and designed the study. PY and YL performed the experimental work. ZM, HL, YM, DH, and HM performed the data analysis. All authors wrote the manuscript and approved the final manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cancer is characterized by increased oxidative stress, an imbalance between reactive oxygen species (ROS) and antioxidants. Enhanced ROS accumulation, as a result of metabolic disturbances and signaling aberrations, can promote carcinogenesis and malignant progression by inducing gene mutations and activating pro-oncogenic signaling, providing a possible rationale for targeting oxidative stress in cancer treatment. While numerous antioxidants have demonstrated therapeutic potential, their clinical efficacy in cancer remains unproven. Here, we review the rationale for, and recent advances in, pre-clinical and clinical research on antioxidant therapy in cancer, including targeting ROS with nonenzymatic antioxidants, such as NRF2 activators, vitamins, N-acetylcysteine and GSH esters, or targeting ROS with enzymatic antioxidants, such as NOX inhibitors and SOD mimics. In addition, we will offer insights into prospective therapeutic options for improving the effectiveness of antioxidant therapy, which may expand its applications in clinical cancer treatment. Howc levels ,7,8.N-acetylcysteine (NAC) and GSH esters [When the high ROS level exceeds non-toxic doses, ROS may cause oxidative damage to macromolecules, such as nucleic acids, proteins, lipids and glucose, resulting in fragmentation of enzymes and structural proteins, membrane damage, gene mutations and even pro-oncogenic signaling activation ,10. IncrH esters ,17, and H esters  and SOD H esters . Some ofH esters .In this review, we will summarize redox homeostasis mechanisms and the relationship between oxidative stress and cancer, providing a detailed description of the rationale for, and recent advances in, antioxidant therapy in cancer. In addition, we also highlight several kinds of antioxidant drugs in pre-clinical and clinical trials, discussing the promise and limitations of antioxidant therapeutic strategies in cancer.•OH), the superoxide radical (O2•−) and hydrogen peroxide (H2O2) [ROS are a class of highly reactive free radicals, such as hydroxyl radical (e (H2O2) ,22. The e (H2O2) ,24. Disr2•−, which can be dismutated to H2O2 [2•− by transferring electrons to molecular oxygen in various subcellular compartments, such as the nucleus [2O2 [2O2 by peroxisomal oxidases in certain mammalian cells [2O2 production in peroxisomes. In addition, a gain-of-function mutation in ACOX1 may further enhance the ROS levels [ROS are prominently generated by transmembrane NOXs and other various oxidases from the mitochondrial electron transport chain (ETC) , endopla to H2O2 ,33. The  to H2O2 . The NOX to H2O2 . They ca nucleus . NOXs-de nucleus . ER is a nucleus ,39. The  nucleus . In resp nucleus ,42,43. I nucleus ,45. As m nucleus ,47. The eus [2O2 . Peroxisan cells . For insS levels . BesidesS levels . UltraviS levels . Cells eS levels . In addiS levels ,54.Increased accumulation of ROS can be eliminated by various enzymatic antioxidant systems including SODs , GSH per2•− into molecular oxygen and H2O2, thus controlling the levels of ROS and limiting their potential toxicity [2•− is dismutated by SODs to form H2O2, which can be decomposed into O2 and H2O by CAT or GPXs [The enzymatic antioxidant system mainly consists of SODs, PRDXs, CAT, PONs and GPXs. Under oxidative stress, these antioxidant enzymes are upregulated or activated to prevent oxidative damage. SODs catalyze the conversion of Otoxicity . Since Stoxicity . SOD1 antoxicity ,64,65. T or GPXs . Several or GPXs ,68. PON2 or GPXs . It was  or GPXs ,71. For  or GPXs .2O2 [2O2. Oxidized TRXs are then reduced by TrxR, with NADPH as a cofactor [Nonenzymatic antioxidants are non-catalytic small molecules that can quench ROS and reduce oxidative stress . The mos2O2 . There a2O2 . TRXs dicofactor . Moreovecofactor . Given tcofactor , thus ma2O2 and O2•−, might function as signaling molecules to cause various signaling pathways to go awry and drive cancer progression [It was demonstrated that oxidative stress is involved in a wide range of pathologies including cancer, and increased production of ROS are common features of cancer cells. Although high ROS levels are cytotoxic and may exert anti-tumorigenic effects via oxidative damage and ROS-dependent death signaling, ROS play critical roles during tumorigenesis and cancer development. Here, we focus on the pro-tumorigenic role of ROS in malignant progression, which may be addressed with antioxidant therapy. The elevated levels of ROS from altered redox homeostasis contribute to the transformation of healthy cells into cancerous cells and enable their survival through two major mechanisms. The first is that ROS directly oxidize macromolecules, such as nucleic acids, proteins, lipids and glucose, resulting in gene mutation and aberrant inflammation . The secgression ,85  or mitochondrial DNA (mtDNA), as well as by causing oxidative damage to biomolecules ,87,88. Ereg) cells and cytotoxic CD8+ T cells in TME can suppress effective tumor immunity and contribute to cancer progression, which is associated with poor response to immunotherapy [2O2 is thought to function as signaling molecules, which might cause metabolic changes in CAFs, such as altered glucose uptake and mitochondrial activity [2•− can suppress T cell-mediated inflammation, thus promoting TAM-mediated immunosuppression and leading to tumor development [In addition to supporting carcinogenesis, ROS were also demonstrated to sustain and accelerate cancer progression via epithelial-to-mesenchymal transition (EMT), which is involved in reprogramming the tumor microenvironment (TME) ,98. The otherapy ,102. In activity ,104. ROSactivity . Furtherelopment .2O2 can dramatically reduce the survival of MCF-7 cells with PRDX1 knockout, showing the potential of pro-oxidants to promote ROS-mediated cell death [Given the important role of ROS in cancer, it follows that modulating ROS levels is a promising anticancer strategy. This may suppress ROS-induced carcinogenesis and cancer progression by inducing oxidative damage and ROS-dependent cell death ,89. Therll death . In addill death , vitaminll death ,110  is the most successful example . It was Il2rg−/− (NSG) mice [NAC is currently one of the most studied antioxidant agents that can be quickly absorbed via the anion exchange membrane and deacetylate to produce cysteine, thus replenishing GSH . NAC canSG) mice . GSH estSG) mice . In addiSG) mice . HoweverAs the most widely used dietary antioxidants, L-ascorbic acid (vitamin C) and α- tocopherol (vitamin E) are of great interest in cancer therapy . Vitamin2•− [2•− and subsequent vascular inflammation in angiotensin II-infused rat model [As mentioned above, the NOX family is a major source of ROS and excessive activation of NOXs can contribute to oxidative stress. Thus, agents that would efficaciously target NOXs to scavenge ROS might hold significant promise for cancer therapy . There a2•− . Moreoveat model ,163. In 2− in vitro and exhibiting anti-inflammatory activity in vivo [Currently, multiple small-molecule global inhibitors that inhibit NOXs or flavoproteins in general, were synthesized, including diphenyleneiodonium (DPI), ebselen and diapocynin . Among t in vivo . In addi in vivo . For ins in vivo . However2•− with a dismutation mechanism. SOD was developed as a drug known as orgotein, to defend against oxidative stress in mammalian cells [SOD is a metalloprotein that can efficiently eliminate Oan cells . The antan cells . It was an cells . In addian cells ,174,175.an cells . Moreovean cells .5+, MnTE-2-pYp5+ and MnTDE-2-ImP5+, have shown high SOD activity that dismutates O2•− to H2O2 [2•− and H2O2, showing protective effects for various types of cancer [Metalloporphyrins have emerged as the most studied SOD mimics, such as Mn porphyrins. Various Mn porphyrin compounds, including MnTM-2-pYp to H2O2 . The pro to H2O2 . The Mn  to H2O2 . In addi to H2O2 . Further to H2O2 . Unlike f cancer .In summary, multiple antioxidant therapeutic strategies were developed for cancer treatment, which can be classified into two different categories of groups according to their targets: enzymatic antioxidants and nonenzymatic antioxidants, both of which have shown potential to act as antioxidant drugs in pre-clinical and clinical research.Because oxidative stress is a well-documented phenomenon in cancer, it is rational that antioxidants can significantly reduce cancer incidence and progression. Although multiple antioxidant therapeutic strategies were explored and some of them are undergoing clinical trial, their efficacy remains unsatisfied. The factors that impede the anticancer activity of antioxidants include: (1) most studies use pharmacological but not dietary doses based on in vitro studies, however, antioxidants may be affected by complex, in vivo conditions; (2) antioxidants might be distributed unevenly in different tissues, and probably cannot function due to the low bioavailability and bio-accessibility in some specific organ; (3) some antioxidants exhibit antioxidant or pro-oxidant properties depending on their concentration and the pressure of oxygen. These factors determine the distinct consequences of the supplementary antioxidants. Moreover, most chemotherapeutic drugs generate high levels of ROS and result in oxidative stress. Treatment with antioxidants in cancer patients might, therefore, lead to an antagonistic effect on chemotherapeutic drug-induced cell death.As discussed above, multiple antioxidants failed to demonstrate efficacy in clinical practice. Given that most antioxidant capacity is attributed to endogenously antioxidant enzymes or antioxidants, we propose that treatment with weak pro-oxidants to boost antioxidant activity might be a promising way for cancer patients, although the underlying biological rationale warrants further investigation and long-term follow-up of interventions are needed. An improved understanding of these mechanisms will facilitate the development of novel therapeutic agents, which might be effective in the prevention and treatment of cancer.


Hairy cell leukemia (HCL) treatment in elderly, frail subjects is still unsatisfactory, and interferons, old-fashioned therapies, can be effectively used in this subset of patients. Here, to the best of our knowledge, we report for the first time an old, frail HCL patient effectively and safely treated with pegylated interferon-α-2a in monotherapy as a first-line treatment. At diagnosis, the patient arrived in a life-threating condition due to severe neutropenia and splenomegaly with high risk of splenic rupture. However, splenectomy was proposed and refused by the patient; therefore, a therapy with pegylated interferon-α-2a was initiated. After six months of therapy, the patient displayed the disappearance of palpable splenomegaly and of peripheral hairy cells at morphological examination without any drug-related adverse event. Our case report supports the use of pegylated interferon-α-2a in monotherapy as an effective and safe alternative therapeutic option in frail, elderly patients not eligible for purine analogous or targeted therapies. BRAF), leading to Ras-independent constitutive activation of the RAF/MEK/ERK pathways and promotion of cell proliferation and survival [Hairy cell leukemia (HCL), a rare, indolent, clonal B-cell disorder, is characterized by the presence of hairy cells with cytoplasmic projections accumulating in the bone marrow (BM), spleen, and peripheral blood, ultimately leading to BM fibrosis, peripheral cytopenia(s), and splenomegaly ,2. Hairysurvival ,6.Historical therapeutic strategies, including corticosteroids, alkylating agents, standard chemotherapy, splenectomy, or recombinant interferon-α, have been replaced by purine analogues, such as cladribine and pentostatin, which have become the first therapeutic choice in fit and young HCL patients ,9,10,11.Interferon-α has been widely used as first-line treatment in the past, with clinical efficacy and safety ,18. To dAn 82-year-old male came under our observation in January 2022 because of progressive pancytopenia and a history of bacterial pneumonia requiring hospitalization and third-generation cephalosporin administration in December 2021. In July 2021, the patient already showed mild thrombocytopenia  and mild neutropenia (white blood cells (WBC), 5900 cells/µL; and absolute neutrophil count (ANC), 1200 cells/µL), with slightly reduced hemoglobin levels (12.7 g/dL). In January 2022, he displayed a worsening of complete blood counts (CBCs) with normocytic anemia , 97 fL); mild thrombocytopenia  and severe neutropenia . At physical examination, his spleen was palpable at the left iliac fossa, and he reported pain and a postprandial fullness sensation at the left hypochondrium. No B symptoms  were referred. Patient’s frailty was evaluated using the frailty index (FI), defined as FI score = Number of health deficits present/Number of health deficits measured, showing a mild frailty  ,20.Morphological analysis was performed on peripheral blood, showing the presence of 50% lymphoid cells with intermediate–high nucleus/cytoplasm ratio, loose chromatin, rare nucleoli, basophil cytoplasm, and peripheral hairy protrusions .+ T lymphocytes; 13% CD19+ B cells with no κ/λ restriction ; 24% CD3−CD56+ natural killer (NK) cells; and 15% CD3+CD56+ NKT cells. CD14+ monocytes were slightly reduced . Flow cytometry immunophenotyping was also performed on peripheral blood, evidencing a B-cell clonal population positive for CD20, CD22, CD25, CD11c, CD103, and CD123 and negative for CD5, CD10, and CD38 . No other alterations were observed, as the remaining 27% of lymphocytes was represented by: 78% CD3BRAF mutation was detected by next-generation sequencing (NGS). BM biopsy confirmed the presence of hairy cells , and HCL diagnosis was made. Total-body CT scan was also performed, and severe splenomegaly , 21 cm; and transversal diameter (TD), 12 cm) was documented. Moreover, large, hypodense, irregular areas within splenic parenchyma were observed, suggestive of initial splenic rupture, supported by the presence of a peri-splenic fluid. Several lymph node enlargements were observed: at hepatic hilum , interaortocaval, and left paraaortic areas  (V600E  1.3 cm) A.Urgent splenectomy was proposed because of high risk of splenic rupture. However, the patient refused surgery, and an alternative therapeutic strategy was quickly decided. Purine analogues were contraindicated, because of recent pneumonia in the setting of severe neutropenia, older age, and comorbidities, including chronic atrial fibrillation, mild heart failure, and type 2 diabetes. Other treatment options were discussed with the patient, who expressed the will to avoid intravenous therapies; therefore, monoclonal antibodies were excluded. Moreover, tyrosine kinase inhibitors (TKIs) were contraindicated because of cardiovascular comorbidities. Consequently, subcutaneous PEG interferon-α-2A was chosen as therapeutic option and was started at a dose of 90 µg once a week with acetaminophen premedication. Antibiotic and antifungal prophylaxis were given with levofloxacin and fluconazole. Growth factors were not administered to favor ANC recovery because of severe splenomegaly and high risk of splenic rupture. + B cells and an increase in CD14+ monocytes . Decreased B cells after treatment might mirror a reduction in certain B cell subpopulations, such as B regulatory cells with immunomodulatory functions, and might be related to physiological immunological reconstitution, as documented in other hematological malignancies, including Hodgkin lymphoma [After three months of therapy, a slight hematological improvement was observed with an increase in hemoglobin levels to 10.4 gr/dL, platelet count to 121,000/µL, and ANC to 830 cells/µL. After six months, hemoglobin levels were normalized (13 gr/dL), and platelets raised to 125,000/µL and ANC to 2230 cells/µL. At physical examination, the spleen was not palpable. No drug-related reactions requiring dose reduction or discontinuation adjustment were documented. Furthermore, no infectious complications were observed. Peripheral blood morphological examination did not show the presence of hairy cells, and the minimal residual disease (MRD) evaluated by multi-parametric flow cytometry was 0.75%. No alterations were observed in the lymphocyte subsets except for a decrease in CD19CT scan re-evaluation was also performed, showing a partial reduction of involved lymph nodes: 23% at hepatic hilum (2 cm vs. 2.6 cm) and 46% of interaortocaval area (0.7 cm vs. 1.3 cm). A significant splenic reduction was documented: 43% reduction from 21 cm of LD at baseline to 12 cm after six months of therapy. Parenchymal hypodensity areas were also reduced, especially those present at the middle third at diagnosis B. DiseasTo date, HCL, a B-cell indolent disease, has been effectively and safely treated with purine analogues in young and fit patients, while the treatment of older and unfit subjects remains unsatisfactory . In our + T cells, macrophages, and NK cells, increase the expression of tumor-associated antigens, and enhance antigen presentation by dendritic cells [+ T cell and NK cytotoxic activity. Therefore, when rituximab is combined with interferon-α-2a during the second phase, tumor cell killing is enhanced likely through antibody-dependent cell mediated cytotoxicity, as demonstrated by the clinical efficacy of interferon-α-2a and rituximab in low-grade lymphomas [The mechanism of action of interferon-α in HCL is still under debate and is likely related to the ability to induce cell differentiation, growth arrest, and apoptosis . Interfeic cells ,25. The ic cells ,27. Moreic cells ,29,30. Iic cells ,29,30,31ic cells . During ymphomas ,33,34. ROur patient arrived under our observation in a life-threating condition due to recent pneumonia in the setting of severe neutropenia and severe splenomegaly with a high risk of rupture. Splenectomy, although not curative, was proposed and refused by the patient. Purine analogues were contraindicated because of the high risk of worsening of clinical conditions and because of the recent pneumonia, severe neutropenia, older age, and comorbidities. Our patient also refused intravenous medications, and monoclonal antibodies, including rituximab, could not be used for induction. Moreover, rituximab was not chosen, because of long-term B-cell deficiency, leading to several infections, including severe coronavirus-19 disease (COVID-19), even though rituximab in monotherapy or in combination with other drugs induces a faster disease remission and hematological recovery with acceptable toxicity ,36,37. OThe pegylated form was chosen because of it has a more favorable pharmacokinetic profile and lower administration frequency than standard interferons, despite the absence of previously published literature showing the efficacy and safety of pegylated formulation in HCL . HematolIn conclusion, we showed that a PEG interferon-α-2A formulation can be effectively and safely used in treatment of unfit, older HCL patients. However, further validation in larger prospective trials is needed.


High-dose Cy showed effects comparable to those achieved by the positive control (silymarin). Severe oxidative stress and inflammatory responses in the liver tissue induced by CCl4 were significantly mitigated by Cy supplementation. The total antioxidant capacity and the activity of superoxide dismutase, catalase, and glutathione peroxidase were increased and the content of malondialdehyde, lipid peroxide, tumor necrosis factor α, interleukin-1β, and interleukin-6 were decreased. Additionally, the Nrf2 and NF-κB signaling pathways, which regulate antioxidative and inflammatory responses, were analyzed using quantitative real-time polymerase chain reaction and western blot assay. Cy treatment not only increased Nrf2 transcription and expression but also decreased NF-κB signaling. Moreover, molecular docking simulation indicated that Cy had high affinity for Keap1 and NF-κB/p65, which may promote nuclear translocation of Nrf2 and inhibit that of NF-κB. In summary, Cy treatment exerted antioxidative and anti-inflammatory effects and ameliorated liver injury by increasing Nrf2 and inhibiting the NF-κB pathway, demonstrating the potential of Cy as a therapeutic agent in liver injury. Acute liver injury has multiple causes and can result in liver failure. In this study, we evaluated the hepatoprotective ability of cyanidin (Cy) and investigated its associated mechanisms. Cy administration significantly and dose-dependently ameliorated acute liver injury induced by carbon tetrachloride (CCl As a critical internal organ, the liver is responsible for multiple physiological functions, including metabolism, detoxification, and immunity. However, the liver is susceptible to damage induced by exogenous materials, such as alcohol, drugs, and other hepatotoxic chemicals, resulting in liver dysfunction ,2. ChemiNumerous studies have shown that acute liver injury is often accompanied by excessive oxidative stress and inflammatory responses . The nucCyanidin  chromenylium-3,5,7-triolis), a naturally occurring flavonoid compound, is found in berries and other fruits . Increas4)-induced mice. Moreover, we measured the antioxidant enzyme activity of SOD, CAT, and glutathione peroxidase (GSH-Px), determined the total antioxidant capacity (T-AOC), analyzed the levels of the oxidative products malondialdehyde (MDA) and lipid peroxide (LPO) and detected the contents of the inflammatory factor tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in Cy-treated mice to assess the antioxidative and anti-inflammatory activity of Cy. Furthermore, the protective mechanism of Cy, including the activation of Nrf2 signaling and inhibition of the NF-κB pathway, was examined using quantitative real-time polymerase chain reaction (qRT-PCR), molecular docking, and western blot analysis. Collectively, our findings indicate that Cy may be a promising therapeutic agent for the safe and effective management of liver injury. Therefore, in this study, we evaluated the hepatoprotective effects of Cy in carbon tetrachloride (CClCy (>98% purity) was purchased from Chengdu Biopurify Phytochemicals Co., Ltd. . Alanine aminotransferase (ALT), aspartate aminotransferase (AST), SOD, CAT, GSH-Px, T-AOC, MDA, and LPO test kits were obtained from Nanjing Jiancheng Bioengineering Institute . Alkaline phosphatase (ALP) and BCA protein detection kits were obtained from Beyotime Biotechnology Co., Ltd. . Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-1β, and IL-6, were purchased from SenBeiJia Biological Technology Co., Ltd. .4 and other chemical reagents were provided by China National Pharmaceutical Group Co., Ltd. .CCl4 (10 mL/kg) in olive oil to induce acute liver injury  were purchased from Gempharmatech Co., Ltd. . All mice were allowed free access to commercial food and sterile water. After acclimation for 7 days, 40 mice were randomly divided into five groups (n = 8): (A) normal control (NC), in which the animals were gavaged with physiological saline; (B) model control (MC), in which the animals were gavaged with physiological saline; (C) Cy-L treatment group, in which the animals were gavaged with Cy (10 mg/kg); (D) Cy-H treatment group, in which the animals were gavaged with Cy (50 mg/kg); and (E) positive control (PC), in which the animals were gavaged with silymarin (50 mg/kg). After pre-treatment with Cy and silymarin for 14 days, all mice, except for those in the control group , were injected intraperitoneally with 0.8% CClr injury . After 1Blood samples were centrifuged  to obtain the sera at 4 °C. The levels of AST, ALT, and ALP were determined using commercial assay kits according to the manufacturer’s instructions. The contents of TNF-α and IL-6 were measured using corresponding ELISA kits.The fixed liver tissues were dehydrated, embedded in paraffin, and sectioned at a thickness of 4 µm. These samples were then stained with hematoxylin and eosin (H&E) staining and photographed using an optical microscope .Apoptosis in liver tissue was further evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Liver sections (4 µm) were dewaxed using water and stained using a TUNEL apoptosis detection kit according to the manufacturer’s instructions . TUNEL-positive cells (green) were detected using a fluorescence microscope . The apoSamples of liver tissue (0.1 g) were homogenized in cold normal saline (1 mL) and then centrifuged  at 4 °C to collect the tissue supernatant. The protein content was measured using the BCA method. The levels of oxidative stress were assessed by analyzing the antioxidant enzyme activities of SOD, CAT, and GSH-Px, determining the T-AOC, and measuring the levels of the oxidative products MDA and LPO, using corresponding kits. Additionally, the levels of inflammatory cytokines  were measured using corresponding ELISA kits.−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as an internal reference.Changes in the mRNA levels of each gene were quantified using qRT-PCR . BrieflyImmunofluorescence staining of the liver was performed to evaluate the levels of TNF-α and IL-6 , and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The stained sections were observed and photographed using a fluorescence microscope. Proteins were extracted form liver segments in each group through RIPA lysis, quantified using a BCA assay kit, and denatured with loading buffer in a boiling water bath. Then, the results were separated by SDS-PAGE and transferred to PVDF membranes. After incubation with primary antibodies Nrf2 (1:1000), NF-κB/p65 (1:2000), p-NF-κB/p65 (1:2000), GAPDH (1:2000) and LaminB1 (1:2000) overnight at 4 °C, the membranes were washed with TBST and incubated with horseradish peroxidase labeled secondary antibodies (1:5000). The protein bands were detected using ECL reagents. https://pubchem.ncbi.nlm.nih.gov/, accessed on 9 March 2022). Those of the target proteins, including NF-κB (PDB code: 1IKN) and Keap1 (PDB code: 4L7B), were downloaded from the RCSB Protein Data Bank . For docking, the ligands were prepared by adding all hydrogen atoms and defining rotatable bonds, and target proteins were applied by extracting the primary ligand, removing water molecules, and adding all hydrogen atoms. The exhaustiveness value was set to 20 to increase the docking accuracy. The best-scoring pose, as determined from the binding energy, was selected, and visually analyzed using PyMOL 2.3.0.Molecular docking experiments were performed to predict the binding mode between small-molecule ligands and biological macromolecules using AutoDock 4.2 . The thrp < 0.05 and ** p < 0.01 were considered the thresholds for significance.All data are represented as individual data points and as the mean ± standard error of the mean. Statistical analysis was performed using SPSS software (version 23.0), and one-way analysis of variance with Tukey’s post hoc test was used for comparisons among multiple groups. All statistical graphs were drawn using Origin software version 2018. * 4. However, preventive administration of Cy remarkably and dose-dependently attenuated the increases in biomarker levels in the MC group. Specifically, in the Cy-H group, compared with that in the MC group, the liver index decreased by 17.1%, and the serum levels of AST, ALT, and ALP decreased by 70.0%, 81.5%, and 84.0%, respectively. These reductions did not significantly differ from those in the PC group . Additionally, the levels of serum TNF-α and IL-6 were used to evaluate the inflammatory response  in the mice are shown in response E,F. Inje4 intoxication. The results of liver H&E staining are shown in 4 stress exposure resulted in an increased number of TUNEL-positive cells in the liver tissue compared to that in the NC group. In the Cy-L, Cy-H, and PC groups, the green fluorescence was less intense, suggesting that cell apoptosis was decreased in the liver tissue, which is consistent with the results of H&E staining. The values of AI were 1.4%, 47.8%, 18.9%, 3.5% and 4.3% in NC, MC, Cy-L, Cy-H and PC groups, respectively. Interestingly, the AI did not significantly differ between the Cy-H and PC groups. The pathological images indicated that Cy ameliorated CCl4-induced liver injury in a concentration-dependent manner.Furthermore, cellular apoptosis in the liver tissue was detected using the TUNEL staining assay, in which cell nuclei exhibited blue fluorescence and fragmented DNA exhibited green fluorescence . As presThe activity of hepatic antioxidant enzymes  in the mice is shown in 4 caused an approximately threefold increase in the levels of MDA (from 3.7 to 9.5 nmol/mg) and LPO (from 9.5 to 31.0 nmol/mg) compared to those in the NC group, indicating the presence of oxidative damage. Conversely, Cy pretreatment prevented abnormal increases in the levels of the oxidation products observed in the MC group. In the Cy-L and Cy-H groups, the MDA levels decreased significantly by 4.2 and 5.6 nmol/mg, respectively, and LPO levels decreased by 7.1 and 20.4 nmol/mg, respectively. The contents of oxidative products in the Cy-H group were not significantly different from those in the NC group.The hepatic levels of the oxidative products MDA and LPO in the five groups are shown in 4 administration resulted in significant inflammation, as demonstrated by the increased levels of hepatic pro-inflammatory mediators. However, compared with the MC group, in which the levels of TNF-α, IL-6, IL-1β were 300.3, 241.9, and 241.9 pg/mg, respectively, the groups pretreated with Cy-L and Cy-H exhibited significantly lower levels of TNF-α , IL-6 , and IL-1β . Interestingly, in the Cy-H group, the levels of hepatic TNF-α, IL-6, and IL-1β were significantly decreased  compared those in the MC group. The decreases observed following Cy-H treatment were slightly lesser in magnitude than those induced by treatment with PC .TNF-α, IL-6, and IL-1β are crucial cytokines that mediate inflammatory responses. As shown in E) and IL-6 (F) in the red channel suggested an excessive inflammatory response in the MC group. Conversely, pretreatment with Cy and silymarin reduced the intensity of red fluorescence, indicating a decrease in inflammatory cytokine levels, which agreed with the results of ELISA. These in vivo findings suggest that Cy mitigated CCl4-induced oxidative stress and inflammation, and this specific mechanism will be investigated in the next section.The levels of TNF-α and IL-6 were further evaluated by tissue immunofluorescence staining analysis, in which blue fluorescence represents the nuclei and red fluorescence indicates inflammatory factors. As shown in The transcription of genes in the Nrf2 antioxidant pathway  in the liver tissue was determined using qRT-PCR A–D. Comp4-induced inflammatory response by suppressing the NF-κB signaling pathway.The relative mRNA levels of genes in the NF-κB inflammatory pathway  in the liver tissue were quantified, as shown in Molecular docking studies are considered a powerful tool for predicting the potential targets of bioactive molecules . The the4-induced toxicity in mice and elucidated its possible mechanism of action. CCl4, an acknowledged hepatotoxin, can attack and damage hepatocytes. CCl4 has been widely employed to establish experimental models of hepatic injury for developing potential treatment strategies [4 caused a remarkable increase in the liver index and in the serum levels of AST, ALT, and ALP, indicating serious liver injury. However, supplementation with different doses of Cy mitigated these increases. In particular, high-concentration Cy had similar effects to the positive control, suggesting that Cy can restore liver function by enhancing cytomembrane stability. The results of pathological observations (H&E and TUNEL staining) corroborated these findings. We analyzed the hepatoprotective activity of Cy against CClrategies . Hepaticrategies . Moreoverategies . In this4-induced liver injury [4 enters hepatocytes, it is metabolized by the cytochrome 450 enzyme family (CYP family), leading to the generation of the trichloromethyl radical (CCl3•) and the trichloromethyl peroxide radical (Cl3COO•). These free radicals, exhibit high reactivity and instability and attack proteins and lipids causing lipid peroxidation and decreasing the levels of cell membrane proteins. The radicals also impair the antioxidant defense system in vivo by depleting reducing agents and inhibiting antioxidant enzymes production. These effects contribute to oxidative stress and induce cell damage and death [Numerous studies have indicated that oxidative stress and inflammatory responses are the main pathogenic factors in CClr injury ,22. In bnd death . Oxidatind death . 2O2) from superoxide radicals, and CAT further degrades H2O2 into water. GSH-Px also plays a role in eliminating peroxides and hydroxyl radicals [4-induced oxidative damage in the liver resulted in decreased antioxidant enzyme activity and increased oxidant production. However, Cy administration restored the antioxidant system in a dose-dependent manner, protecting the liver tissue against CCl4-mediated oxidative stress.Normally, reactive oxygen species (ROS) in organisms are maintained at physiological levels by antioxidant enzymes and non-enzymatic antioxidants in vivo. As important antioxidant enzymes, SOD catalyzes the formation of hydrogen peroxide  seed decreases NF-κB/p65 expression and plasma TNF-α levels and attenuates the metabolic syndrome induced by a sucrose-rich diet [NF-κB, a pivotal regulator of inflammation, usually resides in the cytoplasm in an inactive state. When cells receive inflammatory signals, NF-κB is activated and it translocates into the nucleus, upregulating the expression of pro-inflammatory cytokines  and initiating inflammatory injury . Our expich diet . 4-induced liver injury through antioxidative and anti-inflammatory mechanisms. Specifically, Cy enhanced the activity of antioxidant enzymes , increased the T-AOC, decreased the levels of oxidative products (MDA and LPO), and decreased the levels of pro-inflammatory cytokines . Additionally, Cy, as an inhibitor of Keap1 and NF-κB, enhanced Nrf2 pathway activity by increasing Nrf2 transcription and promoting Nrf2 nuclear translocation, while suppressing NF-κB pathway activity by decreasing NF-κB/p65 expression and inhibiting NF-κB nuclear translocation (Liver diseases are associated with high morbidity and mortality rates worldwide. Our findings suggest that Cy ameliorated CCllocation .


Per) and cryptochrome (Cry) is activated by the heterodimer of the transcription factor circadian locomotor output cycles kaput (Clock) and brain and muscle Arnt-like protein-1 . By binding to E-box sequences in the promoters of Per1/2 and Cry1/2 genes, the CLOCK-BMAL1 heterodimer promotes the transcription of these genes. Per1/2 and Cry1/2 form a complex with the Clock/Bmal1 heterodimer and inactivate its transcriptional activities. Endometrial BMAL1 expression levels are lower in human recurrent-miscarriage sufferers. Additionally, it was shown that the presence of BMAL1-depleted decidual cells prevents trophoblast invasion, highlighting the importance of the endometrial clock throughout pregnancy. It is widely known that hormone synthesis is disturbed and sterility develops in Bmal1-deficient mice. Recently, we discovered that animals with uterus-specific Bmal1 loss also had poor placental development, and these mice also had intrauterine fetal death. Furthermore, it was shown that time-restricted feeding controlled the uterine clock’s circadian rhythm. The uterine clock system may be a possibility for pregnancy complications, according to these results. We summarize the most recent research on the close connection between the circadian clock and reproduction in this review.The circadian rhythm, which is necessary for reproduction, is controlled by clock genes. In the mouse uterus, the oscillation of the circadian clock gene has been observed. The transcription of the core clock gene period ( Bmal1) knockout in arginine vasopressin (AVP) neurons, kisspeptin neurons, GnRH neurons, or the whole body disrupts the timing and pattern of LH secretion, suggesting that the circadian clock system may integrate the HPG axis [Bmal1, circadian locomotor output cycles kaput (Clock), period (Per), and cryptochrome (Cry) within the uterus during pregnancy has been reported [In all mammalian species, including humans, circadian timing is essential for successful female reproduction. For instance, women who have irregular sleep or work schedules have lower fertility and higher chances of miscarriage . SimilarHPG axis ,5. CircaHPG axis ,6,7,8. IHPG axis ,10, and reported . Moreovereported ,7,8. Prereported ,13,14, areported ,16,17. Creported ,20,21,22reported ,24. Metareported ,26. Simireported . Moreovereported ,29. GiveBmal1-knockout mice, Clock mutant mice, and Per- and Cry-deficient animals [The CLOCK-BMAL1 heterodimer induces transcription of Per1/2 and Cry1/2 genes by binding to E-box (CACGTG/T) regions in their promoters. Together with the Clock/Bmal1 heterodimer, Per1/2 and Cry1/2 form a complex that inhibits the transcriptional activity of CLOCK-BMAL1 ,31. Sign animals ,33,34,35 animals ,37,38. T animals . VIP neu animals . It has  animals . The priKiss1 expression in the AVPV [Bmal1 and other clock genes have also been identified in kisspeptin neurons [Female reproduction is under circadian control and temporal information is relayed through the HPG axis ,42,43,44the AVPV . Importa neurons .2-dependent manner, are responsible for controlling LH surge [2 and leptin than SCN signals [Kisspeptin regulates the secretion of sex steroids such as estrogen through the HPG axis . KisspepLH surge . Althoug signals ,58, the  signals ,60,61  was shown to be rescued by P4 supplementation or normal ovarian transplantation, demonstrating that insufficient ovarian P4 production is one of the primary causes of infertility in Bmal1 knockout female mice [Bmal1 in ovarian granulosa cells or theca cells [Bmal1 knockout mice, but not in theca cell-specific Bmal1 knockouts [Consistent with its expression pattern, global rol mice . Global nfertile ,8,71  of uterine regnancy . We foune organs .Per1 and Per2 knockout mice experienced reduced reproductive rates because of estrous cycle irregularities [Per1-Per2 double knockout mice, the follicular reserve was depleted, resulting in infertility [Clock (Clock Δ19/Δ19 mice) were generated to investigate the molecular mechanisms governing circadian clocks [Per and Cry expression, resulting in the loss of the feedback loop for circadian clock genes [Clock Δ19/Δ19 mice are also overweight and develop symptoms of metabolic syndrome under high-fat diet (HFD) conditions [Similarly, larities ,79. Moreertility . Mice win clocks . These mck genes . Clock Δnditions . This obnditions ,84. Fastnditions ,86,87. Rnditions ,88. Thisnditions .The pars tuberalis is situated between the anterior lobe of the pituitary gland and the median eminence. It has been demonstrated that melatonin acts as a photoperiodic signal, synchronizing an endogenous oscillator in the pars tuberalis to the photoperiod . ThyroidBmal1 cause the mistiming of labor onset [Bmal1 knockout mice had 28% more daytime births than control mice, demonstrating that the peripheral circadian clock is involved in the timing of labor [In diurnal primates, labor is often initiated at night, consistent with the increased sensitivity to oxytocin that causes pregnancy-related uterine contractions ,95. Thisor onset . While cor onset , maternaof labor . These dReproduction is critical for species survival. Nevertheless, under certain environmental conditions, reproductive activity is suppressed. Many organisms, together with humans, adaptively reduce reproductive activity during periods of starvation and/or anorexia ,103. InaFeeding rhythms are important for animals because food-entrainable oscillators are located within peripheral tissues, and these peripheral oscillators are independent of the SCN ,111,112.Proper timing of sexual maturation is necessary for reproduction ,128,129.Other factors affecting sexual maturation are endocrine-disrupting chemicals (EDCs). EDCs are substances that can mimic hormones in the body and are found in common household products. EDCs bind to hormone receptors and cause activation or suppression of natural hormones or alter the breakdown of natural hormones, thereby causing changes in normal hormonal signaling. Puberty is a complex developmental stage in which physical changes promote sexual maturation, and this process is sensitive to hormonal disruptions. EDCs have been reported to be involved in pubertal-onset variability  and can In conclusion, elucidating the factors that modify circadian clocks in reproductive organs will provide clues to treating reproductive dysfunction. Moreover, it may suggest strategies for optimizing existing therapeutic interventions. We expect that the appropriate re-establishment of the networks governing circadian rhythms and the reproductive cycle in early life will help prevent future obstetric and gynecological diseases. The influence of circadian rhythms governing protein translation on the regenerative capacity of tissues must be considered in future studies of regeneration.


Long non-coding RNAs (lncRNAs) have been the subject of considerable attention in recent years due to their role in gene regulation. However, the function of lncRNAs remains poorly understood, especially in the context of infection with low-risk human papillomaviruses (HPVs). To further understanding on this issue, we investigated lncRNA expression in HPV-induced common warts.A publicly available high-throughput sequencing dataset for common warts was downloaded from the Gene Expression Omnibus (GEO). lncRNA profiles were generated using the NetworkAnalyst 3.0 workflow, and a list of differentially expressed (DE) lncRNAs in common warts was identified and inputted into the ENCODE, RegNetwork, DisGeNet, and miRNet platforms.CERNA2, LINC02159, SH3PXD2A-AS1, and UNC5B-AS1 genes.A total of 54 lncRNAs were revealed to be significantly dysregulated in common warts. Of these 54 lncRNAs, 24 and 30 were upregulated and downregulated, respectively. The most significantly differentially expressed lncRNAs in common warts included the The current findings suggest that HPV-induced warts impact the host lncRNA transcriptome. To the best of our knowledge, the present study is the first to explore the impact of low-risk HPV infection on lncRNA expression profiles. CERNA2; LINC02159; SH3PXD2A-AS1; UNC5B-AS1.Warts; Human papillomavirus; lncRNA;  The funVerruca vulgaris), which accounts for 70% of warts and is preferentially located on the hands [The HPVs are a family of non-enveloped DNA viruses with a preferential tropism for epithelial tissue . More sphe hands .Despite affecting nearly 1 in 10 people, warts have not been the subject of much genetic research, and current treatments mostly rely on damaging or destroying the infected epithelia , 14. In 22.1The dataset investigated in the present study was obtained from The National Center for Biotechnology Information's (NCBI) Gene Expression Omnibus (GEO) repository (accession number GSE136347). The data contained paired epidermal tissue samples from warts (n = 12) and normal skin (n = 12) that were profiled on the Illumina HiSeq 2500 platform. According to the original study, total RNA was extracted from the samples using an RNeasy Mini Kit , and the purity of the samples was assessed using the Agilent Bioanalyzer  . All the2.2Quality control, filtering, normalization, differential expression analysis, and data visualization were carried out according to the NetworkAnalyst 3.0 workflow , 19, 20.th percentile of genes with the lowest variance  and the 4th percentile of genes with the lowest abundance were removed. Based on this filtering step, 37 genes with low variance and 0 genes with low abundance were excluded from downstream analysis.Both variance and abundance filtering were applied to the dataset. The 15The data was uploaded to the Network Analyst 3.0 web platform and normalized via log2 transformation. The normalized data was displayed in a principal component analysis (PCA) plot and visually inspected for outliers . Differe2.3Gene ontology (GO) analysis was performed as part of the NetworkAnalyst 3.0 workflow to determine which biological processes, cellular components, and molecular functions were enriched in common warts.2.42.4.1As part of the NetworkAnalyst 3.0 workflow, the list of DE lncRNAs were inputted into the ENCODE, RegNetwork, DisGeNet, and miRNet platforms. ENCODE was used to obtain information about interactions between transcription factors and gene targets, while coregulatory interaction data was obtained from RegNetwork. The DisGeNet database was used to determine lncRNA-disease association data, and miRNet was used to predict the interactions between DE lncRNAs and miRNA targets , 22, 23.2.4.2The interaction between DE lncRNA, DE miRNA and DE genes in common warts was carried out using miRNet 2.0, a network-based visual analytic tool for miRNA functional analysis . A total2.5A list of the most differentially methylated (DM) genes  in commo33.13.2No KEGG and Reactome pathways were found to be associated with the 229 significantly DE lncRNAs in common warts. Similarly, gene ontology (GO) term enrichment analysis did not yield any significant GO terms.3.33.4HOTAIR, while MIR4435-2HG is associated with lung neoplasms. HOTAIR lncRNA gene.3 diseases have been found to be associated with 3.4.1Out of 54 DE lncRNA, 28 DE lncRNAs were mapped to 523 miRNAs. The interaction network included 551 nodes and 1,263 edges. Due to the large number of nodes and edges, only the 28 most DE lncRNAs with the common shared miRNAs are presented in Figures 3.4.2The result of the analysis revealed a total of 7,137 nodes, of which 4,423 are genes, 2,686 are miRNAs, and 28 are lncRNAs. To simplify the network visualization, the 28 lncRNAs were extracted with their common shared miRNA (104 nodes) and genes (24 nodes) Figures A and 9B.3.5Merger of the most DE and DM lncRNAs in common warts revealed 21 overlapping lncRNA genes .Table 2L4CERNA2, LINC02159, SH3PXD2A-AS1, and UNC5B-AS1. In common warts, the most downregulated lncRNA was UNC5B-AS1 and the most upregulated lncRNA was MIR100HG.Previous evidence indicated that lncRNAs are deregulated by the high-risk HPV proteins E6 and E7 . Here, wCERNA2, also known as HOST2, was the most significantly DE lncRNA in common warts and was downregulated. CERNA2 acts as an oncogenic lncRNA, and silencing of CERNA2 expression was found to inhibit the proliferative, migratory, and invasive capacities of breast, cervical, gastric, and osteosarcoma cancer cells [CERNA2 was significantly upregulated in psoriatic skin and positively correlated with STAT1, a gene whose expression is inhibited by HPV proteins for episome maintenance and genome amplification [CERNA2 was up-regulated in HPV-positive cervical cancer cells, resulting in the down-regulation of its target microRNA let-7b [er cells , 29, 30.er cells . Comparefication , 33. AddA let-7b .LINC02159, also known as LOC285629, was DE in thyroid cancer, HER2+HR− breast cancer, and metastatic melanoma, and it was identified as a part of lncRNA signatures in colorectal cancer as well as head and neck squamous cell carcinoma [LINC02159 was classified as a low-risk gene for esophageal squamous cell carcinoma, and it is significantly associated with colorectal cancer prognosis [arcinoma , 39, 40.rognosis .SH3PXD2A-AS1 acts as an oncogene in colorectal cancer and esophageal squamous cell carcinoma, with SH3PXD2A-AS1 knockdown resulting in the inhibition of colorectal tumor growth and migration [SH3PXD2A-AS1 was markedly upregulated in psoriatic skin, and it was identified as part of a positive feedback loop that regulated the proliferation of keratinocytes and affected the pathogenesis of psoriasis [Evidence suggests that igration , 44, 45.soriasis , 46.UNC5B-AS1, also known as UASR1, was found to be downregulated in senescent normal oral keratinocytes, [UNC5B-AS1 upregulation increased cell proliferation in breast, cervical, colorectal, oral squamous cell, and papillary thyroid cancers [nocytes, . In cont cancers , 51, 52.MIR100HG overexpression was associated with cetuximab resistance and poor prognosis in colorectal and cervical cancer patients, and it was found to promote cell proliferation in laryngeal squamous cell carcinomas, hepatocellular carcinomas, osteosarcomas, and triple-negative breast cancer [MIR100HG was reported to modulate the transforming growth factor beta (TGFβ) signaling pathway, the latter of which is subjugated by HPV in head and neck squamous cell carcinoma and induces epithelial-to-mesenchymal transition in keratinocytes transformed by HPV16 [t cancer , 59, 60.by HPV16 ,62,63.MALAT1 had the highest number of interactions with microRNAs and protein-coding genes that were previously found to be DE in common warts. Upregulated MALAT1 expression was identified in high-risk HPV-positive cervical squamous tissues and cell lines, and MALAT1 was suggested to modulate cellular apoptosis, proliferation, migration, and invasion [MALAT1 induces epithelial-mesenchymal transition in breast, cervical, colorectal, esophageal, and prostate cancer cells as well as in non-transformed cell lines, namely ARPE-19 cells, HK-2 cells, and lens epithelial cells [MALAT1 expression in cervical cancer cells, whereby MALAT1 overexpression was dependent on the expression of the HPV16 E7 oncoprotein [Our findings showed that invasion , 66, 67.al cells , 75, 76.oprotein .STAT3 gene, which plays a critical role in psoriasis pathogenesis, is essentially activated by high-risk HPVs in both differentiated and undifferentiated keratinocytes [As highlighted above, several of the DE genes in common warts were previously reported to be dysregulated in psoriatic skin. Certain HPV types are highly prevalent in psoriatic skin, and a nationwide population-based cohort study in Taiwan indicated that there is a possible link between HPV infection and the risk of developing psoriasis , 79, 80.inocytes , 81.Likewise, many of the DE genes in our study previously reported to be dysregulated in colorectal cancer. Like psoriasis, HPV positivity resulted in an increased risk of colorectal carcinoma, and HPV infection has been suggested to be a co-factor in the pathogenesis of colorectal cancer , 83. Sma5Our findings indicate that HPV-induced warts impact the expression profiles of certain lncRNA genes. To the best of our knowledge, the present study is the first to explore the impact of low-risk HPV infection on lncRNA expression profiles. Nonetheless, our findings are limited by the relatively small sample size as well as the all-male patient cohort, and they must be validated in a larger cohort. Moreover, more research needs to be carried out in order to determine whether the changes in lncRNA expression were induced as part of the host's response to infection or from the HPV itself.Amneh H. Tarkhan, Mansour A. Alghamdi: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Laith N. AL-Eitan: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Rami Q. Alkhatib: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.King Khalid University through the Small Groups Project (grant number: RGP. 1/66/43). This work was also supported by the Deanship of Research at Jordan University of Science and Technology (177/2017).This work was supported by the Deanship of Scientific Research at Data associated with this study has been deposited at NCBI GEO under the accession number GSE136347.The authors declare no conflict of interest.No additional information is available for this paper.