Dysregulation of the epigenetic enzyme-mediated transcription of oncogenes or tumor suppressor genes is closely associated with the occurrence, progression, and prognosis of tumors. Based on the reversibility of epigenetic mechanisms, small-molecule compounds that target epigenetic regulation have become promising therapeutics. These compounds target epigenetic regulatory enzymes, including DNA methylases, histone modifiers (methylation and acetylation), enzymes that specifically recognize post-translational modifications, chromatin-remodeling enzymes, and post-transcriptional regulators. Few compounds have been used in clinical trials and exhibit certain therapeutic effects. Herein, we summarize the classification and therapeutic roles of compounds that target epigenetic regulatory enzymes in cancer treatment. Finally, we highlight how the natural compounds berberine and ginsenosides can target epigenetic regulatory enzymes to treat cancer. The concept of epigenetics was first introduced in 1942 by Waddington, a British scientist who defined “epigenetics” as changes in the phenotype without underlying genotypic changes to explain altered growth and development  and ginsenosides, natural compounds capable of targeting epigenetic enzymes in cancer. Additionally, we discuss promises and challenges that lie ahead of us.L-methionine (SAM) to the 5′-residue of cytosine (5′-C) in DNA  in DNA , 12, whe) in DNA , 14. Fur) in DNA , 16. Cle) in DNA . DNMTs p) in DNA .DNA demethylation can occur either passively or actively. DNA demethylation or “erasing DNA methylation” can occur passively when DNA is replicated and the modification is not re-established. One example of passive DNA demethylation is the absence of methylation owing to a lack of DNMT1, whereas another is the removal of methyl groups from cytosine (5-mC) by ten-eleven translocation proteins (TETs) in a replication-independent manner. TETs mainly oxidize 5-mC to form 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) , 20. TheL-homocysteine analogs, and miR-29a have been shown to suppress the activity of DNMTs  have been developed for tumor treatment. DNMTis are mainly divided into three types, nucleoside analogs (NAs), non-nucleoside compounds, and natural compounds . Compounof DNMTs –40. Amonof DNMTs . In a phof DNMTs . Interesof DNMTs , 37. Genof DNMTs .Histone methylation, a unique post-translational modification catalyzed by histone MTases (HMTs), occurs at both lysine (K) and arginine (R) residues. Abnormal histone methyl modification plays an important role in the proliferation, apoptosis, differentiation, and invasion of tumor cells. Thus, blocking these abnormal modifications has become a new direction in tumor therapeutics , 66. Thevia the demethylation of H3K9me1/me2  and Jumonji C (JmjC) domain-containing lysine demethylases (JmjC-KDMs). The LSD family includes LSD1/KDM1A and LSD2/KDM1B proteins, which contain the N-terminal Swi3p, Rsc8p, and Moira (SWIRM) domains, a flavin adenine dinucleotide-binding motif (FAD), and a C-terminal amine oxidase domain that is responsible for LSD activity in an FAD-dependent manner . Both LS9me1/me2 . The Jmj9me1/me2 , 72. The9me1/me2 , 74.Histone methylation is a hot topic in tumor epigenetic modification. This modification is associated with the biological behavior of tumor cells and plays a role in the development of tumors. In this section, we focus on a subclass of epigenetic regulators, namely histone methyltransferases. To date, hundreds of HMTs have been identified, including lysine and arginine MTases . SeveralMost HMTs are present as closely homologous pairs. For example, the catalytic SET domains of G9a and GLP share 77% sequence identity and are present as a stoichiometric heterometric complex . In cellvia catalytic subunits enhancer of zeste homologs 1/2 (EZH1/2)  genes  . In DrosX) genes . AberranX) genes , 81. FurX) genes .Inhibitors of EZH1/2 can be classified into three groups according to their basic skeleton structure as follows: those with the pyridone-indazole scaffold, which includes EPZ005687, UNC1999, and GSK343 ; those wDOT1-like protein (DOT1L), an enzyme responsible for H3K79 methylation, does not contain the SET catalytic domain and displays a class I SAM-dependent MTase fold . In cellHOXA9 and MEIS1. Additionally, SYC-522 significantly reduces the expression of CCND1 and BCL2L1, which are important regulators of the cell cycle and anti-apoptotic signaling pathways  family includes nine enzymes divided into three types, type I PRMT, CARM1, and PRMT5. In cells, PRMTs catalyze the methylation of arginine residues on histones. PRMT dysfunction is associated with the occurrence of several cancers.PRMTis are also classified into three types based on their corresponding PRMT type and have been investigated in the early preclinical stage Table 1.via p53 , some of which have entered different clinical stages as anti-tumor drug candidates Table 1.via p53 . Of the via p53   domain, and malignant brain tumor (MBT) domain families . These pChromodomain proteins are further classified into heterochromatin protein 1 (HP1)/polycomb (Pc), chromo-ATPase/helicase-DNA-binding (CHD), chromobarrel domain, and chromodomain Y (CDY) families . Both HPMethyl-lysine reader proteins play important roles in regulating many cellular processes, such as development, the cell cycle, stress responses, and oncogenesis, and have increasingly become the focus of epigenetic research. Inhibitors of methyl-lysine reader proteins, such as MS37452A, SW2_110A, and UCN3866, have been found to inhibit the growth of cancer cells as selective inhibitors of Pc chromobox (CBX) and CDY proteins , 99. Addvia the addition of an acetyl group to the ε-amino of the lysine residue at the midamino end and tail of the histone. This process is dynamically controlled by histone acetyltransferases (HATs), lysine acetyltransferases, and histone deacetylases (HDACs)  Table 2. (HDACs) .There are three major families of HATs, general control non-derepressible 5 (Gcn5)-related N-acetyltransferases (GNATs), p300/CBP, and MYST proteins. p300  and CBP (CREB-binding protein) form a pair of paralogous transcriptional co-activators. Members of the GNAT family include HAT1, yeast Gcn5, and its metazoan orthologs GCN5 and PCAF (p300/CBP-associated factor) . HATs ar2+ for deacetylase activity. The sirtuin family contains seven members , which show no sequence resemblance to members of the classical family and require NAD+ as the cofactor -related protein (sirtuin) families. In humans, members of the classical family include HDAC1, 2, 3, and 8 (class I); HDAC4, 5, 6, 7, 9, and 10 (class II); and HDAC11 (class IV). They share sequence similarity and require Zncofactor , 107.Imbalanced HAT expression and acetylation levels in tumorigenesis make HATs suitable targets for drug development. In preclinical experiments, many small-molecule compounds have been screened as potential HATis to regulate histone acetylation and reduce tumor growth Table 3.Streptomyces that effectively suppressed zinc-dependent HDACs in the preclinical stage , and BRD. PHD finger proteins recognize acetylated, un-acetylated, or methylated histones, with the PHD finger domains in MLL4 (KMT2D) and MLL3 (KMT2C) targeting H4K16 acetylation and involved in the interaction between MLL4/3 and males absent on the first (MOF) (1278). YEATS proteins interact with acetylated histones H3K9, H3K14, and H3K27 .Many BRD proteins are involved in chromatin-remodeling or chromatin-modifying enzymes. BRDs in HATs act as protein–protein interaction modules that specifically recognize acetylated histones to regulate gene transcription, including H4K5, H4K8, H4K12, H4K16, H4K20, H3K14, and H3K36 . At presNatural compounds and their active components have been widely used in traditional medicine in China, Japan, South Korea, and other countries for their various pharmacological effects. Increasing natural compounds have demonstrated high anticancer activity, providing potential candidates for developing multifunctional tumor-targeted drugs. However, their precise mechanisms of action remain unclear. Here, we focus on BBR (C20H18NO4) and ginsenosides, natural compounds that have undergone extensive preclinical investigation and play anti-tumor roles by targeting epigenetic enzymes and ncRNAs Figure 3Coptis, and its derivatives exhibit effective anti-tumor activity , cytochrome P450 3A4 (CYP3A4), and glucose regulated protein 78 (GRP78)  . In addi (GRP78) , 140.Ginsenosides, derived from saponins of ginseng, have a steroid-like hydrophobic backbone connected to one or more sugar moieties and are generally believed to be the major bioactive constituents of ginseng . GinsenoDNMT1 expression to reduce the proliferation of colorectal cancer (CRC) cells by reactivating the epigenetically silenced RUNX3 gene . The Science and Technology Research Project of the Jilin Provincial Department of Education , and Jilin Province Science and Technology Development Project in China (Grant No. 20210204013YY).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.


Osteosarcoma (OSA) is an aggressive bone malignancy. Unlike many other malignancies, OSA outcomes have not improved in recent decades. One challenge to the development of better diagnostic and therapeutic methods for OSA has been the lack of well characterized experimental model systems. Spontaneous OSA in dogs provides a good model for the disease seen in people and also remains an important veterinary clinical challenge. We recently used RNA sequencing and qRT-PCR to provide a detailed molecular characterization of OSA relative to non-malignant bone in dogs. We identified differential mRNA expression of the solute carrier family 2 member 1 (SLC2A1/GLUT1), matrix metallopeptidase 3 (MMP3) and nuclear factor erythroid 2–related factor 2 (NFE2L2/NRF2) genes in canine OSA tissue in comparison to paired non-tumor tissue. Our present work characterizes protein expression of GLUT1, MMP3 and NRF2 using immunohistochemistry. As these proteins affect key processes such as Wnt activation, heme biosynthesis, glucose transport, understanding their expression and the enriched pathways and gene ontologies enables us to further understand the potential molecular pathways and mechanisms involved in OSA. This study further supports spontaneous OSA in dogs as a model system to inform the development of new methods to diagnose and treat OSA in both dogs and people. Canine osteosarcoma (OSA) presents a significant veterinary clinical challenge with an estimated incidence rate of between 13.9–27.2/100,000 dogs, considerably higher than the rate in people, 1–3 cases/annum/1,000,000 people . It sharTP53, is characteristic of OSA  and NRF2 (transcription factor encoded by NFE2L2), which are known to play a role in human OSA, in an OSA canine cohort.More recently our group identified several genes significantly differentially expressed between canine OSA and non-tumor bone tissue . Consistanalysis  by invesAll animal tissue work in this study was approved by the Ethics committee at the University of Nottingham School of Veterinary Medicine and Science and complied with national ethics procedures (permission number - UG 20331). Patients were euthanised under normal veterinary practice under circumstances unrelated to research. Diagnosis of OSA was confirmed by a board certified histopathologist.n = 15) was obtained from Bridge Pathology, UK in the form of formalin fixed, paraffin embedded OSA tissue. The OSA samples were all excised from Rottweilers, 9/15 female, 5/15 male and 1/15 not specified. The females ages ranged between 7-9 years old and 2/9 were entire, and the males were between 6-10 years old and 3/5 were entire. OSA location was 10/15 appendicular, 3/15 axial, 1/15 mixed appendicular/axial, 1/15 not specified. A range of morphologic types were studied including 10/15 osteoblastic; 3/15 chondroblastic or mixed osteoblastic/chondroblastic; and 2/15 suspected giant cell rich. In addition a range of mitotic activity values  were inn = 15) was obtained from Bridge Pathology, UK in the form of formalin fixed, paraffin embedded OSA tissue. OSA samples from a variety of bones including the humerus X2, scapula, femur X3, mandible X2, temporomandibular joint, tibia, maxilla, stifle, carpus and 2 unknown locations were excised from male and female Rottweilers between the ages of 6-11 years old. Tissue was post-fixed in 4% paraformaldehyde for 2 hours, dehydrated through an ethanol series, embedded into paraffin blocks, and sectioned at 7 μm. Immunohistochemistry was carried out using a Leica Novolink Polymer Detection Kit  according to manufacturer's protocols with primary antibodies diluted in fetal calf serum 1:100; anti-SLC2A1(GLUT1) polyclonal unconjugated rabbit antibody , anti-MMP3 polyclonal unconjugated rabbit antibody , anti-NRF2 (NFE2L2) polyclonal unconjugated rabbit antibody  were used to stain proteins of interest. Microscopy was carried out to confirm positive staining cytoplasmic and/or nuclear staining  and systematic random sampling employed to take photomicrographs for H-scoring. Negative controls received no primary antibody and were incubated in fetal calf serum only. Kidney sections from one of the patients were used as positive controls, as the target markers were known to be expressed in the kidney , validated by qRT-PCR . Immunohe kidney –32.n = 10-13 OSA samples) for each antibody. H-scores were calculated using the formula: H-score = [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)] for both cytoplasmic and nuclear staining separately. H-scores based on the resulting 0–300 scale were calculated for each specimen and each protein. The mean, standard error of the mean, minimum, maximum and range of H-scores were calculated. Data was plotted to demonstrate both score distributions and staining intensities. In addition, representative staining scores (based on these samples only) were created as benchmarks to discuss results. H-scores were also classified as low 0–45, moderate 45-90 or high 90+ average scores in order to describe the overall scores. Statistical analysis between cytoplasmic and nuclear H-scores was conducted using paired T-test (SPSS v26). Comparisons between the number of slides with 0, 1, 2, and 3 H-score staining categories in the cytoplasm and nucleus were conducted using chi-square.H-scoring, a well-established semi-quantitative technique for protein expression was used to analyse the samples. It is often considered as one of the “gold standards” for immunohistochemistry evaluation –35. H-scQualitative data was also recorded in order to describe general immunohistochemical staining patterns. Qualitative data was described for both neoplastic areas and, where possible, adjoining areas where no tumor was present. In addition to describing the cell/structure types present and the immunostaining observed, general staining was identified for each sample , both cytoplasmic and nuclear staining were described  and the predominant staining location was identified .n = 47 sections from 13 patients) showed H-score variations between the different patients, however all specimens showed positive immunostaining. Only 1 of the 13 patients showed both low cytoplasmic and low nuclear average scores (7.7%), while 2 of the 13 patients showed both high cytoplasmic and high nuclear average scores (15.4%). Only one patient had a low average GLUT-1 cytoplasmic score, 7 patients had moderate cytoplasmic scores (58.3%) and 5 had high cytoplasmic scores (38.5%). Nuclear scores showed 4/13 patients with low H scores (30.8%), 4/13 at moderate (30.8%) and 5/13 at high (38.5%), overall these were not significantly different to the cytoplasmic staining scores (P > 0.05). The 0-300 cytoplasmic H-scores were slightly higher than the nuclear scores, however the same range for both locations was observed and no significant differences were present , all specimens showed positive immunostaining and less variation was calculated between patients on the 0–300 score in comparison to GLUT1 and NRF2 H-scoring. In total 8/12 (66.7%) patients had a low average nuclear score, while only 1/12 (8.3%) had a high nuclear score. In addition, 50% (6/12) of the patients had a combination of moderate average cytoplasmic scores and low average nuclear scores. Only 1 (8.3%) patient had a low average cytoplasmic score. Low scores were more likely to be observed in the nucleus, whereas moderate and high scores were more frequently observed in the cytoplasm (P < 0.0001). Overall, on the 0–300 scale the cytoplasmic H-scores were higher than those observed in nuclei  exhibited positive MMP3 immunostaining in the extracellular matrix, however osteoid staining was not present in any samples .n = 51 sections from 10 OSA patients) showed considerable variation between patient averages on the 0-300 scale, but positive immunostaining was observed in all specimens. Some patients  demonstrated high average H-scores of both cytoplasmic and nuclear NRF2 staining, some patients demonstrated either high average nuclear staining  or high average cytoplasmic staining . The remaining 50% (5/10) demonstrated moderate levels of both nuclear and cytoplasmic NRF2 staining. In addition, low and high scores were more likely to be present in the nucleus in comparison to the moderate scores which were more frequent in the cytoplasm (P = 0.008). Overall the 0–300 cytoplasmic and nuclear staining H-scores were similar , however the range of H-scores was greater in the nuclear staining , while 8 patients also showed positive nuclear staining in the tunica intima . GLUT1 immunostaining has previously shown an abundance of the protein in blood vessels within the diaphysis of normal long bones, but not in the metaphysis . It has c effect . Our finMMP3 in microarray datasets  in cells, which then trigger DNA damage which leads to apoptosis , 72. TheNFE2L2 in human cancer cells was effective in altering the NFE2L2/NRF2 pathway and improving chemosensitivity (NFE2L2, given in conjunction with cisplatin, improved treatment of OSA (Nuclear staining of NRF2 has been shown in bone metastases of people with OSA , and expsitivity . Oridonisitivity . Tanshinsitivity . A lipost of OSA . These rCanine OSA is divided into several morphologic subclasses: osteoblastic, fibroblastic, chondroblastic, and teleangectatic , howeverOur results have shown that GLUT1, MMP3 and NRF2 are all present in canine OSA from a number of different anatomical locations including the humerus, scapula, femur, tibia, stifle, carpus and the mandible, maxilla and temporomandibular joint. Previous canine OSA studies have shown that tumor location and mitotic index can be correlated with survival time and disease-free interval , 89, theThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by University of Nottingham School of Veterinary Medicine and Science. Written informed consent was obtained from the owners for the participation of their animals in this study.CR and NM: conceptualization. CR, JC, SS, AA, CB, SB, and NM: data curation. CR, JC, JL-R, SB, and NM: formal analysis. CR, SB, and NM: funding acquisition. CR, JC, JL-R, AH, JJ, SS, AA, CB, AB-R, AR, MD, SB, and NM: investigation. CR, JL-R, CB, SB, and NM: methodology. CR, MD, and NM: project administration. CR, MD, SB, and NM: resources. CR, JR, and NM: supervision. CR, AA, and JL-R: validation. CR, JC, AA, CB, and SB: visualization. CR, JC, AH, JJ, CB, AR, and NM: writing—original draft. CR, JC, JL-R, AH, JJ, SS, AA, CB, AB-R, AR, MD, SB, and NM: writing—review and editing. All authors have read and agreed to the published version of the 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.


Pneumoconiosis, coronavirus disease 2019, and idiopathic pulmonary fibrosis all have similar typical pathological changes—pulmonary fibrosis. Pulmonary fibrosis is a chronic lung disease characterized by excessive deposition of the extracellular matrix and remodeling of the lung tissue structure. Clarifying the pathogenesis of pneumoconiosis plays an important guiding role in its treatment. The occurrence and development of pneumoconiosis are accompanied by epigenetic factors  changes, which in turn can promote or inhibit the process of pneumoconiosis. Here, we summarize epigenetic changes and functions in the several kinds of evidence classification (epidemiological investigation,  Pulmonary fibrosis is a chronic lung disease with myriad causes that is characterized by excessive deposition of the extracellular matrix (ECM) and remodeling of lung tissue structure. Pneumoconiosis, coronavirus disease 2019 (COVID-19), interstitial lung disease associated with systemic sclerosis (SSC-ILD), and idiopathic pulmonary fibrosis (IPF) all have such typical pathological changes. Currently, no specific treatment for pulmonary fibrosis exists. If pneumoconiosis occurs, pulmonary fibrosis is almost irreversible, even if there is no longer exposure to dust, and it can develop progressively. Pneumoconiosis is prevalent worldwide, especially in low-income and middle-income countries, and has a higher incidence among gold, iron, and tin mining workers. According to the nature of the mineral dust that causes pneumoconiosis, the disease can be divided into the following categories: (1) silicosis caused by dust containing mainly free silica, which is the most common type of pneumoconiosis; (2) silicate pneumoconiosis caused by silicate-based dust, including asbestos pneumoconiosis, talc pneumoconiosis, cement pneumoconiosis, mica pneumoconiosis, and potter's pneumoconiosis; (3) pneumoconiosis caused by coal dust and carbon-based dust, including coal worker's pneumoconiosis, graphite pneumoconiosis, and carbon black pneumoconiosis; and 4) metal pneumoconiosis caused by metal dust, including aluminum pneumoconiosis, welder's pneumoconiosis, and caster's pneumoconiosis. China is the country most seriously affected by pneumoconiosis in the world, with the highest numbers of dust exposure, new cases of pneumoconiosis, and current cases of the disease. Although the global incidence of pneumoconiosis has decreased in recent years, the number of people living with pneumoconiosis remains high. In China alone, the cumulative number of patients with pneumoconiosis exceeded 6 million as of 2013. In addition, pneumoconiosis cannot be effectively treated, so the economic and health burdens of pneumoconiosis on the country, society, family, and individuals remain severe. The cost of pneumoconiosis for patients in China is approximately 19,000 RMB/person-year  plus 46,000 RMB/person-year (indirect management expenses). The average life expectancy of pneumoconiosis after diagnosis is 32 years. Given the current economic market, the fiscal burden of pneumoconiosis is approximately RMB 2.075 million/person [ metal pnEpigenetics refers to changes in gene expression and function without changing the DNA sequence, and this phenotypic change can be inherited. It plays a key role in many cellular processes, such as gene expression regulation, cell proliferation and differentiation, and chromosome inactivation . EpigeneDNA methylation is the main epigenetic mechanism of gene selective transcription regulation. DNA methylation refers to the process in which DNA methyltransferases (DNMTs) transfer the methyl group of S-adenosylmethionine to a specific base on DNA. In mammals, methylation occurs on the fifth carbon atom of the cytosine (C) of the 5'—C—phosphate—G—3'(CpG) dinucleotide, which is separated from guanine (G) by a phosphate to form 5-methylcytosine (5mC) , 7.The distribution of CpG dinucleotides is not uniform on DNA. The CpG in some areas presents as an aggregated state, usually 1-2 kb, called a CpG island. Approximately 70% of gene promoter regions in mammals contain CpG islands, and most of them are in an unmethylated state. The methylation degree of CpG islands is closely related to gene transcription. Approximately 70-80% of CpG sites not in islands are in a methylation state , 10. GenMaintenance methylation refers to the same methylation modification on the nascent DNA strand as that on the parent strand during cell division. De novo methylation refers to the methylation of unmethylated CpG sites.DNA methylation or demethylation is mainly carried out by numerous enzymes. The role of DNA methylation in mammals is mainly mediated by DNMTs and methyl-CpG-binding proteins (MBPs) –17.DNMTs include DNMT1, DNMT2, and DNMT3. DNMT1, also known as maintenance methyltransferase, is highly expressed in various mammalian tissues. Methylation catalyzed by DNMT1 can be inherited. DNMT2 is also widely distributed in human and murine tissues, but its role is unclear. DNMT3 subtypes include DNMT3a, DNMT3b, and DNMT3L. DNMT3a and DNMT3b are also known as de novo DNMTs. DNMT3a is widely distributed; DNMT3b is mainly distributed in the thyroid, testis, and bone marrow. DNMT3L assists DNMT3a and DNMT3b, because it does not have catalytic activity alone .DNA methylation in the promoter region can directly interfere with the binding of transcription factors to cis-acting elements, leading to a decrease in gene transcription. In addition, DNMT1  and DNMTDNA methylation is a reversible process. DNA demethylation includes (1) passive DNA demethylation, in which maintenance methyltransferases are inhibited or dysfunctional, and the level of DNA methylation gradually decreases with cell division and proliferation; and (2) active DNA demethylation, which is regulated mainly by deaminase activation-induced deaminase (AID)/apolipoprotein B mRNA-editing catalytic polypeptide-like (APOBEC) and translocation t , 21–24. DNA methylation has been extensively studied in pulmonary fibrosis diseases, such as COVID-19, SSC-ILD, and IPF. DNA methylation is also involved in the pathogenesis of fibrosis in multiple organs and systems. For example, DNA methylation can regulate myofibroblast differentiation and apoptosis resistance , 26, leaThe study by de Souza et al.  analyzed2-induced fibroblast to myofibroblast progression. These changes mainly occur in the metabolic process, biological regulation, and biological adhesion pathways. In an experiment by Wang et al. [2 particles, and the genome-methylation level was also reduced in a dose-dependent manner. DNA methylation has not been widely used in pneumoconiosis research and clinical practice, but DNMTs inhibitors, especially 5-azacytidine (5-aza-C) and 5-aza-dC (which are common in cancer treatment), have been used for pneumoconiosis treatment [ACTA2 expression in primary IPF fibroblasts through a chip experiment. MeCP2 plays a key role in the upregulation of ACTA2 by transforming growth factor β (TGF-β) in lung fibroblasts, and 5-azaC can partially block the expression of ACTA2 induced by TGF-β [β [Thy-1 DNA methylation has been extensively studied in other pulmonary fibrosis diseases, and an increase in its methylation plays an important role in pneumoconiosis , 33. In g et al. , 40, thereatment . MeCP2 ireatment  found thby TGF-β . Althougby TGF-β , 43, 44.TGF-β [β . JianghuTGF-β [β , 47 asceTGF-β [β , 47. OthTGF-β [β –50.in vivo and in vitro experiments. Little research on DNA methylation–related treatments for pneumoconiosis exists, but research in other fibrotic diseases, especially IPF, can be used as a reference.Methylation changes do exist in pneumoconiosis, but a full assessment of the specific changes and their effects requires large-scale epidemiological studies and NcRNA refers to RNA that cannot be translated into protein. A small part of ncRNA, called housekeeping ncRNA or infrastructural ncRNA , is expressed constitutively. Expression of most ncRNAs is induced under certain conditions; regulatory ncRNAs can manipulate mRNA expression. Regulatory ncRNAs can be divided into long ncRNA (lncRNA) and short-strand ncRNA (<50 nt) according to their molecular sizes, and short-strand ncRNAs can be divided into microRNA (miRNA), small interfering RNA (siRNA), and piwi-interacting RNA (piRNA) . Circula∗ duplexes. Usually, miRNA∗ is degraded, and miRNA plays a biological role [The classic method of miRNA production involves corresponding independent genes for each miRNA that are transcribed by RNA polymerase II to produce primary precursors and become pri-miRNA. Pri-miRNA is processed in the nucleus by the Drosha-DGCR8 complex into pre-miRNA, which has a hairpin structure. Pre-miRNA is processed by Dicer in the cytoplasm into miRNA/miRNAcal role . In addiThe expression of miRNA also regulated by many factors. As in mRNA, the gene promoter region of miRNA also contains CpG islands, TATA boxes, and transcription initiation elements, and the transcription of miRNA is also regulated by transcription factors, enhancers, and silencers , 51, 52.Many studies have explored miRNA because of the early discoveries about its relevance to various diseases throughout the body. miRNA plays important roles in the occurrence and development of pneumoconiosis, and these roles are summarized in (1) Case-Control Study. Use of miRNA sequencing or microarrays in case-control studies can help identify miRNAs that have been explored and changed significantly. Using a TaqMan low-density core, Yitao [e, Yitao  screenede, Yitao . Huang ee, Yitao  performee, Yitao . Rong ete, Yitao  observede, Yitao  used miRThe difference in genotype not only affects the susceptibility to diabetes and cancer but also seems to affect the susceptibility to pneumoconiosis. In a case-control study of 496 patients with CWP and 513 controls, Wang et al.  discover(2) In Vivo or In Vitro Experiments. In addition to studies that macroscopically examine the role of miRNA in pneumoconiosis, many studies are focusing on pathogenesis of pneumoconiosis; the mainstream view is that respirable productive dust enters the lung and first contacts alveolar macrophages and alveolar epithelial cells. The entry and retention of the vast majority of exogenous irritants  in the lungs will first cause macrophage alveolitis. Macrophages phagocyte the dust, and some macrophages carry the dust into the interlobular septum, which backflows to lymph nodes, blood vessels, and nearby bronchus and other parts. Because the dust cannot be dissolved or discharged, the dust will stay in the lung tissue for a long time and form a dust cell granuloma. With the constant existence of dust and the continuous tissue damage and repair, chronic inflammation and excessive repair processes develop; thus, a large number of myofibroblasts and ECM deposits are produced and eventually lead to fibrosis [fibrosis –105.Macrophages. In the process of pneumoconiosis lung fibrosis, macrophages play an extremely important initial role. Zhang et al. [α and between miR-146a and interleukin- (IL-) 1β by overexpression or inhibition of miRNA in SiO2-treated NR8383 cells [g et al. , 106 foug et al. , 106. Th83 cells , 106. Th83 cells , 106.In vitro experiments discovered that overexpressed miRNA-101 in BMSCs can inhibit autophagy related 4D cysteine peptidase (ATG4D) by regulating the endoplasmic reticulum stress of NR8383 cells and effectively inhibit the autophagy of macrophages caused by silica [Macrophage autophagy is also a hot spot for miRNA changes in pneumoconiosis. Chen et al. –87 ascery silica –87.Fibroblasts. Fibroblasts are the main source of myofibroblasts, and the changes and effects of miRNA in pneumoconiosis-related fibroblasts have been studied the most. Through miRNA sequencing, Zhang et al. screened nine miRNAs in human serum exosomes from case-control studies on silicosis; in this study, the expression of let-7a-5p, let-7i-5p, miR-146b-5p, miR-151A-3p, miR-409-3p, and miR-423-3p decreased, but the expression of miR-96-5p, miR-122-5p, and miR-125a-5p increased [BCL2L1, FAS, MAPK8, and IGF1R [hnRNP K and EZH2 [ncreased , 74, 75.ncreased , 74, 75.ncreased , 74, 75.nd IGF1R , 74, 75.and EZH2 , 74, 75.In vitro TGF-β stimulation of NIH/3T3 showed that miR-486-5p could bind to SMAD2 and participate in the process of pulmonary fibrosis through the TGF-β signaling pathway.Ji et al. , 107 fouβ in vitro showed that miR-293-5p, miR-370-3p, miR-409a-5p, miR-411-3p, miR-555-3p, and miR-1194-3p were consistent with the previous sequencing results. Using miRNA mimics and inhibitors, researchers discovered that miR-370-3p, miR-411-3p, and miR-1193-3p had antifibrosis effects, whereas miR-293-5p had a profibrosis effect [in vivo and in vitro [β/SMAD signaling pathway and alleviating silicosis-related pulmonary fibrosis by stimulating the primary lung fibroblasts of rats with TGF-β [In a study by Gao et al. , 79, 70 s effect , 79. Thein vitro , 79. In th TGF-β , 79.β. Overexpression of miR-542-5p can alleviate fibrosis [Yuan et al.  ascertaifibrosis . Integrifibrosis . Downregfibrosis .Alveolar Epithelial Cells. In addition to macrophages, alveolar epithelial cells are the first cells to directly contact dust. Alveolar type II epithelial cells are transformed into myofibroblasts through the epithelial mesenchymal transdifferentiation (EMT) process, and they are also one of the main sources of myofibroblasts in pneumoconiosis. Jiayu [β [β–stimulated A549 cells. Additional cell experiments proved that miR-34a-5p may play a role in inhibiting EMT by inhibiting SMAD4 [s. Jiayu  discoverJiayu [β . In the Jiayu [β . Using wJiayu [β  found inJiayu [β . These rJiayu [β . The dowJiayu [β . OverexpJiayu [β . Sun et Jiayu [β  detectedJiayu [β , 108 disJiayu [β , 82 deteng SMAD4 , 82.Combined Studies of Different Cells. The research of Zhang et al. [β signaling pathway [g et al. –78, 109 g et al. –78, 109.g et al. –78, 109. pathway –78, 109. pathway –78, 109.β in vitro, and the research demonstrated that miR-449a alleviated pulmonary fibrosis and promoted fibroblast autophagy by inhibiting its target, Bcl2 [in vitro with silica and demonstrated that miR-449a inhibits NOTCH1, which in turn inhibits SNAIL and EMT [Han and Fan et al. , 84, 110et, Bcl2 , 84, 110 and EMT , 84, 110Among the epigenetic changes of pneumoconiosis, miRNA is the most widely studied. A large number of miRNA microarrays and sequencing of human and animal peripheral blood and lung tissues provide biomarkers for disease screening, diagnosis, and prognosis. At the same time, many studies have explored the specific role of miRNA, mainly focusing on miRNA regulation of fibrosis-related target genes. Because of the temporal and spatial specificity of miRNA changes, the search for the key changes of miRNA has become the focus of this kind of research. However, the therapeutic potential of miRNA has not yet been tapped, and the antifibrosis effect remains explored mainly in cell and animal experiments. The treatment of liver fibrosis with miRNA has been widely applied in clinical practice, and clinical trials are exploring miRNA in the treatment of renal fibrosis, but few clinical studies have studied miRNA in the treatment of pulmonary fibrosis , 111. LoThe coding sequence of lncRNA is widely distributed in the genome. Most lncRNAs are produced from the transcription of protein-coding intergenic sequences. A small part of RNA produced by reverse transcription of protein-coding genes or secondary promoters in pseudogenes is called the natural antisense transcript (NAT). These NATs could play a direct role as lncRNAs, or they could bind to the mRNA generated by the transcription of the parent encoding genes to form double-stranded RNA .Some lncRNA expressions are regulated by transcription factors or epigenetics –115, andThere are many types of lncRNA, and their functions are also diverse. The function of lncRNA is related to its location. lncRNAs in the nucleus are mainly involved in the regulation of mRNA and protein synthesis. For example, the combination of lncRNA and the chromatin remodeling complex promotes the formation of a dense chromatin structure, thereby generating gene-silencing regions . lncRNA Like studies of miRNAs, studies of lncRNAs in pneumoconiosis are mainly divided into two topics: (1) lncRNAs that directly regulate target genes related to fibrosis; (2) lncRNAs that compete with miRNAs in binding target genes. The main lncRNA changes involved in pneumoconiosis are summarized in (1) Case-Control Study. Use of an lncRNA microarray or of sequencing technology is conducive to the detection of typical differences in disease variation. Sai et al. [i et al.  used an i et al. . The tari et al. .The genotypes of lncRNAs also have a certain influence on the susceptibility to pneumoconiosis. In a case-control study of 703 patients with CWP and 705 control patients, Wu et al.  genotype(2) In Vivo or In Vitro Experiments. In addition to macroscopically studying the differences of lncRNA in case-control studies, many experiments have also penetrated into the pathogenesis of pneumoconiosis through in vivo and in vitro intervention experiments. The mechanism of lncRNA is mainly studied in two sources of myofibroblasts: fibroblasts and alveolar epithelial cells.Fibroblasts. Cai et al. [β1–induced primary rat fibroblasts [β1–SMAD2/3 signaling pathway [i et al.  sequenceroblasts . In addi pathway .Alveolar Epithelial Cells. A case-control study by Ma et al. in CWP discovered that lncRNA-ATB was generally elevated in patients and was significantly, positively correlated with peripheral blood TGF-β in patients; thus, TGF-β may be a biomarker for CWP [β–induced EMT model using A549 cells and human bronchial epithelial (BEAS-2B) cells and found that the expression of lncRNA-ATB was elevated [ for CWP . Liu estelevated , 100. Adelevated , 100.Yan et al. , 95 deteThe Association of Different Cells. Wu et al. [2 dust–treated macrophages (mouse RAW264.7 and human THP-1 macrophages), and in NIH/3T3 cells and MRC-5 cells treated with TGF-β. miR-489 inhibited the expression of MyD88, IL-1β, and TGF-β in mice and macrophages [u et al. , 93 ascerophages , 93. miRrophages , 93. Therophages , 93.Lei et al.  mined thIn vitro experiments showed that miR-326 inhibited pulmonary inflammation by targeting TNFSF14 in silica-treated lung epithelial cells (HBE and A549) and promoted autophagy activity by targeting PTBP1 in TGF-β–stimulated MRC-5 cells and NIH/3T3 cell models [Xu et al.  found thl models .Because the study of lncRNA started later than that of miRNA, the pathogenic mechanism of lncRNA in pneumoconiosis has not been as thoroughly studied. The application of lncRNA in the treatment of pulmonary fibrosis, cardiac fibrosis, renal fibrosis, and liver fibrosis remains in the exploratory stage and has not been approved clinically. The treatment of lncRNA in pneumoconiosis can be explored by referring to the research on therapeutic targets of other diseases.The occurrence and development of pneumoconiosis involve the changes of multiple genes, and the regulation of gene expression involves many steps. Studies often combine several different mechanisms in series. As mentioned earlier, miRNA expression is regulated by epigenetics, such as DNA methylation, lncRNA, circRNA, and histone modification –59, 138,in vitro showed that macrophages are activated and that circular ZC3H4 (circZC3H4) RNA increased after silica stimulation [in vitro experiments of the murine lung epithelial cell line MLE-12, knockdown of ZC3H4 prevented EMT and migration caused by SiO2 [Yang et al. , 56 detemulation , 56. Cirmulation , 56. In mulation  ascertai by SiO2 . CircZC3 by SiO2 .Pulmonary fibrosis is a typical pathological change of pneumoconiosis. The formation of fibrosis is the basic response of the body to resist pathogens and normal wound healing . In pulmExploration of epigenetic changes in pneumoconiosis is of great value for understanding the pathogenesis and progression of pneumoconiosis, disease surveillance, diagnosis, intervention, and treatment. For example, the development of epigenetic biomarkers with diagnostic value in urine or blood is ideal because it can exempt subjects or patients from the usual invasive tissue sample collection. In addition, epigenetic modulators open up new ideas for the treatment of pneumoconiosis. Clinical data have shown that new epigenetic drugs have good therapeutic potential in human cancers. The first generation of epigenetic modulators approved for clinical treatment includes nonselective DNMT and HDAC inhibitors for myelodysplastic syndrome and cutaneous T cell lymphoma , 143. ln


Although specifically expressed formin genes in TGMS wheat line BS366 named TaFormins (TaFHs) and predicted their functions in inducing stress response. In total, 25 TaFH genes were uncovered, majorly localized in 2A, 2B, and 2D chromosomes. According to the neighbor-joining (NJ) method, all TaFH proteins from wheat and other plants clustered in 6 sub-groups (A-F). The modeled 3D structures of TaFH1-A/B, TaFH2-A/B, TaFH3-A/B and TaFH3-B/D were validated. And different numbers of stress and hormone-responsive regulatory elements in their 1500 base pair promoter regions were contained in the TaFH genes copies. TaFHs had specific temporal and spatial expression characteristics, whereby TaFH1, TaFH4, and TaFH5 were expressed highly in the stamen of BS366. Besides, the accumulation of TaFHs was remarkably lower in a low-temperature sterile condition (Nanyang) than fertile condition (Beijing), particularly at the early stamen development stage. The pollen cytoskeleton of BS366 was abnormal in the three stages under sterile and fertile environments. Furthermore, under different stress levels, TaFHs expression could be induced by drought, salt, abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), indole-3-acetic acid (IAA), polyethylene glycol (PEG), and low temperature. Some miRNAs, including miR167, miR1120, and miR172, interacts with TaFH genes; thus, we constructed an interaction network between microRNAs, TaFHs, phytohormone responses, and distribution of cytoskeleton to reveal the regulatory association between upstream genes of TaFH family members and sterile.Herein, we systematically characterized the TaFHs and miRNA resources for wheat breeding. These findings are, therefore, valuable in understanding the mechanism of TGMS fertility conversion in wheat.Collectively, this comprehensive analysis provides novel insights into The online version contains supplementary material available at 10.1186/s12864-021-07878-7. Eukaryotic cells possess a dynamic actin cytoskeleton that controls cell growth, division, and polarity formation by alternating its global and filamentous state in response to developmental and environmental stimuli . ForminsAtFH1, AtFH5, AtFH6, and AtFH8 have been described in Arabidopsis and their functions explored in vivo. For instance, AtFH1 overexpression in pollen tubes induced the formation of arrays of actin cables, projecting into the cytoplasm from the cell membrane. Interestingly, AtFH1 expression induced tube broadening, growth depolarization, and growth arrest in transformed pollen tubes. With these observations, it was suggested that AtFH1 regulation of actin polymerization could be necessary for the polarized pollen cell growth process [AtFH6 regulated polarized growth by tuning the assembly of actin cables [AtFH8 affects root and root hair development by alternating actin cytoskeleton distribution [ld) gene in 1982, the mutation of which led the mice to fail to“form” proper limbs and kidneys, and a large family of formin homology proteins were related with the field of actin cytoskeleton [formin gene family members are yet to be uncovered in the wheat genome. Also, it is not clear whether formin genes are related to pollen development, especially in the distribution of microspore cytoskeleton in wheat.Also, formins have multiple members in each species; for example, 22 formin family members are present in the Arabidopsis genome. Specifically,  process . The AtFribution . Collectformin gene family members related to pollen cytoskeleton distribution in TGMS wheat. Additionally, miRNAs are one type of small RNAs, which directly mediate the expression of target genes through post-transcriptional negative regulation [formin gene family is scanty. Therefore, there is a need to comprehensively explore the formin gene family in wheat and elucidate the interaction between TaFHs and miRNA; this would reveal their potential roles during anther development inBS366.Wheat is one of the most important staple food crops in China and globally in terms of production and consumption . Given tgulation . Furtherformin genes of wheat (TaFHs), we evaluated their characteristics for the physicochemical, phylogenetic relationship, 3D structure, motif structure, exon/intro structure, chromosomal location, miRNA regulation, and expression patterns in different tissues and different development stages of anthers in the TGMS wheat line, BS366. The results presented will enrich our knowledge of the TaFH genes family and offer the theoretical basis and novel candidate genes for improving and developing the male sterility wheat lines. It is imperative for us to understand the fertility pathway of TGMS.In this study, after identifying the formin genes in T. aestivum L. BLASTP using the formin of A. thaliana proteins as queries and HMMER searches using the formin domain as a query against the T. aestivum L. protein database. NCBI–CDD and SMART were used to confirm whether the formin domains were present. In total, 25 formin genes were identified, denoted as TaFH1 to TaFH10, based on their location on the chromosomes. Based on the physicochemical analysis of the protein sequence encoded by the wheat formin gene, there were significant differences in physical and chemical properties across the members of the wheat formin family. The protein comprised 306–1403 amino acids in length, with a molecular weight of 24.40–153.70 and 5.3–9.86 isoelectric points. Subcellular localization predictions revealed that TaFH proteins functioned in cytoplasmic, nuclear, vacuolation, plasma membrane, chloroplast, and endoplasmic reticulum , which possibly regulated auxin biosynthesis by impeding the downstream genes. Also, tae-miR167a overexpression in Arabidopsis lowered the expression levels of its targets (AtARF6 and AtARF8) and induced male sterility phenotypes [TaFH1-A, TaFH1-B, and TaFH1-D. Tae-miR1120b-3p has been shown to target TaFH3-A and TaFH3-B, and possibly LRR, thereby regulating the jasmonate biosynthesis or auxin response pathway associated with pollen development. A previous study found that the Q gene, involved in bread wheat spike architecture, was suppressed by tae-miR172 [TaFH9-D and regulated floral patterning and development via the transcription factor MADS-box [formin in the related TGMS pathway.To further understand the function of NAs Fig. . We alsoenotypes . Our resTaFH genes responded to stress stimuli, 1.5-kb upstream promoter regions of TaFH genes were scanned for stress-related cis-regulatory elements using the Plant CARE online service. Eight hormone-responsive regulatory elements  associated with ABA, auxin (IAA), methyl jasmonate (MeJA), gibberellin (GA), and salicylic acid (SA) responses were identified in the promoter region of TaFHs . The expression levels of TaFH2 and TaFH4 in the corresponding three stages of stamen development under sterile conditions were lower than those under fertile conditions  under sterile conditions. The expression of TaFH1, TaFH3, TaFH5, and TaFH10 were significantly higher at stage1, but stage 2 under sterile conditions. On the other hand, TaFH6 expression was higher at the early stages of stamen development (stage 1 and 2) under sterile conditions. TaFH7 and TaFH8 showed no obvious expression changes at the three stages under both fertility and sterile conditions. However, TaFH9 expression was lower under sterility conditions than in fertility conditions at stage 1 and 2. These results implied a potential association of TaFH genes with stamen development and fertility transformation via particular pathways.Based on previous reports, the fertility sensitive stage of BS366 was the meiosis stage of microspore mother cells , 16. Herformin gene is highly critical in the development of the pollen cell cytoskeleton [TaFH genes might cause the abnormal pollen cytoskeleton of BS366 under low temperatures.Compelling evidence shows that the pollen cell cytoskeleton of BS366 is abnormal at low temperature, and the skeleton , 16. In cis-element of TaFH, we found that TaFH had different types and numbers of regulatory elements. To affirm the function of TaFH, we conducted a qRT-PCR assay to examine the expression pattern of the formin genes in the leaves of BS366 following different hormone treatments , gibberellin (GA), indole-3-acetic acid (IAA), methyl jasmonate (MeJA), and salicylic acid (SA) are the main plant growth regulators that exert critical functions in plant growth and metabolism. By exploring the nts Fig. .Fig. 8Sformin genes slightly responded to the ABA, GA, IAA, MeJA, and SA treatments. Specifically, TaFH3, TaFH4, TaFH5, TaFH7, TaFH8, TaFH9, and TaFH10 were no obvious expressed changing under abscisic acid-induced stress, the expression of TaFH1 and TaFH6 was initially inhibited but later rose and peaked at 8 h, after which it began to show a wave-like decreasing trend and then rose again. Under auxin (IAA) stress, TaFH1, TaFH2, TaFH3, TaFH4, TaFH5, TaFH6, TaFH7, TaFH8, and TaFH10 were initially overexpressed, but their expression later decreased. The expression of TaFH1, TaFH2, and TaFH8 peaked at 4 h, whereas TaFH9 expression was impeded and then increased, and later peaked at 12 h. The expression of TaFH2, TaFH3, TaFH4, TaFH5, TaFH6, TaFH7, TaFH8, TaFH9, and TaFH10 demonstrated a certain upward trend under gibberellin stress. The expressions of TaFH2 and TaFH10 rose significantly at 2 h after gibberellin stress, then decreased and increased again. TaFH3, TaFH4, TaFH5, TaFH6, TaFH7, TaFH8, and TaFH9 showed a wave-like increasing trend, then decreased and rose again. The expression of TaFH4, TaFH5, TaFH6, TaFH7, and TaFH8 peaked at 24 h, whereas that of TaFH9 peaked at 4 h. Under methyl jasmonate stress, TaFH1 expression was inhibited but later increased at 12–24 h. The expression of TaFH2, TaFH3, TaFH4, and TaFH5 was inhibited within 24 h, and that of TaFH6 and TaFH10 showed an upward and downward trend, then peaked at 24 h. Further, the expression of TaFH7 was inhibited at 2 h, then increased and later decreased. TaFH8 expression peaked at 8 h, increased at 2 h, but then decreased, increased, and then decreased again at 12 h; the relative expression peaked at 2 h. The expression of TaFH9 was enhanced, then inhibited, and peaked at 8 h. Under salicylic acid stress, the relative expression of TaFH2, TaFH5, TaFH7, TaFH8, TaFH9, and TaFH10 was higher at 2 h compared to 0 h. TaFH2 expression peaked at 2 h, while TaFH5 peaked at 8 h. The expression of TaFH7, TaFH8, TaFH9 and TaFH10 all peaked at 4 h. Besides, the relative expression of TaFH1, TaFH3 and TaFH6 decreased at 2 h, whereas that of TaFH1 decreased at 8 h. Also, the expression of TaFH3, TaFH4, and TaFH6 peaked at 4 h. These results demonstrated that formin genes might be central to hormone regulation in the BS366 wheat.According to the results, all the formin gene family to cold stress, drought, and high salt, we employed qRT-PCR to analyze the expression pattern of the formin genes in the leaves of BS366 under low temperature (10 °C), salt (NaCl), and drought stress (PEG) treatments.To evaluate the response mechanism of the TaFH2, TaFH3, TaFH4, TaFH5, TaFH8, TaFH9, and TaFH10 was inhibited under cold stimulation, but later rose. The expression of most TaFH genes peaked at 12 h; however, the expression of TaFH1, TaFH6, and TaFH7 rose and then decreased post-cold stimulation. High salt stress impeded the expression of TaFH1, TaFH4, TaFH7, TaFH8, TaFH9, and TaFH10, whereas the expression of TaFH2, TaFH3, TaFH5, and TaFH6 showed an opposite trend. Under abiotic drought stress, except for TaFH7 and TaFH8, the expression of TaFH2, TaFH3, and TaFH9 peaked at 8 h and then decreased. These results indicate that TaFH genes might respond to different plant hormones and participate in a complex regulatory network in thermo-sensitive male sterile line BS366.Initially, the expression of profilin and Arp2/3 during cell division. Also, they participate in vegetative and reproductive plant growth [formin genes revealed different expression of TaFH1, TaFH2, TaFH3, TaFH4, TaFH5, TaFH6, TaFH9 and TaFH10 under sterile and fertile conditions at these stages. Therefore, we concluded that the formin genes TaFH1, TaFH2, TaFH3, TaFH4, TaFH5, TaFH6, TaFH9, and TaFH10 might affect the cytoskeleton, resulting in abnormalities during meiosis and thus male sterility in TGMS BS366.Formins initiate actin assembly and control key cytoskeleton genes. They regulate the nucleation of actin and mediate t growth . Previout growth  found abARF6 and ARF8, which regulates jasmonate biosynthesis by impeding downstream genes, thereby affecting pollen development. In rice, miR167 potentially targets ARF6, 8, 12, 17, and 25 genes. Notably, ARF8 can interact with OsGH3–2. Besides, OsmiR167 responds to exogenous auxin via its target gene OsARF8. Abnormal OsGH3–2 expression induces morphological aberrations, including short plants and small panicles [AtARF6 and AtARF8 upon its translation into Arabidopsis, and influenced the synthesis of IAA and JA, thereby regulating the development of anthers [MiRNAs are endogenous, small, non-coding RNAs, exerting critical roles in the regulation of hormone signaling pathways and cellular functions during developmental processes . Of notepanicles –26. Studpanicles , 28. Tae anthers .formins, we found that taemiR167a targeted TaFH2-A, TaFH2-B, and TaFH2-D; taemiR167b targeted TaFH1-A, TaFH1-B, TaFH1-D, TaFH2-A, TaFH2-B, and TaFH2-D; whereas taemiR167c-5p targeted TaFH2-A, TaFH2-B, and TaFH2-D. As such, we suggested that taemiR167 might regulate the formin genes in pollen-sensitive stages and influence BS366 fertility; leading to abnormal anther dehiscence. Moreover, the gene FBA  was predicted to be the target of miR1120. Of note, miR1120 could be regulated by ABA under low-temperature treatment, which affected the expression of FBA, then regulated the sugar metabolism to mediate ATP generation [OsFH5 mutants were characterized by aberrant inflorescence. The cytological analysis demonstrated that OsFH5 mutants exhibited severe cell elongation defects and abnormal microtubule arrays [TaFH3  were retrieved from the wheat genome Ensemble database . To identify members of the formin genes family from T. aestivum, we adopted two methods: Firstly, the hidden Markov model (HMM) profile of the formin (accession number in Pfam: PF06507) was downloaded from the Pfam database (http://pfam.sanger.ac.uk/) [T. aestivum proteome using HMMER3.0 [T. aestivum L. proteome via BLAST. Genes collected from the above two methods were integrated, and duplicates were eliminated manually. All protein sequences of the candidate formin genes were verified using domain analysis programs NCBI–CDD (https://www.ncbi.nlm.nih.gov/cdd/) and SMART , the gene structure with intro-exon boundaries of TaFH gene family using the Gene Structure Display Server (GSDS). Basic physical and chemical parameters of TaFH proteins, including amino acid length, theoretical isoelectric point, and molecular weight, were obtained from an online program, ProtParam (https://web.expasy.org/protparam/).The whole-genome sequences of .ac.uk/)  and usedHMMER3.0 . AdditioPhyscomitrella patens , Selaginella moellendorffii , Populus trichocarpa , Sorghum bicolor , Brachypodium distachyon , Arabidopsis thaliana , and Oryza sativa  were acquired from the downloaded data (ftp://ftp.ensemblgenomes.org/). MUSCLE software (http://www.drive5.com) was applied for amino acid (aa) alignments. A phylogenetic tree was generated via the MEGA X software using the neighbor-joining (NJ) method and 1000 bootstrap tests and visualized using the online software tool EvolView [The formin coding sequences for EvolView .The 3D structure of wheat formin proteins was constructed by homology modeling method, which based on the resolved of homologous protein. The Phyre 2 server was been used to obtain the 3D structure and further visualized by PyMOLWin.formin gene on the wheat chromosomes was mapped to IWGSC RefSeq v1.1 (cv. Chinese_Spring) using blast programs (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The centromere locations were retrieved from IWGSC (www.wheatgenome.org). Then, using the MapInspect tool (http://mapinspect.software.informer.com/), their locations were drawn onto the physical map of each chromosome. The graphic of chromosomal distribution was refined using Adobe illustrator.The location of each TaFH genes were predicted via a web-based psRNATarget server using default parameters [MiRNAs and their targets in rameters . They werameters .cis-acting elements in the 1500 bp upstream promoter regions of the identified TaFH genes were analyzed via PlantCARE (http://bioinformatics.psb.ugent. be/webtools/ plantcare/html/). All of these sequences were used to identify the cis-acting elements by the recently released T. aestivum genome database .The TaFH genes during pollen development in BS366, we sampled the anthers at three stages: stage 1 (secondary sporogenous cells had formed); stage 2 ; and stage 3 (meiotic division stage) [The thermo-sensitive genic male sterile (TGMS) wheat line BS366 was planted in the experimental fields in Beijing  and Nanyang  and managed conventionally. To analyze the roles of n stage) . For tisn stage) . In briePollen cell staining was performed as described by Wang et al.  and Xu eTaFH genes, total RNA was isolated from wheat tissues using TRIzol reagent  following the manufactures’ instructions. First-strand cDNA synthesis was performed using a PrimeScriptTM RT Reagent kit with gDNA Eraser . Next, qRT-RCR assay was conducted on an Eco Real-time PCR system  using SYBR® Permix Ex TaqTM , as described previously [For expression analysis of eviously .TaFH genes were determined using the comparative threshold cycle method 2-△△CT [18S gene served as the reference control. All the qRT-PCR reactions were conducted in three biological replicates. The primer used for qRT-PCR were conserved for all three homologues and designed using Primer premier 5.0 program and are listed in Table Relative expression levels of d 2-△△CT . Wheat 1Additional file 1: Figure S1. Phylogenetic relationship and motif structure of wheat formin proteins. The phylogenetic tree of TaFH proteins constructed from a complete alignment of 25 wheat formin proteins using MEGA 6.0 by the neighbor-joining method with 1000 bootstrap replicates. Domain distribution of TaFH proteins were investigated using the MEME web server.Additional file 2: Figure S2. Exon-intron structures of TaFH genes. Exons are represented by green boxes and introns by blank lines.Additional file 3: Figure S3. Three-dimensional structure of representative TaFH protein.Additional file 4: Figure S4. The flowchart of TaFH gene family with the treatment of BS366.Additional file 5: Table S1. The interaction of miRNAs with TaFH genes. Table S2. The primers of the qRT-RCR in TaFHs.


Brassica napus is minimal. Furthermore, the new reference Brassica napus genome offers an excellent opportunity to systematically characterize this gene family in B. napus. In our study, 21 BnaBZR genes were distributed across 19 chromosomes of B. napus and clustered into four subgroups based on Arabidopsis thaliana orthologs. Functional divergence analysis among these groups evident the shifting of evolutionary rate after the duplication events. In terms of structural analysis, the BnaBZR genes within each subgroup are highly conserved but are distinctive within groups. Organ-specific expression analyses of BnaBZR genes using RNA-seq data and quantitative real-time polymerase chain reaction (qRT-PCR) revealed complex expression patterns in plant tissues during stress conditions. In which genes belonging to subgroups III and IV were identified to play central roles in plant tolerance to salt, drought, and Sclerotinia sclerotiorum stress. The insights from this study enrich our understanding of the B. napus BZR gene family and lay a foundation for future research in improving rape seed environmental adaptability.BRASSINAZOLE RESISTANT (BZR) are transcriptional factors that bind to the DNA of targeted genes to regulate several plant growth and physiological processes in response to abiotic and biotic stresses. However, information on such genes in  It is well known that a group of transcriptional factors, BRASSINAZOLE RESISTANT (BZR), bind to specific sequences to convey environmental stimuli in regulating plant tolerance to various stresses . BZR, alve genes . These ithaliana . At the t stress . Moreovet stress . Furthert stress . Subsequve stage . Additiostresses . These mArabidopsis, six members of the BZR gene family were identified, AtBZR1, AtBZR2, AtBEH1, AtBEH2, AtBEH3, and AtBEH4. The dominant mutants of bzr1-D and bes1-D displayed a delay in flowering with wider dark green leaves and showed upregulated expression of BR-responsive genes in A. thaliana  did not exhibit any noticeable phenotype, while sextuple mutant  shows abnormal tapetum development, and male sterility  and during stress conditions. In which genes belonging to subgroups III and IV were identified to play central roles in plant tolerance to salt, drought, and S. sclerotiorum stress. In addition, we also explore some bna-miRNA, such as bna-miRNA395, bna-miR171, and bna-miR159, which are stimulated by the environmental cues, suggesting that different bna-miRNAs direct post-transcriptional regulation of BnaBZR gene family during biotic and abiotic stresses. Insights from these findings provide a framework for future studies in improving rape seed environmental adaptability.This study comprehensively described the B. napus BZR genes were downloaded from the brassica genome browser GENOSCOPEAtBZR genes from A. thaliana genome browser TAIRAt1G19350, At1G75080, At3G50750, At4G36780, At4G18890, and At1G78700). Furthermore, the amino acid sequences from B. oleracea and B. rapa were acquired from the Brassica database BRAD,AtBZRs as a query. The protein sequence with 80% sequence similarity was uploaded to ScanPrositeA. thaliana, B. oleracea, B. napus, and B. rapa to confirm the conserved domain. Physiochemical characteristics of the BnaBZRs proteins were investigated by the ProtParmThe proteins sequences of the BZR gene. Functional conserved motifs were examined by using the MEMEThe NJ (Neighbor-joining) method in MEGA X was utilized to plot the evolutionary tree. The tree’s validity was verified by performing 1,000 boot replications, and the Newick format of the phylogenetic tree was further annotated and visualized by the iTOLB. napus genome was employed to inspect the chromosomal distribution of the 21 BnaBZRs, and the map was drawn using the TBtools program  and synonymous mutation (Ks) of a single codon were also calculated by Ka/Ks Calculator 2.0 (The genome feature file (GFF) of the  program . Moreove toolkit . The colator 2.0 .BnaBZRs, we used the DIVERGE 3.0 program  were obtained from the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan. The seeds were sprouted on moistened filter paper under the listed conditions: 70% relative humidity and 20 ± 5°C, 16 h light/8 h dark at a light intensity of 50 μmol/m2/s. To investigate the expression profile of BnaBZRs in B. napus, tissues from the adult plant, such as mature-siliqua, root, stem, seed, leaf, flower, flower-bud, and shoot-apex, were collected and stored at −80°C for further experiment. Furthermore, for salt treatment, the three-week-old seedlings were immersed into 400 mM sodium chloride . After reaching a final concentration (400 mM), the leaf samples were collected at 2, 8, and 16 h post-treatment, while for drought treatment, 25% PEG solution was applied to simulate a drought stress-like condition. The leaf tissues were harvested at 2, 8, and 16 h post-treatment. The collected leaves were instantly frozen into the LN2 (liquid nitrogen) and kept at −80°C. To extract RNA and synthesize cDNA, we employed the same procedure as described in our prior study (B. napus Actin gene (geneBank ID: XM013858992) was used. The expression patterns of BnaBZRs were measured by the 2−∆∆Ct method described by t-test was performed to find the significant difference. The graph was generated using GraphPad Prism8.0 software  from the A. thaliana genome browser and then used it as a query in BLASTP program in the B. napus genome browser GENOSCOPE  was among 46.64–85.28, which shows that members of BnaBZRs were unstable. In addition, the isoelectric point (pI) values were between 5.14 and 9.81, indicating BnaBZRs were highly basic, and the negative GRAVY (grand average of hydrophobicity) value shows that these proteins are hydrophilic in nature. The amino acids encoded by the BnaBZR proteins are varied from 102aa to 400aa, in which the BnaA01BEH3 amino acid sequence was the shortest contained only 102 amino acids; however, the amino acid length of BnaC08BZR2-1 was the highest, around 400 amino acid residues. The molecular weight ranges from 10,990 KD to 44,156 KD, and the aliphatic index (AI) was between 48.12 and 73.3. Moreover, the subcellular localization signals of BnaBZRs were predicted by Plant-mPLoc. As transcriptional regulatory proteins, all BnaBZRs signals were detected in the nucleus  analysis was performed using the coding and genomic sequence of the B. napus and A. thaliana. As shown in BnaBZRs. Similar to the A. thaliana BZR gene family, most members in the BnaBZRs subfamily had the same number of exons and intron. For instance, most members of group I, group II, and group III have two to three exons and one to two introns, except for BnaA09BEH2, which contains four introns. Conversely, group IV contained one to three exons and only one intron, except for BnaA07BEH4, with no intron. Results from this analysis indicate that group I, II, and III might receive the original genes because, in the previous studies, it has been reported that the gain of introns is far slower than the intron loss during duplication  and Type II (ϴII) functional divergence utilizing the CodeML package in DIVERGE v2.0  was selected to detect specific amino acid residues among the BnaBZR family  have two, five, two, four, five, and three orthologous copies in the B. napus genome, respectively  and synonymous (Ks) ratios for each pair of duplicated BnaBZR proteins , indicating BnaBZR experienced immense selective pressure across different sites. Additionally, the models M7 (beta) and M8 (beta + ɯ > 1) were also compared, which considered a very stringent test of positive selection  and TGA-box (cis-element take part in ABA responsiveness) were only found in BnaA06BZR1 and BnaC05BZR1. A-Box (cis-element involved in the development process), CCGTCC-Box (cis-acting element activates the meristem development), and DRE-core (regulates drought and cold-responsive gene expression) were detected in BnaC05BZR2, BnaC08BZR2-1, and BnaC06BZR2. Furthermore, OCT (cis-element involved in cell proliferation), GCN4-motif (cis-element involved in nitrogen-related response), chs-CMA2a (light-responsive element), and DRE1 (regulates drought and cold-responsive gene expression) were detected in BnaC06BZR2, BnaC07BEH1, and BnaA01BEH2, respectively. GATT-motif, SARE , 3-AF1 binding site (light-responsive cis-element), CARE (involved in ABA response), and LAMP-element were absent in all BnaBZRs except in BnaC08BZR2-2, BnaA07BZR2, BnaA03BEH1, BnaC07BEH2, and BnaC07BEH3, respectively. Similarly, AT1-motif, Box-III, motif-I, and CTAG-motif were only found in BnaC01BHE3. In addition, CAG-motif, ATC-motif, and dOCT (cis-element involved in cell cycle responses) were detected in BnaC01BEH2 and BnaC03BEH3. Overall, light-responsive cis-elements were the most abundant in all BnaBZRs promoters, which indicates the transcriptional activity of the BnaBZRs might be induced by light. Additionally, cis-elements related to hormone responses, such as auxin (AUX), gibberellins (GA), ABA, and salicylic acid (SA), were predicted, in which cis-element involved in ABA responses were the most common in all BnaBZRs. Moreover, stress and defense responsive cis-elements, including ARE (anaerobic regulatory element), WUN-motif (regulates wound-related response), LTR (associated with cold response), and STRE, which activated by the several stress condition, especially heat shock, nutrient deficiency, and osmotic stress were also predicted. Results from this analysis showed that the BnaBZRs contain various kinds of development and stress-related responsive elements, indicating that the BnaBZRs might control plant physiology in response to several phytohormones and environmental stresses.To further examine the individual role of detected , but the, by using the transcriptomic data available in BnTIR database http://yanglab.hzau.edu.cn/BnTIR. As shown in BnaBZR gene family exhibited higher expression in sepal, petal, seed, lower stem, middle stem, upper stem, root, vegetative rosette, cotyledon, filament, and siliqua, while no or mild expression was observed in pollen and bud  including mature-silique, shoot-apex, seed, leaf, flower-bud, flower, stem, and root by qRT-PCR. As shown in BnaBZRs (BnaA06BZR1 and BnaC06BZR2) were consistently expressed in all selected tissues, except for BnaA06BZR1, which shows relatively lower expression in flower-bud. Additionally, the increased expression level of the BnaCO6BZR2, BnaC07BEH1, BnaA03BEH3, and BnaA02BEH4 was mainly observed in the mature siliqua . The non-significant association between transcriptome data and qRT-PCR might be related to harvesting of siliqua at 38 and 44 days after flowering, respectively. Therefore, the fold change should not be expected to have same for both datasets. Overall, most members of the BnaBZRs expressed differently in the plant tissues, suggesting the key roles of BnaBZRs in various tissues during plant development.To further explore the potential functions of the  and bud . In deta siliqua . In contBnaBZR gene family in response to the environmental stresses, we investigated their expression levels in drought, salinity, cold, and S. sclerotiorum stresses using publicly available RNA-seq data (CRA001775 and SRP311601). As shown in BnaA03BEH3 and BnaC06BEH4 were induced. In contrast, the expression pattern of the four BnaBZRs  was peaked at 8 h of drought treatment. Under salinity treatment, the expression pattern of the BnaA03BEH3 and BnaC07BEH3 was first induced at 4 h but then reduced in 24 h of salinity treatment. Furthermore, under cold treatment, the RNA transcripts of the group IV members BnaC03BEH3, BnaA02BEH4, and BnaA07BEH4 were upregulated during 24 h of cold treatment. However, BnaA07BEH4 and BnaC06BEH4-induced expression was only observed in S. sclerotiorum infection  treated with different stresses, such as drought and salinity  was targeted by the bna-miRN291 and bna-miRN285. However, except for the bna-miRN285/BnaC07BEH1 and bna-miR164/BnaA02BEH4, the majority of the bna-miRNA-targeted genes was expected to be silenced by the cleavage inhibition. Furthermore, we have also predicted the UPE value, which is within 8.94–21.43, needed to unpair the secondary structure around the target site. Nevertheless, most of the bna-miRNA identified in this study, such as bna-miRNA395  enrichment analysis. Our results showed that a total of 43 GO terms were categorized into three groups, BP , CC (cellular component), and MF ,” “biosynthetic processes ,” “gene expression (GO:0010467),” and “cellular process (GO:0050794),” whereas BnaC05G0163000ZS, BnaA06G0135100ZS, BnaA02G0233100ZS, BnaA01G0099000ZS, and BnaC03G0707200ZS might be involved in plant tolerance to stresses . Under the CC category, all BnaBZRs were in the nucleus (GO:0005634), while some of the genes, such as BnaA09G0609500ZS, BnaC08G0463100ZS, BnaC05G0163000ZS, BnaA06G0135100ZS, BnaC06G0273700ZS, BnaA07G0248700ZS, BnaC08G0253000ZS, BnaA03G0416600ZS, and BnaC07G0389100ZS, were also predicted in the cytosol (GO:0005829) and cytoplasm (GO:0005737) category, respectively. Additionally, in the MF category, all BnaBZRs were significantly enriched in binding  together with “organic cyclic compound binding (GO:0097159)” ” . ResultsBnaBZRs, we used the sequencing data of 159 accessions of rape seed from the B. napus genomic browser . . BnaBZRse family . Howeverso shown , which dBrassica rapa, Glycine max, Tomato, Eucalyptus grandis, and Petunia hybrid  crop, originated from the natural hybridization of the B. oleracea and B. rapa  can be found in the article/RS conceived and planned the experiments and wrote the manuscript. RG, LL, and YS extracted the RNA and performed the qRT-PCR analysis. K-MZ and JW analyzed the data. X-LT revised and supervised the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by the Jiangsu Agriculture Science and Technology Innovation Fund (CX(21)2009).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.


Accordingly, ATP gated P2X receptors expressed on tumor cells, immune cells, and neurons play a vital role in modulating tumor development, invasion, progression, and related symptoms. P2 purinergic signaling is involved in the development of different lung cancer-related symptoms. In this review, we summarize recent findings to illustrate the role of P2X receptors in tumor proliferation, progression, metastasis, and lung cancer- related symptoms, providing an outline of potential anti-neoplastic activity of P2X receptor antagonists. Furthermore, compared with opioids, P2X receptor antagonists appear to be innovative therapeutic interventions for managing cancer symptom clusters with fewer side effects.Symptoms associated with lung cancer mainly consist of cancer-associated pain, cough, fatigue, and dyspnea. However, underlying mechanisms of lung cancer symptom clusters remain unclear. There remains a paucity of effective treatment to ameliorate debilitating symptoms and improve the quality of life of lung cancer survivors. Recently, extracellular ATP and its receptors have attracted increasing attention among researchers in the field of oncology. Extracellular ATP in the tumor microenvironment is associated with tumor cell metabolism, proliferation, and metastasis by driving inflammation and neurotransmission  Lung cancer is one of the most common types of cancer, annually causing about 1.8 million deaths worldwide . The numvia protease releasing and cytoskeletal remodeling, playing a prometastatic role in cancer (Accumulated adenosine triphosphatez (ATP) and other extracellular nucleotides shape tumor microenvironment (TME), significantly influencing cancer proliferation, progression, tumor/immune-cell cross-talk, and related symptoms , 13. Accn cancer –25. A stn cancer . To unden cancer , 27. Colvia P2X pathways. Thus, dissecting the neuro-immune pathways via P2X receptors may provide new therapeutic strategies in cancer treatment.More data demonstrated that ATP, its hydrolyzation products, ectonucleotidases, , and purinergic receptors play a significant role in the modulation of the TME immune component. Extracellular nucleotides and P2 purinergic signaling drive the recruitment of inflammatory cells  and adjust immunomodulation on tumor sites . The purvia high dosage ATP leads to the opening of a larger conductance membrane pore, which in turn induces tumor cell death and inhibits tumor growth  and afferent sensory neurons   , 54 partl nerve) , 56. Sevl nerve) –59. Datal nerve) , 61. A-3l nerve) . Similarvia macrophages , a short-term promoter of neuropathic pain . P2X7 rerophages . Conversrophages . Differerophages , 68–71. rophages .The knockdown of P2X7 receptors with the utilization of selective or specific antagonists  benefits cancer pain by reducing the release of cytokine, such as Il-1β , 72, 73.Kyushu University and Nippon Chemiphar Co. Ltd initiated a Phase 1 clinical trial of NC-2600 in 2016 to elucidate its efficacy in chronic neuropathic pain . Although P2X7 antagonists, such as AZD9056 and CE-224,535 were well tolerated in Phase 1 clinical studies, they showed insignificant efficacy in treating rheumatoid arthritis in Phase 2 clinical trials. Another P2X7 antagonist, AZD9056, has the therapeutic potential to decrease Crohn’s disease activity index along with improvement in chronic abdominal pain, as shown in a Phase 2a clinical study of Crohn’s disease Table 1.A persistent cough is a common symptom affecting over 50% of patients with lung cancer with a considerably adverse impact on quality of life . Cough cvia stimulation of P2X3 and P2X2/3 receptors expressed on the vagal sensory neurons central to the cough reflex  is one of the cancer cluster symptoms that affects up to 90% of patients with lung cancer . This divia activation of glial cells and central sensitization in the spinal cord  nal cord . Similarnal cord , 103 in nal cord , 105 or nal cord , 107. Ennal cord , 109. Synal cord . These cnal cord , cachexinal cord , and depnal cord , which mnal cord , 72, 73.via sensory neurons and evoke a sensation of fatigue. Moreover, in an animal model, ATP released from fatigued muscle activates muscle macrophages, which subsequently release IL-1β to produce hyperalgesia. The blockade of P2X4 receptors in muscle inhibits the development of such hyperalgesia  and stimulate/modulate reflexes . Various cancer-related symptoms may share similar pathogenic mechanisms. To date, a significant clinical investigation has established a role for P2X receptor antagonists in the treatment of chronic cough and cancer-related pain. Thus, a basis has been formed for the further evaluation of potential therapeutic roles for P2X antagonists in the treatment of other cancer-induced symptoms.YM, ZG, and WY drafted the manuscript. NZ, PD, and RC designed and revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by grants from the National Natural Science Foundation of China (81870079) and National Outstanding Youth Incubation Program of Guangzhou Medical University(2020-207).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.


We aimed to systematically identify the possible risk factors responsible for severe cases.We searched PubMed, Embase, Web of science and Cochrane Library for epidemiological studies of confirmed COVID-19, which include information about clinical characteristics and severity of patients’ disease. We analyzed the potential associations between clinical characteristics and severe cases., chronic obstructive pulmonary disease (COPD) , malignancy , and chronic liver disease . Acute respiratory distress syndrome (ARDS) , shock  and acute kidney injury (AKI)  were most likely to prevent recovery. In summary, patients with severe conditions had a higher rate of comorbidities and complications than patients with non-severe conditions.We identified a total of 41 eligible studies including 21060 patients with COVID-19. Severe cases were potentially associated with advanced age (Standard Mean Difference (SMD) = 1.73, 95% CI: 1.34–2.12), male gender (Odds Ratio (OR) = 1.51, 95% CI:1.33–1.71), obesity , history of smoking , hypertension , diabetes , coronary heart disease , chronic kidney disease (CKD) , cerebrovascular disease Patients who were male, with advanced age, obesity, a history of smoking, hypertension, diabetes, malignancy, coronary heart disease, hypertension, chronic liver disease, COPD, or CKD are more likely to develop severe COVID-19 symptoms. ARDS, shock and AKI were thought to be the main hinderances to recovery. The World Health Organization (WHO) has officially declared the outbreak as a pandemic and a public health emergency [ly 2.22% , 4.Some researchers have suggested that there are several factors possibly responsible for the severity of COVID-19, such as hypertension, diabetes and smoking –7. ThereThe literature search was performed using international databases PubMed, Embase, Web of science and Cochrane Library using the search terms: (“COVID-19” OR “SARS-Cov-2”) AND : The specific search strategy is listed in To minimize bias, two authors (LXY and ZXR) independently screened titles and abstracts and extracted potentially eligible articles. The full texts of selected articles were then carefully assessed according to the inclusion and exclusion criteria. Disagreements were resolved by discussion, with the third researcher (LQ) to reach a consensus.Inclusion criteria:(1) case-control studies or cohort studies; (2) articles reporting the clinical characteristics and the severity of disease in patients diagnosed with COVID-19; (3) articles reporting the severity of COVID-19 and details of related factors; (4) articles reporting the specific grading standards of the severity of COVID-19.The definition of severe disease was based on clinical symptoms, i.e., patients having severe dyspnea, extremely low oxygen saturation, respiratory distress or requiring mechanical ventilation, ICU admission or death.The exclusion criteria: (1) review articles, letters, comments or opinions; (2) samples of less than 50; (3) incomplete information or full texts unavailable.All included studies were retrospective cohort studies so, the Newcastle-Ottawa Scale (NOS) was used to evaluate quality . The majTwo independent authors (LXY and ZXR) performed the data extraction by using standardized forms, which included date of publication, authors, region, number of included patients, sex, body mass index (BMI), smoking habits, comorbidities, complications, and severe or non-severe COVID-19.2 statistic. We used a Mantel-Haenszel random effects model to calculate effect sizes. In order to discover the potential sources of heterogeneity, we performed subgroup analyses and meta-regression analysis according to study location (Hubei province and outside Hubei province) and the median ages of patients. The stability of results was assessed by conducting a sensitivity analysis which omitted each study in turn. Funnel plots were used to assess for publication bias and asymmetry was taken to indicate bias. In this study, P values <0.05 were regarded as indicating statistical significance for the summary OR and SMD. In order to quantify the effects of risk factors on COVID-19 patients, we calculated population attributable fraction (PAF) by using the attributable fraction formula:Pc is the prevalence of the risk factors in the population [We calculated odds ratios (ORs) in the dichotomous variables and standard mean difference (SMD) in the continuous variables with the 95% confidence intervals (CIs) and assessed publication bias using Stata 12.0. Heterogeneity was measured by Ipulation . In thisth, 2020 and March 13th, 2021 were included, all of which were retrospective cohort studies. In total, 21060 patients were included in the meta-analysis. The characteristics of all the included studies are shown in The complete literature-search process is displayed in The results of the quality evaluation of the included retrospective cohort studies are displayed in We identified 15 risk factors in three categories  that have impact on the severity of confirmed cases. All statistically significant factors in our analysis are summarized in 2 = 50.8%) [2 = 98.6%) [2 = 30.6%) [2) in patients with severe disease  [The demographic characteristics including sex, age, smoking history and BMI were pooled for meta-analysis and the results are displayed in = 50.8%) , 22–51. = 98.6%) , 41–51 w= 98.6%)  and Kim = 98.6%) . Patient= 30.6%) , 48, 50. = 0.0%) , 30, 35.2 = 68.4%) [2 = 52.6%) [2 = 59.9%) [2 = 11.9%) [2 = 28.4%) [2 = 67.9%) [2 = 55.6%) [2 = 0.0%) [2 = 79.4%) [= 68.4%) , 47, 51,= 52.6%) , 49, 51.= 11.9%) , 41, 43,= 28.4%) , 50, 51,= 67.9%) , 22–51, = 55.6%) , 47–51 a = 0.0%) , 47, 50.= 79.4%) , 38, 45 2 = 48.7%) [2 = 27.4%) [2 = 40.7%) [2 = 0.0%) [2 = 39.0%) [2 = 0.0%) [2 = 75.0%) [2 = 0.0%) [The results shown in = 48.7%) , 26, 44,= 27.4%) , 44, 51  = 0.0%) , 22, sec= 39.0%) , 25, arr = 0.0%) , 12 and = 75.0%) , 24, 51  = 0.0%) , 24 did We calculated the population attributable risk (PAR) of the population infected with COVID-19. PAR reflects the proportion of severe COVID-19 that can be attributed to exposure to a risk factor. We performed subgroup analyses to identify the possible sources of heterogeneity of some outcomes. We tried to analyze several factors such as study location, publication date, sample size and patients’ average age of which study location gave a relatively meaningful result as shown in By excluding every article in turn, we conducted a sensitivity analysis and there was no evident change in either the significant outcomes or in heterogeneity. To assess the risk of publication bias, we drew a funnel plot for each factor. The results, displayed in 2), patients with advanced age or a smoking history faced a greater risk of having severe COVID-19. This finding has also been supported by some previous studies [This study has examined the potential risk factors of severe COVID-19 such as demographic characteristics, comorbidities and complications to assist clinicians with allocation of medical resources. The higher the ORs of the risk factors, the greater the risks they bring. Our analysis has shown that males, obese patients  and AKI (7.4%) were also high which showed that they possibly make a considerable contribution to the severity of COVID-19, but there are no specific clinical guidelines to prevent them. Therefore, more attention should be given to some other factors like obesity (7.1%) and diabetes (6.5%) since they can to some extent be prevented through practical guidelines  and publWe should acknowledge that there are some limitations which may affect the accuracy of the outcomes and should be considered. First, some studies did not explicitly show their criteria for severe patients and non-severe patients, meaning the actual severity of patients with these two outcomes may not be consistent with those studies. Second, detecting significant heterogeneity in some analyses, we chose to use a random effects model to process data but did not identify the clear sources of heterogeneity through subgroup analyses. Third, the majority of the patients came from China, so they may not be representative of all patients across the world and so results should be carefully evaluated and accepted cautiously. The conclusion can be updated when more studies from outside China are published. Forth, we have tried to include studies from different cites but some articles may still include duplicated cases since they seldom mentioned the detailed composition of patient groups.In summary, patients with COVID-19 who have the following characteristics: male gender, advanced age, a history of smoking, obesity, hypertension, diabetes, malignancy, coronary heart disease, hypertension, COPD, CKD and chronic liver disease, were more likely to develop the severe form of the disease. The emergence of complications like shock, ARDS, AKI generally increased the risk of the disease developing into the severe condition.S1 Checklist(DOC)Click here for additional data file.S1 FigFunnel plot of (A) sex, (B) smoking history, (C)age, (D) BMI, (E) Chronic kidney disease, (F) Chronic obstructive pulmonary disease, (G) Coronary heart disease, (H) Malignancy, (I) Cerebrovascular disease, (J) Hypertension, (K)Diabetes, (L) Chronic liver disease, (M) Acute respiratory distress syndrome (ARDS), (N) Shock, (O) Acute kidney injury (AKI) and severe COVID-19 disease for the assessment of publication bias.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Rectal inflammatory myofibroblastic tumor (IMT) is an extremely rare mesenchymal tumor characterized by a mixture of spindle-shaped myofibroblasts or fibroblasts and inflammatory infiltration of lymphocytes and plasma cells. To date, only 8 cases of rectal IMT have been reported. Herein, we report an additional case of rectal IMT in a 28-year-old woman.A 28-year-old woman presented with abdominal pain and hematochezia.Colonoscopy showed a 3.0-cm subepithelial tumor with central ulceration, covered by white exudate in the rectum. Rectal magnetic resonance imaging revealed a 4.0 × 3.0-cm-sized well-defined subepithelial tumor in the right wall of the rectum, with suspicious right perirectal fat infiltration.Laparoscopic anterior resection was performed. Microscopic examination of the surgical specimen revealed bland-looking spindle cells intermingled with lymphoplasma cells. Immunohistochemistry and fluorescence in situ hybridization showed anaplastic lymphoma kinase positivity and anaplastic lymphoma kinase positivity rearrangement. Rectal IMT was confirmed based on histological, immunohistochemical, and fluorescence in situ hybridization findings. The patient was doing well without evidence of tumor recurrence 1 year after the surgery.Rectal IMT, despite its rarity, should be considered in the differential diagnosis of rectal cancer. Second, an accurate histopathologic diagnosis and complete surgical resection can be the most important approaches to offer a chance for the cure of rectal IMT. The occurrence of IMT arising from the colorectum was first described by Coffin et al. in 1995. Subsequently, approximately 60 cases of colorectal IMT have been documented in the literature. The frequency of its occurrence was in the order of the ascending colon, transverse colon, cecum, sigmoid colon, descending colon, and rectum.Inflammatory myofibroblastic tumor (IMT) is a rare solid tumor characterized by the proliferation of spindle-shaped myofibroblasts and inflammatory infiltration of lymphocytes and plasma cells.–13 Herein, we report an additional case of rectal IMT in a 28-year-old woman and review the literature pertaining to this condition.Rectal IMT is extremely rare. To date, only 8 cases of rectal IMT have been reported.23 , hemoglobin level of 9.0 g/dL , mean corpuscular volume of 86.1 fL , mean corpuscular hemoglobin level of 28.3 pg , mean cell hemoglobin concentration of 32.9 g/dL , platelet count of 328,000/mm3 , serum albumin level of 4.1 g/dL , aspartate aminotransferase level of 29 U/L , alanine aminotransferase level of 40 U/L , alkaline phosphatase level of 88 U/L , and total bilirubin level of 0.4 mg/dL . The coagulation profiles were within normal limits. The C-reactive protein (CRP) level was increased to 8.67 mg/dL . Tumor markers including serum carcinoembryonic antigen, carbohydrate antigen (CA) 19-9, and CA 125 were all within normal limits. To determine the cause of hematochezia, we performed a colonoscopy. A 3.0-cm subepithelial tumor with central ulceration, covered by white exudate, was found in the rectum, 9 cm above the anal verge  was found in the rectum without regional or distant metastasis. We surgically removed the lesion for diagnostic and therapeutic purposes. Laparoscopic anterior resection was performed. Macroscopically, the specimen revealed a yellowish fungating mass with central ulceration covered with whitish mucus materials. Microscopic examination of the surgical specimen revealed bland-looking spindle cells intermingled with lymphoplasma cells  years and included 5 men and 4 women.Rectal IMT is extremely rare. In this report, a review of the medical literature disclosed 9 reported cases (including the present case) of IMT arising from the rectum Table .[6–13] IThe presenting symptoms of rectal IMTs were abdominal pain (6 of 9 cases), hematochezia (5 of 9 cases), weight loss (4 of 9 cases), and constipation (3 of 9 cases). The common laboratory findings were elevated CRP level or erythrocyte sedimentation rate (5 of 9 cases) and anemia (4 of 9 cases). Tumor markers, including serum carcinoembryonic antigen, (CA) 19-9, and CA 125, were within normal limits (3 of 9 cases). Abdominal pain, hematochezia, elevation of CRP level or erythrocyte sedimentation rate, and anemia were the most common findings in rectal IMTs. The most common colonoscopic finding was a large rectal mass with central ulceration, similar to our case. These clinical and colonoscopic findings resemble those of rectal cancers. Therefore, the primary diagnosis of rectal IMT is difficult.In our study, no cases were diagnosed as IMT with colonoscopic biopsy. However, the dramatic difference in the prognosis and treatment between rectal cancer and rectal IMT stresses the importance of differential diagnosis in rectal tumors. All cases, including ours, were diagnosed and treated with surgical resection. Therefore, surgery to obtain adequate tissue is necessary to achieve a satisfactory diagnosis and treatment. The prognosis of IMT is good and is associated with distant metastasis in approximately 10% of cases. In our study, the mean follow-up time was 33.4 months (1 month–7 years). Two patients showed relapse during follow-up. One patient underwent reoperation, and 1 died of metastasis.IMT is a slow-growing, benign tumor with a potential risk of recurrence. Therefore, the World Health Organization has classified IMT as an intermediate neoplasm.,15 Absent ALK gene rearrangement was associated with a higher overall age, subtle histologic differences, and death from disease or distant metastases, suggesting a favorable prognostic indicator.,16,17 In our study, ALK gene rearrangement was detected in 4 cases, negative in 2 cases, and not evaluated in 3 cases. Among the 2 ALK-negative patients, 1 died of multiple metastases, including the liver, bladder, prostate, and pelvic floor. However, the role of ALK gene rearrangement as a prognostic indicator of rectal IMT remains unclear because of the limited number of patients with this disease.ALK gene rearrangement on chromosome 2p23 is a well-known oncogenic driver in non-small cell lung cancer and has also been identified in approximately 50% of IMTs.4Rectal IMT, despite its rarity, should be considered in the differential diagnosis of rectal cancer. Second, an accurate histopathologic diagnosis and complete surgical resection can be the most important approaches to offer a reasonable chance for the cure of rectal IMT.Conceptualization: Young-Eun Joo.Investigation: Hyung-Hoon Oh.Resources: Hyung-Hoon Oh, Young-Eun Joo.Supervision: Young-Eun Joo.Writing – original draft: Hyung-Hoon Oh, Young-Eun Joo.Writing – review & editing: Hyung-Hoon Oh, Young Eun Joo.