Numerous Ca2+ chelating, storage, and transport mechanisms are required to maintain normal cellular physiology. Ca2+-binding proteins, mainly calmodulin and calbindins, sequester free intracellular Ca2+ ions and apportion or transport them to signaling hubs needing the cations. Ca2+ channels, ATP-driven pumps, and exchangers assist the binding proteins in transferring the ions to and from appropriate cellular compartments. Some, such as the endoplasmic reticulum, mitochondria, and lysosomes, act as Ca2+ repositories. Cellular Ca2+ homeostasis is inefficient without the active contribution of these organelles. Moreover, certain key cellular processes also rely on inter-organellar Ca2+ signaling. This review attempts to encapsulate the structure, function, and regulation of major intracellular Ca2+ buffers, sensors, channels, and signaling molecules before highlighting how cancer cells manipulate them to survive and thrive. The spotlight is then shifted to the slow pace of translating such research findings into anticancer therapeutics. We use the PubMed database to highlight current clinical studies that target intracellular Ca2+ signaling. Drug repurposing and improving the delivery of small molecule therapeutics are further discussed as promising strategies for speeding therapeutic development in this area.Intracellular Ca Low hydration energy, high polarizability, relative flexibility of coordination sites and bond length, and large concentration gradient across cellular membranes (100 nM intracellular to 2 mM extracellular) due to low intracellular levels make it the ion of choice at the core of cellular signaling in prokaryotes and eukaryotes alike i as evident by its role in modulation (activation or inactivation) of Ca2+ pumps (such as PMCA and SERCA) and Ca2+ channels  i gradient required for cellular motility. Migrating endothelial cells have higher expression of PMCA at their leading edges to maintain low basal [Ca2+]i levels thereby, preventing continued activation of Myosin Light Chain Kinase (MLCK) and extended contraction of the cell membrane at the migration frontier [2+]i extrusion allows the proliferation of pancreatic beta cells i signaling was later confirmed by independent work of two groups using high-throughput RNAi screens to identify inhibitors of thapsigargin evoked CRAC current (ICRAC) i can exert anti-migratory and antiangiogenic effects on tumor-derived endothelial cells. Two separate studies have shown that treatment of Breast Tumor-derived Endothelial cells (BTEC) and Renal Tumor-derived Endothelial Cells (RTEC) with 100 mM ATP leads to a biphasic increase in [Ca2+]i—an initial transient release from ER followed by SOCE and then a prolonged release rise due to Ca2+ influx via P2X7R and P2Y11R [2+]i then activates adenylate cyclase10/cAMP/EPAC-1 pathway to disrupt actin cytoskeleton and thus, inhibit migration of tumor-derived endothelial cells.The somewhat controvertible, but the generally accepted, role of ligand-gated Cac for P2 ,229,230.n tumors . ATP binoduction ,232. Theon (EMT) . For insd P2Y11R ,235. Thi2+ ion efflux machinery at the ER-PM junction during tumor formation or progression also requires attention. PMCA and Na+/Ca2+ Exchanger (NCX) lead a concerted effort on a normal cell surface to expel excessive free cytosolic Ca2+ ions and thereby prevent cytotoxicity i transporters too can be leveraged for killing cancer cells. It is well known that calmodulin regulates the slow inactivation of CRAC currents by direct interaction with the STIM1 SOAR domain, thereby disrupting the STIM1-ORAI1 complex [2+ elevation) for tumor survival, growth, migration, and invasion [The anomalous signaling, localization, and mobilization of Caproteins . It is a tissues ,359,360. tissues . It was  tissues . Some of kinases . For ins kinases ,365,366. kinases . Direct  kinases ,369,370. complex . It has  complex . On the  complex . CaM andinvasion ,377,378.2+-binding proteins that are altered by cancer cells [2+ ion-dependent processes including proliferation, differentiation, cell adhesion, migration, intercellular interactions, immune response, and apoptosis [2+ signaling with the Wnt pathway as the potential downstream effector [2+-binding protein is shown to attract dendritic cells and macrophages for phagocytosis [2+ ion buffer which has dominant expression in neurons has so far only been found to protect osteosarcoma cells from apoptotic stimuli [Calreticulin (CRT) and calbindin are other major EF-hand containing Caer cells ,381,382.poptosis ,389,390.poptosis ,394,395.poptosis ,397. Evepoptosis ,399. Thepoptosis ,401. Foreffector ,403. In effector . Studieseffector ,407,408.ocytosis . In glioocytosis . In pancocytosis . Very li stimuli .2+ signaling [2+ ion uptake by mitochondria. Thus, plummeting Ca2+ ion transport from ER to mitochondria in transformed cells portends tumor progression.Aberrant cellular metabolism, a hallmark of a wide variety of tumors, stems from the dysfunction of mitochondrial pathways for ATP production . The shiignaling ,415,416.2+ ion transport, ER stress, ER Ca2+ release, mitochondrial morphology regulation, cellular bioenergetics, apoptosis and survival, autophagy, and ROS generation [2+ ion release, and thus crucial for autophagy and apoptosis induction [2+ ionrelease into mitochondria creates an imbalance in ATP/ADP ratio that derepresses AMPK/mTOR/ULK-1 axis and initiates autophagy flux (based on nutrient starvation) [2+ ion leak channels as less Ca2+ storage in the ER manifests into poor ion supply for ATP production [2+ ion release into the mitochondria, ergo inducing autophagy [2+ ion release into the mitochondria partly by interacting with Bcl2 which then dissociates from IP3R, thereby restoring the ER-mitochondrial Ca2+ ion transfer [2+ ion transport by upregulating the expression of tumor suppressor PML (promyelocytic leukemia) that blocks phosphorylation and inactivation of IP3R by p-Akt [ER-mitochondria junctions or Mitochondrial-Associated Membranes (MAMs) are hubs for proteins that are integral to vital processes such as phospholipid synthesis and translocation, mitochondrial Caneration . IP3R, anduction ,418,419.rvation) . Conversoduction ,422. By oduction ,424,425.oduction ,427. Remoduction . In highoduction ,430,431.utophagy ,433,434.transfer . Chemothby p-Akt ,436.2+ ion transport has emerged as an integral component of the crosstalk between autophagy and apoptosis [2+ ion uptake via MAMs triggers intrinsic apoptosis pathways. Thus, tumor suppressors and proapoptotic molecules like BRCA1 or PTEN bind to IP3R to boost Ca2+ ion uptake at MAMs while tumor promoters and anti-apoptotic molecules such as Bcl2 or Bcl-xL repress mitochondrial Ca2+ ion overload by binding to and inhibiting the channel activity [2+ ion transporting complex at MAMs are also exploited by cancer cells [2+ uptake channel, MCU with its regulators MICU1/2 act as the key mediators of Ca2+-based mitochondrial functions [2+ ion uptake via MCU and its absence causes cell death via mitochondrial overload, tumor cells are widely observed to have elevated levels of these proteins, although expression of MCU is subtype-dependent [The ER-mitochondria Capoptosis . Inhibitactivity ,439. VDAer cells ,441. It er cells ,443. Proer cells ,444. Asier cells ,446,447.unctions ,449. Becependent ,450. Forependent ,452. On 2+ signaling at lysosomal membranes and surfaces of acidic vesicles provides a functional scaffolding for endocytic traffic and autophagy, thereby directly influencing cellular health [Car health ,454.2+ signaling [2+ ion release channels in tumor invasiveness [2+ ion signals via TPCs on lysosomal membranes, and this promoted G0/G1 cell cycle transition, tumor vascularization, focal adhesion kinase (FAK) formation, and migration. Both in vitro and in vivo silencing or pharmacological inhibition of TPC1/2 have also been shown to block invasion and migration of urinary bladder carcinoma (T24), hepatoma (HUH7), and mammary carcinoma [Alteration of such signaling in cancer contributes to cancer hallmarks such as uninhibited growth, angiogenesis, and metastasis ,456,457.ignaling . Initialsiveness . The stuarcinoma ,460. Nguarcinoma ,461.2+ ion release channel expressed on lysosomal membranes and is known to finely control autophagy [2+ activated transcription factor, TFEB is noticeably correlated with cellular malignancy [2+ ion release followed by calcineurin activation that subsequently dephosphorylates TFEB and releases it from 14-3-B-B; free TFEB is then translocated to the nucleus to evoke transcription of autophagy-related genes [TRPML1 is another Cautophagy ,462. Prelignancy ,465,466.ed genes . In shored genes . TFEB uped genes . Althouged genes .2+ signaling involves a smorgasbord of proteins that chelate or transport Ca2+ ions across various cellular compartments and thereby assist in signal induction, relay, or integration. With Ca2+ signaling being pivotal for vital cellular processes, distortion of this machinery can be highly advantageous to cancer cells. Although there has been a steady upward trend in the number of published studies that delineate the functional exploitation of various intracellular Ca2+ signaling components in tumors, limited headway has occurred in translating those research outcomes into clinical applications. To demonstrate this, we first utilized the PubMed database to get a list of all the publications within the realm of intracellular Ca2+ in cancer. Using the advanced search feature, we selected “intracellular Ca2+” and “cancer” as the keywords to look for in all fields  of PubMed articles. We got more than 11,000 studies (not shown). As the published articles between 1960 and 1990 only accounted for less than 0.1 percent of the total, we set our year range for subsequent searches as 1990–2020. To minimize the occurrence of non-relevant articles in our dataset , we used stricter filtration criteria [2+ molecules, Revised Filter 2 was determined to be a sub-set of Revised Filter 1 [2+ signaling molecules in focus was that Revised Filter 2 had a lower percentage of articles on “CaSR or Vitamin D” amongst all the ones with main Ca2+ signaling molecules from the endoplasmic reticulum or its interorganellar junctions [Intracellular Cacriteria A. Based Filter 1 B–D. A no2+ and Vitamin D on cancer (31%) [2+ signaling player in the endoplasmic reticulum or its interorganellar junctions accounted for less than 10 percent of all the published clinical articles. We further evaluated from the main set of published articles the number of studies that used antineoplastic agents (mainly chemotherapeutics) by searching for appropriate keywords. There are 819 articles in the main set that had utilized antineoplastic agent(s) to address a hypothesis related to the role of a major Ca2+ signaling molecule in cancer [2+ signaling molecules as major components of research. Studies with bisphosphonates and not chemotherapeutics were predominant in the antineoplastic group of articles followed by the ones that utilized dietary Ca2+ and/or Vitamin D [2+ signaling.Clinical studies that were extracted from the list of articles in Revised Filter 1 were labeled in PubMed with one or more of these terms— Clinical Study, Clinical Trial, Clinical Trial Phase I, Clinical Trial Phase II, Clinical Trial Phase III, Clinical Trial Phase IV, Comparative Study, Controlled Clinical Trial, Meta-Analysis, Multi-Center Study, Randomized Controlled Trial, Systematic Review. Two-hundred-and-seventy-seven clinical studies were identified with most of them concentrated between year ranges 1991 to 1996 and 2010 to 2017 (with approximately 10 or more studies published each year) A. With oer (31%) B. Transln cancer C. Out ofitamin D D. Among 2+ signaling proteins is currently in the early stages. Some of the examples include RP4010 , Synta66 , CAD204520 , and SKF96365  [2+ signaling could be increased by leveraging the advancements made in the nanocarrier-based targeted drug delivery systems. For example, taking from the design of Antp-LP4 related peptides (VDAC1 inhibitors), Venetoclax (a Bcl2 inhibitor) can be encapsulated in inorganic (such as gold nanoparticle or quantum dots) or organic nanocarriers (such as liposomes) coated with transferrin to specifically target cancer cells that demonstrate significant surface expression of transferrin receptors [2+ signaling [2+ signaling in cancer. Several FDA-approved anticancer agents  and drugs against other disease conditions  can impact Ca2+ signaling machinery [2+ signaling See Research using small molecule drugs targeting numerous Calinical) ,475,476.linical) . Toxicitlinical) ,480,481.eceptors ,482,483.ignaling . Drug reachinery ,485. Som2+ in tumor cells, the vast clinical potential of targeting key players in this area remains underappreciated. Whether it is through redesigning existing therapeutics or developing novel treatments for directly modulating the intracellular Ca2+ levels, greater efforts are needed to find ways to reverse the hijack of intracellular Ca2+ signaling in cancer.Even after decades of research on intracellular Ca


Glioma is a highly fatal malignancy with aggressive proliferation, migration, and invasion metastasis due to aberrant genetic regulation. This work aimed to determine the function of transmembrane protein 60 (TMEM60) during glioma development. The level of TMEM60 in glioma tissues and normal tissues and its correlation with glioma prognosis were checked in The Cancer Genome Atlas (TCGA) database. The levels of TMEM60 in glioma cell lines and normal astrocytes were determined by quantitative real-time PCR and western blotting assay. TMEM60 knockdown and overexpression were conducted, followed by detection of cell viability, migration, invasion, and apoptosis. CCK-8 and colony formation assay were adopted to detect cell viability proliferation. Transwell assay was performed to measure cell migration and invasion. Cell apoptosis was evaluated by flow cytometry. The alternation of key proteins in the PI3K/Akt signaling pathway was measured by western blotting. TMEM60 expression was significantly higher in glioma tissues than that in the healthy control and was correlated with poor overall survival of patients. The protein and mRNA levels of TMEM60 were both elevated in glioma cell lines in comparison with the normal cell lines. Elevated level of TMEM60 led to enhanced proliferation, migration, and invasion and suppressed cell apoptosis. TMEM60 promoted the activation of PI3K/Akt signaling. Our data suggested that TMEM60 plays an oncogenic role in glioma progression via activating the PI3K/Akt signaling pathway. Glioma is the most commonly occurring brain malignancy in adults, exhibiting highly aggressive feature and grave prognosis –3. NumerOver the past decades, the developed therapeutic manners including surgical operation and chemo- and radiotherapy have successfully achieved partial remission in glioma patients, yet there still exist some patients who show slight response, and the 5-year relative survival is only 5% , 10, 17.In this present work, we, for the first time, confirmed the elevated expression of transmembrane protein 60 (TMEM60) in glioma tissue samples and cell lines. We evaluated cell viability, migration, invasion, and apoptosis after knockdown or ectopic expression of TMEM60 and determined the changes on the PI3K/Akt pathway. Our basic experimental findings provided novel evidences to uncover the TMEM60/PI3K/Akt signaling in glioma and supplied a notable perception concerning the regulatory axis in glioma advancement and novel therapeutic approaches for glioma.https://gepia2.cancer-pku.cn/#help). The expression of TMEM60 in tumor and nontumor tissues and the correlation between TMEM60 and GBM (glioblastoma multiforme) or LGG (brain lower grade glioma) prognosis were analyzed.We performed gene profiling of glioma patients from the Cancer Genome Atlas (TCGA) and healthy donors from the GTEx database by using the online website GEPIA , glioma cell line TJ905, GOS-3, U87MG, and SHG-44 were obtained from ATCC . SVGP12 and U87 were cultured in EMEM . HA was cultured in Astrocyte Medium . GOS-3 and TJ905 were cultured in DMEM . SHG-44 was cultured in RPMI 1640  . All celTo generate the lentiviral shRNA constructs against human TMEM60, the TMEM60 shRNA sequences were cloned into the pLKO-puro vector. The sequences of shTMEM60 are as follows: shTMEM60-1, 5′-GAGTAACCCATGTAAATTACT-3′; shTMEM60-2, 5′-CGAGCTGGACTATCTGTGACT-3′. pLKO.1, pVSVG, pREV, and pGAG were cotransfected into HEK293T cells for 24 h, and cell culture media were collected. The full-length TMEM60 sequences were cloned into the pCDH-puro vector. pCDH, pSPAX.2, and pMD.2G were cotransfected into HEK293T cells for 24 h, and cell culture media were collected. The viruses were used to infect cells in the presence of polybrene. Forty-eight hours later, SHG-44 and U87MG cells were cultured in a medium containing puromycin for the selection of stable clones. The clones stably knocking down or overexpressing TMEM60 were identified and verified by western blotting .μl CCK-8 (5 mg/ml) was added to each well, and the cells were incubated for another 4 hours. After that, the absorbance values at 450 nm were measured by using a microplate reader . For colony formation assay, the cells were suspended as single cells and seeded in 6-well plates  and incubated for 10 days to form colonies [Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay and colony formation. For CCK-8 assay, cells were transfected with indicated oligonucleotides and seeded in 96-well plates at a density of 5,000 cells per well. After incubation for 24, 48, and 72 hours, 20 colonies . The colβ, p-GSK3β, mTOR, p-mTOR, p70S6K, p-p70S6K, 4E-BP1, p-4E-BP1, and β-actin, at 4°C overnight. The next day, the protein bands were incubated with the corresponding horseradish peroxidase- (HRP-) coupled secondary antibodies, and enhanced chemiluminescence (ECL) visualization was performed on a gel image system (Bio-Rad) [The total proteins were extracted from cells after homogenization with RIPA lysis buffer (Beyotime), quantified by using a BCA assay kit (Beyotime). An equal amount of proteins was separated by SDS-PAGE, shifted to polyvinylidene fluoride (PVDF) membranes, and blocked with 5% skim milk in PBST for 2 hours, followed by incubation with primary antibodies against TMEM60, PARP, cleaved PARP, caspase-3, cleaved caspase-3, p-AKT, AKT, GSK3Bio-Rad) , 31.U87MG and SHG-44 cells were trypsinized, counted, and seeded in 6-well plates for 24 hours. Cells were cultured for 10 days to form visible clones. The clones were then washed, fixed, and stained with crystal violet, captured, and counted , 33.The migration and invasion of glioma cells were determined by Transwell assay . In brief, cells were placed into top chambers of 24-well plates with a serum-free medium, and the lower chambers were filled with complete medium . After 4Flow cytometry was used to evaluate apoptosis of glioma cancer cells after TMEM60 knockdown. Glioma cancer cells were plated in 6-well plates with TMEM60 overexpression or vector as control. After double staining with fluorescein isothiocyanate- (FITC-) Annexin V and propidium iodide was performed using an FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's recommendations , the celt test or one-way analysis of variance (ANOVA) were conducted to compare differences between two or multiple groups. The P value <0.05 was considered statistically significant.Data in this study were presented as mean ± standard deviation (SD) of at least three independent experiments and processed by using the GraphPad software (USA). The student's We first identified the correlation between TMEM60 and glioma. The analysis on glioma patient samples and healthy donors from TCGA and the GTEx database showed that the levels of TMEM60 in glioma were remarkably elevated in GBM and LGC tumor tissues, compared with those in healthy tissues, and this elevated level of TMEM60 was correlated with poor overall survival of patients Figures . MoreoveThe abnormal overexpression of TMEM60 indicated that it may facilitate glioma progression . To veriThe high aggressiveness of glioma cells was also exhibited by cell migration and invasion . Here, wAfter determination of cell proliferation and metastasis, we detected the apoptosis of U87 and SHG-44 cells . As showβ, mTOR, p70-S6K, and 4E-BP1 (Figures β, mTOR, p70-S6K, and 4E-BP1 (Figures Studies have proved that the PI3K/AKT signaling contributed to aggressiveness of cancers, including glioma. Here in this work, we investigated whether TMEM60 modulates glioma cell behaviors through regulating the PI3K/AKT signaling pathway. Knockdown of TMEM60 suppressed the activation of PI3K/AKT signaling, as was manifested by decreased phosphorylation of Akt, GSK-3 Figures . Moreove Figures . TherefoIn the present work, we evaluated the role of a rarely reported gene TMEM60 in glioma carcinogenesis. We analyzed the expression of TMEM60 in patients with glioma and healthy donors and spotted its upregulation in glioma tissues compared with the nontumor tissues. The overall survival analysis also unraveled the correlation between high TMEM60 level with poor prognosis. It is well known that glioma is a highly aggressive malignancy with rapid proliferation . MoreoveTo determine the specific role of TMEM60 in glioma carcinogenesis, we conducted knockdown and overexpression of TMEM60 in glioma cell lines and confirmed that TMEM60 overexpression led to enhanced cell viability, proliferation, migration, and invasion, as well as decreased cell apoptosis, which is consistent with the abnormal low level of TMEM60 in glioma tumor sections. Also, the knockdown of TMEM60 exerted opposite effects on GBM cell phenotypes. All these findings suggested that TMEM60 plays a potential oncogenic role in glioma , 44. We Due to the abnormal cellular functions, elevated levels of PI3K and Akt are poor prognostic indicators for patients with malignancies , 64, 65.Taken together, we determined the aberrant expression of TMEM60 in glioma tissue samples and cell lines compared with the normal control. TMEM60 play an oncogenic role in glioma by promoting glioma cell proliferation, migration, and invasion and impairing cell apoptosis via activating the PI3K/Akt signaling pathway.


The transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily of channels, acts as ‘polymodal cellular sensor’ on primary sensory neurons where it mediates the peripheral and central processing of pain, itch, and thermal sensation. However, the TRPA1 expression extends far beyond the sensory nerves. In recent years, much attention has been paid to its expression and function in non-neuronal cell types including skin cells, such as keratinocytes, melanocytes, mast cells, dendritic cells, and endothelial cells. TRPA1 seems critically involved in a series of physiological skin functions, including formation and maintenance of physico-chemical skin barriers, skin cells, and tissue growth and differentiation. TRPA1 appears to be implicated in mechanistic processes in various immunological inflammatory diseases and cancers of the skin, such as atopic and allergic contact dermatitis, psoriasis, bullous pemphigoid, cutaneous T-cell lymphoma, and melanoma. Here, we report recent findings on the implication of TRPA1 in skin physiology and pathophysiology. The potential use of TRPA1 antagonists in the treatment of inflammatory and immunological skin disorders will be also addressed. Transient receptor potential (TRP) channels are polymodal cation channels primarily permeable to calcium, which work as cellular sensors implicated in many physiological functions, ranging from pure sensory activities, such as nociception and temperature sensation, and homeostatic functions, such as osmoregulation, to many other functions, such as muscle contraction and vasomotor control . In mammEmerging evidence suggests that multiple TRPs are involved in the regulation of the cutaneous functions. Apart from their prominent expression and role in nociceptive neurons, where they mediate the peripheral and central processing of pain, itch, and thermal sensation ,18,19,20The skin-localized sensory afferents are involved in the neuronal processing of multiple sensory modalities. Aβ-fibers with thickly myelinated axons, thus fast conduction velocities and low activation thresholds, are the predominant class of fibers responsible for sensing light touch. A subpopulation of C-fibers is responsible for gentle touch and light forces similar to Aβ-fibers ,25,26. SA specific subset of C-fiber and Aδ- fiber nociceptors is exclusively sensitive to the selective TRPV1 agonist, capsaicin, the pungent ingredient in hot chili peppers, and thereby defined as ‘capsaicin-sensitive’ sensory neurons. TRPV1-expressing neurons comprise a subgroup of neurons defined as peptidergic because of their ability to produce neuropeptides, including the calcitonin gene-related peptide (CGRP) and tachykinins, such as substance P (SP) and neurokinin A (NKA) ,29, whicAllium sativum) [The most prominent feature of the TRPA1 resides in its unique sensitivity for several exogenous and endogenous agonists which, based on their structure, activate the channel covalently, or modulate its activity in a different way. A number of naturally occurring TRPA1 agonists mainly found in alimentary sources include herbs and spices, such as cinnamaldehyde, contained in the cinnamon oil extracted from the Cinnamomum , severalsativum) . Other rsativum) ,35, chemsativum) ,37,38, gsativum) , lidocaisativum) , propofosativum) ), and lasativum) ,43,44). sativum) , acetaldsativum)  and crotsativum)   , resultiNon-reactive compounds which are unable to modify the channel covalently include compounds from plant origin, such as menthol , thymol,The last ten years have witnessed a series of discoveries that have placed the TRPA1 channel as a major sensor of oxidative, nitrative, and carbonylic stress for the peripheral nervous system. Reactive oxygen (ROS), nitrative (RNS), and carbonylic (RCS) species have shown the ability to gate TRPA1 on peripheral terminals primary sensory neurons, thereby signaling pain, and neurogenic inflammation. ROS activate TRPA1 through a cysteine oxidation or disulfide formation , whereas2O2) [-), superoxide (O2-) [2) activate TRPA1 channels [2 (15-d-PGJ2), PGA2 and PGA1, and 8-isoprostane-PGA2 are formed. Cyclopentenone PGs and iso-PGs have been recognized as TRPA1 activators [2S), produced by cysteine metabolism and endowed with vasodilatatory and other properties [Among ROS, TRPA1 activators comprise hydrogen peroxide (H2O2) ,39,58, hde (O2-) . Among Rde (O2-) ,60, and channels ,61,62,63tivators ,65. Finaoperties , has alsoperties .Growing evidence has revealed that TRPs are actively involved in the regulation of skin physiology ,75,76,77In human skin, immunoreactivity for the TRPA1 channel has been detected in both keratinocytes and melanocytes . It was The production of the stratum corneum is one of the main roles of epidermal keratinocytes. Some studies showed that the administration of TRPA1 activators as well as the application of cold stimuli to the skin of mice, in which the epidermal barrier was mechanically disrupted, accelerated the rate of barrier regeneration . ConversAtopic dermatitis (AD) and allergic contact dermatitis (ACD) are common inflammatory skin diseases characterized by skin barrier disruption and an inflammatory response dominated by T helper 2 (Th2) cells and related products, such as interleukin (IL)-4, IL-5, and IL-13 ,90. PrurTRPA1 contributes to the pathogenesis of chronic  and acutA TRPA1-dependent pathway of itch in AD has been firstly identified by using a murine model of the disease induced by IL-13 . In thisAn additional study revealed that in a different murine model of AD induced by 2,4-dinitrochlorobenzene (DNCB), genetic deletion of TRPA1 attenuated pathological findings of AD, including ear thickness, epidermal hyperplasia and pruritus, and dermal infiltration by mast cells, Th2 cytokines, and macrophages . MoreoveIn chronic allergic itch, multiple pathways of TRPA1 activation have been shown. These include a keratinocyte-neuron axis based on the release of thymic stromal lymphopoietin  and periPsoriasis is a common chronic inflammatory skin disease, characterized by erythema, skin thickness, and scaling . PrurituMore recently, the role of TRPA1 in psoriasis has been explored. It was originally reported that in a murine model of imiquimod (IMQ)-induced psoriasis-like lesions, topical application of the drug was associated with elevated expression of TRPA1 in affected skin areas . SimilarCutaneous T-cell lymphomas (CTCL) are a heterogeneous group of primary cutaneous lympho-proliferative disorders, including mycosis fungoides (MF) and Sézary syndrome (SS) as the most common clinical presentations . PrurituChronic histamine-independent pruritus occurs in a wide variety of other cutaneous and systemic diseases. A common skin disease characterized by pruritus is scabies, a contagious parasitic infestation caused by the mite Sarcoptes scabiei hominis . In the A common systemic cause of itch is that related to liver dysfunction, which in turn causes an elevation of circulating bile acids (BA). Remarkably, one study reported the co-expression of the G-protein-coupled BA receptor 1 (TGR5) and TRPA1 in cutaneous afferent neurons in mice . TRG5 acA study reported TRPA1 over-expression in the epidermis of patients with bullous pemphigoid, a rare autoantibody-mediated blistering disease characterized by intense pruritus, compared to healthy skin, but channel expression did not significantly correlate, neither with eosinophil dermal infiltration nor with the severity of pruritus  Figure .Finally, a distinctive cause of pruritus induced by physical factors is that following burn injuries. Recently, one study revealed that mRNA levels of TRPA1 as well as TRPV4 were increased in the skin of itching burn scars . HoweverThe hypothesis that TRPA1 is implicated in chronic neuropathic ,128,129,Here we showed that TRPA1 may exert a number of functions in different physiological and pathological skin processes. Increasing evidence indicates a major role of TRPA1 in histamine-independent itch occurring in chronic inflammatory skin diseases such as AD, liver dysfunction, or neoplastic diseases, such as cutaneous T-cell lymphoma, thus making these conditions suitable areas of investigation for drugs that target TRPA1. Among the various TRPA1 antagonists that have shown selectivity toward TRPA1, only five have been tested in clinical trials for the treatment of pain or other conditions  ,137,138.www.clinicaltrial.gov) using the key words “TRPA1 and skin” or “TRPA1 and pruritus” finds only one study on the effects of L-menthol, a substance that does not directly target TRPA1, as a topical counter-irritant on cutaneous pain and hyperalgesia provoked by topical application of the TRPA1-agonist trans-cinnamaldehyde in healthy human volunteers. Despite robust evidence of TRPA1 involvement in pruritic skin diseases, reasons explaining the delay in translating TRPA1 antagonists from the pre-clinical to the clinical setting, remain unclear. However, other therapeutic strategies are worth exploring.A recent review identified 28 patent applications for TRPA1 antagonists from 2015 to 2019. Among them, some have been successfully tested in pre-clinical models of skin diseases such atopic dermatitis . In contPetasites hybridus), and parthenolide, a major constituent of Tanacetum parthenium, TRPA1 channel and the TRPA1 expressing neurons undergo to a dose-dependent neuronal desensitization which may account for the relief of pain and neurogenic inflammation of the two plant extracts [It is noteworthy that multiple molecules can selectively activate TRPA1 inducing long-lasting desensitization of the channel. Crotalphine has been shown to inhibit a chemically induced inflammatory hypersensitivity in mice via the desensitization of TRPA1-peptidergic nerve endings . In alleextracts ,143.TRPA1 remains a field of active investigation for other skin conditions. For example, TRPA1 proteins have been found in melanoma cell lines ,145,146,Finally, increasing evidence supports a role for TRPA1 in fibrosis associated with systemic diseases ,151. Acc


However, the role of miRNAs in the mechanism of tea plant flower sterility remains unclear. Therefore, we performed miRNA sequencing on the flowers of fertile male parents, female parents, and sterile offspring.ATD1A, SPL, ACA1, ACA2, CKB22 and MADS2.A total of 55 known miRNAs and 90 unknown miRNAs were identified. In the infertile progeny, 37 miRNAs were differentially expressed; 18 were up-regulated and 19 were down-regulated. miR156, miR157, miR164, miR167, miR169, miR2111 and miR396 family members were down-regulated, and miR160, miR172 and miR319 family members were up-regulated. Moreover, we predicted that the 37 differentially expressed miRNAs target a total of 363 genes, which were enriched in 31 biological functions. We predicted that miR156 targets 142 genes, including We detected a large number of differentially expressed miRNAs in the sterile tea plant flowers, and their target genes were involved in complex biological processes. Among these miRNAs, the down-regulation of miR156 may be one of the factor in the formation of sterile floral buds in tea plants.The online version contains supplementary material available at 10.1186/s41065-021-00188-8. Tea is one of the most popular and widely consumed beverages in the world. As such, producers expect tea varieties to have high yield . HoweverMicroRNAs (miRNAs) are a type of endogenous, non-coding, single-stranded RNA molecule composed of about 21 to 25 nucleotides (nt) in length that exist widely across organisms and are quite conserved . GeneralSPL9 and SPL10, and was regulated by miR156, contrary to the expression pattern of miR156 [ARF6 and ARF8 in specific floral organs, but different isoforms showed differences in the ability to inhibit ARF6 and ARF8 expression [ARF10, ARF16 and ARF17 [Studies have shown that a number of miRNAs, including miR156, miR159, miR160, miR164, Mir166/165, miR167, miR169, miR172, miR319, miR390, and miR399, are related to flower development. It has been reported that miR156, miR172 and miR390 are involved in the regulation of flowering time; miR156 was up-regulated at the juvenile stage to prevent flowering, followed by a decrease in expression with plant growth ; miR172 f miR156 ; mutationd ARF17 .In our study, we selected Foxiang2 (FBH), Fudingbaicha (MBH), and their hybrid sterile progeny (ZDH) because of the self-incompatibility of tea plants. We performed small RNA sequencing on flowers, analyzed the differentially expressed genes, and predicted the target genes of different miRNAs. Through the analysis of the data, we identified the key miRNAs related to tea plant sterility and provided reference information for research on the mechanism of tea plant sterility .C. sinensis (L.) O. Kuntze) used in this experiment were ‘Foxiang2’ (FBH), ‘Fudingbaicha’ (MBH) and sterile hybrid (ZDH), were collected from the Tea Research Institute of Yunnan Academy of Agricultural Sciences. The floral organ of ‘Foxiang2’ and ‘Fudingbaicha’ were complete bisexual flowers. The hybrid offspring of sterile flower buds consist of multiple dwarf pistils, the petals cannot expand normally, the filaments were short, and no pollen in the anthers . The Small RNA Sample Pre-Kit was used to construct the library. Briefly, with total RNA as the starting material, the special structure of the 3′ and 5′ ends of Small RNA were used to add linkers to both ends of the Small RNA, followed by reverse transcription to synthesize cDNA to construct the library. The effective library concentration was greater than 2 nM. Agilent 2100 was used to detect the insert size of the library and Illumina HiSeq 2000 sequencing was performed. All of the experiments were performed in triplicate. In order to ensure the quality of information analysis, the following was removed: low-quality reads, reads with less than 10% base information, reads contaminated with 5′ linkers, reads without 3′ linker sequences and inserts, 3′ linker sequences, and polyA/T/G/C reads.The Bowtie software package locates the small RNA after length screening to the reference sequence, and analyzes the distribution of small RNAs on the reference sequence . Then, tTo obtain the corresponding relationship between miRNA and target gene, the target genes of known and novel miRNAs were predicted using the miRNA target gene prediction software psRobot . Gene OnP-values were adjusted using the Benjamin and Hochberg approach for controlling the false discovery rate. The genes with P-value < 0.05 found by DESeq were differentially expressed [We performed statistical analysis on the expression of known and new miRNAs in each sample. The input data of differential expression is the read count data obtained from miRNA expression level analysis. For the biological replicates, DESeq2 based on negative binomial distribution was used for analysis. In the comparison of the two samples, the binomial distribution was emphasized, and A1 and A2 were the total number of reads. Define M = (log2A1-log2A2) and A = (log2A1 + log2A2/2). DESeq2 calculates the difference expression based on the MA-plot . The resxpressed .Total RNA was isolated using TRlpure reagent  according to the manufacturer’s instructions. cDNA was synthesized from total RNA using a PrimeScript RT reagent kit . The obtained cDNA was used as a template in a SYBR green-basedq-PCR reaction kit . miR165a-3p was used for normalization, and the primers are shown in Table In order to explore the role of miRNAs in the regulation of tea plant flower development, we used high-throughput sequencing to construct miRNA libraries of FBH, MBH and ZDH. The Q20 percentage of raw data exceeded 98%, and the Q30 percentage was above 96%, with the ratios of GC content above 47%. As a result, a total of 13,976,247, 16,644,199, and 13,976,692 clean reads were obtained for FBH, MBH and ZDH, respectively. The length of plant small RNA ranged from 18 ~ 30 nt, and a total of 11,704,253, 14,693,758 and 13,045,116 small RNA sequence read were identified for FBH, MBH and ZDH, respectively (Table Arabidopsis in the miRBase 21.0 database (http://www.mirbase.org/ftp.shtml). A total of 55 known miRNAs were identified and divided into 27 miRNA families. Among these miRNA families, miR156 and miR396 contained four members, miR159, miR166, miR169, miR172 and miR399 contained three members, miR157, miR160, miR164, miR167, miR169, miR170, miR171, miR319, miR390, miR395, miR398 and miR858 contained 2 members, and miR162, miR165, miRK2111, miR393, miR394, miR403, miR408 and miR8175 contained 1 member  targeted by miR160a-5p were predicted; nine floral homeotic protein (AP2) and two ethylene-responsive transcription factor (RAP2–7) co-targeted by miR172a, miR172c and miR172e-3p were predicted; two transcription factors (GAMYB) targeted by miR319c were predicted; two calcium-transporting ATPases (ACA12) co-targeted by miR396a-5p and miR396b-5p were predicted; and one ABC transporter I family member (ABCI11) co-targeted by miR172a and miR172c were predicted. In addition, we predicted that miR156 has 142 target genes, including three ATPase family AAA domain-containing proteins (ATD1A), four Squamosa promoter-binding proteins (SPL), two calcium-transporting ATPases (ACA1 and ACA2), two Cyclin-dependent kinases (CKB22), and Floral homeotic protein (MADS2). These results indicated that the differentially expressed miRNAs regulate complex biological processes.Among the target genes, three auxin response factor genes (We randomly selected 12 miRNAs for RT-qPCR analysis to verify the accuracy of the small RNA sequencing. Among these miRNAs, miR156a-5p, miR156j, miR157a-5p, miR157d, miR164a and miR164c-5p were down-regulated, and miR319, amiR319, cmiR172a, miR172e-3p, miR160a-5p and novel_126 were up-regulated in ZDH (Fig. As the reproductive structure of plants, flowers are the key to species reproduction. Flower development is regulated by external factors, such as the environment, and the plant’s genetic information . MicroRNArabidopsis, miR160 participates in the auxin-related signal transduction pathway through targeted regulation of the ARF10, ARF16 and ARF17 genes, and regulates the growth and development of flowers [TCP24 gene in Arabidopsis destroys the cell wall of anther endoderm hierarchy, leading to male sterility [In  flowers . miR172  flowers , 28. Ove flowers . The abs flowers . In addi flowers . Overexpterility . In contArabidopsis petals and sepals by regulating the expression of the SPL2 gene [SPL8 gene deletion in Arabidopsis results in small anthers, less pollen, and reduced fertility [AP2 [CUC1 and CUC2, which can promote the development of floral organs [CUC2 and CUC1, floral organ fusion occurred, and the number of pistil and stamen were reduced [miR156 inhibits the expression of the SPL transcription factor family to regulate the formation and development of floral organs , and 11 PL2 gene . SPL8 geertility . Notablyity [AP2 , and miRity [AP2 . Our resl organs . In doub reduced . We idenmiR159 and miR319 have overlapping effects in the regulation of flower development, regulating flowering time and anther development . In addiSPL. In contrast to the previous reports, up-regulation of miR172 did not inhibit AP2 expression. In addition, miR160, miR164, and miR319 were also involved in regulating the formation of sterile floral buds. Our study identified a number of important miRNAs related to tea plant fertility, these results provided a basis for further elucidating the mechanism of sterile floral buds in tea plant.In conclusion, Fig. Additional file 1: Table S1. Identification of known miRNAs. Table S2 Identification of novel miRNAs.


The SET and MYND domain-containing (SMYD) gene family comprises a set of genes encoding lysine methyltransferases. This study aimed to clarify the relationship between the expression levels of SMYD family members and the prognosis and immune infiltration of malignant tumors of the digestive system.The Oncomine, Ualcan, Kaplan–Meier Plotter, cBioPortal, Metascape, and TIMER databases and tools were used to analyze the correlation of SMYD family mRNA expression, clinical stage, TP53 mutation status, prognostic value, gene mutation, and immune infiltration in patients with esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), and stomach adenocarcinoma (STAD).In ESCA, the mRNA expression of SMYD2/3/4/5 was significantly correlated with the incidence rate, that of SMYD2/3 with the clinical stage, that of SMYD2/3/4/5 with TP53 mutation status, that of SMYD2/4/5 with overall survival (OS), and that of SMYD1/2/3/4 with relapse-free survival (RFS). In LIHC, the mRNA expression of SMYD1/2/3/4/5 was significantly correlated with the incidence rate, that of SMYD2/4/5 with the clinical stage, that of SMYD3/5 with TP53 mutation status, that of SMYD2/3/4/5 with OS, and that of SMYD3/5 with RFS. In STAD, the mRNA expression of SMYD2/3/4/5 was significantly correlated with the incidence rate, that of SMYD1/4 with the clinical stage, that of SMYD1/2/3/5 with TP53 mutation status, that of SMYD1/3/4 with OS, and that of SMYD1/3 with RFS. Furthermore, the function of SMYD family mutation-related genes in ESCA, LIHC, and STAD patients was mainly related to pathways, such as mitochondrial gene expression, mitochondrial matrix, and mitochondrial translation. The expression of SMYD family genes was significantly correlated with the infiltration of six immune cell types and eight types of immune check sites.SMYD family genes are differentially expressed and frequently mutated in malignant tumors of the digestive system . They are potential markers for prognostic prediction and have important significance in immunity and targeted therapy. Esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC) and gastric cancer (GC) are the main malignant tumors of the digestive system, accounting, respectively, for 5.3, 8.2, and 8.2% of cancer-related deaths worldwide . TherefoAn imbalance of immune effector cells in the tumor microenvironment contributes to malignant tumor cell immune escape. In recent years, tumor immunotherapy has received extensive attention in a variety of solid tumors and has been regarded as an important treatment method . For exaThe occurrence and development of malignant digestive tract tumors is a complex process. In the past, some studies have reported the expression pattern of SMYD family members in some cancer patients and its correlation with prognosis. However, the entire SMYD family has not been so far systematically investigated in malignant digestive tract tumors. Therefore, we conducted a comprehensive analysis of the SMYD family based on public data reposited in various large databases to determine its role in malignant digestive system tumors.1 is currently the world’s largest oncogene microarray database and integrated data mining platform, which integrates RNA and DNA-seq data from gene expression omnibus (GEO), The Cancer Genome Atlas (TCGA), and published literature sources. To date, ONCOMINE contains 65 gene expression datasets comprising nearly 48 million gene expression measurements form over 4700 microarray experiments. We used the cancer microarray database (without TCGA data) to analyze the mRNA expression levels of SMYD family members in ESCA, LIHC, GC, and normal esophagus, liver, and stomach tissues  = Top 10%,” “data type = mRNA,” output Disease Summary for SMYD family.Oncomine tissues  and summ2 is a comprehensive and interactive online data analysis website based on relevant data found in TCGA database, including gene expression data of 184 ESCA, 371 LIHC, and 415 GC patients. The portal’s user-friendly features allow to perform: (1) analyze relative expression of a query gene(s) across tumor and normal samples, as well as in various tumor sub-groups based on individual cancer stages, tumor grade, race, body weight, or other clinicopathologic features, (2) estimate the effect of gene expression level and clinicopathologic features on patient survival; and (3) identify the top over-and under-expressed (up and down-regulated) genes in individual cancer types. We used the UALACAN database to evaluate the expression levels of SMYD family members in ESCA, LIHC, gastric adenocarcinoma (STAD), and normal esophagus, liver, and stomach tissues, and determine the correlation between clinical stage and TP53 status , this study included 80 ESCA patients, 371 LIHC patients, and 375 GC patients, but patients miss expression values and lack complete clinical data. We divided patient samples into high- and low expression groups according to the best cut off of the expression level of SMYD family members, and used the Kaplan–Mayer Plotter to analyze the overall survival (OS) and relapse-free survival (RFS) of ESCA, LIHC, and STAD patients. Enter SMYD1/2/3/4/5 in the “Gene symbol” of the “Start KM Plotter for pan-cancer” module, select Auto select best cut off in the “Split patients by” module, select OS, RFS in the “Survival” module, and then select ESCA, LIHC, and STAD to generate survival curves, and use Kaplan–Meier method to draw survival curves. A P-value of <0.05 was considered statistically significant.The Kaplan--Meier Plotter4 integrates data from large-scale cancer research projects, such as TCGA and the International Cancer Genome Consortium (ICGC), whose gene data types cover somatic mutations, DNA copy number changes, mRNA and microRNA expression, DNA methylation, protein and phosphorus protein abundance, and provides visual and multidimensional cancer genomic data (z-scores relative to diploid samples (RNA Seq V2 RSEM),” “Enter a z-score threshold ±1.8,” “Select Patient/Case Set: Samples with mRNA data (RNA Seq V2),” enter SMYD1/2/3/4/5 in “Enter Genes” to generate a mutation frequency visualization chart, and then select the top 10 ESCA, LIHC, and STAD genes significantly related to SMYD family gene mutations in “Co-expression” module for enrichment analysis after removing duplicates.The cBioPortalmic data . This st5 is a gene list analysis tool. It integrates data from over 40 types of biological information databases for gene annotation and analysis, and provides a unique protein–protein interaction (PPI) network analysis function. We used the “Multiple Gene list” module of the Metascape tool to perform gene annotation and enrichment analyses on the genes obtained from the cBioPortal that were highly related to ESCA, LIHC, STAD, and SMYD family member mutations  enriching “Biological Processes,” “Cellular Components” and “Molecular Functions” and “KEGG pathways” classification. To further capture the relationships between the terms, a subset of enriched terms were selected and rendered as a network plot, where terms with a similarity >0.3 were connected by edges. We selected the terms with the best P-values from each of the 20 clusters, with the constraint that there were no more than 15 terms per cluster and no more than 250 terms in total. The network was visualized using Cytoscape  enter CD274, CTLA4, GZMB, HAVCR2, LAG3, PDCD1, TIGIT, TNF,” “Gene Symbols: (X-axis) enter SMYD1/2/3/4/5,” “Correlation Adjusted by: Tumor Purity,” generate the scatter plot of immune test site correlation, which is statistically significant using Spearman’s rho value. A P-value of <0.05 is considered statistically significant.TIMERck sites , 2017a. We measured the mRNA expression of the five SMYD family members in 20 cancer types, and compared them with those of normal tissues using the Oncomine database . AccordiP = 0.97E-01), but the expression of SMYD2/3/4/5 was significantly higher than that of normal tissues . The expression of SMYD1/2/3/4/5 in LIHC tissues was significantly higher than that of normal tissues , whereas SMYD1 expression in STAD tissues was not significantly different from that of normal tissues (P = 0.143E-01). However, SMYD2/3/4/5 expression was significantly higher than that of normal tissues  ] .P = 1.92E-01). However, SMYD2/3/4/5 expression was significantly higher than that of normal tissues . The expression of SMYD1/2/3/4/5 in LIHC tissues was significantly higher than that in normal tissues , whereas SMYD1 expression in STAD tissues was significantly lower than that in normal tissues (P = 6.60E-03). Furthermore, SMYD2/3/4/5 expression was significantly higher than that in normal tissues  ] . In summWe determined the association between the mRNA expression levels of different SMYD family members and the clinical stage and TP53 mutation status of patients with ESCA, LIHC, and STAD using the UALCAN data mining website. Clinical stage correlation analysis showed significant expression differences in SMYD1 between stage-1 and stage-2, and stage-1 and stage-3 in STAD patients, in SMYD2 between stage-1 and stage-2, and stage-2 and stage-3 in ESCA patients, and between stage-3 and stage-4 in LIHC patients. Significant differences in SMYD3 expression were found between stage-1 and stage-2, and stage-1 and stage-3 in ESCA patients, and in SMYD4 between stage-1 and stage-3, stage-2 and stage-3 in LIHC patients, and between stage-1 and stage-3 in STAD patients. Significant differences in SMYD5 expression were found between stage-1 and stage-3, stage-1 and stage-3 in LIHC patients . NotablyP = 0.033; SMYD4 HR 2.51 (1.24–5.07), P = 0.0083; SMYD5 HR 0.4 (0.16–0.95), P = 0.032], SMYD1/2/3/4 were all significantly correlated with RFS , SMYD3/5 were significantly correlated with RFS , P = 0.00024) , P = 0.01; SMYD4 HR 0.66 (0.47–0.91), P = 0.011], SMYD1/3 were significantly correlated with RFS   0.33 (1.11–0.96), = 0.024) . In LIHC0.00024) . In STAD 0.0077] . OverallP = 0.939, P = 0.133, P = 0.146; PFS, P = 0.289, P = 0.146, P = 0.369)  . Next, w= 0.369) . After dWe used the 124 genes significantly related to SMYD family mutations for GO and KEGG enrichment analyses . GO enriThe prospect of immunotherapy is broad. PD-1 inhibitors are immunotherapeutic drugs used in the treatment of ESCA, LIHC, and GC. Therefore, we used the TIMER website to verify the correlation between the expression of SMYD family genes and immune cell infiltration and immune check sites in ESCA, LIHC, and STAD patients. We found that the mRNA expression of SMYD family members in ESCA, LIHC, and STAD patients was significantly correlated with six kinds of immune cells, including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. Specifically, in ESCA, SMYD1 was positively correlated with macrophages, SMYD2 was negatively correlated with CD4+ T cells, and SMYD3 was positively correlated with macrophages and negatively correlated with neutrophils and dendritic cells. SMYD4 was positively correlated with macrophages, whereas SMYD5 was negatively correlated with neutrophils and dendritic cells . In LIHCIt has been established that the SMYD gene family plays an important role in tumors. However, the mechanism underlying the function of different SMYD family members in malignant digestive system tumors remains largely unknown. We conducted a comprehensive analysis of mRNA expression differences, clinical stage and TP53 correlations, prognostic value, mutation, functional enrichment analysis, PPI analysis, immune cell infiltration, and correlation of immune check sites using mining and analysis of major online database websites, to explore the role of SMYD family members in malignant digestive system tumors. To the best of our knowledge, this is the first comprehensive analysis of the prognostic value of SMYD family members in malignant digestive system tumors and their relationship with immune infiltration.Each SMYD family member has its own unique structural domain, cell functions and tissue distribution. A large number of studies have found that the SMYD family mainly regulates the transcription and translation of oncogenes or tumor suppressor genes, affecting tumor transcription regulation, chromosome remodeling, DNA damage repair, and signal transduction by: (1) promoting histone methylation, forming a transcription mplex with RNA polymerase II, specifically recognizing the promoter region of target genes, and promoting the transcriptional activation of downstream target genes, and (2) promoting non-histone protein methylation, directly binding with molecular chaperones, regulating key tumor signaling pathways and downstream target genes, and affecting the malignant characteristics of tumors .SET and MYND domain-containing 1 protein, a regulator of heart and skeletal muscle, is the most unique SMYD family member. Subtypes A and B are expressed in striated muscle, while subtype C is expressed in CD8+ cells . SMYD1 cIncreased SMYD2 expression has been significantly associated with the low survival rate of patients with esophageal squamous cell carcinoma, revealing the carcinogenic potential of SMYD2 . In BC, in vitro  are responsible for tumor development, metastasis and recurrence. SMYD4 is closely related to CSCs. It has been reported that SMYD4 binds to miR-135a and activates the expression of Nanog by regulating the methylation of its promoter, contributing to the conversion between CSCs to non-CSCs. SMYD4 has not been extensively studied in cancer. Only in BC, SMYD4 exerts anti-tumor effects through local inhibition of PDGFR-A . We founSET and MYND domain-containing 5 has been identified as a key regulator of BC cell cancer metastasis. SMYD5 inhibits Toll-like receptor 4 (TLR4) expression in macrophages through H4K20me3, thereby regulating immune system balance . We founHigh SMYD family mutation frequency was found in ESCA, LIHC, and STAD patients, with total mutation rates of approximately 57, 51, and 43%, respectively. Previous studies have shown that SMYD3 expression is increased in KRAS-mutated cancer, which may be due to the regulative effect of KRAS on SMYD3 gene transcription or protein stability. SMYD3 silencing may reduce the progression of advanced cancer rendering SMYD3 a potential therapeutic target for cancer patients with KRAS mutations . Using GThe SMYD family is also closely related to immune infiltration. Our study has some limitations, such as the use of database-retrieved data, lack of real world verification using cell, animal, and tissue studies, or investigations of the relevant underlying molecular mechanism and clinical application in ESCA, LIHC, and GC treatment. In conclusion, our study identified a high frequency of SMYD family mutations and differential SMYD family gene expression in malignant digestive system tumors, indicating that the SMYD family may provide potential prognosis prediction markers, and immune or targeted therapy targets.www.oncomine.org; http://ualcan.path.uab.edu/analysis.html; www.kmplot.com; www.cbioportal.org; http://metascape.org; https://cistrome.shinyapps.io/timer/.Publicly available datasets were analyzed in this study. This data can be found here: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.DL and MN: conceptualization. QJ and PK: methodology. SS and CW: formal analysis. SY and ZQ: investigation. DL and SZ: writing – original draft preparation. XW: writing – review and editing. ES: supervision; LW: project administration. LL: funding acquisition. 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.


Mast cells (MCs) act as primary effectors in inflammatory and allergic reactions by releasing intracellularly-stored inflammatory mediators in diseases. The two major pathways for MC activation are known to be immunoglobulin E (IgE)-dependent and -independent. Although IgE-dependent signaling is the main pathway to MC activation, IgE-independent pathways have also been found to serve pivotal roles in the pathophysiology of various inflammatory conditions. Recent studies have shown that human and mouse MCs express several regulatory receptors such as toll-like receptors (TLRs), CD48, C300a, and GPCRs, including mas-related GPCR-X2 (MRGPRX2). MRGPRX2 has been reported as a novel GPCR that is expressed in MCs activated by basic secretagogues, neurokinin peptides, host defense antimicrobial peptides, and small molecule compounds  and leads to MC degranulation and eicosanoids release under in vitro experimental condition. Functional analyses of MRGPRX2 and Mrgprb2 (mouse ortholog) indicate that MRGPRX2 is involved in MC hypersensitivity reactions causing neuroinflammation such as postoperative pain, type 2 inflammation, non-histaminergic itch, and drug-induced anaphylactic-like reactions. In this review, we discuss the roles in innate immunity through functional studies on MRGPRX2-mediated IgE-independent MC activation and also the therapeutic potential of MRGPRX2 inhibitors on allergic and inflammatory diseases. Mast cells (MCs) are at the forefront of exposure to environmental factors and are known to be important players and regulators of innate immune responses to pathogens ,2. MCs iTwo main pathways for MC activation are known: immunoglobulin E (IgE)-dependent and IgE-independent ,6. IgE-dIn this review, we will firstly outline the characteristics and downstream signaling of MRGPRX2, a very unique GPCR without parallel, and secondly discuss how MRGPRX2 activation may contribute to the etiology of neurogenic inflammation and pain, type 2 inflammatory reactions such as atopic dermatitis (AD), chronic urticaria (CU), allergic contact dermatitis (ACD), non-histaminergic itch, and small molecule compound-induced pseudoallergy. Finally, we propose that inhibiting the inappropriate activation of MRGPRX2 in MCs may lead to new therapeutic approaches for many allergic and inflammatory diseases.MRGPRs were cloned as a GPCR family with 35% homology to the proto-oncogene MAS1 gene and were reported to be sensory neuron-specific GPCR [MRGPRX1-X4) and MrgprD-H [MrgprA genes, 11 MrgprB genes, and 13 MrgprC genes in mice. Within the MRGPR family, proenkephalin A gene products such as bovine adrenal medulla (8–22) peptide were first identified as ligands for MRGPRX1 [MRGPRX2 gene expression [fic GPCR . In humaMrgprD-H ,24, but edulla 8– peptide  MRGPRX1 . In 2005 MRGPRX1 . The phypression . Howeverpression ,27,28. Bpression ,29. Furtpression ,30. In apression . The pecpression . In addipression . This impression , while tpression . Based opression ,35, suggMrgpra1 being expressed in the neurons and Mrgprb2 in MCs [Mrgprb2 and Mrgprb3, which are expressed in MCs as observed in human MRGPRX2, recognize SP, hBDs, and pseudoallergic inducers [Mrgpra1 and Mrgprb2 have been reported as mouse orthologs that recognize the MRGPRX2 ligand SP ,36, with2 in MCs , so it cinducers ,20,37. Tinducers . The caninducers . Therefo2+ mobilization and suppresses forskolin-induced intracellular cAMP elevation after MRGPRX2 ligands stimulation /AKT, MAPKs such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2), p38), and nuclear factor-κB (NF-κB) are involved in cytokine production from MCs during allergy [It is generally known that GPCR ligands act on the hydrophobic pocket formed by the extracellular loops and the adjacent transmembrane regions of the GPCRs, converting them into cellular responses via G-proteins, β-arrestins, and other downstream effectors . Furthermulation  [17,41,4n in MCs ,42. Storse model , support allergy ,45,46,47 allergy ,49,50, a allergy ,51. IL-3 allergy . These s allergy ,42,51,53 allergy ,55 and t allergy . On the  allergy . Thus, M allergy . The lacMCs are predominantly located in close proximity to peripheral nerve endings compared to other innate immune cells, making them the first cells to respond to sensory nerve activation. MCs are also involved in the mobilization of a variety of innate immune cells, furthering the inflammatory cascade and sensitization of peripheral afferents . The croMrgprb2 receptor is expressed on MCs, but not expressed on DRG neurons. Mrgprb2-KO mice implied that SP-induced infiltration of other immune cells  was suppressed in Mrgprb2-KO mice and also the effect was NK-1-independent [Postoperative pain has both inflammatory and neuropathic components. Effective postoperative analgesia and sedation are needed to increase patient satisfaction and decrease postoperative morbidity and mortality. . In periependent . Therefoependent . The facependent  has led Migraine, a transient headache, is thought to be caused by activation of the trigeminal nerves, consisting of the neurons innervating the cerebral vessels whose cell bodies are located in the trigeminal ganglion. Trigeminal afferents lead to the release of vasoactive peptides, causing neurogenic inflammation . ChronicTAC1 and the temperature-sensitive ion channel TRPV1 are expressed in the neuropeptide-producing nociceptors [AD or eczema is a chronic, recurrent, itchy, inflammatory skin disease. The incidence of this disease has increased over the past 30 years, with 10–20% of children affected . Althougiceptors . Serhan iceptors . When thiceptors . Furthericeptors . This moiceptors . SEB useiceptors . This reiceptors . MBP, aniceptors . MRGPRX2iceptors ,32. Thesiceptors .In CU, in which wheals occur daily over the entire body for at least 6 weeks, the release of various vasoactive autacoid mediators, proteases, chemokines, and cytokines from MCs plays an important role in inducing the wheal response . Approxi+ effector T cells, which are initiated in the lymphoid organs during the sensitization phase, are recruited to the skin after re-exposure to the haptens. Since CD4+ regulatory T cells are involved in the suppression of CD8+ effector T cells, ACD is considered to be a disruption of cutaneous immune tolerance to haptens [+ T cells [ACD is considered to be a skin inflammation caused by repeated skin exposure to contact allergens or non-protein chemicals called haptens ,96. ACD  haptens ,96. On t T cells . Mrgprb2 T cells ,98,99, sIn patients with RA, an increased number of activated MCs is observed in synovial tissue and fluid, and MC activation has been linked to the potentiation of inflammation in the pathogenesis of RA ,101,102.It is widely accepted that environmental and host factors are intricately intertwined in the pathogenesis of UC, and events such as disruption of the mucosal barrier, changes in the healthy balance of the intestinal microbiota, and the abnormal stimulation of the intestinal immune response trigger the onset of the disease . SubstanHistamine is considered the classic itch substance, but most types of chronic itch are not ameliorated by antihistamines, so that non-histaminergic and neural mechanisms in chronic itch conditions have been studied ,116. As Non-allergic drug hypersensitivity, one of the specific side effects, is also called pseudoallergic reaction or anaphylactoid reaction . PseudoaMRGPRX2 plays an important role not only in relation to diseases but also as a biological defense mechanism in mast cells ,139. MCsMRGPRX2 is thought to play important roles in the above-mentioned diseases by inducing neurogenic inflammation, and thus, MRGPRX2 inhibitors are expected to improve these diseases. MRGPRX2 inhibitors can be divided into three categories: (1) direct inhibition of MRGPRX2, (2) inhibition of MRGPRX2 downstream signaling, and (3) other mechanisms .Angelica Dahurica radix), and roxithromysin (a derivative of erythromycin) were shown to bind to MRGPRX2 in molecular docking studies and surface plasmon resonance (SPR) and inhibit C48/80 or SP-induced passive cutaneous anaphylaxis (PCA) in mice [Paeonia Lactiflora), quercetin (a plant flavonoid), and genistein , which were found to bind MRGPRX2 only in the docking model, also suppressed C48/80-induced PCA in mice [QWF is a tripeptide antagonist of NK-1, a canonical receptor for SP which is a representative neuropeptide of the MRGPRX2 ligands. QWF has also been reported to act as an MRGPRX2 antagonist. QWF competitively inhibits SP binding not only to human MRGPRX2 but also to Mrgpra1 and b2 . QWF inh in mice ,143,144. in mice ,146,147. in mice ,160,161. in mice . It has  in mice . Since b in mice  has allo in mice ,146,147.Mrgpr family forms clusters in the genome and the Mrgpr family shows common reactivity to ligands [For the purpose of ameliorating acute drug-induced pseudoallergic reactions in MCs, it will be useful to develop high-affinity MRGPRX2 inhibitors for a wide range of MRGPRX2 ligands. DNA aptamer has been reported as a method to obtain high-affinity MRGPRX2 inhibitors . Nucleic ligands , it is n2+ concentration and suppresses MC activation. It also suppresses MRGPRX2 expression on the plasma membrane, and thus, MRGPRX2 is inhibited through a complex mechanism [2+ concentration and activation of MAP kinase. However, it may also inhibit MRGPRX2 activation and MC activation by interacting with the basicity of MRGPRX2 ligands [Recently, inhibitory compounds for MRGPR2 with anti-inflammatory properties by blocking the above-mentioned MRGPRX2 downstream signaling have been reported. Resveratrol, a polyphenol found in grapes, red wine, and peanuts, increases the expression of nuclear factor erythroid-derived 2-related factor (Nrf2) and induces heme-oxygenase 1 (HO-1), which has antioxidant effects . Since techanism . In addiechanism . In addiechanism . Lactic  ligands . TherefoCD300f (leukocyte mono-immunoglobulin-like receptor 3), a known inhibitory regulator of IgE- and LPS-stimulated MC activation, suppresses MRGPRX2-mediated MC activation . CD300f High-affinity MRGPRX2 inhibitors are expected to be applied to areas where there are few effective therapeutic agents, such as postoperative pain, migraine, and drug-induced acute pseudoallergic reactions. The key is to obtain a specific antagonist with high affinity for MRGPRX2, which has the peculiar ligand recognition ability. Although molecular modeling experiments have enabled us to predict the 3D structure of MRGPRX2 for designing agonists , the exaSo far, rodents have made significant contributions to the elucidation of the MRGPX2 function in vivo. However, the results from the Mrgprb2-KO mouse may be re-examined, because, as mentioned above, the Mrgpr family of receptors may compensate for the lost function. The rat-sized monkey or common marmoset  is becoming an attractive animal model in biomedical research . It may The development of high-affinity MRGPRX2 inhibitors is expected to make a significant contribution to the treatment of neurogenic inflammation, type 2 inflammation such as atopic dermatitis and chronic urticaria, and non-histaminic itch. They may also be a useful drug for unexpected, drug-induced pseudoallergic reactions. It will be necessary, however, to find a way to preserve the mast cell defense mechanism of MRGPRX2.


Bos taurus and Bos indicus suffer with high incidence of infertility/subfertility problems; however, the etiology remains poorly understood. The uncertain predictability and the inability of semen evaluation techniques to maintain constant correlation with fertility demand for alternate methods for bull fertility prediction. Therefore, in this study, the global differential gene expression between high- and low-fertile crossbred bull sperm was assessed using a high-throughput RNA sequencing technique with the aim to identify transcripts associated with crossbred bull fertility. Crossbred bull sperm contained transcripts for 13,563 genes, in which 2,093 were unique to high-fertile and 5,454 were unique to low-fertile bulls. After normalization of data, a total of 776 transcripts were detected, in which 84 and 168 transcripts were unique to high-fertile and low-fertile bulls, respectively. A total of 176 transcripts were upregulated (fold change > 1) and 209 were downregulated (<1) in low-fertile bulls. Gene ontology analysis identified that the sperm transcripts involved in the oxidative phosphorylation pathway and biological process such as multicellular organism development, spermatogenesis, and in utero embryonic development were downregulated in low-fertile crossbred bull sperm. Sperm transcripts upregulated and unique to low-fertile bulls were majorly involved in translation  and ribosomal pathway. With the use of RT-qPCR, selected sperm transcripts (n = 12) were validated in crossbred bulls (n = 12) with different fertility ratings and found that the transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes was significantly (p < 0.05) lower in low-fertile bulls than high-fertile bulls and was positively (p < 0.05) correlated with conception rate. It is inferred that impaired oxidative phosphorylation could be the predominant reason for low fertility in crossbred bulls and that transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes could serve as potential biomarkers for fertility in crossbred bulls.Crossbred bulls produced by crossing  Male fertility is of great importance in dairy cattle breeding industry because semen from a single bull is utilized to breed several thousands of females. The most accurate method for testing the bull fertility is insemination of many fertile females, but this method is time-consuming and expensive, and only a lesser number of males can be tested at any given time . TherefoBos taurus males with Bos indicus females suffer with high rates of infertility/subfertility; however, the etiology remains poorly understood. Reportedly, a greater proportion of crossbred bulls were culled due to infertility/subfertility and poor semen quality; the average ejaculate rejection rate was around 55% in crossbred bulls  and performed in accordance with relevant guidelines and regulations.n = 12) of known fertility status were procured from Kerala Livestock Development Board, Mattupetty, Kerala, India. The CRs of the experimental bulls are shown in the n = 4 samples) were individually subjected to high-throughput RNA-seq analysis as detailed below.Cryopreserved semen straws of Holstein Friesian crossbred bulls  as described by Escherichia coli DNA polymerase I. The second strand of cDNA was synthesized using second strand enzyme mix and buffer. The double-stranded cDNA fragments were purified using 1.8× AMPure beads. The purified double-stranded cDNA was end repaired to ensure that each molecule was free of overhangs and has 5′ phosphates and 3′ hydroxyls before the adaptor ligation. The adaptor ligated DNA was then purified using AMPure beads and enriched with specific primers, compatible for sequencing on to the Illumina platforms. The final enriched library was purified and quantified by Qubit® Fluorometer, and the size was analyzed by a bio-analyzer.Total RNA (1 μg) was used to enrich mRNA using NEB Magnetic mRNA Isolation Kit . The transcriptome library was prepared using NEB ultra II RNA library prep kit  and sequenced using Illumina NextSeq 500  paired-end technology. The enriched mRNA was fragmented (approximately 200 bp) using fragmentation buffer. Random hexamer primers were then added and hybridized to complementary RNA sequences. These short fragments were used as templates to synthesize the first strand of cDNA using reverse transcriptase and dNTP. The DNA–RNA hybrids produced during first strand cDNA synthesis are converted into full-length double-stranded cDNAs using RNase H and n = 2) and low-fertile (n = 2) bulls were sequenced using Illumina NextSeq 500 sequencing system  to generate paired-end 76 bp reads. The sequence analysis was done using the online server tool Galaxy1  program, and the reads were then processed with Cutadapt tool   (Bos taurus UMD 3.1.94/Btau8), and the samples were sorted with aligned sequences using Samtools    . Between high- and low-fertile groups, significant changes in transcript expression, splicing, and promoter were studied using Cuffdiff .The cDNA of high-fertile  . Processgalaxy4) , all then 2.0.2) . The mapn 2.0.1) . With th2.2.1.2) , the pre2. The total number of sperm transcripts expressed between high- and low-fertile populations was plotted using online tool Venny (Version 2.1.0). Selected differentially expressed spermatozoa transcripts were generated as heat map using Clustvis (r programming) , >−1 (downregulated in low fertile), and between >±1 and < ± 1 . The transcripts with FPKM present only in high-fertile group were considered as unique to high-fertile bulls, while transcripts with FPKM present only in low-fertile group were classified as unique to low-fertile bulls. All the raw data were uploaded and available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under PRJNA516089ramming) .Uniport3 and the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources (v6.8)4 and categorized as molecular function (MF), biological process (BP), cellular component (CC), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The top 10 BPs, CCs, and MFs were plotted as a donut pie chart using Highcharts5. Pathway enrichment was carried out using Custer Profiler and enriched KEGG functions. Interaction of genes and detailed network analysis of combined GO categories and pathway analysis was performed using ClueGo (Version 2.5.4) and Cluepedia (Version 1.5.4) plugins in the open source Cytoscape (Version 3.7.1)6 platform (B. taurus as background.Gene ontology (GO) classification of sperm transcripts was done using platform . A netwoTPT1, PFN1, ZNF706, CRISP2, MDB4, TNP2, ADIPOR1, TNP1, IQCF1, RACK1, TMSB10, and TSSK6) were selected based on fold change, FPKM, and functional relevance to sperm fertilizing potential  based on the analysis using Genorm, Normfinder, Delta Ct, and the comprehensive ranking by RefFinder . The difference was considered as significant when p < 0.05.Twelve genes  and TPT1 were highly upregulated, whereas ZNF706 was highly downregulated in low-fertile bulls. The top 10 transcripts unique to the high- and low-fertile populations after excluding the non-coding RNAs are listed in With the use of Illumina Next Seq-500 RNA sequencing, the total raw data generated for the study were 115 million reads. Processing of raw data resulted in 97 million reads, which was mapped against the pulation . Since tle bulls , while 1le bulls , in whicA complex pool of coding and non-coding RNAs was also observed in the sperm transcriptome. Among the total transcripts , coding RNAs, non-coding RNAs, pseudogenes, and processed pseudogene were 13,002, 88, 375, and 98, respectively. Among the non-coding RNAs, miscellaneous RNA (misc_RNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and ribosomal RNA (rRNA) were 48, 21, 12, and 7, respectively. After normalization, a total of 776 transcripts were detected, in which coding RNA (known/categorized), coding RNA (unknown/uncategorized), and non-coding RNA were 585, 148, and 43, respectively. Among the non-coding RNAs, misc_RNA, snRNA, snoRNA, and rRNA were 19, 12, 6, and 6, respectively. A total of 61 protein coding ribosomal mRNAs were observed among the total transcripts after normalization.PYCARD, APCS, and ENSBTAG00000039963) and 3 KEGG pathways  sperm transcripts upregulated in the low-fertile population revealed their involvement in 16 MFs, 39 BPs, and 24 CCs  indicated that they are highly involved in the ribosome pathway  12.23%,  between high- and low-fertile bulls is given in RACK1 gene. To understand the variability in the expression of the 12 genes, the geometric mean with 95% confidence interval was calculated for high- and low-fertile bulls, and the results are shown in ZNF706, CRISP2, TNP2, and TNP1) were significantly (p < 0.05) downregulated in low-fertile bull spermatozoa. Correlation analysis revealed that expression levels of ZNF706, CRISP2, TNP2, and TNP1 genes were positively and significantly (p < 0.05) related to bull fertility , has a role in apoptosis, cellular differentiation, and sperm functions (RPL14) is abundant in the testis of Bos taurus and Bos indicus bulls (RPS8) is abundant in human sperm (PFN1) modulates actin and is involved in oocyte maturation, fertilization, embryo development (DDX39B) and poly(A) binding protein cytoplasmic 1 (PABPC1) are involved in RNA metabolism (CD74 molecule (CD74) controls the antigen presentation for immune responses and reported in the testes of chickens  may be related to zinc that is essential for male fertility and to zinc-containing metalloenzymes that are associated with sperm functions (PRKCA 1 (PICK1) is necessary for cytoskeletal organization (ANKRD9) is involved in lipid metabolism (RUNDC34) and LYR motif-containing 4 (LYRM4) are involved in protein biosynthesis. A family with sequence similarity 71 member F1 (FAM71F1) was detected in Leydig cells and downregulated in azoospermic males (EPOP) is a novel gene that has not been reported in bull spermatozoa.The highly downregulated sperm transcripts in low-fertile crossbred bull were unctions . Proteinnization  and acronization , and itsnization . Its dowtabolism  and downtabolism . RUN domic males . ElonginTSSK6, C12H13orf46, FABP3, and IQCF1. However, in RT-qPCR analysis, we found that TSSK6 and IQCF1 were expressed in both high- and low-fertile bull spermatozoa, but the level of expression was lower in low-fertile bulls. With the available knowledge, although it is difficult to explain this paradoxical situation, in NGS data processing, it may be possible that reads of a particular transcript might be discarded during the process of removal of low-quality reads. Testis-specific serine kinase 6 (TSSK6) is involved in protein phosphorylation, sperm chromatin condensation (C12H13orf46) is unknown. Fatty acid-binding protein 3 (FABP3) plays a role in remodeling of member polar lipids in spermatogenesis (IQCF1) is localized in the acrosome and involved in tyrosine phosphorylation, motility, capacitation, and acrosome reaction  and thymosin beta 10 (TMSB10). TMSB10 is abundant in the embryos of high-fertile sires , spermatogenesis , cell differentiation , in utero embryonic development , and oxidative phosphorylation pathway , which are extremely vital, are downregulated in low-fertile crossbred bull sperm. Oxidative phosphorylation is essential for the sperm to synthesize ATP and produce energy in all mammals , located in the acrosome and sperm tail (CRISP2 protein levels were found in azoospermia (TNP1 and TNP2) have a role in motility, chromatin integrity, and nucleo-histone to nucleo-protamine transition  can be found below: The animal study was reviewed and approved by Institute Animal Ethics Committee, SRS of ICAR-NDRI.MAP: methodology, experiment, writing – original draft, and data curation. AK: conceptualization, project administration, supervision, writing – review, and editing. JE, PN, and AS: methodology and data curation. MS: data curation and bioinformatic analysis. EK: methodology and writing – original draft. TD: formal analysis, writing – review, and editing. All authors contributed to the article and approved the submitted version.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.


TPX2, CENPF, BUB1, PLK1, KIF2C, AURKB, CDKN3, BUB1B, HMGA2, CDK1, ASPM, and CKS1B) and 2 decreased genes (TACC1 and MYH10) were associated with all the three above processes. Importantly, 2 (CDKN3 and CKS1B) out of the 11 increased genes (except HMGA2) are previously uncharacterized ones in LUAD and can potentially be prognostic markers. Moreover, PLK1 could be a promising therapeutic target for LUAD. Besides, protein–protein interaction network analysis showed that CDK1 and CDC20 were the hub genes, which might play crucial roles in cell proliferation of LUAD. Furthermore, transcriptional regulatory network analysis suggested that the transcription factor E2F1 could be a key regulator in controlling cell proliferation of LUAD via expression modulation of most cell cycle-, cell division-, and cell proliferation-related DEGs. Finally, trichostatin A, hycanthone, vorinostat, and mebeverine were identified as four potential therapeutic agents for LUAD. This work revealed key regulators contributing to cell proliferation in human LUAD and identified four potential therapeutic agents for treatment strategy.Lung adenocarcinoma (LUAD) is one of the most common and malignant cancer types. Abnormal cell proliferation, exemplified by cell cycle and cell division dysregulation, is one of the most prominent hallmarks of cancer and is responsible for recurrence, metastasis, and resistance to cancer therapy. However, LUAD-specific gene regulation and clinical significance remain obscure. Here, by using both tissues and cells from LUAD and normal lung samples, 434 increased and 828 decreased genes of biological significance were detected, including 127 cell cycle-associated genes (95 increased and 32 decreased), 66 cell division-associated genes (56 increased and 10 decreased), and 81 cell proliferation-associated genes (34 increased and 47 decreased). Among them, 12 increased genes ( After activation and proliferation in vitro, they were reinfused to patients to stimulate anti-tumor immune effect or kill tumor directly. For example, blocking the immune checkpoint-related genes PD-1, PD-L1, and PD-L2 were adopted in anti-PD therapy to enhance T cell-mediated anti-tumor immunity (Lung adenocarcinoma (LUAD) is one of the most common and malignant cancer types, accounting for more than 50% of lung cancer. Targeted therapy and immunotherapy are two of the major ways used in LUAD treatment. The therapeutic targets usually include growth factor receptors of lung cancer cell membrane , specific kinases  in signal transduction pathways, and specific genes related to proliferation, division, invasion, and metastasis of LUAD  from LUAD and normal lung samples, predicted cell cycle-, cell division-, and cell proliferation-associated prognostic markers, determined key regulators involved in cell proliferation, and investigated their protein–protein interaction (PPI) and transcriptional regulatory (TR) networks from a system view. This study provided new clues for our understanding of human LUAD pathogenesis, as well as biomarkers for diagnosis and prognosis, and potential targets for immunotherapy.https://www.ncbi.nlm.nih.gov/geo/)   databaseov/geo/) . Paired-ov/geo/)  was usedov/geo/)  and a lohttp://bonsai.hgc.jp/~mdehoon/software/cluster/index.html) (Gene Cluster 3.0 (ex.html)  is an opp-value of ≤0.05. The Database for Annotation, Visualization and Integrated Discovery (DAVID) tool (https://david.ncifcrf.gov/) (All DEGs were detected using an absolute fold change (FC) threshold of ≥2.0 and a rf.gov/)  is a toohttp://amigo.geneontology.org/amigo)   databaseTotal RNA was extracted from both cells (95D and H1299) and patient tissues  using TRIzol Reagent (Thermo Fisher Scientific) and reverse transcribed into cDNA using oligo-dT shortly. PCR primers Table 1 https://thebiogrid.org/) (https://www.ebi.ac.uk/intact/) (The BioGRID database (id.org/)  depositeintact/)  is a freintact/) .http://www.maayanlab.net/ESCAPE/) (https://www.grnpedia.org/trrust/) (https://www.encodeproject.org/)   is a TR trrust/)  is a manct.org/)  is a comwww.broad.mit.edu/cmap) is a drug discovery database  and selecting “HG-U133A” from the “Select a GeneChip Array” menu. The probe setlists of upregulated genes and downregulated genes were loaded into the cMAP web server as query signatures.The Connectivity Map  and 943 downregulated genes (blue points in the left panel) in cancer tissues   and cellhttps://david.ncifcrf.gov/)  v6.8 (rf.gov/)  was emplrf.gov/) –35, and rf.gov/) –38. The rf.gov/) –41.TPX2, CENPF, BUB1, PLK1, KIF2C, AURKB, CDKN3, BUB1B, HMGA2, CDK1, ASPM, and CKS1B) and 2 downregulated (TACC1 and MYH10) genes participated in all three processes (HMGA2 and 2 downregulated (http://gepia2.cancer-pku.cn) (CENPF and BUB1 was observed in two cell lines (H1299 and 95D). Some other genes were also upregulated in patients’ tissues or cell lines, for example, TPX2, CDKN3, HMGA2, ASPM, and CKS1B. However, due to the heterogeneity of limited samples, no significance was detected. The qRT-PCR method estimates gene expression by detecting fluorescence intensity, while the RNA-Seq technology directly reflects the reads count in cells. Besides, the large sample size of the RNA-Seq dataset should eliminate internal errors raised by diversity to a more acceptable level. We thus performed further analyses based on the RNA-Seq result.We paid special attention to the DEGs that are involved in cell cycle, cell division, and cell proliferation S3, beca-pku.cn) . The addTPX2, CENPF, BUB1, PLK1, KIF2C, AURKB, CDKN3, BUB1B, CDK1, ASPM, and CKS1B), as well as HMGA2, showed significant difference (p ≤ 0.05) between high and low groups . No significance was found for the two downregulated genes (TACC1 and MYH10). The increased expression of these cell cycle-, cell division-, and cell proliferation-associated genes in LUAD tissues with a poor prognosis forecasts crucial roles in malignant proliferation and carcinogenesis. All the 11 significantly increased genes could be potential biomarkers for the diagnosis of cell proliferation. Among them, CDKN3 and CKS1B are previously uncharacterized ones in LUAD, which could be promising prognostic markers. Both expression and survival situation of the serine/threonine-protein kinase PLK1 were different between patients with LUAD and normal humans, suggesting that PLK1 could be a therapeutic target for LUAD treatment.To determine whether the above upregulated DEGs involved in cell cycle, cell division, and cell proliferation could be used as prognostic markers for LUAD diagnosis, we performed overall survival (OS) analysis by employing GEPIA2. All 11 genes , dotted purple line , and dotted red line   and post-translational modification (such as HSP90AA1 andHSPA8) were found in the network (The network module is a cohesively linked group of genes or their products (represented by nodes in the graph), which densely interacted internally and sparsely linked with the rest of the network . Network modules , 47. CDC modules , 49. AllTo further verify the functions of proteins in the network, ClueGO functional analysis was carried out  and p53 E2F1 was closely and positively correlated with MYBL2, TYMS, FOXM1, CHAF1B, NDC80, PRC1, CDK1, AURKB, CCNB1, EZH2, DDX11, NUSAP1, CDKN2A, and FEN1   that could control the expression of cell cycle-, cell division-, and cell proliferation-related DEGs mentioned above    and downregulated (TACC1 and MYH10) genes were loaded into the cMAP web server as query signatures. This resulted in four promising therapeutic agents for LUAD, including trichostatin A, hycanthone, vorinostat, and mebeverine (https://pubchem.ncbi.nlm.nih.gov), as shown in To discover potential bioactive molecules/compounds that could suppress cell proliferation of LUAD, the probe set lists of upregulated genes  LUAD and wild-type LUAD may be slightly different. However, due to the insufficient mutation information, this work is laid aside for the moment.TPX2, CENPF, BUB1, PLK1, KIF2C, AURKB, CDKN3, BUB1B, CDK1, ASPM, and CKS1B) involved in all three biological processes  could be used as prognostic markers for LUAD, namely, 9 previously reported prognostic markers  and 2 promising prognostic markers (CDKN3 and CKS1B). These genes play crucial roles in the pathogenesis and development of various cancers. For example, TPX2 promotes metastasis in LUAD - and CDK -associated genes were obviously enriched and closely interacted with each other. Most CDC- and CDK-associated genes were increased in LUAD, including the hub genes CDK1 and CDC20, suggesting the important roles of these genes. Some CDK inhibitors have been approved by the FDA and used in cancer treatment. For example, ribociclib targeting CDK4/6 is used in breast cancer therapy  for the treatment of LUAD. Trichostatin A is a histone deacetylase inhibitor, which was reported to inhibit breast cancer and glioma proliferation by inducing cell cycle arrest and apoptosis , 70. A sPLK1 could be a promising therapeutic target for LUAD. Network analysis showed that CDK1 and CDC20 may play crucial roles in cell proliferation of LUAD. Also, we found that E2F1 can mediate the expression of most cell cycle-, cell division-, and cell proliferation-related DEGs. This work revealed key regulators that contribute to cell proliferation in LUAD and identified four potential therapeutic agents for treatment strategy.In summary, we detected DEGs between LUAD and normal lung samples using both tissues and cells. A total of 434 increased and 828 decreased genes were found in LUAD. Special attention was paid to cell cycle-, cell division-, and cell proliferation-associated genes. Eleven increased genes involved in cell cycle, cell division, and cell proliferation in LUAD can be used as prognostic markers. The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Shanghai Chest Hospital [KS1740]. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Ethical review and approval was not required for the animal study.KW, RL, CP, and LL designed the study and revised the manuscript. KW, MZ, and JW wrote, analyzed, and interpreted the data. PS carried out qRT-PCR experiment. PS, JL, and HJ revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a special project in the framework of the National Key Research and Development Program (NKRDP) , National Natural Science Foundation of China , National and Provincial Multi-disciplinary Cooperative Diagnosis and Treatment Capacity Building Project for Major Diseases (Lung Cancer), Shanghai Municipal Science and Technology Major Project (2018SHZDZX01), Shanghai Science and Technology Commission Experimental Animal Research Project (18140903800), Shanghai Pujiang Program (15PJD021), Program for scientific and technological innovation from the Science and Technology Commission of Shanghai Municipality , Shuguang Planning of Shanghai Municipal Education Commission of (16SG14), Program of Shanghai translational medicine collaborative innovation center cooperation , Shanghai Municipal Commission of Health, Scientific Research Program of Traditional Chinese medicine (2020JP009), Shanghai Jiao Tong University Affiliated Chest Hospital Major Project (YJXT20190106), and Shanghai Transportation Collaborative Self-help Project of Collaborative Innovation Center of University Translational Medicine .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. 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