Yolk sac tumour (YST) is the second most common ovarian germ cell tumour and usually presents in children and young women. However, tumours rarely occur as malignant gynaecological tumours with YST components.We present one case of endometrioid carcinoma and clear cell carcinoma with YST components and two other cases of YSTs associated with high-grade serous carcinoma of the ovary in females. After surgery and adjuvant chemotherapy, the patient with endometrioid carcinoma had progressive disease and died 20 months later, and the other two were still alive at the last follow-up.To our knowledge, these mixed neoplasm associations are unusual, and these cases illustrate the diagnosis and prognosis of YST associated with malignant gynaecological tumours, emphasizing early recognition and aggressive treatment. Yolk sac tumour (YST) is the second most common ovarian germ cell tumour and usually occurs in infants and adolescents , 2. ThesA 36-year-old female presented with abdominal distension for over half of a month. Ultrasonography showed a large pelvic mass measuring 2.5*2.5*2.4 cm. A work-up showed α-fetoprotein (AFP) > 1000 ng/ml and cancer antigen 125 (CA125) 28.8 U/mL. Abdominal enhanced CT revealed an intrauterine-cervical canal mass, presence of multiple nodules and greater omentum thickening in the pelvis and fluid in the left fallopian tube.We performed laparotomy with total abdominal hysterectomy (TAH), bilateral salpingo-oophorectomy (BSO), pelvic lymphadenectomy (PLN), para-aortic lymphadenectomy (PALN) and tumour debulking. The immunohistochemical markers were positive for AFP, Glypican-3 (GLP3), SALL-like protein 4 (SALL4), PMS2, MSH6, MLH1, MSH2, P16, oestrogen receptor (ER), progesterone receptor (PR), Ki-67, P53, NapsinA, cytokeratin (CK), EMA, and CDX2  and were negative for octamer 4 (OCT-4) and CD30, which is consistent with endometrioid carcinoma and clear cell carcinoma with a yolk sac component (G2)  regimen was prescribed for 3 cycles. The AFP level was 107.1 ng/mL after the 3rd cycle of chemotherapy. However, positron emission tomography-computed tomography (PET-CT) revealed intrahepatic metastatic lesions. Therefore, we suggested performing liver ablation and changing the chemotherapy regimen. Unfortunately, she had progressive disease and died 20 months later.A 55-year-old postmenopausal woman was admitted for vaginal bleeding and abdominal distension. A CT scan demonstrated a cystic solid mass in the bilateral adnexal region with minor ascites. The preoperative AFP was 1190 ng/mL and CA125 was 272.4 U/mL, while carcinoembryonic antigen (CEA) and CA199 were within the normal range. TAH, BSO, PLN, PALN and omentectomy were performed.Pathology revealed high-grade serous carcinoma arising from the right ovary with a YST component. Her FIGO stage was IIa. Immunohistochemical staining showed that the germ cells were positive for AFP, GLP3, SALL4, CK, CK7 and EMA and were negative for OCT-4, Napsin A, P16, PR and ER Fig. .After surgery, she received 6 courses of chemotherapy with the TP regimen. The serum AFP levels were normal after the first chemotherapy cycle. At 2 years postoperatively, she had a recurrent vaginal stump. Therefore, the patient received Niraparib maintenance treatment following reoperation and chemotherapy. At the time of the latest available follow-up, the patient remained clinically and radiologically free of disease.A 60-year-old woman presented with a complaint of abdominal distension. Before visiting our hospital, she was found to have masses in the left adnexa and right middle abdomen, with massive effusion in the abdominal cavity and pelvic cavity. A recent ultrasound examination showed cystic solid masses measuring 18.1*13.2*10.5 cm and 7.4*5.4*5.4 cm, with obvious blood flow signal in the upper right side of the uterus and left the attachment area, respectively. The tumour marker results were as follows: serum AFP 11233.0 ng/mL, CA125 773.2 U/mL and CA153 39.1 U/ml. The patient underwent exploratory laparotomy. There was 5500 mL of yellow fluid in the abdominal cavity, and the right ovary was replaced by a tumour measuring 20*15*15 cm in size. Most of the tumour was removed, and the fast-frozen pathology result was a malignant tumour. TAH, BSO, PLN and omentectomy were performed.Pathologic examination demonstrated an AFP-producing ovarian germ cell tumour (YST) with high-grade epithelial carcinoma (serous and clear cell carcinoma), FIGO stage Ic. Immunohistochemical staining was performed to confirm the histological diagnosis, and the tumour cells were positive for AFP, GLP3, SALL4, CK, CK7, EMA and Ki-67 but negative for OCT-4, NapsinA, P16, ER and PR (Fig. The patient was treated postoperatively with six cycles of chemotherapy consisting of TP. The serum levels of these markers decreased to normal values after four cycles of chemotherapy. At the current follow-up, there were no signs of tumour recurrence.YST is a malignant germ cell tumor characterized by endodermal differentiation and usually occurs in young patients , 2. YST Pathologic analyses have revealed that YSTs can be pure or mixed with other germ-cell or epithelial tumors, including embryonal carcinoma, clear cell carcinoma, adenocarcinoma, and serous adenocarcinoma –10. In oAFP is synthesized mainly by the liver and yolk sac and is a useful diagnostic tumor marker for hepatocellular carcinomas . HoweverBecause YSTs associated with gynecological malignant tumors are extremely rare, there are no guidelines for treatment. At present, a combination of comprehensive surgery and platinum-based adjuvant chemotherapy is the main means of treatment. Previous studies revealed that YST components mixed with other tumors may be less responsive to chemotherapy . CurrentFor inoperable patients with advanced-stage ovarian cancer, neo-adjuvant chemotherapy is a safe and effective alternative. The TP regimen for every 3 weeks remains the gold standard therapeutic regimen . MoreoveOn the other hand, preoperative frailty assessment is fundamental to predict adverse outcomes and to offer them a personalized therapeutic strategy in gynecologic oncology , 29. ModWe reported three cases of YSTs associated with malignant gynaecological tumours in females. Because mixed YST-carcinoma is a rare malignant neoplasm with high mortality, complete surgical staging combined with chemotherapy may improve the survival rate. In the future, further studies are needed to explore new diagnostic and effective management strategies for YSTs associated with malignant gynaecological tumours.


Plant senescence is the last stage of plant development and a type of programmed cell death, occurring at a predictable time and cell. It involves the functional conversion from nutrient assimilation to nutrient remobilization, which substantially impacts plant architecture and plant biomass, crop quality, and horticultural ornamental traits. In past two decades, DNA damage was believed to be a main reason for cell senescence. Increasing evidence suggests that the alteration of epigenetic information is a contributing factor to cell senescence in organisms. In this review, we summarize the current research progresses of epigenetic and epitranscriptional mechanism involved in cell senescence of plant, at the regulatory level of DNA methylation, histone methylation and acetylation, chromatin remodeling, non-coding RNAs and RNA methylation. Furthermore, we discuss their molecular genetic manipulation and potential application in agriculture for crop improvement. Finally we point out the prospects of future research topics. As known, cell senescence in plant can occur in predicable space and time during a plant’s lifespan, for example in root cap cell of root, treachery cell of vascular, aerenchym cell and leaf mesophyll cells, petal cell, tapetum cell of anther, macrospore cell, aleuronat layer cell of seed, as well as hypersensitive cell , where CG methylation is located in the gene promoter and the body, while non-CG methylation is at TEs for regulating gene expression, ensuring transposon silencing and maintaining genome stability . Moreove3DNA methylation at the fifth carbon of cytosine residues represents a conserved epigenetic mark in eukaryotes that has key function in regulating gene transcription, transposon silencing and genome stability . In plan3.1Arabidopsis shoots aging by using a methylation-sensitive DNA fragmentation (MS-AFLP) assay. They showed a global declining in DNA methylation levels during Arabidopsis shoots aging, simultaneously accompanying by a downregulation of CMT3 and MET1 methyltransferase genes, and an upregulation of demethylase genes including ROS1, DME, DML2, and DML3  promoter, led to global DNA hypomethylation and developmental defects including reduced fertility, failed flowering, or greatly delayed plant senescence (Arabidopsis leaves of demeter-like 3 (dml3) mutant that lost the DEMETER-like DNA demethylase, relative to wild-type at three developmental stages, namely NS (non-senescent), ES (early senescence)), and LS (late senescence). While a reduction of genome-wide methylation levels at CG context was observed at stage transition from NS to LS in wild-type, loss of DML3 led to significant increase of DNA methylation at the promoters of several senescence-associated genes, thereby suppressing their expression and delaying leaf senescence. These results demonstrated that DML3 activating DNA demethylation and expression of selected senescence-associated genes modulated leaf senescence  displayed a faster senescence progression and enhanced nitrogen remobilization from the leaves. Loss of methylation in CHG and CHH contexts at W-box element targeted by WRKYs as well as a core motif recognized by bZIP transcription factors (ACGTG) was observed in ddc. Differentially methylated regions at ROS1 promoter were related to down-regulation of ROS1 with the progression of senescence  displayed abnormal DNA methylation and developmental tissues like delayed or absent heading or growth defects  inhibited leaf growth, accelerated leaf and sepal senescence, and produced dark green fruit. Gene expression analysis revealed that the SlELP2L-deficient plants had up-regulated gene expression level of DNA methyltransferases, implying that SlELP2L may play a role in DNA methylation in tomato  gene, and the methylation status of NMR19-4 was associated with repressed PPH expression and delayed leaf senescence. Epi-allelic variations at key senescence associated genes have also been demonstrated to regulate leaf senescence in several other crop species. In maize, alterations in DNA methylation were observed during critical life event transitions when silencing the Mutator-Don Robertson transposable elements and were proposed to contribute to whole-plant senescence  as well as lysine 9 acetylation on histone 3 protein (H3K9ac) are active histone modifications associated with leaf senescence. Two silence marks, lysine 27 dimethylation and trimethylation , are also linked to leaf senescence .4.1Arabidopsis, that senescence-associated genes (SAGs) correlates with elevated levels of H3K4me3 and H3K9ac, while the inhibition of senescence-downregulated genes is related to H3K27me3 (The combination of chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq methods has been employed to explore the genome-wide landscape of histone modifications and gene transcription during leaf senescence. H3K27me3 . Zhang e4.2WRKY53, a key regulator of leaf senescence, levels of H3K4me3 and H3K4me2 were observed significantly increased at the coding regions and 5’ end of WRKY53. The overexpression of SUVH2, a histone methyltransferase, however, suppressed the transcriptional initiation of WRKY53 and additional SAGs, thereby leading to a delay of leaf senescence , a H3K27me3 demethylase, directly accelerated the expression of ten specific SAGs, and loss-of-function of REF6 postponed leaf senescence by increasing H3K27me3 levels at all the target SAGs. Recently, in tomato plant overexpressing SlJMJ4, a histone H3K27 demethylase, resulted in premature senescence and enhanced dark-and ABA- induced leaf senescence , the Arabidopsis HDA6 mutant exhibited higher levels of acetylated H3 and prolonged leaf lifespan  by removing H3 acetylation marks, thereby promoting aging in Arabidopsis  of nutrient recycling-related genes , the D1 synthesizer attenuator PDIL1-2 of photosystem II protein, as well as WRKY53 and ELF4. This recruitment removes H3K9ac from their promoter region leading to the repressing of gene expression and the delaying both leaf senescence and flowering during the early stage of plant development  and deacetylases (HDACs). The earliest study exploring the relationship of histone deacetylases and leaf senescence is AtHD1 . Transgemalities . AtHDA6,lifespan . AtHDA9,bidopsis . AtHDA15bidopsis . Furtherelopment . In ricesistance . Howeverrichment .4.4HvS40, which encodes a potential regulator of leaf senescence, is significantly up-regulated. At the onset of leaf senescence, the regions of promoter and coding sequence of HvS40 enriched H3K9ac, but declined the enrichment of H3K9me2  and aging  in crop plants were identified in impact of histone modification enzymes. For example, during leaf senescence, the expression of the barley gene  H3K9me2 . Under dnescence . Conversnescence . In ricend aging . Zhong e . These complexes catalyze changes in nucleosome position, occupancy, or/and composition to regulate DNA and core histones interactions, thereby remodeling chromatin locally or globally to adopt or impede regulatory proteins. On the basis of ATPase/helicase like regions (Snf2 domain) characteristics, chromatin remodeling proteins have been briefly classified into six groups and 24 subfamilies, each with specific functional properties . Snf2 faectively . Roles oectively . This pa5.1Chromatin conformation is dynamically changed during cell senescence induced by age and environmental cues . Ay et a5.2ORE7/ore7-1D) with lower expression of the activated ORE7 genes was used in this study to analyze the senescent phenotypes. ORE7/ore7-1D mutant showed a delayed senescence phenotype as well as globally altered gene expression. Moreover, more reticular chromatin distribution and intensely nuclear bodies than wild-type plants were observed in ORE7/ore7-1D mutant. It is possible that ORE7 binds to AT-rich DNA sequences and modifies the chromatin architecture in the nucleus, then leading globally altered gene expression. In addition, a subset of hormones  related genes was down-regulated in ORE7/ore7-1D mutant, suggesting ORE7 repressed the signaling pathway of hormones during plant senescence  domain, showed delayed leaf senescence during dark-induced senescence and natural senescence. drd1-6 mutants exhibited higher photosynthetic parameters and lower expression levels of SAG12 and chlorophyll degradation-related genes than wile-type plants during dark-induced senescence. Moreover, the transcript levels of 180-bp centromeric (CEN) repeats and pericentromeric repeats termed transcriptionally silent information (TSI) showed slower and lesser increase in the drd1-6 mutant than in the wile-type plants. Similarly, ddm1-2 also exhibited a longer stayed green phenotype and higher photochemical efficiency than wild-type plants  and ATP-dependent DNA helicase DDM1 (DECREASED DNA METHYLATION 1), were identified as regulators of leaf senescence . drd1-6 e plants . TherefoNAC083, NAC032, NAC019, PIF4, WRKY6 etc. with BRM  and CHR19 (ETL1) were shown to be upregulated during senescence  is one member of the two catalytic ATPase subunits of the SWI2/SNF2 complex. It directly targets to a large amount of SAGs . The H3Kwith BRM , suggestwith BRM , while tnescence . Further6Arabidopsis and rice to retain chromatin and regulate the structure of chromatin and gene expression through interaction with H3K4me3-labeled gene sites , including miRNAs, siRNAs and piRNAs; medium-sized RNAs (50-300 nt), including snoRNAs and snRNAs; and long non-coding RNAs (lncRNAs) over 200 nt in length . miRNAs ne sites . LncRNAsne sites .6.1Caenorhabditis elegans, marked the beginning of miRNA research, and since then, researchers have identified thousands of miRNAs in both plants and animals. These miRNAs play diverse roles in regulating a wide range of biological processes  gene expression. It targets seven out of nine Arabidopsis GRFs  are short, non-coding RNAs that are highly conserved and typically range in length from 20 to 24 nucleotides. The discovery of lin-4, the first miRNA, in 1993 , in nemarocesses . miRNAs rocesses . Althougrocesses . These rrocesses . Only a  Populus . AtORE1 nescence ; MIR164 nescence . AtKIR1,nescence ; and thenescence . miR396 sis GRFs . The miRsis GRFs ; miR319 sis GRFs . A receneam SAGs . High-thnescence .Arabidopsis. MIM775 transgenic lines exhibited accelerated senescence during post-submergence recovery, while miR775-overexpresing (Oe1) and galt9 transgenic lines showed delayed senescence. The expression of SAG29, SAG12, and ORE1 decreased in miR775-Oe1 and galt9 transgenic lines, while their expression enhanced in MIM775-1 lines, suggesting that the miR775-GALT9 module regulated the expression of SAGs to control senescence during post-submergence recovery in Arabidopsis. In crop plants, very limited miRNA was reported their detail functions related to leaf senescence. A novel tomato miRNA known as SlymiR208 was identified. When it is overexpressed, the expression of SlIPT2 and SlIPT4 was significantly reduced, and endogenous CK concentrations in leaves was declined, leading to premature leaf senescence, which was in concurrence with the phenotype of Isopentenyltransferases 4 (IPT4)-silenced lines, showing early leaf aging  and the NAC family gene BGIOSGA016313 through osa-miR5809  were targeted by microRNAs. Notably, the majority of differently expressed target genes of DElncRNAs were related to the membrane component, with almost half of these genes involved in transporting organic material. These findings strongly suggest that DElncRNAs play a crucial role in substance transport across membranes during nodule senescence  signals, thereby regulating alternative splicing of NSRs-targeted pre-mRNA to influence root development and senescence  is a recently discovered class of epigenetic regulator involved in regulating gene expression during plant development and in response to stresses. Dynamic landscapes of long noncoding RNAs were observed in ng aging . Above, -miR5809 . Furthernescence . Anothernescence . In addif plants . Up to n7RNA modification, such as N6-methyladenosine (m6A) and 5-methylcytosine (m5C), are another kind of epigenetic regulation. With the advances in technology of mass spectrometry, next-generation sequencing, and genome mapping, detection of mRNA modifications has become more precise. Up to now, more than 170 various RNA modifications were reported. The increasing reports are mostly focused on chemical modifications of specific mRNA nucleotides like m6A and m5C, which can enhance or reduce the binding activity of the regulatory factors, such as transcription factors, RNA binding proteins, and non-coding RNAs. Here, we try to summarize the chemical mRNA modifications, specifically m6A affect plant senescence .Arabidopsis, this complex contains two core methyltransferases and several accessory proteins. The former includes mRNA adenosine methylase MTA and mRNA adenosine methylase MTB, which are orthologue of METTL3 and METTL14, respectively.The latter includes FKBP12 INTERACTING PROTEIN 37KD (FIP37) and HAKAI. m6A methyltransferase deposits m6A on the region closed to stop codons of mRNAs and the region with a RRACH sequence in 3′UTRs, respectively, like in mammalian, responding for most of m6A enrichment of mRNA. Another m6A methyltransferase FIO1 that is orthologue of METTL16, can add m6A modifications on the regions with a YHAGA (Y = C/U) motif of coding sequence transcript around stop codons or with a RRACH in 3’UTRs, affecting all m6A levels in plant (N6-methyladenosine (m6A) is the most widespread, plentiful and conserved internal cotranscriptional modification in eukaryotic RNAs, especially within higher eukaryotic cells. m6A modification is modified by the m6A methyltransferases (writer) and removed by the demethylases (erasers). It is recognized by m6A-binding proteins (readers) . In planin plant , which iin plant . NeverthArabidopsis flowering (Recent research suggests that the specificity of m6A with its target could be developed through interaction of m6A writers and other writer-associated proteins such as RNA binding protein (RBP), named m6A recruiters with specific transcripts in plants. For examples, it has reported that the RBP FCA interacted with MTA, MTB, and FIP37 in the complex and acted m6A enrichment on the antisense transcript COOLAIR of noncoding RNA during lowering . The m6Alowering . Moreovelowering . Althouglowering . In darklowering  shows silowering .Arabidopsis, including roots, rosette leaves, and flowers showed that the fractions of transcripts apparently enriched m6A modifications in senescent tissues, and m6A enriched level is much higher than transcript altered level, suggesting that m6A may be more important regulator in organ differentiation and programed cell death process , alternative polyadenylation (APA), protein folding, translation, localization, RNA transport, and RNA decay . We summ-mutants  , MET1 and ROS1 were significantly regulated at the transcription level in dark-induced leaf senescence  are specifically modified SAGs? How do these writers and erasers determine their targets in distinct physiological procedure and various tissues and organs, at different developmental stage, or under multiple stresses? How do reader proteins recognize their targets (SAGs) and carry out their roles in following RNA metabolic processes? At the same time, when we are applying epigenetomic and epitranscriptome editing in crop biotechnology, numerous challenges must be faced. First, it is required to develop a novel techniques to analyze DNA modification, histone modification and RNA modification dynamics from the tissue levels to the cellular levels even at single-nucleotide resolution or at single-amino acid resolution. For example, DNA/RNA epigenetic modified sites at single-base resolution are largely unknown in crops, the techniques of TCTA-seq, miCLIP, MAZTER-seq, m6A- SAC-seq, and Nanopore DRS are necessary to be developed to accurately detect DNA/RNA methylation and interrogate modified sites at single-nucleotide resolution in crops. The biological actions of erasing/writing epigenomic marks at specific SAGs, histone epigenetic mark targeted to a specific SAGs locus are mostly unidentified in plants, especially, epitranscriptome mark at SAGs transcript is blank yet. Although many epigenetic enzymes and factors of DNA or RNA modifications or histone modification have been reported in several crops (Over the last decade, there have been significantly progressed in the mechanistic understanding of epigenetic modifications and epitranscriptome, including dynamics of H3K4me3 and H3K9ac, dynamics of non-coding RNAs, m6A dynamics during tissue and organ development and under stresses in the model plant al crops , their iYZ: Writing – original draft. DH: Writing – original draft. YM: Writing – original draft, Writing – review and editing.


Background: The association between Killer cell lectin like receptor B1 (KLRB1) and cancer has been reported, but the roles of KLRB1 in breast invasive carcinoma (BRCA) has not been fully revealed.Methods: Our study utilized the Cancer Genome Atlas (TCGA), Kaplan-Meier (K-M) Plotter, and TIMER databases to investigate the expression and clinical relevance of KLRB1 in BRCA and to explore its roles and mechanism in BRCA progression using gene set enrichment analysis, CCK-8, migration, apoptosis, and western blotting. We examined the relationship between KLRB1 expression and the BRCA immune microenvironment, using data from TCGA, and Gene Expression Profiling Interactive Analysis (GEPIA) databases and validated these findings in K-M Plotter databases.+ T cells, CD4+ T cells, neutrophils, dendritic cells, among others), cell markers, and immunotherapy.Results: A significant decrease of KLRB1 expression was observed in BRCA patients. BRCA patients with low KLRB1 levels were associated with older age, advanced disease stage, HER2-positivity, poor prognosis, and a decreased survival probability compared to the high-expression group. Increased KLRB1 expression levels were correlated with inhibition of breast cancer cell proliferation, migration, and invasion, as well as promotion of cell apoptosis, possible through regulation of the NF-κB, PI3K/AKT, and TNF signaling pathways. Moreover, the study also indicated that decreased KLRB1 expression correlated with tumor purity, immune score, and immune cell infiltration (B cells, CD8Conclusion: Decreased KLRB1 expression in BRCA is associated with poor prognosis and immune microenvironment. This study also highlights KLRB1 as a potential molecular marker for poor prognosis in BRCA patients, and therefore, it may provide clinical implications for the management of patients with BRCA. Breast cancer (BC) is a prevalent malignancy among women worldwide, posing a substantial risk to their lives –4. Breasin vitro and the anti-tumor function of KLRB1 in vivo [Studies have reported a significantly higher number of differentially expressed genes during BRCA progression –10. For  in vivo , whereas in vivo . Xu and  in vivo , 15. HowKLRB1 levels were found to be significantly reduced in non-paired BRCA tissues when compared to normal breast tissues . MoreoveKLRB1 expression levels were significantly decreased in tissues of BRCA patients at T2 and T4 stages compared to those at the T1 stage , 2B. In In the K-M Plotter database, a survival analysis revealed that decreased KLRB1 levels were correlated with a decrease in OS, DMFS, and RFS in BRCA patients with various estrogen receptor (ER) and HER2 statuses, intrinsic and PAM50 subtypes, and lymph node statuses . The KLRUnivariate COX regression analysis demonstrated that M stage, ER status, N stage, PAM50 status, T stage, and decreased KLRB1 level were adverse factors affecting the survival of BRCA patients . MultivaGSEA analysis revealed that KLRB1 was involved in various biological functions and pathways related to immune response, and cancer –4C. ThesSignificantly elevated KLRB1 levels were observed in the KLRB1 overexpression group of MDA-MB-231 and HS578T cells –5C. More+ T cells, CD4+ T cells, neutrophils, and dendritic cells , immune score (r = 0.786), and estimate score (r = 0.725) levels. We also observed that reduced KLRB1 levels were positively correlated with the T cells, cytotoxic cells, B cells, dendritic cells, Th1 cells, iDC, TFH, pDC, CD8 T cells, aDC, NK CD56dim cells, TReg, Tem, T helper cells, macrophages, neutrophils, Tcm, mast cells, eosinophils, NK CD56bright cells, and NK cells, and negatively correlated with Th17 cells, Th2 cells, and Tgd cells . Moreoveic cells .The TIMER database analysis illustrated a positive correlation between KLRB1 expression and immune-cell markers such as PDCD1 (r = 0.731), CD274 (r = 0.438), and CTLA4 (r = 0.648). Moreover, KLRB1 expression exhibited a positive correlation with the expression of PDCD1 (r = 0.624), CD274 (r = 0.317), and CTLA4 (r = 0.541) under condition of tumor purity, and PDCD1 (r = 0.731), CD274 (r = 0.441), and CTLA4 (r = 0.643) under condition of age in BRCA. We also observed positive correlations between KLRB1 expression and PDCD1 , CD274 , and CTLA4  levels in BRCA subtypes basal, luminal, and HER2, respectively. Using the GEPIA database, we discovered similar positive correlations between KLRB1 expression and PDCD1, CD274, and CTLA4 levels in normal breast and BRCA tissues as presented in BC remains a significant cause of morbidity and mortality among patients . ConsequRecent literature has indicated gene involvement in the progression of BRCA, as evident from multiple studies , 21, 22.+ T cells, CD4+ T cells, neutrophils, and dendritic cells in patients with BRCA and its subtypes basal, HER2, and luminal. Furthermore, KLRB1 expression was positively correlated with immune cell markers such as PDCD1, CD274, and CTLA4. K-M survival analysis indicated that OS and PFS were better in cancer patients treated with PDCD1, CD274, and CTLA4 with elevated KLRB1 expression. In addition, GSEA results showed that KLRB1 was significantly associated with PD-L1 expression and PD-1 checkpoint pathway in cancer. These findings suggest that KLRB1 plays an important role in the progression of BRCA using PD-1 and PD-L1 and may have significant clinical value in BRCA immunotherapy.Immunotherapy is a promising treatment that can improve the quality of life and survival time of cancer patients –28. For This study aimed to evaluate the biological roles of KLRB1 in BRCA through database analysis and basic research, providing a new candidate molecule for treating BRCA patients with positive clinical value. However, further research is needed to confirm the relationship between KLRB1 expression and clinical features, cancer subtypes, and prognosis using clinical tissue samples of BRCA patients, and explore the signaling mechanisms of KLRB1 in BRCA progression in the future. In brief, our results reveal that KLRB1 levels are significantly lower, and decreased KLRB1 expression correlates with age, T stage, pathological stage, HER2 positivity, and poor prognosis in BRCA. Therefore, reduced KLRB1 expression is a potential biomarker for poor prognosis in BRCA patients. Conversely, increased KLRB1 expression could reduce BRCA cell proliferation, migration, and invasion, while promoting apoptosis. Furthermore, KLRB1 expression levels are significantly correlated with tumor purity, immune infiltration, and immunotherapy-related markers such as PDCD1, CD274, and CTLA4. Therefore, KLRB1 may have clinical implications in predicting the prognosis of BRCA patients.Decreased KLRB1 expression in BRCA is associated with poor prognosis and immune microenvironment. This study also highlights KLRB1 as a potential molecular marker for poor prognosis in BRCA patients, and therefore, it may provide clinical implications for the management of patients with BRCA.The transcriptome data of FPKM patients were downloaded from TCGA database official website. Among them, there were 113 cases of normal breast tissue and 1113 cases of BRCA tissue in TCGA database. 113 cases of normal breast tissues and 113 cases of BRCA tissues were paired, and then the data were combined and sorted out to identify the expression levels of KLRB1 in normal breast tissues and BRCA tissues by using the differential analysis. The transcriptome data of patients with FPKM were obtained from the official website of TCGA database. The database contained 113 cases of normal breast tissue and 1113 cases of BRCA tissue. We paired 113 cases of normal breast tissues with 113 cases of BRCA tissues by combining and sorting out the data to determine the expression levels of KLRB1 in normal breast and BRCA tissues through differential analysis methods.P < 0.05 being considered statistically significant.Clinical data for a total of BRCA patients were obtained from the TCGA database. We also grouped the clinical characteristics  of BRCA patients to determine the statistical significance of KLRB1 levels within each group, with ROC curves are commonly used to assess the diagnostic value of genes –31. ThusP < 0.05 was used for inclusion in the multivariate COX regression analysis. Following the multivariate COX regression analysis, results were filtered using P < 0.05, and KLRB1-related nomograms were constructed based on the multivariate COX regression analysis results.We employed univariate COX regression analysis to assess the relationship between HER2 status, T stage, M stage, PR status, N stage, ER status, PAM50 status, KLRB1 levels, and OS, DSS, and PFI of cancer patients based on data from the TCGA database. A significance threshold of P < 0.05 being used as the significance threshold.The TIMER database provides the means to analyze the relationship between genes and tumor purity as well as immune cell infiltration in BRCA and its subtypes. Specifically, we utilized the gene module in the TIMER database to examine the association between KLRB1 levels and immune cell levels, as well as tumor purity. Furthermore, the correlation analysis was used to investigate the relationship between KLRB1 levels and BRCA immune cell markers, with P < 0.05.The GEPIA database is an online analytical tool that utilizes data from both the TCGA and Genotype-Tissue Expression (GTEx) databases. To confirm the relationship between KLRB1 levels and BRCA immune cell markers, we utilized the correlation analysis module data available on the GEPIA database, with a statistical significance value of P-value of less than 0.05 was used as the significance threshold.We utilized the GSEA analysis method to investigate the biological functions and pathways involved in a gene –31. In tMDA-MB-231 and HS578T cells were cultured with RPMI-1640  basal medium supplemented with 10% fetal bovine serum. BC cells were maintained at 37°C and passaged every 3–4 days. The pCDNA3.1 expression vector was synthesized by GeneCopoeia (China). MDA-MB-231 and HS578T cells were cultured in 6-well plates at appropriate densities for 24 hours, following which KLRB1 overexpression and control vectors were respectively introduced using Lipofectamine 3000 . RNA and protein were collected 24 hours after transfection, and KLRB1 expression levels were confirmed in MDA-MB-231 and HS578T cells using RT-PCR and Western blotting.For RNA extraction, Trizol solution was added to 6-well plates containing MDA-MB-231 and HS578T cells, after which the BC cells were lysed using a pipetting gun and transferred to a new centrifuge tube. Standard procedures were applied, and this involved the addition of chloroform, isopropanol, 75% ethanol and DEPC treated water. The RNA concentration was determined by calculating the absorbance ratio detected with a spectrophotometer. Reverse transcription was performed according to the instructions provided in the Takara kit, and PCR amplification was conducted to synthesize KLRB1 cDNA. GAPDH was utilized as the internal control. The KLRB1 primers used were ATCTCTTCCTCGGGATGTCTGTCAG (Forward primer) and AGGATGT CACTGAAACACTCAACCC (Reverse primer).A cell lysis solution was added to MDA-MB-231 and HS578T cells in 6-well plates, with the lysate being completely mixed with the cancer cells using a pipetting gun and subsequently transferred to a new centrifuge tube. Protein concentrations were calculated for cells via the BCA method before proceeding with standard electrophoresis, electroporation, primary antibody incubation, secondary antibody incubation, and exposure sequences. The relative expression levels of KLRB1 in control and KLRB1 overexpression cells were subsequently calculated. Protein concentrations for primary antibodies, including GAPDH  and KLRB1 , were maintained at 1:3000 and 1:5000, respectively, during incubation.2 for additional 2 hours. The absorbance values of MDA-MB-231 and HS578T cells were determined using a spectrophotometer , and statistical analysis was performed to compare the control and KLRB1 overexpression groups. The experiments were performed in triplicate.The CCK-8 assay is a common method for assessing the proliferation ability of cancer cells. In this study, MDA-MB-231 and HS578T cells, which had demonstrated good growth after cell transfection, were counted and seeded at a density of 3000 cells/well in 96-well plates. The proliferation ability of these cell lines was evaluated as per the manufacturer’s instructions for the CCK-8 kit. Briefly, a CCK-8 solution of 10 ul/well was added at 24, 48, and 72 hours, and the cells were then incubated at 37°C and 5% COFollowing transfection, MDA-MB-231 and HS578T cells in good growth condition were collected using trypsin without ethylenediaminetetraacetic acid. The collected cells were centrifuged and washed once with phosphate buffer, before being stained with 500 μl of buffer, 1 μl of Annexin V-PE, and 5 μl of 7-AAD . The effects of KLRB1 overexpression on the apoptosis of MDA-MB-231 and HS578T cells were evaluated using flow cytometry, under dark conditions. The experiment was conducted three times.When MDA-MB-231 and HS578T cells in good growth condition after transfection had reached 80–90%, a 0.2 ml gun tip was used to create a straight line along the bottom of the 6-well plate, after which the cells were washed with a phosphate buffer solution. Control and KLRB1 overexpressing BC cells were then photographed and labeled using microscopy at this point, as well as at 24 h in the future. The experiment was replicated three times to ensure accuracy. Transwell assays were performed on MDA-MB-231 and HS578T cells after transfection. Basolateral chambers were balanced with 600 μL of DMEM containing 10% FBS, 200 μL of MDA-MB-231 cell or HS578T cell suspension in FBS-free DMEM were cultivated into the apical chambers. Culturing for 24 h, the transwell chambers were fixed with 4% paraformaldehyde for 10 minutes, stained with 0.1% crystal violet for 15 minutes, and washed with PBS for three times. An inverted fluorescence microscope was used to observe the transmembrane cells.t-test. Pearson correlation analysis was applied to investigate the relationship between KLRB1 gene and BRCA immune cell infiltration. The figure was displayed through GraphPad Prism and R languages, and a statistical significance level of P < 0.05 was considered as the criterion for determining significance.To determine the expression levels of the KLRB1 gene in BRCA tissues, the Wilcoxon rank sum test was employed. Moreover, the prognostic and diagnostic values of the KLRB1 gene were analyzed through survival analysis and ROC analysis. Differences in cell proliferation, apoptosis, and migration ability between the control group and KLRB1 overexpression group were determined using the The data of TCGA, K-M plotter, GTEx, Xiantao Academic, and GEO databases were obtained or by contacting the corresponding authors.Supplementary Figures


As the most common arrhythmia, atrial fibrillation (AF) is associated with a significantly increased risk of stroke, which causes high disability and mortality. To date, the underlying mechanism of stroke occurring after AF remains unclear. Herein, we studied hub genes and regulatory pathways involved in AF and secondary stroke and aimed to reveal biomarkers and therapeutic targets of AF-related stroke.The GSE79768 and GSE58294 datasets were used to analyze AF- and stroke-related differentially expressed genes (DEGs) to obtain a DEG1 dataset. Weighted correlation network analysis (WGCNA) was used to identify modules associated with AF-related stroke in GSE66724 (DEG2). DEG1 and DEG2 were merged, and hub genes were identified based on protein–protein interaction networks. Gene Ontology terms were used to analyze the enriched pathways. The GSE129409 and GSE70887 were applied to construct a circRNA-miRNA-mRNA network in AF-related stroke. Hub genes were verified in patients using quantitative real-time polymerase chain reaction (qRT-PCR).EIF4E3, ZNF595, ZNF700, MATR3, ACKR4, ANXA3, SEPSECS-AS1, and RNF166 were significantly associated with AF-related stroke. The hsa_circ_0018657/hsa-miR-198/EIF4E3 pathway was explored as the regulating axis in AF-related stroke. The qRT-PCR results were consistent with the bioinformatic analysis.We identified 3,132 DEGs in blood samples and 253 DEGs in left atrial specimens. Co-expressed hub genes of EIF4E3, ZNF595, ZNF700, MATR3, ACKR4, ANXA3, SEPSECS-AS1, and RNF166 have potential as novel biomarkers and therapeutic targets in AF-related stroke. The hsa_circ_0018657/hsa-miR-198/EIF4E3 axis could play an important role regulating the development of AF-related stroke.Hub genes  Cardioembolic stroke often results in severe neurological deficits and disability . One of With the development of novel technologies, including microarray and next generation sequencing, the exploration of underlying molecular mechanisms behind AF-related disease becomes possible. Xie, Y et al. reported higher level of miR-641 and miR-30e-5p in serum exosomes in AF related stroke patients than in AF patients . What’s However, no research has been performed to clarify the hub genes and underlying circRNA-miRNA-mRNA regulatory networks in AF-related stroke.Serum miR-106 and MYL4 levels are closely related to the prevalence of atrial fibrillation, which can reflect the risk of thromboembolism in patients with atrial fibrillation and can be used as a biological indicator to predict the prognosis of patients with atrial fibrillation.In this study, we aimed to identify AF-related differentially expressed genes (DEGs) and cardiogenic stroke-related DEGs. The DEGs of AF-related stroke was further identified, and the key modules and hub genes were explored. Finally, we identified differentially expressed miRNA (DEMis) and differentially expressed circRNA (DECircs) and constructed a circRNA-miRNA-mRNA network to elucidate the molecular mechanisms of AF-related stroke.https://www.ncbi.nlm.nih.gov/geoprofiles). The mRNA expression profile GSE79768 was performed on Platform GPL570 with paired left atrial (LA) and right atrial specimens obtained from patients with persistent AF (n = 13) or sinus rhythm (n = 13), which described the mechanisms of AF-related LA remodeling, arrhythmogenesis, and thrombogenesis. The mRNA expression profile GSE58294 was performed on Platform GPL570 containing plasma samples from patients with cardioembolic stroke (n = 69) and healthy controls (n = 23). Subjects were analyzed at three time points: less than 3 h, 5 h, and 24 h after stroke. The mRNA expression profile data GSE66724 was based on peripheral blood cells in eight patients with AF and stroke and eight AF subjects without stroke (same anticoagulation and arrhythmia therapy). The miRNA dataset GSE70887 was performed on Platform GPL19546, containing LA biopsies of two sinus rhythm and four persistent AF patients. The circRNA dataset GSE129409 was performed on GPL21825 with heart tissues from three AF patients and three healthy controls. The flowchart of this study is shown in Fig 1.Microarray datasets of stroke and AF patients were downloaded from Gene Expression Omnibus (P < 0.05 and log FC > average (abs(log FC)) + 2 * SD (abs(log FC)) [P < 0.05 were used for filtering AF-DEGs. The |log FC| > 0.62 and P < 0.05 were used to identify stroke-DEGs. The |log FC| > 0.273 and P < 0.05 were used to identify AF-related stroke-DEGs. As per DEMis and DECircs, the cutoff values for log FC were 1.14 and 2.09, respectively. Volcano maps and heatmaps were also drawn by the plot and pheatmap package in the R studio to exhibit all DEGs.The limma package in R  was appllog FC)) . The expThe co-expression network of DEGs was constructed based on the GSE66724 microarray dataset using the R package “weighted correlation network analysis (WGCNA) .” The so The ROC curve was then plotted and the AUC was calculated to evaluate the capability of selected genes to distinguish AF-related stroke patients and AF patients without stroke. Only genes with an AUC > 0.8 were considered hub genes. The comparative toxicogenomic database (http://ctdbase.org/) was used to analyze possible relationships between hub genes and nervous or cardiovascular diseases .DEG1 was confirmed by intersecting AF-DEGs and stroke-DEGs. Then, genes in the module of interest by WGCNA were intersected with AF-related stroke-DEGs, and DEG2 was obtained. Thereafter, DEG1 and DEG2 were merged to obtain AF-related stroke genes (DEG3). The venn plot of these three DEG datasets were showed in P < 0.05 were considered significantly enriched. Subsequently, the AmiGO database  was used to analyze the GO consortium for filtered hub genes, verify their accuracy, and annotate their biological functions.Functional annotation of Gene Ontology (GO) ,16 was phttps://string-db.org/) was applied with a confidence score > 0.4. Cytoscape software  was used to visualize and analyze the biological networks and node degrees [To calculate the interactions between molecules in AF-DEGs, stroke-DEGs, and WGCNA modules, the online database STRING  (https:/ degrees .http://www.mirdb.org), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw), and TargetScan (http://www.targetscan.org), were used to predict the targets of DEMis. The targeted genes fulfilling the qualification of at least two databases were selected. Predicted genes were further filtered by matching the hub genes selected above. Then, the miRNA-mRNA pairs were determined. The miRNA targets of DECircs were predicted using the online database circBank (http://www.circbank.cn/), and they were validated by the DEMis selected above. Then, the circRNA-miRNA pairs were determined. Cytoscape was used to visualize this circRNA-miRNA-mRNA network.Three online databases, including miRDB  to inhibit RNA degradation. This study was approved by the Medical Ethics Committee of Huashan Hospital, Fudan University. All patients participating in this study provided written informed consent before blood collection.−ΔΔCT method was applied to analyze the results. All sequences for RNA primers were purchased from Sangon Biotech, Shanghai, China.The total RNA was extracted from blood samples with an RNAprep pure Blood Kit . Total RNA was quantified with a NanoDrop spectrophotometer 2000  and then reversely transcribed into cDNA using random primers with the PrimeScript™ RT Reagent Kit . A quantitative real-time polymerase chain reaction (qRT-PCR) was carried out with TB Green™ Premix Ex Taq™  on a CFX-96 real-time PCR System . Expression data was normalized by GAPDH, and the 2S1 Table). In GSE58294 (stroke mRNA dataset), we identified 348 upregulated and 445 downregulated genes at <3 h, 555 upregulated and 632 downregulated genes at 5 h, and 525 upregulated and 629 downregulated genes at 24 h. Here, we defined 566 co-expressed DEGs at the three time points mentioned above as the stroke-DEGs (S2 Table). In GSE66724 (AF-related stroke mRNA dataset), we identified 195 upregulated and 133 downregulated genes (S3 Table). In GSE70887 (AF miRNA dataset), we identified 12 upregulated and 9 downregulated miRNAs (S4 Table). In GSE129409 (AF circRNA dataset), we identified 270 upregulated and 162 downregulated circRNAs (S5 Table). Heatmaps of the top 30 AF-DEGs and stroke-DEGs are shown in Fig 2A–2D.After data pre-processing, we screened the DEGs of the datasets GSE79768, GSE58294, GSE66724, GSE70887, and GSE129409. In GSE79768 (mRNA dataset of AF), we identified 160 upregulated and 93 downregulated genes , GSE70887 , and GSE129409 .The volcanic diagram of all genes and the expression heatmap of the top 30 DEGs in GSE66724 . Based on the scale-free topology criterion, a soft-thresholding power of 5 was selected . Five modules were identified for further analysis. The cluster dendrogram of the modules is shown in Fig 4D, while the clustering of module eigengenes is provided in Fig 4E. Moreover, we analyzed the association of gene modules by comparing AF with stroke and AF without stroke (. The turquoise module showed the highest positive correlation (r = 0.34). Therefore, we identified the turquoise module as the key one for further analysis. A total of 432 genes were included in the turquoise module (S6 Table). Moreover, we illustrated in turquoise the module membership and gene significance for AF with stroke  . In addition, genes in the turquoise module were overlapped with DEGs in GSE66724. Fourteen genes (DEG2), including ZNF595, BC044596, MATR3, LOC389765, PDCD4, SEPSECS-AS1, GRINA, ELANE, TVP23B, AMFR, ROGDI, ZNF700, PLVAP, and MYL4, were hub gene candidates. We overlapped these two gene sets (DEG3) and obtained 21 genes: ZNF595, MATR3, PDCD4, SEPSECS-AS1, GRINA, ELANE, TVP23B, AMFR, ROGDI, ZNF700, PLVAP, MYL4, RNF166, ACKR4, EIF4E3, PDZK1IP1, HTRA1, NELL2, BEX2, ANXA3, and SLC51A.A hierarchical clustering tree was created based on the dynamic hybrid cut with a scale-free network and topological overlaps , Toll-like receptor 8 , complement C3 , cathepsin S , keratin 5 , myeloid cell nuclear differentiation antigen , snail family transcriptional repressor 2 , caspase 1 , and purinergic receptor P2Y13 . The top 10 hub genes in stroke-DEGs included G protein subunit gamma transducin 1 , phospholipase C gamma 1 , platelet factor 4 , AKT serine/threonine kinase 1 , gamma-glutamyl hydrolase , catenin beta 1 , junction plakoglobin , major histocompatibility complex, class II, DQ alpha 1 , LCK proto-oncogene , and adenylate cyclase 4 . Fig 6C showed that DEG1 owned 9 genes, including RNF166, ACKR4, EIF4E3, PDZK1IP1, HTRA1, NELL2, BEX2, ANXA3, SLC51A.We identified 155 and 43 nodes from the PPI network of AF-DEGs and stroke-DEGs, as shown in http://genemania.org/). As shown in Fig 7A, hydrolase activity, secretory granule lumen, azurophil granule and primary lysosome were the main enriched terms.We performed interactomics and GO functional enrichment analysis on DEG3 based on GeneMania , we filtered eight hub genes of AF-related stroke (AUC > 0.8), including EIF4E3, ZNF595, ZNF700, MATR3, ACKR4, ANXA3, SEPSECS-AS1, and RNF166. The database showed that hub genes targeted several nervous system and cardiovascular diseases . Results showed that the expression of EIF4E3, ANXA3, and hsa_circ_0018657 was significantly higher in AF-related stroke patients than those in AF patients without stroke, which was consistent with the bioinformatic analysis. The expression of MATR4, ACKR4, RNF166, and miR-198 in AF-related stroke patients was lower than those in AF patients without stroke.The expression levels of eight hub genes, As a serious public health concern, AF has shown an increasing incidence and prevalence in the elderly population, which is associated with elevated risks of cerebrovascular disease events and deaths –22. Dischsa_circ_0018657/hsa-miR-198/EIF4E3 axis as an important regulatory pathway in the development of AF-related stroke. These results were further validated in our qRT-PCR experiments.In this study, a series of bioinformatics analyses were carried out to filter hub genes of AF-related stroke. We first took the overlap of DEGs from AF and stroke patients to obtain DEG1. Then, WGCNA was used to identify key modules associated with AF-related stroke to obtain DEG2. The overlap of DEG1 and DEG2 (DEG3) was considered to include important genes in the development of AF-related stroke. Then, the PPI network of AF- and stroke-related DEGs was constructed, and we filtered eight hub genes based on ROC analyses. Those hub genes could facilitate the prevention of AF-related stroke and provide novel therapeutic targets. We also investigated the biological processes, cellular components, and molecular functions of the DEGs, revealing that these genes are significantly associated with the regulation of the BMP signaling pathway, smooth muscle cell proliferation, and extracellular matrix disassembly. Finally, we constructed a circRNA-miRNA-mRNA network of AF-related stroke and screened out the EIF4E3 was explored as a potential molecular signature for AF patients with a high probability of stroke occurrence, which may play an important role in the development of AF-related stroke. IF4E3 belongs to the EIF4E family as a translational initiation factor that interacts with the 5-prime cap structure of mRNA and recruit mRNA to the ribosome. Interestingly, although considered to regulate the nervous system directly or indirectly, EIF4E3 also has a role in the cardiovascular system. Mrvová, S and colleagues performed bioinformatics analyses of expressed sequence tags and the 3’-UTRs of the main transcript splice variants of the translational initiation factor EIF4E3 and showed that EIF4E3 mRNAs have a great potential in heavy post-transcriptional regulation [EIF4E3 truncated transcript variants were mainly found in the brain. However, EIF4E3 also promotes angiogenesis in the region surrounding myocardial infarction [In the present study, gulation . EIF4E3 farction .et al. explored peripheral blood mononuclear cell microRNA profiles from coronary artery disease patients and found that miR-198 exhibited a high expression level in unstable angina pectoris patients compared with such levels in stable patients [miR-198 could play an important role in the development of AF-related stroke by binding to the 3’UTR of EIF4E3.Along with genetic susceptibility, thrombus vulnerability is another main reason for AF-related stroke. miRNAs have also been proposed as potential biomarkers for vulnerable plaques. Hoekstra, M patients . Sepramapatients , which ihsa_circ_0018657, EIF4EBP2 was also found to participate in brain dysfunction. Martín-Flores, N showed the significant association of SNP rs1043098 in the EIF4EBP2 gene with the onset of dyskinesia induced by L-DOPA administration [EIF4EBP2 expression was downregulated compared to those in healthy controls [hsa_circ_0018657 could also play a part in cerebral disease, which needs further verification. As a miRNA sponge, hsa_circ_0018657 could regulate miRNA-198 by targeting EIF4E3. The role of this axis in the development of AF-related stroke deserves further exploration.As the host gene of stration . In multcontrols . Thus, hZNF595 and ZNF700 belong to the zinc finger protein family, whose members function as transcription factors that can regulate a broad variety of developmental and cellular processes. In schizophrenia (SCZ) patients, nonsense de novo mutations of ZNF595 are common. Data from genome-wide association studies suggested that common variants in the ZNF595 gene may be associated with SCZ and SCZ-related traits [ZNF700, which is relevant in Down syndrome brain development, providing a novel framework for further studies on epigenetic changes and transcriptional dysregulation during chromosome 21 neurogenesis [d traits . Pathoged traits –33. Whilogenesis .MATR3 gene encodes a nuclear matrix protein, which is proposed to stabilize certain mRNA species. Previous studies proved that mutations in MATR3 cause hereditary amyotrophic lateral sclerosis [MATR3 was significantly associated with neuronal development, synaptic plasticity, and memory-related processes [MATR3 encodes the nuclear matrix protein MATR3, which is strongly expressed in the neural crest, developing heart, and great vessels [MATR3 expression appear to cause similar left ventricular outflow tract defects in humans and mice. ACRK4 is a member of the G protein-coupled receptor family and is a receptor for C-C type chemokines. ACKR4 binds the homeostatic chemokines CCL19, CCL21, CCL25, and CXCL13 and has been attributed scavenging properties [ACKR4 was upregulated in the border/infarct area after myocardial infarction, and knocking out ACKR4 protected against adverse ventricular remodeling in mice post-infarction, indicating that ACKR4 may be a novel therapeutic target to ameliorate cardiac remodeling [ANXA3 is recognized as a regulator of cerebral ischemia/reperfusion injury. The upregulation of ANXA3 could promote cell viability, decrease cell apoptosis, and reduce the production of inflammatory cytokines in neurons after oxygen-glucose deprivation [ANXA3 would promote repair and healing of the myocardium after infarction by the activation of the PI3K/Akt signaling pathway [The clerosis –40. In arocesses . In addi vessels . Thus, soperties . The expmodeling . ANXA3 irivation . The sil pathway . Thus, t pathway .in vitro and in vivo experiments should be carried out to validate our results.This study also has several limitations. First, this study was based on microarray analysis, and gene expression may be not directly equivalent to protein expression. Second, data for the analysis of AF-related stroke mainly refers to persistent AF patients. Although persistent AF was most hazardous in stroke cases, other AF forms should be studied in the future. Finally, although we performed qRT-PCR to verify the expression levels of genes, more EIF4E3, ZNF595, ZNF700, MATR3, ACKR4, ANXA3, SEPSECS-AS1, and RNF166 may link AF and secondary stroke. The hsa_circ_0018657/hsa-miR-198/EIF4E3 pathway could be an important regulating axis in AF-related stroke. In addition, the hub genes TLR4, TLR8, C3, CXCR2, KRT5, MNDA, SNAI2, CASP1, and P2RY13 may be associated with AF recurrence and maintenance. GNGT1, PLCG1, PF4, AKT1, GGH, CTNNB1, JUP, HLA-DQA1, LCK, and ADCY4 may be associated with stroke.The hub genes of S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Cancer is a disease with ecological and evolutionary unity, which seriously affects the survival and quality of human beings. Currently, many reports have suggested Gas6 plays an important role in cancer. Binding of gas6 to TAM receptors is associated with the carcinogenetic mechanisms of multiple malignancies, such as in breast cancer, chronic lymphocytic leukemia, non-small cell lung cancer, melanoma, prostate cancer, etc., and shortened overall survival. It is accepted that the Gas6/TAM pathway can promote the malignant transformation of various types of cancer cells. Gas6 has the highest affinity for Axl, an important member of the TAM receptor family. Knockdown of the TAM receptors Axl significantly affects cell cycle progression in tumor cells. Interestingly, Gas6 also has an essential function in the tumor microenvironment. The Gas6/AXL pathway regulates angiogenesis, immune-related molecular markers and the secretion of certain cytokines in the tumor microenvironment, and also modulates the functions of a variety of immune cells. In addition, evidence suggests that the Gas6/AXL pathway is involved in tumor therapy resistance. Recently, multiple studies have begun to explore in depth the importance of the Gas6/AXL pathway as a potential tumor therapeutic target as well as its broad promise in immunotherapy; therefore, a timely review of the characteristics of the Gas6/AXL pathway and its value in tumor treatment strategies is warranted. This comprehensive review assessed the roles of Gas6 and AXL receptors and their associated pathways in carcinogenesis and cancer progression, summarized the impact of Gas6/AXL on the tumor microenvironment, and highlighted the recent research progress on the relationship between Gas6/AXL and cancer drug resistance. Cancer Currently, many reports have suggested TAM receptors and ligands play an important role in cancer. TAM receptor family members include Tyro3, Axl, MerTK (collectively known as TAM) . The TAM22.1The TAM ligand family includes human growth inhibitor specific 6 (GAS6), PROS1, LGALS3, Tulp-1 etc . GAS6 is2.2via non-inflammatory reactions, and affect natural killer cell differentiation and platelet aggregation etc.  and vascular cell adhesion molecules (VCAM) , which c3Upregulation of Gas6/AXL is associated with carcinogenesis in multiple malignancies and shortens overall survival, and may be involved in tumor cell proliferation, migration, apoptosis, and maintenance of tumor stem cells through multiple signaling pathways Figure 13.1Post-binding activation of AXL and GAS6 is correlated with enhanced proliferation and survival in multiple tumors, including prostate, colorectal, gastric and renal cancers, and osteosarcoma –13. The 3.2AXL is considered a driver of tumor metastasis. AXL activity highly contributes to the acquisition of migratory potential in cells . Tumor m3.3via BAD (BCL2-associated cell death agonist) phosphorylation and ERK1/2  on tumor development Figure 24.1AXL is widely synthesized by endothelial cells and could promote angiogenesis by regulating the production of VEGF and PDGF, thus participating in the mediation of normal and tumor vascular systems. For instance, suppression of AXL reduces the expression of Tie2 and VEGFR-2 , leading4.2Substantial evidence shows GAS6/AXL signaling is important in the promotion of an immunosuppressive TME. The GAS6/AXL axis regulates many tumor immune-related biomolecules, e.g., major histocompatibility complex I (MHC-I) and programmed death ligand-1 (PD-L1) in tumor cells , and the4.2.1+ T cells, inducing them to recognize tumor cells and secreting perforin and granzyme. Evidence indicates AXL is negatively associated with MHC-I molecules. Rothlin et al. found that TAM deficient mice have elevated in vivo amounts of MHC-I in myeloid cells, confirming for the first time an association between MHC-I and Axl  lyse tumor cells by presenting MHC-I antigen epitopes to CD8 and Axl . Jeon an and Axl . Aguiler and Axl . Of more and Axl . In summ4.2.2PD-L1 is produced by a variety of tumor cells, and its interaction with its receptor induces pathways for blocking T cell activation  to evade4.2.3+ T cells and NKs  in various tumor settings is important for homeostasis under physiological conditions. The main mechanisms involve macrophage polarization and efferocytosis of apoptotic cells , 97.TAM receptor-mediated efferocytosis has tumor-promoting functions such as immunosuppression, tumor metastasis, and resistance to therapeutics . Gas6/AXvia strong induction of the JAK/STAT1 pathway  AXL may be a potential mediator of T-cell rejection, increasing tumor cell invasion and metastasis and suppressing the immune response by enhancing T-cell rejection . The mec4.3.4via IL-2, a powerful growth factor. After GAS6 addition to a co-culture system comprising CD4+CD25-T cells and CD4+CD25+ Tregs, T-cell proliferation was reduced as well as IL-2 expression. After Axl knockout, Foxp3 expression in Tregs was decreased and IL-2 expression was increased. Therefore, Gas6 can inhibit CD4+ T cells by depleting IL-2 or inhibiting IL-2 production (Regulatory T cells (Tregs) regulate immune evasion, considered the primary mechanism of evasion from immune surveillance . Tregs ioduction . These foduction .5Tumor cell resistance is an important issue in cancer therapy, often leading to failed treatment or recurrent disease. Besides its roles in survival, proliferation and migration, AXL expression is a possible mechanism underlying resistance to immunotherapy, chemotherapy and molecularly targeted therapies. AXL may lead to innate or acquired resistance to chemotherapy, immunotherapy, molecularly targeted therapies and even radiotherapy , 118.5.1Hong et al. found that chemotherapeutic agents such as etoposide (VP-16) and cisplatin induce AXL upregulation in resistant acute leukemia, as a potential mechanism of chemoresistance . Wang an5.2Widespread overexpression of AXL is also found in tumors following resistance to various targeted therapies , 123, re5.3As described in Section 3 of this paper, the Gas6/AXL pathway regulates the immune microenvironment by modulating important components of the immune microenvironment, including the tumor’s vascular system; critical biomarkers such as MHC-I molecules and PD-L1; important cytokines such as IL-4, CCL3-5 and G-CSF; and key immune cells such as phagocytes, DCs, NK cells, effector T cells and Tregs. In addition, it induces the formation of an immunosuppressive microenvironment, suppresses the host’s antitumor immunity, and mediates tumor immune escape. Therefore, the relationship between Gas6/AXL signaling and resistance to immunotherapy deserves further attention. By comparing the transcriptomes of PD-1-responding and non-responding tumors, Hugo et al. found that AXL is upregulated in non-responding tumors  and may The above evidence provides a theoretical basis for the development of AXL-related drugs in combination with conventional therapeutic modalities based on synthetic lethality in the context of tumor resistance to therapy. Given the role of AXL in cancer growth and metastasis as well as its relatively low expression in normal tissues compared with tumor tissues, AXL represents a highly potential therapeutic target in cancer therapy. Currently, a series of therapeutic drugs targeting AXL have been developed, including small molecule inhibitors , 127, mo6Roles for Gas6/AXL signaling in cancer development and progression, and shaping of the tumor microenvironment are gradually being revealed. Recently, substantial resources have been deployed to develop broad therapies targeting Gas6/AXL in cancer . CurrentGuarantor of integrity of the entire study: XP, QZ and LL. Study conception and design: XP, QZ and LL. Literature search: XZ, DP, RW, JZ, YL, YW, NZ. Clinical studies: XZ, DP, RW, JZ, YL, YW, NZ. Experimental studies/data analysis: XZ, DP, RW, JZ, YL, YW, NZ. Statistical analysis: XZ, DP, RW, JZ, YL, YW, NZ. Manuscript preparation: All authors. Manuscript editing: All authors. All authors contributed to the article and approved the submitted version.


It shows higher expression in several cancers involving breast, pancreatic, prostate, melanoma, leukemia, and neuroblastoma, indicating it raises the survivability of cancerous cells. In various cancers including gastric cancers, and neuroblastoma, TRPM2 is known to conserve viability, and several underlying mechanisms of action have been proposed. Transcription factors are thought to activate TRPM2 channels, which is essential for cell proliferation and survival. In normal physiological conditions with an optimal expression of TRPM2, mitochondrial ROS is produced in optimal amounts while regulation of antioxidant expression is carried on. Depletion of TRPM2 overexpression or activity has been shown to improve ischemia–reperfusion injury in organ levels, reduce tumor growth and/or viability of various malignant cancers like breast, gastric, pancreatic, prostate, head and neck cancers, melanoma, neuroblastoma, T-cell and acute myelogenous leukemia. This updated and comprehensive review also analyzes the mechanisms by which TRPM2-mediated Ca2+ signaling can regulate the growth and survival of different types of cancer cells. Based on the discussion of the available data, it can be concluded that TRPM2 may be a unique therapeutic target in the treatment of several types of cancer.The transient receptor potential melastatin subfamily member 2 (TRPM2), a thermo and reactive oxygen species (ROS) sensitive CaVideo AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01149-6. Drosophila first recognized the trp gene through visual transduction mutation cyt. It also works as a lysosomal Ca2+ release channel  polymerase (PARP) because of DNA injury i indicating a positive feedback regulation of TRPM2 i is present, but perhaps not in resting [Ca2+]  .2+] in the mitochondrial matrix, probably, implying that during anoxia Ca2+ may be released into the cytoplasm of the cells. The mitochondrial respiratory chains produce most ROS in cells cyt, H2O2, cADPR (Cyclic adenosine 5′-diphosphate ribose), NAADP (Nicotinic acid adenine dinucleotide 2’-phosphate), and extra- and intracellular pH of the cells i i . The ranal cells . Either termined . Determitermined . Proper termined , clotrimtermined , ACA 152+cyt, H2termined , 2-APB [termined , antioxitermined , 113. Totermined , 124. Motermined  and arou of CD38 .2+ entry into different cell types subjected to ischemic reperfusion injury. Inhibitors of Ca2+ entry through TRPM2 have been shown to prevent IR injury in case of heart, liver, brain and/or kidney. Because it has been recognized that ROS-initiated stimulation of TRPM2 takes part together with acute and chronic liver injury, considerable additional investigation is required to explain the mechanisms engaged as well as the situations under which pharmacological hindrance of TRPM2 can be a selective clinical approach to diminish ROS-initiated liver injury. In the case of cancers, the expression pattern of TRPM2 in various kinds of cancer suggests that TRPM2 can stimulate tumor survival. Inhibited TRPM2 has been known to increase cell death along with augmenting sensitivity to chemotherapeutic agents including doxorubicin in several malignancies with neuroblastoma [A lot of work has been done on the activation of TRPM2 channels by ROS for different cell types including neuronal cells, liver cells, pancreatic, and cardiac cells. Now it is well-known that TRPM2 shows a crucial function in Cablastoma , 98, gasblastoma , T cell blastoma , triple-blastoma . The pre