Anguilla anguilla), where pituitary extract from carp (CPE) or salmon (SPE) is applied to override a dopaminergic inhibition of the neuroendocrine system, preventing gonadotropin secretion and gonadal development. The present study used either CPE or SPE to induce vitellogenesis in female European eel and compared impacts on egg quality and offspring developmental competence with emphasis on the maternal-to-zygotic transition (MZT). Females treated with SPE produced significantly higher proportions of floating eggs with fewer cleavage abnormalities and higher embryonic survival. These findings related successful embryogenesis to higher abundance of mRNA transcripts of genes involved in cell adhesion, activation of MZT, and immune response  throughout embryonic development. The abundance of mRNA transcripts of cldnd, foxr1, cea, ccna1, ccnb1, ccnb2, zar1, oct4, and npm2 was relatively stable during the first eight hours, followed by a drop during MZT and low levels thereafter, indicating transfer and subsequent clearance of maternal mRNA. mRNA abundance of zar1, epcam, and dicer1 was associated with cleavage abnormalities, while mRNA abundance of zar1, sox2, foxr1, cldnd, phb2, neurod4, and neurog1 (before MZT) was associated with subsequent embryonic survival. In a second pattern, low initial mRNA abundance with an increase during MZT and higher levels persisting thereafter indicating the activation of zygotic transcription. mRNA abundance of ccna1, npm2, oct4, neurod4, and neurog1 during later embryonic development was associated with hatch success. A deviating pattern was observed for dcbld1, which mRNA levels followed the maternal-effect gene pattern but only for embryos from SPE treated females. Together, the differences in offspring production and performance reported in this study show that PE composition impacts egg quality and embryogenesis and in particular, the transition from initial maternal transcripts to zygotic transcription.Low egg quality and embryonic survival are critical challenges in aquaculture, where assisted reproduction procedures and other factors may impact egg quality. This includes European eel ( The aquaculture sector has expanded rapidly and further development relies on diversification and on closing the life cycle for aquatic species in captivity ,2. HowevIn oviparous teleosts, embryonic and early larval development is influenced by intrinsic properties of the gamete, as well as extrinsic factors, e.g. ambient conditions during egg incubation and larval culture ,7,8. Whiccna1, ccnb1, ccnb2, npm2), cell division , cell adhesion , microRNA regulation (dicer1), pluripotency regulation, cell signaling, and activation of MZT  [c3, igm, il1β) appear to play a role in early protection and organogenesis of the embryo and larva until the adaptive immune system is fully developed [neurod4, neurog1) to establish the nervous system during embryonic stages, which to date have been mainly studied in the model species zebrafish (Danio rerio) [Studies of molecular ontogeny during early embryogenesis have documented a tight relationship between the abundance of specific maternal mRNA transcripts, egg quality, and embryonic developmental competence –23. Embr2, phb2) ,10,17. T2, phb2) –27. Alsoeveloped –31. On teveloped ,32. Thiso rerio) –35.The incorporation of these maternal transcripts into the cytoplasm occurs during oocyte development. In oviparous female teleosts, the reproductive developmental processes are regulated by gonadotropins (GTH), i.e. follicle stimulating hormone (FSH) and luteinizing hormone (LH), in combination with maturation-inducing steroid (MIS), and maturation-promoting factor (MPF) . Here, tAnguilla anguilla) and the Japanese eel (A. japonica) are high-value species in aquaculture and the development of hatchery technology for closed-cycle production would be of great value. Their complex diadromous life cycle, including long migrations to their spawning area, makes this difficult. During the silvering process that marks the onset of the spawning migration, intrinsic inhibitory mechanisms at the brain-pituitary level arrest puberty at an early stage [Anguillid eels belong to the group of aquaculture species, which do not reproduce in captivity. In particular, the European eel , 20 mg kg-1 BW (week 4–6), 30 mg kg-1 BW (week 7–9), and 40 mg kg-1 BW (week 10–16) [A. rostrata) [A. dieffenbachii) [A. australis) [For female eels, assisted reproduction protocols commonly use pituitary extracts (PE) from carp (CPE) or salmon (SPE) as source of FSH and LH to induce vitellogenesis, while follicular maturation is completed by administration of a MIS, e.g. 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) ,45. The -1 BW ,48,49, wk 10–16) ,51. Manyk 10–16) , includiostrata) , and thenbachii)  or used stralis) ,55. In sstralis) ,57. PituAgainst this background, we aimed to elucidate effects of CPE and SPE on European eel reproductive success and offspring quality. We compared the effects of constant dose treatment schemes on reproductive parameters including egg characteristics, fertilization success, occurrence of cleavage abnormalities, embryonic survival, and hatch success in a standardized experimental design. Gene expression analyses were performed to assess mRNA transcript abundance of genes in ovary, unfertilized eggs, and embryos at regular intervals throughout development.-1, Sigma Aldrich, Germany).All fish were handled in accordance with the European Union regulations concerning the protection of experimental animals (Dir 2010/63/EU). Eel experimental protocols were approved by the Animal Experiments Inspectorate (AEI), Danish Ministry of Food, Agriculture and Fisheries (permit number: 2015-15-0201-00696). Each individual fish was anesthetized before tagging, biopsy, and stripping of gametes, and euthanized after stripping  or at the end of the experiment  using an aqueous solution of ethyl p-aminobenzoate  were caught at Lower Bann, Toomebridge, Northern Ireland, while in 2017, 26 female silver eels  were caught in Klitmøller Å, Lake Vandet, Denmark. The female eels were transported to the EEL-HATCH experimental facility in Hirtshals, Denmark, using an aerated freshwater tank. On arrival, fish were randomly distributed into replicated 1150 L tanks, connected to two Recirculating Aquaculture Systems (RAS), at a density of 10–15 females per tank, where one RAS unit was allocated for each hormonal treatment. In both years, female eels were equally distributed into the two hormonal treatment groups. Hence, 27 females were allocated to CPE treatment  and 24 females to SPE treatment .In both trials, male eels originated from Stensgård Eel Farm, where they were raised from glass eels on a formulated diet  at a temperature of ~23°C. In 2016, experiments comprised 60 male eels , and in 2017, 88 males . After transport to the facility, males were randomly distributed in four tanks (485 L) connected to a RAS unit at a density of ~15–22 eels per tank.For acclimatization to oceanic conditions, salinity was gradually increased from 10 to 36 PSU over 14 days using Blue Treasure Aquaculture Salt , while temperature was adjusted from ~16°C to 20°C. Subsequently, each individual was tagged with a passive integrated transponder (PIT tag) in the dorsal muscle, while initial length and weight were recorded. During the experiment, male and female broodstock were maintained at ~20°C and ~36 PSU under 12 h—12 h light regime, with a 30 min twilight in the morning and evening to resemble the Sargasso Sea photoperiod.-1 initial BW for 10–21 weeks [-1 current BW to induce follicular maturation and ovulation [-1, Sigma-Aldrich, Missouri, USA). Subsequent rearing occurred in a temperature incubator at 18°C [-1, Sigma-Aldrich, Missouri, USA) were stocked with eggs/embryos and incubated as above. 3 flasks stocked with ~2500 eggs/embryos were used to follow embryonic development and 3 flasks stocked with ~600 eggs/embryos were used to analyze hatch success.After acclimatization, vitellogenesis in the female broodstock was induced by weekly intramuscular injections of either CPE or SPE, each at 18.75 mg kg21 weeks . Salmon 21 weeks ,59, and 21 weeks ,60. Aftevulation . Prior tvulation , and thevulation . Eggs wevulation ,64. Afte at 18°C  and 36 PFor each female, initial length and weight, weekly weights and weight at DHP injection were recorded as well as the number of weeks until spawning and the time between priming and DHP were recorded. The weight of stripped eggs  was recorded prior to fertilization and unfertilized eggs were sampled for dry weight (3 × 0.1 mL) and gene expression (see below). For dry weight, samples were kept in the oven at 60°C for 24 h and weighed. At 0.5 hpf, the amount of floating eggs (%) was determined in a 25 mL volumetric column. Digital images were used throughout the experiment to document oocyte, egg, and embryonic development. Here, all images were taken with a Nikon Eclipse 55i microscope equipped with a Nikon digital sight DS-Fi1 Camera, while analyses used NIS Elements image software . A digital image of the ovarian biopsy was taken, when females were primed or DHP was given, and oil droplet size was measured subsequently. Ten of the largest oil droplets from ten oocytes at average stage were measured per female. In order to calculate fertilization success and embryonic survival, sub-samples from the embryonic development flasks were taken at 2, 3, 4, 5, 6, 7, 8, 16, 24, 32, 40, and 48 hpf and digital images were obtained. Fertilization success was measured from the digital images taken at 4 hpf, where eggs were categorized as fertilized when > 4 blastomeres could be observed and fertilization success was calculated as the percentage of fertilized divided by the total number eggs (obtained from the floating layer) . At the thαa, foxr1, npm2, zar1, phb2, c3, igm, il1β) were retrieved from previous studies  were designed on the basis of the coding sequences of the closely related species A. japonica, publicly available in Genebank, National Center for Biotechnology Information (NCBI). Primers for 12 genes  were designed based on the most accurate assembly and annotation of the European Eel genome [https://dataverse.no/dataset.xhtml?persistentId=doi:10.18710/L7GO8T. Here, gene prediction and exon coordinates were determined by the authors with Augustus v 2.4 [https://blast.ncbi.nlm.nih.gov/Blast.cgi). Subsequently, from all samples, the expression of the 24 genes was analyzed with two technical replicates, using the qPCR BiomarkTM HD system (Fluidigm) based on 96.96 dynamic arrays (GE plates), as previously described [ccna2, cei, thαa, igfr-1b). Further analysis of gene expression was carried out according to the 2-ΔΔCt method, in relation to a random unfertilized egg sample, according to [Samples for gene expression were taken from the ovarian tissue of the female after spawning, and from the unfertilized eggs and embryos at 2, 4, 8, 24, 32, and 48 hpf. The samples were stored in Eppendorf vials filled with RNA later, then kept in the fridge at 4°C for 24 hours and subsequently at -20°C until analysis. RNA was extracted using the NucleoSpin RNA kit  according to manufacturer’s instructions. RNA concentration and purity was analyzed by spectrophotometry using Nanodrop One . From the resulting total RNA, 1 μg was transcribed using qScript cDNA Synthesis Kit , following the manufacturer’s instructions. This included a step to permanently inactivate all trace levels of DNase activity using PerfeCta DNase I (RNase free) . Primers of eight genes . Gene e10 or arcsine square-root-transformed. Alpha was set at 0.05. Tukey analysis was used to compare least-squares means between treatments. Akaike (AIC) and Bayesian (BIC) information criteria were used to assess which covariance structure fitted the data most appropriately [Data were analyzed using SAS Statistical Software . Prior to analysis, residuals were tested for normality (Shapiro–Wilk test) and homogeneity of variances . Data deviating from normality or homoscedasticity were logpriately . The effpriately . The effFor female and quality parameters, ANOVA models were first run testing the effect of broodstock series , hormonal treatment and their interaction. While initial length (p < 0.0001) and weight (p < 0.0001) differed between female broodstock from the two locations, quality parameters were similar between female broodstocks, including fertilization success (p = 0.7832), cleavage abnormalities (p = 0.6711), embryonic survival (p = 0.4538), and hatch success (p = 0.6258). Data from the two experiments were therefore pooled. Comparing the two resulting treatment groups, no significant difference was found in the initial weight between CPE (686. 3 ± 49.7 g) and SPE females . Likewise, the initial length between females from CPE (70.1 ± 1.6 cm) and SPE (70.3 ± 1.8 cm) treatment did not differ (p = 0.952).Overall, 51 females were included in the combined analyses of the two trials. Female information and reproductive success for each treatment are shown in The number of stripped eggs relative to body weight (p = 0.926) and the dry-weight of unfertilized eggs (p = 0.865) did not differ between CPE and SPE females. The percentage of floating eggs was higher in females from the SPE treatment than in females from the CPE treatment (p = 0.038), but the fertilization success of eggs from the floating layer (p = 0.868) and egg size at 4 hpf (p = 0.428) did not differ. Embryonic survival was higher for SPE treated females than CPE . For CPE, the relationship was significant for 7 genes  with six of these overlapping between treatments. For all genes, the relationship was best described by a positive linear regression.Detailed results of gene expression analyses are shown in dcbld1, epcam, oct4, and igm, which are associated with cell adhesion, MZT activation, and immune response, showed significantly higher mRNA levels in offspring from SPE than from CPE treated females. In contrast, cdhr2, cldnd, foxr1, and ccnb1, associated with cell division, cell cycle control, and cell adhesion, showed significantly higher mRNA levels in offspring from CPE than from SPE treated females. Significant variations in patterns of mRNA abundance throughout embryonic development were observed for all genes, with the exception of cdhr2 and dcbld1. Here, cldnd, foxr1, cea, ccna1, ccnb1, ccnb2, zar1, oct4, and npm2 showed a similar pattern with relatively stable abundance during the first eight hours until MZT , grass carp (Ctenopharyngodon idella), Atlantic halibut (Hippoglossus hippoglossus), and gourami (Osphronemidae), the expression of genes for both FSH and LH increases with the progression of ovarian development and peaks during the spawning season [Oncorhynchus keta), a semelparous species with synchronous oocyte development, while CPE is derived from common carp, an iteroparous species with asynchronous oocyte development. In the case of anguillid eels, evidence is accumulating that they are batch spawners with asynchronous or group synchronous oocyte development [Assisted reproduction techniques are commonly used in aquaculture to enhance reproductive success including a variety of hormonal treatments depending on the targeted stage of the reproductive cycle . Resultig season –80. It ielopment ,60,81. Ielopment ,83 and aelopment . Such spGadus morhua) [Limanda ferruginea) [Scophthalmus maximus) [During reproductive development, mRNA transcripts as well as nutrients (e.g. yolk and vitamins) are incorporated into the growing oocytes . Materna morhua) , yellowtruginea) , turbot maximus) . The obsdcbld1 and epcam differed between treatments, with higher mRNA abundances in embryos of SPE treated females. Although expression of dcbld1 has been associated with cell adhesion during embryonic development, insights into its role are still limited [dcbld1 transcript levels in Atlantic cod eggs with larger than 100-fold differences among females. However, when all females were included in the analysis, no relationship was found between dcbld1 abundance and egg quality. In our study, dcbld1 transcript abundance also varied considerably among offspring from individual females, but differed between treatments. The relation between transcript abundance in unfertilized eggs and in the ovary indicated a maternal effect, which in combination with higher mRNA abundance in SPE compared to CPE females may relate to the incomplete cell adhesion observed, compromising the development in European eel embryos.Abundance of mRNA transcripts of two genes related to cell adhesion,  limited . Rise et limited  found hiepcam has been associated with cell adhesion, migration, proliferation, differentiation and signaling [epcam is required for epithelial morphogenesis during epiboly [epcam to eggs and embryos with a positive effect of high transcript levels on performance of embryos obtained from SPE treated females. A negative relationship with cleavage abnormalities was found for CPE. These results indicate that high mRNA abundance of this gene is also important for normal development in European eel embryos.The second gene, ignaling –91 with ignaling  and zebrignaling . Thus, i epiboly  as well  epiboly . In our oct4 in medaka (Oryzias latipes)[Oreochromis niloticus) [oct4, sox2 (soxb1), and nanog transcripts are responsible for activating zygotic expression and initiating the clearance of maternal mRNA through activation of microRNA, hence they are considered fundamental for successful MZT [oct4 in ovaries, eggs, and embryos, with highest abundance in the SPE treated group, suggesting that maternally inherited oct4 plays a similarly important role in successful MZT and survival during embryonic development in eel. Transcript levels of oct4 in later embryonic stages were linked to higher hatch rates in both treatments, which indicates that oct4 assumes an additional role during later embryonic development and successful hatching.Studies of the expression patterns of  latipes)–98, Nileloticus) ,100, andloticus) –105 havesful MZT . We founSparus aurata) [Labeo rohita) [igm has been associated with higher larval survival. Maternal antibody transfer improved the protection against pathogenic attacks for developing embryos of zebrafish [igm mRNA in European eel via the ovary to the eggs. Higher mRNA levels in embryos obtained from SPE treated females might indicate a strengthened immune-readiness, due to maternally derived immune factors. Our results showed maternal transfer of c3 and il1β transcripts. While il1β exhibited relatively stable abundance throughout embryonic development, c3 followed a different pattern increasing towards hatch, indicating a potential role for early larval stages.During early development, teleost fish rely exclusively on their innate immune system, until their adaptive immune system is sufficiently developed . Materna aurata)  and Indi rohita) , maternaebrafish . In Euroebrafish , with ancldnd, foxr1, cea, ccna1, ccnb1, ccnb2, zar1, oct4, npm2, which all are genes involved in early developmental functions, such as cell adhesion, cell division, cell cycle control, oocyte-embryo transition, and MZT activation. This pattern compares to maternal-effect genes known from other teleost species [sox2, neurod4, neurog1, phb2, and c3, primarily genes involved in neurogenesis but also in cell signaling and immune response. This pattern of starting transcription during MZT is common for genes involved in processes such as organogenesis [cdhr2, epcam, dicer1, igm, il1β, genes involved in cell adhesion, microRNA regulation, and immune response. It is notable that a deviating pattern between treatments was observed for dcbld1 transcripts, which appeared to follow the maternal-effect gene pattern only for embryos obtained from SPE treated females.Overall, the mRNA abundance of 20 genes revealed three main patterns of differential expression throughout development. In the first pattern, high abundance in early embryonic development (until 8 hpf) was observed, followed by a drop in mRNA levels after the MZT (between 8 and 24 hpf), likely demonstrating transfer and subsequent clearance of maternal mRNA. This pattern was found for  species . In the ogenesis . In the ldnd, a member of the family of claudins, which are known for their importance in generating tight junctions between cells in teleosts [ldnd mRNA was found to be highly abundant during early embryonic development in zebrafish [foxr1 has been shown in zebrafish [Oryzias melastigma) [Monopterus albus) [foxr1 transcript abundance for embryonic survival around MZT was recently suggested for zebrafish [Oncorhynchus mykiss), abundance of ccna1 might be linked to embryonic developmental competence [zar1, first shown to be critical for the oocyte-to-embryo transition in mice [Genes whose transcripts followed pattern one included cteleosts –112. Cldebrafish . This waebrafish , freshwaastigma) , rice fis albus) , and Eurs albus)  possiblyebrafish , which iebrafish ,116,117.mpetence . In this in mice , also ap in mice ,120, and in mice .sox2 has been shown to be responsible for the successful activation of the MZT in zebrafish, together with oct4 and nanog [sox2 in our study are in accordance with this, indicating a similar function in European eel. For phb2, a previous study on European eel found that the abundance of transcripts was higher in a “high hatch group” compared to a “low hatch group” [neurod4 and neurog1 are key players [neurod4 and neurog1 during late embryonic development and hatch success, which calls for more detailed research on the function of these two genes during eel embryogenesis.Within the second pattern of mRNA abundance, nd nanog . The exph group” . Our resh group” . Another players –35. In odicer1 has been ascribed an essential role in microRNA (miRNA) synthesis during embryonic development. It has mainly been investigated in zebrafish [dicer1 transcripts led to slower growth rates and shorter survival [dicer1 transcripts in rainbow trout embryos suggest an important role during early development [Representing the third pattern, ebrafish ,122, whesurvival , as wellsurvival ,124. Higelopment . This iszar1, epcam, dicer1 and cleavage abnormalities as well as between mRNA levels before MZT (8 hpf) of seven genes, zar1, sox2, foxr1, cldnd, phb2, neurod4, neurog1 and later embryonic survival (48 hpf) indicated maternal mRNA transfer and the importance of all these genes for successful development in European eel embryos. The expression of five genes, ccna1, npm2, oct4, neurod4, neurog1 during later embryonic development (32 hpf) was associated with hatch success and corroborated the importance of their transcription during the transition from maternal mRNA control to zygotic transcription.The association between mRNA levels at 2 hpf of three genes, Assisted reproduction protocols developed for anguillid eels typically use repeated administration of pituitary extract for induction of vitellogenesis. Ours is the first study that compared differences in egg quality and embryonic developmental competence between carp and salmon pituitary extracts. In two successive trials, using a constant weekly dose, a higher proportion of female broodstock responded to CPE treatment, however a higher proportion of SPE treated females produced viable offspring. The lower embryonic developmental success of CPE treated females was associated with abnormalities in cell cleavages during early embryogenesis. These findings point to differences in constituents of the carp and salmon pituitaries applied, affecting female response, oocyte development during vitellogenesis, and final maturation. Complementing gene expression analyses showed that differences in embryonic survival were related to differences in mRNA transcript abundance of eight genes involved in cell adhesion, cell division, cell cycle control, MZT activation, and immune regulation. Thus, the differential impact of CPE and SPE appeared to be related to the variability in mRNA abundance in the eggs including maternal transcripts known to be important for healthy embryonic development. Differential expression patterns during embryonic development were observed for 20 genes involved in key mechanisms showing either increasing or decreasing expression profiles around the MZT. The mechanisms regulating the transfer of mRNA to the developing oocytes are still poorly known, however follicular development may be affected by differences in content, composition or affinity of gonadotropins, FSH and LH, as well as other pituitary hormones affecting the deposition of mRNA transcripts into the oocytes. Unravelling the influences of hormonal factors in PEs may prove important to developing novel treatment protocols. Better understanding of the physiology and ontogeny of maternal and embryonic mRNA transcript abundance of different genes during embryogenesis will alleviate early development failure in teleost species in aquaculture.


Breast cancer stem cells (BCSCs) are the minor population of breast cancer (BC) cells that exhibit several phenotypes such as migration, invasion, self-renewal, and chemotherapy as well as radiotherapy resistance. Recently, BCSCs have been more considerable due to their capacity for recurrence of tumors after treatment. Recognition of signaling pathways and molecular mechanisms involved in stemness phenotypes of BCSCs could be effective for discovering novel treatment strategies to target BCSCs. This review introduces BCSC markers, their roles in stemness phenotypes, and the dysregulated signaling pathways involved in BCSCs such as mitogen-activated protein (MAP) kinase, PI3K/Akt/nuclear factor kappa B (NFκB), TGF-β, hedgehog (Hh), Notch, Wnt/β-catenin, and Hippo pathway. In addition, this review presents recently discovered molecular mechanisms implicated in chemotherapy and radiotherapy resistance, migration, metastasis, and angiogenesis of BCSCs. Finally, we reviewed the role of microRNAs (miRNAs) in BCSCs as well as several other therapeutic strategies such as herbal medicine, biological agents, anti-inflammatory drugs, monoclonal antibodies, nanoparticles, and microRNAs, which have been more considerable in the last decades. Approximately 25% of cancers in women and 15% of cancer deaths are caused by BC. About 0.8–1% of all BCs are due to male breast carcinoma, although the incidence of BC in men has increased recently. It has been estimated that the prevalence of BC in the world will reach 2.3 million by 2030 . The prevalence of BC is increasing; according to the recent report 2020), 279,100 new cases and 42,690 deaths of BC have been estimated in the United States , epithelial specific antigen (ESA), and aldehyde dehydrogenase (ALDH) activity in different types of BC. Unlike CD24, known as a differentiated BC cells marker, CD44 has been identified as a stemness marker on the surface of BCSCs. CD44 is bonded to hyaluronic acid (HA) and extracellular matrix proteins such as osteopontin (OPN) and matrix metalloprotease (MMP)  and closed  configurations. Closed configuration has a vital role in BC progression and CSC survival through activation of NFκβ, Akt, and focal adhesion kinase (FAK) signaling (BCSCs express variety of specific markers such as CD44se (MMP) . Severalse (MMP) . Howeveres of BC , 47, 48. markers . Frequen4−/CD24+ . ALDH ac markers , 51. ALD markers . To dete markers . A regul markers . The oth markers . A recenignaling .It has been reported that the use of radiation to destroy cancer cells after surgery may convert differentiated cancer cells to CSCs through the expression of CSC markers such as Oct4/Sox2/KLF4. Therefore, in some cancer cases, radiation is not recommended, as it can involve recurrence and metastasis . Hypoxiaε (PKCε) is mediated by hyaluronan and regulates human breast tumor cells and BCSC functions. Activated PKCε increases phosphorylation of Nanog, a stem cell marker. Phosphorylated Nanog is translocated into the nucleus and increases miR-21 expression and decreases tumor suppressor program cell death protein 4 (PDCD4) expression. Along with this process, inhibitors of apoptotic proteins (IAPs) and MD11 are upregulated and then leads to antiapoptosis and chemotherapy resistance in BCSCs , Notch, Wnt/β-catenin, and Hippo signaling have been implicated in stemness maintenance and regulation of self-renewal, metastasis, and therapeutic resistance into CSCs , 56–61. in BCSCs . The othin BCSCs . In addiin BCSCs   and HER2. In this way, activated ERK stimulates MAPK interacting kinase (MNK) signaling, and then, MNK activates XIAP, its downstream protein. XIAP is an apoptotic inhibitor protein that is upregulated in inflammatory and aggressive BC and links NFκβ signaling activity to MAPK signaling . On the Nanog and KIF4 via ZFP36L1 phosphorylation, which has been known as a Nanog and KIF4 mRNA binding protein -dependent manner. Chemotherapy in an HIF1-dependent manner regulates the expression of dual specificity phosphatase (DUSP9) and DUSP16. Reciprocally, increased expression of DUSP9 inhibits ERK and decreased expression of DUSP16, which activates p38 signaling pathway. ERK inhibition leads to increased expression of Nanog through decreased phosphorylation of FoxO3, and p38 activation stabilizes transcripts of stemness genes such as  protein   phenotype through induction of cell apoptosis. The role of PI3K/Akt in BCSCs may be mediated by HER2, which makes tumors more aggressive through unknown mechanism . HER2 dyOn the other hand, inflammation may show a critical role in the relapse of BC through the NFκB–IL-6 pathway, which is involved in BCSC characterization and recurrence of tumor progression. NFκB regulates interleukin-6 (IL-6) and helps in the maintenance of BCSC and conversion of non-CSC to CSC. In addition, some alterations in PI3K/mTOR signaling has been detected in triple negative BC. PI3K/mTOR inhibition through PI3K/mTOR inhibitor or TORC1 inhibitor increases mammosphere formation, a CSC enrichment model, and expression of CSC markers as well as fibroblast growth factor 1 (FGF1) and Notch1 expression in CSCs through increased mitochondrial metabolism . Hence, + to ER− transition is mediated by genetic and epigenetic changes. Proliferation and expansion of ER− CSCs could be driven by ER+ BC cells. The secretion of FGF in ER+ BC is through ER signaling, by induction of EGF receptor, Notch, and Wnt signaling through FGF paracrine manner in ER− CSCs (+ cells mediates binding of Receptor activator of NFκB (RANK) with its ligand, RANKL, as a paracrine signal transduction in PR-negative CSCs  and Has2 antisense (HAS2-AS) transcripts, high mobility group AT-Hook 2 (Hmga2), and EMT inducing transcription factors such as Snail, Snai1, Hmga2, and fibronectin 1 /interleukin-like EMT inducer (ILEI), an oncogenic protein. Interaction of ILEI with LIFR, its receptor, promotes cytokine receptor signaling through STAT3 activation to drive EMT and metastasis . Besidesnectin 1 . In addiof BCSCs .Wnt and Notch signaling pathways are other signaling pathways involved in cancer stem cell maintenance , 61. WntIt has been shown that there is an association between estrogen signaling and Notch signaling in ERα+ BC cells, which is mediated by Delta-like protein 1 (DLL1), Notch ligand. Upregulation of Notch signaling by overexpression of DLL1 promotes proliferation, migration, angiogenesis, and cancer stem cell phenotypes in ERα+ BC cells. This function for DLL1 is specific in ERα+ BC cells. Thus, it suggests that estrogen and estrogen signaling stabilizes DLL1 through inhibition of ubiquitylation and degradation of DLL1 .In BCSCs, activated Notch signaling by jagged1 leads to Akt phosphorylation and IκB kinase α (IKKα), NFκB, and Notch1 activation. Recruitment of IKKα, NFκB, and Notch1 to antiapoptotic gene cIAP-2 promoter increases expression of cIAP-2  Figure . Along w+ samples express high levels of vimentin and Snail. It suggests that the hedgehog signaling pathway and SHH/GLI1 axis are involved in EMT, migration, and invasion of BCSCs  are a class of proteins implicated in inflammatory signaling and tumorigenesis . TLRs arCAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in BCSCs  such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) may regulate radiotherapy sensitivity. Decreasing cellular cholesterol through treatment with cholesterol-lowering agents such as HDL decreases radiotherapy resistance, whereas VLDL decreases radiotherapy sensitivity .EGFR and HER2, two growth factor receptors that have been overexpressed in BCSCs, have an important role in radiotherapy resistance of BCSCs. EGFR and HER2 inhibitors such as GW572016 may make BCSCs sensitive to radiotherapy as well as inhibit proliferation through inhibition of receptor phosphorylation . The theMetastasis has been described by two potential ability of BCSCs, migration, and invasion. As mentioned above, not only EMT is an important process in chemotherapy and radiotherapy resistance but also it is implicated in metastasis of BCSCs. Snail, a transcriptional repressor, is one of the most important inducer of EMT through repression of E-cadherin expression. In addition, the expression of snail maintains malignancy phenotypes of BCSCs such as metastasis. Therefore, it could be suggested that snail is a potential target to prevent metastasis of BCSCs . Along wExtracellular matrix protein 1 (ECM1) is an important prognosis marker for the invasion of breast cancer as well as migration and drug resistance. In addition, ECM1 controls the expression of genes involved in EMT and BCSC maintenance through induction of Mucin1 (MUC1). The cytoplasmic tail of MUC1 has a physical association with β-catenin that stabilizes and increases posttranslational expression of β-catenin. Therefore, altered expression of β-catenin changes the expression of genes related to BCSC phenotypes such as invasion and EMT progression .CD36, a member of the scavenger receptor family with binding to its receptor, thrombospondin-1 (TSP-1) plays an important role in metastasis of BCSCs through several pathways. CD36 binds to TSP-1 and TGF-β1 and promotes proliferation and migration of BCSCs. On the other hand, it can interact with NFκB and STAT3, which express genes related to carcinogenesis. In addition, CD36 has a main role in BCSC metastasis through uptaking fatty acids and regulating 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) enzyme and β-oxidation of fatty acids . FurtherThe role of CD44 in cancer progression, invasion, metastasis, and angiogenesis of BCSCs is remarkable. There are several ligands such as hyaluronic acid, osteopontin, and matrix metalloprotease that bond to CD44. Binding ligands to CD44 induces cleavage of CD44 at extracellular and transmembrane domain sites by membrane-type 1 matrix metalloprotease (MT1-MMP) and presenilin-1/g secretase, respectively. Then, the intracellular domain fragment of CD44 (CD44-ICD) displaces into the nucleus and activates expression of genes related to migration, invasion, metastasis, and angiogenesis of BCSCs .Cell survival mediated by hypoxic condition after radiotherapy is contributed to the angiogenesis of CSCs . In bothThe role of CD44 has been confirmed in angiogenesis as well as metastasis. CD44 binds to its ligand, hyaluronic acid, as well as VEGF through its binding domains and increases angiogenesis .miRNAs are introduced as one of the non-coding RNAs classes that suppress gene expression through inhibition of translation. Several studies suggest that several miRNAs regulate stemness phenotypes of BCSCs through targeting genes involved in signaling pathways. Substantial shreds of evidence support that resistance of BCSCs to chemotherapy and radiotherapy have been associated with metastasis and relapse of BC after treatment . TherefoViola odorata extract, a herbal medicine, decreases stemness potency and cell viability of BC-cell-derived mammospheres as well as induction of apoptosis , an active component of  pathway .p53, p21, p16, p15, BRCA1, and BRCA2, and reduced expression of ABCG2 through demethylation mechanism  and AD-01 (one of the its derivatives) are novel biological agents that have an effective role in inhibiting tumor growth initiation, angiogenesis, and metastasis of BC cells as well as BCSCs through targeting CD44 pathway and downregulating DLL4, a Notch ligand, and Notch4 , 119. Diechanism .Developing anti-inflammatory drugs and monoclonal antibodies could be useful for preventing proliferation, metastasis, and drug resistance of BCSCs. Aspirin (ASA) prodrug is one of the anti-inflammatory drugs that has shown antineoplastic activity without toxicity through targeting NFκB–IL6 pathway and cyclooxygenase (COX)/prostaglandin in BCSCs , 124. InPreviously, it has been shown that EGFR is a critical player in tumor initiation, metastasis, and drug resistance of BC and BCSCs. Therefore, implication of EGFR monoclonal antibody such as Cetuximab effectively could be suggested as a useful strategy to reduce BCSC population . BevacizRecently, nanoparticles have been an interesting approach for targeting cancer cells especially CSCs. Specific phenotypes of CSCs can be used for designing nanoparticles. Among nanomaterials, mesoporous silica particles have demonstrated efficient drug delivery in cancer therapy. It has been designed based on glycolytic phenotype and Notch activity of CSCs. Uptaking mesoporous silica nanoparticles accompanied with γ-secretase inhibitor by BCSCs targets Notch signaling pathway and eliminates stemness properties of BCSCs.miRNAs are the most attractive topics in BCSCs. They have been implicated in targeting BCSCs through several signaling pathways. +/CD24−, ALDH activity, and ESA or CD326 (EpCAM), CD133, CD166, Lgr5, CD47, and ABCG2 in different types of BCs. BC cells' conversion to BCSCs due to genetic and epigenetic changes leads to deregulation of several signal transduction pathways such as MAP kinase, PI3K/Akt/NFκB, TGF-β, Wnt/β-catenin, Notch, hedgehog, and Hippo signaling. Deregulation of these signaling pathways and other factors leads to several stemness phenotypes such as chemotherapy and radiotherapy resistance, EMT, invasion, migration, and angiogenesis. Additionally, the role of miRNAs in stemness features of BCSCs is remarkable. A variety of miRNAs upregulated in BCSCs are contributed to stemness phenotypes of BCSCs. Recently, implicating novel treatment strategies such as herbal medicine, biological agents, anti-inflammatory drugs, monoclonal antibodies, nanoparticles, and microRNAs that target different signaling pathways and other deregulated genes directly or indirectly could be effective for targeting BCSCs.BCSCs are one of the main reasons for tumor development and recurrence of BC after treatment. These cells have some specific markers: CD44SY and SS: conception, providing the data and design, manuscript writing. FS, MN, AG, and KG: conception and final approval of 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.


The results of our study indicate that the CK5/6 positive subgroup exhibited high gene expression signature related to aggressive behavior and exhibited worse clinical outcome.A number of urinary bladder urothelial carcinoma (UB UC) mRNA-based classification systems have been reported. It also has been observed that treatment response and prognosis are different for each molecular subtype. In this study, cytokeratin (CK)5/6 and CK20 immunohistochemistry (IHC) were performed, and IHC-based subgroup classification was applied. UB UC was classified into CK5/6 single-positive (SP), CK20 SP, double-positive (DP) and double-negative (DN) subgroups, and transcriptional analysis was performed. The results of gene ontology (GO) terms and functional analysis using differentially expressed genes indicate that, CK5/6 SP and DP subgroups were enriched in cell migration, immune activation, interleukin 6-Janus kinase-signal transducer and activator of transcription 3 (IL6-JAK-STAT3) signaling pathway and tumor necrosis factor-α signaling via the nuclear factor-κB (NF-κB) signaling pathway signature gene. In addition, compared with the other subgroups, the DN subgroup showed inhibited cell movement, cell migration, and cell activation. Furthermore, in survival analysis, the CK5/6 SP subgroup was significantly associated with poor progression-free survival ( Bladder cancer worldwide is the 10th most common form of malignant tumor and has the 14th highest cancer-associated mortality .Recently, several studies have been published that utilized next-generation sequencing (NGS) analysis to analyze and classify UB UC according to gene expression ,4,5. WhiMolecular subtypes for UB UC have been established recently, and studies attempting to demonstrate their clinical significance are actively in progress . MoleculIn the mRNA-based subtype classification, high expression of KRT5 is classified as the basal type and high expression of KRT20 is classified as the luminal type ,5,16,17.Thirty cases of MIBC in patients who underwent a radical cystectomy between 2016 and 2018 and for whom we were able to acquire fresh frozen tissues were included. With these samples, we subdivided the cases into four subgroups based on CK5/6 and CK20 IHC expression. The CK5/6 single-positive (CK5/6 SP) subgroup was entified . Three cases were selected from each subgroup, and mRNA sequencing was performed on fresh frozen tissue samples of 12 patients. The mean age of patients was 68.7 years  at diagnosis, and the male-to-female sex ratio was 10:2. Seven patients were in stage IIIA and five patients were in stage IIIB according to the 8th edition of the TNM staging system of the American Joint Committee on Cancer (AJCC) . Lymph nWe additionally performed CD44, CK14, GATA3, FOXA1 and P53 IHC in 12 cases. In CD44, a basal-type marker, IHC expression was positive in all CK5/6 SP and DP subgroup cases and mostly negative in CK20 SP and DN subgroups. In CK14, another basal-type marker, IHC expression was positive in one case in the DP subgroup and two cases in the CK5/6 SP subgroup and was negative in all remaining cases. In GATA3, a luminal-type marker, IHC expression was positive in all three CK20 SP subgroup cases and negative in all three CK5/6 SP subgroup cases. FOXA1 IHC expression was positive in two cases in the CK20 SP subgroup and one case in the DN subgroup and was negative in all remaining cases, including CK5/6 SP and DP subgroup cases. P53 IHC expression showed a wide range from negative to greater than 95% positive expression.p-value < 0.05 and |fold change| ≥2 as the cut-offs, we identified 38 differential expression genes (DEGs) between the DP and CK20 SP subgroups, 98 DEGs between the DP and CK5/6 SP subgroups, 433 DEGs between the DP and DN subgroups, 183 DEGs between the CK20 SP and CK5/6 SP subgroups, 256 DEGs between the CK20 SP and DN subgroups, and 614 DEGs between the CK5/6 SP and DN subgroups. In total, 1062 DEGs were identified. Compared with the DP, CK5/6, and DN subgroups, the CK20 SP subgroup had 10, 77, and 174 upregulated genes, respectively, and 28, 106 and 82 downregulated genes, respectively. Compared with the DP and CK 20 SP and the DN subgroups, the CK5/6 SP subgroup had 43, 106, and 429 upregulated genes, respectively, and 55, 77, and 185, downregulated genes, respectively. Compared with the DP, CK20 SP and CK5/6 SP subgroups, the DN subgroup had 79, 82 and 185 upregulated genes, respectively, and 354, 174 and 429 downregulated genes, respectively. A Venn diagram of DEGs in the four major comparison conditions is shown in RNA sequencing data were analyzed from 12 MIBC tissues. Using adjusted The results of Gene Ontology (GO) analysis between each subgroup are summarized in When comparing the CK5/6-positive and CK5/6-negative groups, we identified 226 DEGs. Compared to the CK5/6-negative group, the CK5/6-positive group had 169 upregulated genes and 57 downregulated genes. The CK5/6-positive group was enriched with cell migration, immune response, MAPK signaling pathway and TNF signaling pathway associated GO terms. Compared to CK5/6- negative tumors, many DEGs  associated with previously mentioned GO terms were upregulated in CK5/6-positive group tumors .The ingenuity pathway analysis (IPA) results showed that some functions related to immune response and cell migration were activated in the CK5/6 SP and DP subgroups . RegulatGene set enrichment analysis (GSEA) confirmed that the interleukin 6-Janus kinase-signal transducer and activator of transcription 3 (IL6-JAK-STAT3) signaling pathway, inflammatory response and TNF-α signaling via nuclear factor-κB (NF-κB) signaling pathway were significantly enriched in the DP and CK5/6 SP subgroups.  However,We compared the expression patterns of gene signature markers between four subgroups . ExpressThe clinicopathological and demographic characteristics of the 189 patients for whom we performed IHC analysis are summarized in The follow-up period ranged from 1 to 277 months, the median follow-up period was 16 months, and the median survival period was 92 months. Of a total of 189 patients, clinical follow-up data for 185 patients were available. During the follow-up period, disease progression was found in 95 cases, and death occurred in 128 cases.p = 0.008). The Kaplan–Meier analysis also showed that the high expression of CK5/6 was associated with unfavorable PFS (p = 0.005) (p = 0.028). However, there was no significant difference in overall survival (OS) according to IHC-based classification (p = 0.709) or IHC expression .We performed the Kaplan–Meier survival analysis according to IHC-based classification. Of the four IHC based subgroups, the CK5/6 SP subgroup had the worst progression-free survival (PFS) (= 0.005) . In contIn recent years, research on molecular-based UB UC subtype classification has been actively underway. As gene expression profiles were reported to show different disease progressions, responses to chemotherapy and survival rates, the UB UC molecular subtype classification became a necessary step in UB UC diagnosis and treatment planning. In MIBC, the basal type is known to arise from basal and stem cells of normal urothelium, and the luminal type is known to arise from terminally differentiated superficial umbrella cells . During Previous studies utilized NGS analysis to assess gene expression patterns and reported differences in CK expression patterns between molecular subtypes. In this study, we took a different approach in case selection. Cases of the CK5/6 SP, CK20 SP, DP and DN subgroups were selected based on CK IHC results, and gene expression profiles were evaluated with these cases. Furthermore, based on IHC, CK5/6 and CK20 protein expression areas were accurately evaluated before tissue collection, which led to more precise tumor tissue collection.MIBC cases with sufficient fresh-frozen tissues were included, which resulted in 30 samples classified to four subgroups based on CK5/6 and CK20 IHC results. Nine cases in the DP subgroup and five cases in the DN subgroup among 30 cases may indicate a relatively high proportion of DP and DN subgroups in UB UC molecular subtype classification. The DP and DN subgroup cases may be in a stage of transition between molecular subtypes or they may be subtypes completely independent from CK5/6 SP and CK20 SP subgroups. However, research regarding this aspect of subtype classification has not been presented to date. Compared to the CK5/6 SP and CK20 SP subgroups, the DP subgroup showed a relatively high expression of basal- and luminal-type markers, which gave us the impression of a mixed phenotype. This subgroup showed the strongest immune signature genes expression. However, in GO, IPA, and GSEA functional analysis, the DP subgroup did not show a significant difference from the CK5/6 SP subgroup. Based on these results, the DP subgroup was expected to be close to the CK5/6 SP subgroup in tumor characteristic aspects. The DN subgroup, in every comparison with the three other subgroups, showed the highest number on GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with significant differences. The DN subgroup also showed mostly low gene expression on biological signature gene cluster expression analysis. In GO, IPA, and GSEA functional analysis, the DN subgroup showed no significant difference from the CK20 SP subgroup, except for “activation of cells’ associated molecules”, in the IPA result. The DN subgroup is the most similar to the CK20 SP subgroup among the three other subgroups, but we think it is a unique subtype that shows differences in gene expression from the other subgroups.Expression of p63-associated genes was high in the CK5/6 SP and DP subgroups. TP63, which is a transcription factor associated with basal/stem cells in the urothelium, is known to be activated in the basal-type MIBC and to regulate basal gene expression signature [In comparison between subgroups, expression of TP53-like signature genes was the lowest in the DN subgroup and varied among cases. Six cases, which showed diffusely strong positive results on IHC, showed high expression of TP53-associated gene mRNA. Association between TP53 mRNA expression and TP53 IHC protein expression showed a positive correlation.Expression of immune response-associated genes was high in the DP subgroup, and the CK5/6 SP, DN, and CK20 SP subgroups followed in respective order. Dividing the genes into two groups based on CK5/6 IHC results, immune response GO term associated DEGs were more enriched and associated genes were more upregulated in DP and CK5/6 SP subgroups than in DN and CK20 SP subgroups. Additionally, on IPA analysis, immune activation was more upregulated in DP and CK5/6 SP subgroups than in the other subgroups. In a previous study in our group, it was found that programmed cell death-ligand 1 (PD-L1) expression was enriched in the BASQ subtype positive for CK5/6 and CK14 on IHC . These rExpression of EMT marker was enriched in the DP and CK5/6 SP subgroups. The EMT is the process of epithelial or endothelial cells acquiring mesenchymal phenotypes, and is associated with tumor progression and metastasis process ,25,26,27Expression of cell adhesion markers showed low expression of tight junction-related genes TPJ2 and CLDN4 and high expression of desmosome-related genes DSC3 and PKP1, gap junction-related genes GJB3 and GLB4, and epithelial integrin genes ITGA6 and ITGB4 in the CK5/6 SP subgroup. These results imply that the CK5/6 SP subgroup shows increased expression of cell adhesion, especially basolateral cell adhesion-related genes. This result is in keeping with previous studies, which reported high cell adhesion gene expression signature in the mRNA-based basal subtype, including urobasal B and SCC-like subtype .Expression of the MAPK and TNF signaling pathways was enriched in the DP and CK5/6 SP subgroups. Most of the DEGs associated with these signaling pathway were upregulated in the DP and CK5/6 SP subgroups.On GSEA analysis, the IL6-JAK-STAT3 signaling pathway and TNF-α signaling via NF-κB signaling pathway were significantly enriched in DP and CK5/6 SP subgroups. In contrast, the DN subgroup was found to be less sensitive to this pathway. The IL6-JAK-STAT3 signaling pathway is upregulated in various types of cancer and its hyperactivation is associated with adverse clinical outcome ,33,34. FOn IPA analysis, compared to the other subgroups, the DN subgroup showed downregulated cell movement, cell migration, and cell activation. Such cellular functions, which are associated with cell motility, are closely related to cancer invasion and cancer metastasis ,38,39,40Different molecular subtypes were reported to have different prognoses and chemotherapy sensitivity in MIBC. In addition, we also found that the IHC-based classification has prognostic value. Compared with the CK5/6-negative subgroup (CK20 SP and DN subgroups), the CK5/6-positive subgroup (CK5/6 SP and DP subgroups), which implies the high EMT, IL6-JAK-STAT3 signaling pathway, TNF-α/NF-κB signaling pathway and immune gene expression signature, show adverse disease progression outcome, and this result is in keeping with the findings of previous studies [In the personalized precision medicine era, subgrouping of patients based on molecular characteristics has considerable influence on selecting therapeutic regimens and forecasting therapeutic responses and prognoses. In addition, mRNA sequencing analysis is a good method to examine phenotype characteristics of a tumor, however, in this study we utilized an IHC-based classification that is simpler and more affordable. We examined the molecular characteristics of subgroups based on IHC classification. In summary, we found that the CK5/6-positive subgroup showed a high gene expression signature related to aggressive behavior and showed worse clinical outcomes.In total, MIBC tissues from 30 patients who underwent a radical cystectomy at Seoul National University Hospital (SNUH) from 2016 to 2018 were included in this study. First, 30 fresh frozen tissue samples from 2016 to 2018 were investigated. The section containing prominent tumor tissue was selected, of which one section was prepared as a formalin-fixed paraffin-embedded (FFPE) tissue block, and the corresponding symmetric section was fresh frozen with liquid nitrogen and preserved in −70 °C until tumor excision. A total of 30 samples were classified to four subgroups based on IHC results for CK5/6 and CK20. In each subgroup three representative cases were selected, and RNA sequencing was carried out with a total of 12 fresh frozen tissue samples. All 12 patients were included in the prospective studies. Written informed consent was obtained from all patients enrolled in this mRNA analysis group.Additionally, 189 MIBC patients who underwent transurethral resection of the bladder or radical cystectomy at SNUH or Seoul Metropolitan government-Seoul National University Boramae Medical Center were included in the survival analysis group. A total of 189 FFPE block tissue samples from 2004 to 2010 were investigated. A tissue microarray (TMA) block was prepared from FFPE tissue blocks . Two cores (2 mm in diameter) containing invasive tumor areas were obtained from each case. This study was approved by the Institutional Review Board (IRB) of SNUH .IHC for CK5/6, CK20, CK14, GATA3, FOXA1, CD44 and TP53 was performed with an automatic immunostainer , following the manufacturer’s instructions. The primary antibodies against CK5/6 , CK20 , CK14 , CD44 , GATA3 , FOXA1  and p53  were used. Immunohistochemical positive control tissues were tonsil tissue for CK5/6 and CD44, duodenal mucosa epithelium for CK20, squamous cell carcinoma tissue for CK14, urothelial carcinoma tissue for GATA3, gastric mucosa epithelium for TP53, and breast cancer tissue, previously confirmed with positive expression for FOXA1.The full-section IHC staining was performed on all 12 cases, and in each case, two different invasive tumor areas were investigated. Additionally, the same IHC was carried out for 189 MIBC tissue TMA blocks.The evaluation results of CK5/6, CK20, CK14, GATA3 and FOXA1 IHC were derived from a previously published study . BrieflyAfter IHC results were identified on the FFPE tissue block, mRNA was extracted from the corresponding location of the symmetric fresh-frozen MIBC tissues. A 3-mm-sized plunger was used for punching out the fresh-frozen tissue. RNA library was assembled with TruSeq RNA Access Library Prep Kit, and base sequence analysis was carried out with Illumina HiSeq 2500 platform .  Paired demultiplexed fastq files were generated, and initial quality control was performed using FastQC . The adaptor sequences were removed by using Trimmomatic program and trimmed data were mapped to the reference genome (UCSC hg19) using the HISAT2 and Bowtie2. Previously known gene/transcripts were assembled with the StringTie program. Raw data were normalized and, mRNA expression data were presented as reads transcript per million and were transformed into log 2 volume values for the analysis.p-value < 0.05 and |fold change| ≥2). The analyses were performed by biological process, molecular function, and cellular component, which are the GO subcategories. Functional annotation based on the KEGG database was implemented. Additionally, the functional analyses were performed with the use of IPA.In this study, DEGs were identified with DESeq2. Statistical analysis was performed on selected genes whose median count for each gene was greater than 5 in at least one comparison combination. Functional analysis of DEGs was performed using GO and the KEGG pathway . Two-sided


Glycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal  interactions . Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential. Glycyrriza uralensis Fischer, Glycyrrhiza glabra Linné or Glycyrrhiza inflata Batalin, which are herbaceous perennial plants of the bean family Fabaceae native to the western Asia and southern Europe1. Besides its ecological values for windbreak and sand fixation, both roots and shoots of licorice compose specialized bioactive compounds/molecules with pharmaceutical potential. Licorice root extracts have been used in herbalism and traditional medicine and presented anti-carcinogenic3, anti-inflammatory, anti-fungal, anti-piroplasmic and cytotoxic activities4. More recently, glycyrrhizin, the most important bioactive triterpenoid saponin in licorice roots, is under the consideration for treating COVID-19 infection caused respiratory syndrome5. And licorice shoots are a kind of high-quality forage grass because of their high content of coarse fiber and flavonoid6. Their beneficial effects on human health has made licorice a valuable trade item. However, these bioactive compounds in licorice with pharmaceutical interest, such as glycyrrhizin or flavonoid, are typically in minute quantities7. Therefore, a better understanding of pathways associated with biosynthesis, regulation, and accumulation of these phytochemicals becomes a key step to reach the pharmaceutical potential of licorice10.Licorice or liquorice is the common name of 12. In addition, accumulation of valuable bioactive constituents in many functional plants is tissue-specific and meanwhile, only happens at specific developmental stages. One well-known example are ginsenosides in ginseng, which accumulate specifically in roots and rhizomes in “mature” plants, while little could be detected at juvenile stage in these perennial plants13. Consistently, the expression of regulatory genes and biosynthetic genes of ginsenosides is also spatio-temporal specific14. A growing number of studies have been conducted to screen genes involved in the same metabolic pathways by co-expression network16. Spatio-temporal gene expression profiling may provide important clues for future functional analyses of genes in non-model organisms.Spatio-temporal gene expression is the activation of genes within specific tissues of an organism at specific times during development (WIKIPEDIA). Many key genes only express in certain tissues and at certain developmental stages in response to both internal and external cues to ensure the accomplishment of each step in plant life cycles18. Because of the accuracy and sensitivity of RT-qPCR suitable internal references for data normalization in RT-qPCR analysis are prerequisite to obtain reliable results19. Expression of suitable reference genes should be constant under the experimental conditions to be tested in a specific research19. Housekeeping genes, due to their stability and indispensable function for survival, are the typical first choice for reference gene selection21, and stably expressed genes in RNA-seq experiments might also be good candidates. That being said, no “universal” reference gene has been verified to be stably expressed across all given experimental conditions23. Consequently, selection of appropriate reference genes is required for a standardized RT-qPCR procedure following the MIQE  guidelines24.Reverse transcription quantitative real-time PCR (RT-qPCR) is a method for accurate expression analysis and comparisons of small numbers of genes among various experimental samples25, and accumulated to higher levels in summer than in winter26. Spatio-temporal spcific expression of related biosynthetic and regulatory genes should be the cause of these spatial-temporol accumulation of bioactive compounds. For example, the key genes in glycyrrhizin biosynthetic pathway, β-amyrin synthase (β-AS), β-amyrin 11-oxidase (CYP88D6), 11-oxo-β-amyrin 30-oxidase (CYP72A154), are mainly expressed in roots and rhizomes, whereas no transcripts were observed in leaves27. Therefore, the screening of reference genes across different tissues or throughout different developmental stages, more importantly, the spatio (tissue)-temporal  interactions is of great importance for the study of gene functions in licorice.In licorice, the types and contents of many bioactive compounds varied remarkably among different tissues at different developmental stages. Glycyrrhizin, the most important bioactive triterpenoid saponin in licorice roots, are predominantly accumulated in roots and rhizomes28 previously studied reference genes under the drought stresses in G. glabra, information concerning suitable reference genes is still lacking in licorice, especially for congeneric species, G. uralensis or G. inflata.Selection of reference genes, as of now, has been focused on a single factor/dimension, i.e., time  or space (tissue). However, the expression of genes is constantly under the influences of multiple factors/dimensions. Here, to better understand the spatio-temporal gene expression patterns in licorice, we investigated the expression profiles of 14 candidate reference genes in this study. Although MaroufiGlycyrrhiza species, and finally, we summarized the reference genes previously used within Leguminosae plants.Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual conditions. To examine this hypothesis, we 1) evaluated the stability of 14 candidate genes, which were stably expressed housekeeping genes derived from our RNA-seq analysis; 2) selected optimal reference genes under the conditions of different tissues, developmental stages, and tissue × developmental stages, respectively; 3) compared the suitable reference genes under different experimental conditions and between the two congenic G. uralensis and G. inflata) were grown in the test field at the Northwest Biological Agricultural Center, Chinese Academy of Sciences . Roots, rhizomes and leaves were collected in April (returning green), May (rapid growth and flowering), July (seed setting), and October (aging)  Fig. . All samTotal RNA was isolated using HiPure Total RNA Mini Kit  according to the manufacturer’s instruction. The RNA quality and concentration were measured with agarose gel electrophoresis and spectrophotometer . Removal of genomic DNA contamination and first strand cDNA synthesis were performed using PrimeScript RT reagent Kit with gDNA Eraser .Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), β-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2),) were selected in previous studies29, and the other four (ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), R3H domain protein 2 (R3HDM2) were selected from a RNA-seq dataset (SRA accession: PRJNA574093). RT-qPCR primers were designed using PrimerQuest Tool, INTEGRATED DNA TECHNOLOGIES (IDT) (https://sg.idtdna.com/Primerquest/Home/Index) based on the following parameters: Melting temperature (Tm) of 59–65 ℃; GC content of 45–55%; optimum length of 17–30 bp and amplicon length of 50–200 bp. All primers were synthesized by TSINGKE Company . Detailed information listed in Table A total of 14 candidate reference genes was included in this study, in which ten, (The RT-qPCR were carried out in 384-well blocks using TB Green Premix Ex Taq II (Tli RNaseH Plus)  on LightCycler 480  according to manufacturers’ instructions. Three technical repeats were carried out for each sample.(−1/slope) − 1] × 100. Expression stability of the 14 candidate reference genes were evaluated by four Microsoft Excel-based computational programs, geNorm30, NormFinder31, BestKeeper32 and Delta CT33. geNorm method ranks the expression stability by M value for each reference gene, and the smaller the M value, the more stable the gene. Based on NormFinder, the gene expression stability was calculated by the SV value. BestKeeper calculates the stability of candidate genes by the Pearson correlation coefficient (r), while the stability of the genes is evaluated by the pair-wise comparisons.The slope of the standard curve of a cDNA tenfold dilution series was constructed to calculate the PCR amplification efficiency (E), and the E value was obtained according to the equation E = [1034. Based on the rankings from the Microsoft Excel-based computational programs, RefFinder assigns an appropriate weight to an individual gene and calculates the geometric mean of their weights for the overall final ranking.The RefFinder, a comprehensive system to integrate the currently available major computational programs  to compare and rank the stability of candidate reference genes, was used for the overall ranking of the candidate reference genesGlycyrrhiza species were also analyzed. The results were visualized by Venn Diagrams, and it was plotted using the OmicShare tools, a free online platform for data analysis (www.omicshare.com/tools).The top three most suitable reference genes selected by RefFinder under the conditions of different tissues, developmental stages, and tissue × developmental stages were compared; and the suitable reference genes under the same experimental condition between the two congenic β-amyrin synthase , is a key gene in glycyrrhizin biosynthesis and mainly expressed in root organs27, we thus chose to validate the reliability of the selected reference genes in different treatment conditions. The primers of β-AS used for RT-qPCR were listed in Table β-AS was analyzed by seven normalization ways, including the most stable, the top two most stable, the top three most stable, the least stable, the top two least stable, the top three least stable, and all the candidate reference genes. One way ANOVA was carried out to evaluate the expression level of β-AS under different normalization conditions .To confirm the suitability of the reference genes recommended in the present study, we measured the differential expression of a specific licorice gene with a known expression profile under different tissues. Licorice Reference genes reported previously in Leguminosae species was selected by searching “NCBI” or “Web of Science” with the key words “Leguminosae” and “reference gene”. The top three most suitable reference gene was considered to calculate the recommendation frequency for developmental stages and tissues, respectively, among Leguminosae species.ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2) were determined by a single PCR product of expected size, and further confirmed by a single peak in the melting-curve analysis. The efficiency of the PCR amplification (E) was calculated from the standard curve by making a dilution series with mixed samples. The E value of the reported reference genes ranged from 67.28% (CAC) to 116.84% (CYP) and correlation coefficients varied from 0.9526 (CAC) to 0.9976 (CYP)  for RT-qPCR analysis. The E value of the selected candidate reference genes from RNA-Seq data ranged from 90.63% (CAC) to 107.58% (CYP) and correlation coefficients varied from 0.9887 (CS) to 0.9963 (COPS3)  values, and the transcripts of these genes showed different levels of abundance in a Tables  and S2. ata Fig.  and S4. TIF1 had the lowest Cq both in G. uralensis (mean Ct of 20.30) and G. inflata (mean Cq of 19.94), indicating the highest expression level of TIF1 in the two species, while EF1 (mean Cq of 27.50) or DREB (mean Cq of 25.56) was expressed at low levels in G. uralensis or G. inflata, respectively  of 6.74%) and G. inflata (CV of 2.92%), while surprisingly, a commonly used reference gene, ACT, was the most variable across all samples (CV of 10.45%) in G. uralensis (Table TIF1 (9.60%) was the most variable across all samples in G. inflata  were tested Fig. . In G. uG. uralensis and G. inflata. In G. uralensis, the top three most stable candidate reference genes were CAC, R3HDM2, ABCC2 identified by geNorm, DNAJ, CAC, R3HDM2 by NormFinder, COPS3, TIF1, UBC2 by BestKeeper, and CAC, R3HDM2, DNAJ by Delta CT . When pairwise variations Vn/n + 1 < 0.15, it means that an addition reference gene (n + 1) is not necessary. For all the experimental conditions in G. uralensis, among which CAC and R3HDM2 showed the highest recommended frequency   Vass., Glycine max (L.) Merr., Hedysarum coronarium L., Lens culinaris Medic., Lupinus angustifolius L., Medicago sativa L., Phaseolus vulgaris Linn., Vigna angularis (Willd.) Ohwi et Ohashi) under different experimental conditions, including different tissues or different experimental stages. In this study, we added G. uralensis and G. inflata to this list.Reference gene selection has been reported in 12 Leguminosae species  had been studied under different developmental stages, among the 22 reference genes recommended, eukaryotic elongation factor  and tubulin  were the most choices, and the frequency of recommendation in the 10 species were 15.15% and 12.12%, respectively . From the results of our survey, we found EF1, TUB and UBQ are commonly used housekeeping genes, which have been identified as the most suitable ones at different developmental stages in several species. EF had been selected as the most suitable reference gene at different developmental stages in E. songoricum36, G. max37, and C. arietinum38. TUB was the optimal reference gene at different experimental stages in E. songoricum36, G. max37, and H. coronarium39. While UBQ was validated as the most stable reference gene at different developmental stages in C. tetragonoloba40, H. coronarium39, L. angustifolius41. For the different tissues in Leguminosae, the most stable reference gene candidates were ACT, EF and UBQ. Among them, ACT was recommended as a stable reference gene in different tissues of C. tetragonoloba40, E. songoricum36, G. max42, P. vulgaris43. EF was recommended in C. obtusifolia44, G. max37, M. sativa45, and UBQ was recommended in G. inflata . We found CAC was the most stable reference gene when all factors taken account in G. uralensis, and COPS3 was the optimal reference gene with the highest recommended frequency in G. inflata -temporal  interactions . Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis.In this study, we evaluated the expression of 14 candidate reference genes across different tissues  at various developmental stages  in the two congeneric medicinal plants, Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.In addition, we also documented the reference genes that have been used in RT-qPCR analyses among 12 different Leguminosae plants under the same biotic conditions with current study, i.e., tissue and/or developmental stage. Among the 115 genes have been tested, even the routinely used reference genes showed variable expressions under different experimental conditions. Therefore, to avoid the misinterpretation of RT-qPCR results, a thorough evaluation of reference genes is strongly recommended. More importantly, given that biosynthesis of bioactive compounds is typically spatio-temporal dependent, the selection of suitable reference genes should follow suit. Based on previous studies and our current results, we concluded (1) transcriptome is a rich reservoir for selecting stably expressed candidate reference genes, (2) customized design, especially the interaction among the experimental conditions, is warranted for searching suitable reference genes in any given species, and (3) without validation study, gene(s), including housekeeping genes, could lead to ambiguous results, especially in non-model species. Finally, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Supplementary Information 1.


Dictyostelium discoideum, the function of Kinesin-8 remains elusive. We present here some biochemical properties and localization data that indicate that this motor (DdKif10) shares some motility properties with other Kinesin-8s but also illustrates differences in microtubule localization and depolymerase action that highlight functional diversity.The functional organization of microtubules in eukaryotic cells requires a combination of their inherent dynamic properties, interactions with motor machineries, and interactions with accessory proteins to affect growth, shrinkage, stability, and architecture. In most organisms, the Kinesin-8 family of motors play an integral role in these organizations, well known for their mitotic activities in microtubule (MT) length control and kinetochore interactions. In  Dictyostelium discoideum through a gene knockout of the DdKif10 isoform, and were unable to detect any significant phenotypic consequences of its removal .2, 1 mM EGTA), 6 μM of motor was added to paclitaxel-stabilized MTs (0–25 μM) in 200 μL reactions, allowed to bind 15 min (20 °C), and sedimented at 150,000g (15 min). Supernatants were removed, pellets were resuspended in 200 μL BRB80, and aliquots were run on 7.5% SDS-gels and Coomassie stained. Band intensities were quantitated, and curve fittings were performed using FIJI [Protein from these cells was affinity purified following the methods in . Twice-cing FIJI . ATPase ing FIJI .The MT gliding assay and cellular imaging were performed as described in . Fixed ckif8 (DDB_G0284471) [kif10, bp +47 to +763) were amplified with primers containing 5′ Not1 and 3′ Sal1 restriction sites, and cloned in reverse orientation relative to the DdActin15 promoter in pLD1A15SN [kif8, +343 to +714; and Ddkif10, +400 to +763) were amplified with primers containing 5′ Not1 and 3′ Mlu1 restriction sites and were cloned downstream but in a forward orientation adjacent to the initial segment. The constructs were sequenced to confirm orientation, junctions, and PCR fidelity. The expression of a single RNA transcript that folds into a hairpin, with a large unpaired loop  was predicted. The constructs were introduced into Ddkif8 or Ddkif10 null cells [The long hairpin constructs were assembled using PCR-generated fragments as outlined in ll cells , and theD. discoideum coding regions that challenges simple BLAST comparisons . However, important here is that even more local comparisons do not reveal significant similarity to either HsKif18A or ScKip3, particularly within regions of their tail known to contain a second, non-ATP sensitive MT binding domain (HsKif18A 777-898aa [DdKif10 is a 1238aa (139 kDa) polypeptide with an amino-terminal motor domain, a central region with two prominent predicted coiled-coil motifs, and a carboxy terminal tail that likely contains cargo-binding regions ,18. The 77-898aa , ScKip3 77-898aa ).D. discoideum for analysis (D. discoideum clones that had detectable expression for either the full length or the motor-only domain constructs. A mid-length construct (725aa) containing a bit over half of the DdKif10 polypeptide did express as expected, and we were able to isolate this motor fragment for further analysis.We developed three expression constructs to produce different-size tagged versions of DdKif10 in analysis . Despite725) readily bound to MTs in an ATP-sensitive fashion (n = 71). MTs were seen to attach to the coverslip, glide smoothly over a distance, then readily detach and diffuse away. There was no evidence of MT bundling (with or without ATP) or MT depolymerization activity in similar mix and match assays.The 110 kDa carboxy-terminal GFP fusion product of the mid-length construct . Similar RNAi strategies have proven effective in targeting the functional activities of the essential DdKif6 gene [We also tested the idea that DdKif10 and DdKif8 cooperate in some capacity to organize the interphase MT array, providing MT pushing forces as a counterbalance to dynein-mediated pulling . We exprif6 gene  and multif6 gene ,26,27. HKinesin-8 motors are generally considered slow, processive, and accumulate at the MT plus ends where they effect tip dynamics . Our res725 motor is similar, in part, to the latter scenario, broadly found in the cytoplasm and absent from the nuclear volume during interphase, but as cells enter mitosis, it appears to migrate into the semi-closed nuclear compartment and form punctate enrichments in the immediate spindle region. However, in contrast, our in vivo imaging indicates that the DdKif10725 motor does not accumulate on interphase or mitotic MTs. We recognize we are unable to express a full-length version with a proper tail domain, but MT decoration is also seen with a variety of expressed motor/neck versions similar in scale to DdKif10725, and in multiple organisms [In some metazoans ,36,37, Krganisms ,38.D. discoideum spindles are not consistent among the mitotic cells we have observed, and thus we cannot say if they represent D. discoideum kinetochores or the spindle overlap region that Kinesin-8 targets in other organisms [D. discoideum; however, gene knockouts or knockdowns of this isoform have no obvious effect on cell growth that would implicate a major role in cell division.The punctate structures seen on or near rganisms . We cannS. cerevisiae motor (ScKip3) [725 motor, during interphase, mitosis, or in vitro assays. Within the motor domain, the loop 2 region of Kinesin-8 and -13 has been implicated in MT interaction and/or managing MT dynamics, and more recently in facilitating stable interactions with the plus ends of K-fiber MTs [D. discoideum MTs show plus end tip dynamics [As a function of their affinity to MTs and particularly the plus ends, Kinesin-8 motors are generally known to effect MT dynamics, either through direct depolymerization or through effecting MT catastrophe . In most(ScKip3) ,33 and t(ScKip3) ,36 motor(ScKip3) ,37. We diber MTs ,40,41. Diber MTs . This didynamics ,42, overD. discoideum, acting in sum to support the radial MT array [Finally, our previous work suggests that DdKif10 collaborates with DdKif8 (Kinesin-4) to counterbalance dynein pulling activity during interphase in MT array ,18. LaseMT array , and we MT array ,25,26,27Dictyostelium remains an enigma. On the one hand, it is a bona fide MT-based motor with movement and catalytic properties, but it does not show localization or some of the functional activities shared with isoforms from other organisms that would readily illustrate a cellular role during interphase or mitosis. It seems unlikely that a motor would persist in a genome without an impactful function, and thus these results underscore the challenges in studying individual members of a diverse gene family.Despite strong consistent phenotypes in multiple organisms, the function of the Kinesin-8 isoform in