Previous work showed a role of BNIP3 in myocardial remodeling and progression to HFrEF. We utilized a multiomics approach to unravel BNIP3-related molecular mechanisms in the pathogenesis of HFrEF. BNIP3 knockdown in HFrEF improved glycolysis, pyruvate metabolism, branched-chain amino acid catabolism, and oxidative phosphorylation, and restored endoplasmic reticulum (ER)–mitochondrial (mt) calcium and ion homeostasis. These effects of BNIP3 on cardiac metabolism were related to its interaction and downregulation, and/or phosphorylation, of specific mt-proteins involved in the aforementioned metabolic pathways, including the MICOS and SLC25A families of carrier proteins. BNIP3 affected ER–mt-calcium and ion homeostasis via its interaction-induced VDAC1 dimerization and modulation of VDAC1 phosphorylation at Ser104 and Ser241, and the downregulation of LETM1. At the ER level, BNIP3 interacted with the enzyme SERCA2a and the PKA signaling complex, leading to the downregulation of SERCA2a and PKA-mediated Ser16 phospholamban phosphorylation. Additionally, BNIP3 attenuated AMPK and PRKCE activity by modulating AMPK phosphorylation at Ser485/491 and Ser377 residues, and PRKCE phosphorylation at Thr521 and Thr710 residues. BNIP3 also interacted with sarcomeric, cytoskeletal, and cellular transcription and translation proteins, and affected their expression and/or phosphorylation. In conclusion, BNIP3 modulates multiple pathobiological processes and constitutes an attractive therapeutic target in HFrEF. Heart failure with reduced ejection fraction (HFrEF) is associated with eccentric myocardial remodeling, increases in LV volumes, and a depressed LV ejection fraction (LVEF), secondary to complex structural and molecular changes at the cellular level . These cThe Bcl-2 nineteen kilodalton interacting protein 3 (BNIP3) has been shown to play a role in mitophagy and apoptosis ,6,7,8. BThe production of adenoviruses (Ad) encoding for Null (control), BNIP3, or BNIP3 shRNA (ShBNIP3) was performed by Vector Biolabs , as previously described . The pro2, 95% air at 37 °C. Subsequently, they were washed once with M199 medium to remove unattached and dead cells. The ACM were then transfected with Ad-Null, Ad-BNIP3, or Ad-ShBNIP3, with an infectious dose of 100 multiplicity of infection, for 36 h to allow for sufficient gene transduction before the following experiments were performed.The isolation of rat adult cardiac myocytes (ACM) was performed in male Sprague Dawley rats weighing 250–350 g, as previously described . All reaThe ACM were loaded with the red mt-superoxide indicator  and tetra-methylrhodamine methyl ester perchlorate  for 10 min at 37 °C to assess mt-ROS and mt-membrane potential, respectively. Cells were then washed with M199 medium and baseline live images were taken at 20× magnification using an Olympus IX71 fluorescent microscope . Subsequently, cells were stressed with phenylephrine  and imaged at 2 h and 12 h following PE stress. Photomicrographs were taken from at least 5 fields per condition per group. Fluorescence intensity was analyzed in ImageJ. Data were obtained from at least 3 independent experiments.4 rod-shaped ACM were added to each chamber of the Oroboros instrument. Glutamate (10 mmol/L) and malate (2 mmol/L) were added to produce NADH and to activate the ETC complex I (state 2-LEAK phase). ADP (2.5 mmol/L) was then added to maximally stimulate respiration, in the presence of glutamate and malate, to assess the OXPHOS phase (state 3). Next, succinate (5 mmol/L) was added to produce FADH2, which, together with glutamate and malate, allow for the assessment of complex I and II activity combined, and, therefore, maximal OXPHOS capacity. Afterwards, Rotenone (1 μmol/L) was added to inhibit complex I and measure complex II activity only. Oligomycin (2 μmol/L) was then added to inhibit ATP synthase activity (state 4 respiration), followed by the addition of carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (2 μmol/L FCCP) as an uncoupler to assess electron transfer system (ETS) capacity. Thereafter, Antimycin A (5 μmol/L) was added to inhibit complex III activity for the determination of residual O2 consumption. Finally complex IV activity was measured by adding N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (0.5 mmol/L TMPD). All reagents were purchased from Sigma-Aldrich . Data were obtained from 7 independent experiments.Mitochondrial respiration was assessed and analyzed with an Oroboros oxygraph-2k, using an appropriate respiration buffer  at high sample dilutions. The titration method of substrates, inhibitors, and uncouplers was utilized for the measurement of state 2 and 3 of respiration and respiratory capacity at multiple levels of the respiratory chain, as previously described ,16. BrieAll procedures involving the handling of animals were approved by the Animal Care and Use Committee of Tulane University, and adhered with the Guide for the Care and Use of laboratory Animals published by the National Institutes of Health. Sprague Dawley rats weighing 180–200 g underwent ascending aortic banding (AAB), as previously described . The effProtein extraction from frozen LV and right ventricular (RV) myocardia was performed as previously described . Followi2-transformed, stripped of those matched to multiple proteins, and weighted by an adaptive intensity weighting algorithm. Differentially expressed proteins across experimental groups were determined by applying a permutation test with Benjamini–Hochberg correction for multiple comparisons [p-value of <0.05 was considered significant. Phosphopeptide quantification was performed in Scaffold PTM , as previously described [Protein and phosphoprotein identification was performed in Proteome Discoverer , as previously described . We usedparisons . An unadescribed , the resescribed .2-transformed, MS data were exported from Scaffold or Scaffold PTM, and were then uploaded to Qlucore bioinformatics software for the visualization and presentation of the analyzed proteomic or phosphoproteomic data in Scaffold or Scaffold PTM, respectively. Data visualization was accomplished via the generation of heat maps, PCA plots, and Venn diagrams. Bioinformatics analysis of the aforementioned datasets was performed in Ingenuity Pathway Analysis (IPA) bioinformatics software by utilizing IPA’s “Core Analysis” function with a cutoff p-value of <0.05 for each of the two-group comparisons: ShLuc vs. Sham and ShBNIP3 vs. ShLuc. Analyzed datasets were then compared using IPA’s “Comparison Analyses” function to yield the most enriched canonical pathways and upstream regulators that were shared between the two-group comparisons. Protein–protein interaction (PPI) networks and enriched reactome pathways, by q-value, of the commonly identified mt-proteins in both, LV and RV, proteomic datasets that were differentially downregulated in ShLuc vs. Sham and differentially upregulated in ShBNIP3 vs. ShLuc, was performed in the STRING database. A q-value of < 0.05 was considered significant.The normalized, raw, logIPA analysis content information for the LV and RV proteomic datasets were the following: Analysis Creation Date: 17 September 2021; Content version: 65367011  and 68752261 (Release Date: 6 September 2021), respectively; Analysis IDs: 38776621 and 38833098, respectively.Sample preparation for the non-targeted metabolomics and acyl-carnitine LC–MS analysis was performed as described elsewhere . A total2-transformed. Statistical analysis was performed in Qlucore using one-way ANOVA and multiple comparison correction by Tukey. A p-value of <0.05 was considered statistically significant. Data visualization  of the statistically analyzed dataset was also performed in Qlucore. Bioinformatics analysis of the non-targeted metabolomics datasets was performed by utilizing IPA’s “Core Analysis” function with a cutoff p-value of <0.05 for each of the two-group comparisons: ShLuc vs. Sham and ShBNIP3 vs. ShLuc. Analyzed datasets were compared using IPA’s “Comparison Analyses” function, as described above. Enrichment pathway analysis was performed in MetaboAnalyst v5.0 bioinformatics software.MS raw data were uploaded to Qlucore bioinformatics software, and then logIPA analysis content information for the metabolomics dataset analyzed in Qlucore were the following: Analysis Creation Date: 28 September 2021; Content version: 68752261 (Release Date: 6 September 2021); Analysis ID: 38841804.The co-immunoprecipitation (Co-IP) experiment was performed as previously described . Brieflym/z at a resolution of 60,000 at 200 m/z. The automatic gain control target for the full scan was set to 3e6. The MS/MS scan was operated in top 20 mode using the following settings: resolution of 15,000 at 200 m/z; automatic gain control target 1e5; maximum injection time 19 ms; normalized collision energy at 28%; isolation window of 1.4 Th; charge state exclusion unassigned, 1, >6; dynamic exclusion 30 s. The MS raw files were analyzed and searched against rat or human protein databases using Maxquant (1.6.2.6). The parameters were set as follows: the protein modifications were carbamidomethylation (C) (fixed), oxidation (M) (variable), phospho (STY) (variable); the enzyme specificity was set to trypsin; the precursor ion mass tolerance was set to 10 ppm; MS/MS tolerance was 0.5 Da. Label-free quantification was used to quantify protein expression among the different samples. MS raw data were then uploaded and log2-transformed in Qlucore for data visualization. Differentially expressed proteins across experimental rat HFrEF and Sham groups was determined by applying Student’s t-test with a cutoff p-value of <0.05 vs. <0.1. Bioinformatic analysis of the statistically analyzed rat BNIP3 interactome dataset was performed by utilizing IPA’s “Core Analysis” function with a cutoff p-value of <0.05 vs. <0.1. The two analyses were then compared using IPA’s “Comparison Analyses” function, as described above. The PPI networks and enriched reactome pathways, by q-value, of the identified BNIP3 interacting proteins in rat or human LV myocardium, and those that were commonly identified in both, was performed in the STRING database. A q-value of < 0.05 was considered significant.Mass Spectrometry: The full scan was performed on a Q Exactive Orbitrap mass spectrometer between 300–1650 p < 0.05) and 38908520 .IPA analysis content information for the rat BNIP3 interactome, HFrEF vs. Sham, were the following: Analysis Creation Date: 19 October 2021; Content version: 68752261 (Release Date: 6 September 2021); Analysis ID: 38908519  in the LV and RV proteomic datasets are presented in Regarding the upstream regulators, BNIP3 knockdown in HFrEF enhanced the activity of the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), La-related protein 1 (LARP1), and the microRNAs miR-30c-5p, miR-155-5p, and miR-16-5p, whose activities were attenuated in ShLuc vs. Sham. BNIP3 knockdown in HFrEF also attenuated the activity of the lysine-specific demethylase 5A (KDM5A), MAPK kinase 4 (MAP4K4), X-box-binding protein 1 (XBP1), transforming growth factor beta-1 (TGF-β1), transcription regulator protein BACH1, cell tumor antigen p53 (TP53), and the microRNA miR-9-5p. A western blot analysis of the molecular marker PGC-1α D showed To further validate the effect of BNIP3 on mt-function, i.e., mt-membrane potential and mt-reactive oxygen species (ROS) production, we conducted functional studies in isolated ACM in vitro using the fluorophores TMRM and MitoSOX. BNIP3 overexpression (OE) in ACM, under baseline/normal conditions, attenuated mt-membrane potential and enhanced the production of mt-ROS vs. Ad-Null (control group) A,B. More2-fold change and the variance in biological samples in Sham, ShLuc, and ShBNIP3 groups of the differentially abundant metabolites in ShLuc vs. Sham. The PCA plot indicates that the highest variance exists between Sham and the HFrEF biological samples. Moreover, the ShBNIP3 biological samples are separated from the ShLuc biological samples, and are closer to Sham, suggesting the improvement of some of these metabolites in the ShBNIP3 group toward Sham. Heat maps and PCA plots  or inhibition (blue bars), in ShLuc vs. Sham and ShBNIP3 vs. ShLuc, respectively. UDP-N-acetyl-D-glucosamine biosynthesis, CMP-N-acetylneuraminate biosynthesis, and glutamine degradation were predicted to be activated, while as citrulline metabolism, glycolysis, and BCAA catabolism were predicted to be inhibited/downregulated, in ShLuc vs. Sham. The glucosamine and acetylneuraminate biosynthesis pathways were inhibited in ShBNIP3 vs. ShLuc. Moreover, BNIP3 knockdown in HFrEF was associated with the upregulation of the pentose phosphate pathway, sucrose degradation, leucine degradation, and glycolysis. Collectively, these results are in agreement, and complement the directional changes of the enriched metabolic pathways seen in the proteomic datasets. Enrichment pathway analysis  in the LV and RV phosphoproteomic datasets. The orange and blue color intensities represent the z-score-based extent of upregulation/activation or downregulation/inhibition, respectively. PKA, cardiac β-adrenergic, and AMPK signaling were amongst the highest enriched signaling pathways in the two-group comparisons, LV > RV. These signaling pathways were predicted to be downregulated in ShLuc vs. Sham and upregulated in ShBNIP3 vs. ShLuc, LV > RV. Cardiac β-adrenergic networks  LV myocardium for some of the BNIP3 interacting proteins with biological functions involved in OXPHOS, metabolism, calcium cycling and homeostasis, and muscle contraction and translation regulation. The heat maps are presented based on differential expression in HFrEF vs. Sham with p-value cutoff of < 0.05, between 0.05 and 0.1, and of > 0.1. The sarcoplasmic/endoplasmic reticulum calcium ATPase isoform 2a (SERCA2a) and the mt-proton/calcium exchanger protein, LETM1, were part of the BNIP3 interactome. LETM1 regulates calcium efflux at the inner mt-membrane, while SERCA2a pumps the cytoplasmic calcium back into the sarcoplasmic reticulum during the relaxation phase of the cardiac cycle. The SERCA2a and LETM1 expression was decreased in ShLuc vs. Sham and increased in ShBNIP3 vs. ShLuc , and AMPK. Both AMPK and PRKCE have been shown to regulate mt-metabolic pathways and mt-function, while PKA regulates both mt-function and calcium cycling at the ER–mt interface. The effect of BNIP3 extended beyond metabolism to include the regulation of the MICOS and the mt-SLC25A family of carrier proteins, mt-translation/elongation, cellular translation, ER stress and UPR pathways, oxidative stress, and apoptosis. Moreover, there was interaction of BNIP3 with sarcomeric and cytoskeletal proteins and/or modulation of their function by post-translational phosphorylation. The major effects of BNIP3 are largely summarized in the schematic drawing A,B, and Our data suggest that glycolysis, FA metabolism, and mt-metabolic pathways  were all downregulated in HFrEF, as previously described ,25,26,27C. elegans was found to control mt-morphology and to directly activate the mt-apoptotic program by sensing the metabolic status of mitochondria [Additional to its direct interaction with and modulation of mt-metabolic pathways, BNIP3 interacted with and its knockdown in HFrEF improved the expression of the mt-carrier proteins SLC25A3, SLC25A11, and attenuated p-S46-SLC25A4, with directional changes towards Sham. These mt-carrier proteins are also known to affect mt-energetics and metabolism by shuffling phosphate, 2-oxoglutarate/malate, and ADP/ATP across the inner mitochondrial membrane, respectively. Recent work has shown that loss of SLC25A3 function induced remodeling of the cardiac mt-protein acylome . The loschondria . The effchondria ,31. The chondria . Furtherchondria ,34,35. BPrevious work has shown multiple effects of BNIP3 on mitochondria, ranging from the downregulation of the nuclear and mt-encoded ETC complexes to the regulation of mitophagy and apoptosis ,37,38,39Previous work has shown that BNIP3 regulates ER–mt-calcium homeostasis by promoting the dimerization of the VDAC channels . In thisThe mt-sodium/calcium exchanger (NCLX) is essential for the regulation of mt-calcium homeostasis and efflux, and cellular viability, as previously shown ,45. The Our study shows an important role of BNIP3 in regulating cellular signaling that modulates mt-function and calcium cycling in the heart. The cAMP-dependent protein kinase catalytic subunit alpha, PRKACA, and its regulatory subunit, PRKAR2A, were identified as part of the BNIP3 interactome. BNIP3 knockdown improved p-S339-PRKACA, i.e., enhanced its activity, as previously shown , and imp2 and attenuated p-S485/491-AMPKα, with directional changes towards Sham. Although the effect of AMPKα2 phosphorylation at Ser377 is not known, its phosphorylation at Ser485/491 exerts an inhibitory effect in the heart, as previously shown [BNIP3 knockdown in HFrEF improved AMPKα signaling, and that of its downstream effector PGC-1α, both of which were downregulated in HFrEF. BNIP3 knockdown enhanced p-S377-AMPKαly shown . AMPK plly shown  and metaly shown ,70. Simily shown , has beely shown ,73,74, aly shown . Our finly shown . In neurly shown , calciumly shown , and chaly shown . AnotherWhile the effects of BNIP3 on the regulation of mt-function and metabolism, calcium homeostasis, and signaling at the ER–mt interface, were explicit and coherent, the mechanistic insights by which BNIP3 affects ER stress and UPR, sarcomeric and cytoskeletal proteins, and cellular transcription and translation, are less clear. BNIP3 interacted with the chaperone protein HSP90B1 and the disulfide isomerase protein, P4HB, which were upregulated in HFrEF and are upregulated under conditions of ER-stress-induced UPR and ER-associated degradation (ERAD). The transcription factor X-box-binding protein 1 (XBP1), which regulates the UPR during ER stress, was also activated in HFrEF vs. Sham. BNIP3 knockdown in HFrEF attenuated their expression/activity, thereby suggesting an improvement in ER stress. This is likely a secondary effect of BNIP3-mediated restoration of ER calcium content and calcium homeostasis ,81, rathIn summary, BNIP3 modulates important pathobiological processes involved in myocardial remodeling and the pathogenesis of HFrEF. We show important molecular mechanisms by which BNIP3 modulates ER–mt signaling, calcium homeostasis, and metabolic remodeling. Our findings also provide suggestions of BNIP3′s role in the regulation of cellular transcription and translation, sarcomere function, and myocardial cytoskeleton. We believe that the potential ability to target BNIP3, and to reverse multiple pathobiological processes involved in the pathogenesis of HFrEF, is a novel finding. This makes BNIP3 an attractive molecular target for the treatment of HFrEF. Our results also provide a framework for future mechanistic investigation and potential therapeutic interventions. We acknowledge the fact that our study has limitations in terms of addressing all the signaling pathways and mechanisms related to BNIP3 that were not elucidated in the bioinformatics analysis, such as BNIP3 interaction with Parkin for the induction of mitophagy. Moreover, the presented work does not differentiate which of the findings are a direct effect of BNIP3 or the consequence of improvement in mitochondrial function and signaling. A more in depth mechanistic work is warranted to address this issue.


Dietary DHA of 0.42% was beneficial to the growth of common carp fed plant-derived diets;Dietary DHA ameliorated the muscular fatty acid nutrition, and textural firmness of common carp fed plant-derived diets; mstna expression was decreased by dietary DHA;Myogenic regulatory factors expression was significantly elevated, while Dietary DHA modification to the muscular textural firmness of common carp may be attributed to the inhibition of FoxO1 pathwaysCyprinus carpio L.), three plant-derived diets with varying DHA levels  were prepared to feed juveniles  for 8 weeks, and the muscular texture, fibers density, and transcriptome were analyzed. The results showed that the growth performance, muscular DHA content, fibers density, and texture of the fish fed diets D2 and D3 were significantly ameliorated compared with the fish fed diet D1. The muscular transcriptome profiles indicated that the up-regulated genes of fish fed dietary DHA mainly in response to muscle proliferation, as well as the FoxO pathway, were significantly enriched in the D2 and D3 groups. Consistent with this, the Quantitative Real-Time PCR (qRT-PCR ) assays indicated that the expression of myogenic regulatory factors  was up-regulated in the high-DHA groups. Additionally, the expression of foxo1 (inhibitor of myofiber development) mRNA was down-regulated, while its negative regulatory pathway (MAPK and PI3K) was activated in the D2 and D3 groups. The results suggested that the DHA supplementation is beneficial to modifying the muscular textural firmness of common carp fed plant-derived diets, which could be attributed to the inhibition of FoxO1 pathways.Omega-3 fatty acids have a positive effect on the muscle textural firmness of fish, while the intrinsic mechanism is poorly understood. To investigate the potential mechanism of textural modification caused by dietary docosahexaenoic acid ( DHA) in common carp ( In recent years, with the increase of plant feedstuffs in the aquafeeds, the muscle quality, such as n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) contents and textural firmness, was impaired in cultured freshwater fish ,2. AlthoMyod and Myog genes in the skeletal muscle of rats [The muscle textural firmness of fish is closely associated with the myofiber number and size, which are determined by muscle fiber development . The con of rats . Additio of rats . Moreove of rats . These mCyprinus carpio L.) is characterized by high productivity, low feed inputs, and suitabability for multiple culture patterns, having a long history of being cultured in China [Common carp . Using soybean meal, cottonseed meal, and rapeseed meal as the main protein sources, and soybean oil, DHA purified oil (contains 83.15% DHA) as the lipid sources, three diets (D1–D3) containing isonitrogenous (31%) and isolipidic contents (7%) were formulated. Diets D1–D3 were supplemented with DHA purified oil at 0% (control diets), 0.5%, and 1.0%, respectively. The preparation of diets was carried out, as we previously reported in detail [Common carp juveniles  were provided by local fisheries . Before the rearing experiment, all fish were fed the control diets for 2-week to acclimatize them to the feeding conditions. Then, 360 healthy fish  were randomly assigned to 9 cylindrical aquaria with a diameter and depth of 1 m and 40 fish per aquaria. During the 8-week trial, fish were fed to satiation 3 times a day , half of the water in the aquaria was changed daily (6:30 p.m.), the water temperature was kept at 25 ± 2 °C, and oxygen saturation was maintained by aeration. Meanwhile, the photoperiod was maintained at 12 h light/12 h dark.After the 8-week trial, all fish were fasted for 24 h and anesthetized with MS-222 (10 mg/L) before sampling. To analyze the growth performance, all fish in each tank were individually weighed. Blood, muscle, and viscera samples were randomly collected from nine fish per aquaria (27 fish per group). Particularly, the blood was sampled from the caudal vasculature and stood for 30 min at 4 °C before being centrifuged for 10 min at 4 °C, 3500 rpm, and then the serum was rapidly frozen and stored at −80 °C until analysis. Then, the body length, as well as the body, splanchnic, and liver qualities were measured for evaluating the morphological parameters. The left side muscle was isolated from fish, and rapidly preserved at −80 °C until the transcriptome and gene expression analysis. The other side muscle was sampled for the detection of flesh quality parameters, histological fiber, and ultrastructure. In addition, the approximate composition of whole fish was detected by randomly collecting another fish per aquaria.Weight gain rate (WGR), feed conversion rate (FCR), and specific growth rate (SGR) were analyzed to evaluate the growth performance. The morphological parameter was evaluated by hepatosomatic index (HSI), viscerosomatic index (VSI), and condition factor (CF). These above parameters were assessed using standard formulations as previously reported .The approximate components of the whole fish and diets were measured through the assay method described in our previous study . BrieflyAccording to the common method, as previously reported , the lipThe serum samples were kept at a low temperature during testing. The serum biochemical indices, including total protein (TP), albumin (ALB), glucose (GLU), total cholesterol (CHO), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), free fatty acid (NEFA), alkaline phosphatase (AKP), acidic phosphatase (ACP), superoxide gasification enzyme (SOD), catalase (CAT), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px), were measured via the use of an enzyme-linked immunosorbent assay (ELISA) method. All these indicators were analyzed according to the detection scheme of mercantile reagent kits .Fish lipid quality/flesh lipid quality (FLQ), polyunsaturated fatty acid/saturated fatty acid ratio (PUFA/SFA), n-6/n-3 PUFA, index of thrombogenicity (IT), and hypocholesterolemic/hypercholesterolemic ratio (HH) are the indices for evaluating the muscle nutrition of fatty acids. The values of these above indices were calculated based on the data of FA contents in muscle using standard formulations, as previously reported .The muscular textural properties, like tenderness, hardness, resilience, chewiness, etc., were evaluated by a Texture Analyzer , with the specific parameters set according to our previous study . The musFor histological observation, the muscle samples were cut into 8-micron slices (8 μm) by a cryostat microtome, and cold-fixed in 10% buffered formalin for 10 min. Then, the sections were stained with modified oil-red O , followed by light microscopy observations . For ultp values, the method of Benjamini and Hochberg was adopted to calculate the false discovery rate (FDR) and the differently expressed genes were identified when FDR ≤ 0.05, |logFC| ≥ 1. These transcripts were considered to be down- or up-regulated when the log2 fold change was ≤1 or ≥1, respectively. In addition, with the use of Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the function of differently expressed genes (DEGs) was explored.The total RNA of the white muscle was isolated from three fish per tank and pooled into a mixture sample, and three samples per dietary group were prepared for transcriptome analysis. RNA Sequencing Library Preparation and Deep Sequencing were performed by a Biomedical Technology Company . The statistical analysis of transcript levels was assessed by the DESeq R software package. Based on the myog, myod, mrf4, mrf5, mstna, mstnb, pi3k, pdk, sgk, ras, raf, mapk, and foxo1, related to myofiber development and FoxO signaling pathways were detected by qPCR. RNA extraction, cDNA synthesis, and qPCR assays were executed as described previously [−ΔΔCT method, with the 18S rRNA as the internal control [To verify the accuracy of RNA-Seq, data were verified by qRT-PCR of RNA samples of the transcriptome. The expression of 13 differently expressed genes, including  control . The prip < 0.05.All data were subjected to one-way ANOVA and Duncan’s test by SPSS 22.0 , and expressed as mean ± standard error of means (SEM). When the difference between groups was significant, Tukey’s multiple comparisons test was executed and considered significant with p < 0.05), while no obvious differences were measured in FCR, HIS, VSI, and CF among the three groups (p > 0.05). Similarly, there were no significant differences in the proximate composition of the whole body among the three treatments (p > 0.05).As shown in p < 0.05) (p < 0.05), while dietary DHA had no significant influence on the serum TP, NEFA, and MDA contents, as well as the serum SOD, CAT, and GSH-Px activities (p > 0.05).Serum TG, LDL, HDL, and GLU levels, as well as AKP and ACP enzymatic activities, showed significant differences among the four dietary DHA treatments ( < 0.05) . Comparep < 0.05). Particularly, higher levels of muscular ALA, EPA, and DHA were observed in the fish treated with diets D2 and D3 (p < 0.05), and the n-3 PUFA contents were enhanced 1.37–1.99-fold compared with fish fed the D1 diet. While significantly low muscle LA and n-6 PUFA contents were detected in the D2 and D3 groups (p < 0.05). In the aspect of the nutritional indices of FA in muscle (p < 0.05).The FA profiles of muscle were shown in n muscle , the nutp > 0.05), while the muscle WHC was significantly ameliorated by dietary DHA (p < 0.05). Additionally, the gumminess of textural properties in muscle was elevated in the D2 and D3 dietary treatments compared with the D1 dietary treatment (p < 0.05) (p > 0.05).The muscular proximate composition, edible quality, and textural properties were shown in  < 0.05) . Althougp < 0.05) (p < 0.05) (p < 0.05).The histological characteristics of the muscle, including myofiber diameter and density, myofiber sarcomere length, and fillet lipid distribution, were shown in  < 0.05) A–D, wher < 0.05) A–C,E. ThTo explore the potential molecular mechanisms of changes in the myofiber caused by dietary DHA, the muscle transcriptome was analyzed. As shown in myod, mrf5, myog, mrf4), myosin, and mapk, were up-regulated in the muscle of D2 and D3 dietary treatments (p < 0.05), while 11 DEGs, including the mstn and RNA-binding protein 24, were down-regulated in the fish treated with D2 and D3 diets.The biological functions of DEGs were identified by the gene ontology (GO) enrichment analysis. As shown in egfr, grb2, sos, ras, raf, mek, and erk) and PI3K (pi3k and sgk) pathway components were up-regulated, and the AMPK pathway was down-regulated with the D2 and D3 dietary treatments. Additionally, the down-regulation of foxo was detected in the D3 treatment compared with that in the D1 treatment (p < 0.05).The top 15 enriched KEGG pathways between D1 versus D2 , D1 versp < 0.05), while, compared to the D1 group, 14 DEGs, including the FoxO pathways, were inhibited in the D2 and D3 groups (p < 0.05).In addition, 43 DEGs related to the FoxO signaling pathway also showed a distinct expression pattern among the different groups . Specifimyog, myod, mrf4, mrf5, mstna, mstnb, pi3k, pdk, sgk, raf, mek, erk, and foxo1) related to myofiber development and FoxO signaling were selected for qPCR confirmation. The consistency of RNA-Seq and qRT-PCR results data , MAPK and PI3K pathway components  in the muscle of D2 and D3 dietary treatments(high-DHA contents) were higher (p < 0.05), while the foxo1 and mstna mRNA levels in the muscle of D2 and D3 dietary treatments were lower (p < 0.05), and the mstnb mRNA displayed similar levels to those found among the three treatment groups.To verify the reliability of the transcriptome profile, 13 key genes (lts data  indicateCtenopharyngodon idellus) found that the WGR, SGR, feed efficiency (FE), and protein efficiency were improved when the dietary LC-PUFAs content increased from 0% to 0.52% [Megalobrama amblycephala) [Carassius auratus gibelio) [Oreochromis niloticus) [Micropterus salmoides) [Like other freshwater fish species, common carp can synthesize LC-PUFA, using C18 PUFA as a substrate (like LA and ALA) through a cascade of fatty acid desaturase (Fads) and elongase of very-long-chain fatty acids (Elovls). Thus, its essential fatty acids (EFA) are LA and ALA . While ito 0.52% . While icephala) , gibel cgibelio) , Nile tiloticus) , and larlmoides) . This inCyprinus carpio var. specularis), golden pompano (Trachinotus ovatus), California Yellowtail , and hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatu) [Channa argus), and pirarucu (Arapaima gigas) were attenuated by the plant-derived diets [In addition to ameliorating the growth performance, dietary n-3 LC-PUFA also had a positive influence on the lipid metabolism of fish ,24. Receceolatu) ,24,25,26ed diets ,28. In ted diets ,29.Schizochytrium sp. (rich in DHA) level [Sparidentex hasta) were significantly improved in the FO dietary treatment [Sparus aurata) fed with the plant oil (VO) diets showed lower n-3/n-6 ratio, FLQ, and AI than those given FO dietary treatment. These results indicate that the muscular FA nutritional value of fish can be significantly elevated by the supplementation of n-3 LC-PUFA.Compared to the EFA requirement of growth and health, maintaining fatty acid nutrition needs more EFA in cultured fish . In our A) level . Additioreatment . Álvarezreatment  indicateSolea senegalensis), the fiber density in the VO dietary treatment was decreased, while the fast-twitch fiber diameter and dorsal muscle cross-sectional area were increased, compared with fish of FO dietary treatment (high n-3 LC-PUFA contents) [In addition to the muscle FA nutrition, the myofiber structure and muscle texture characteristics were also ameliorated by dietary DHA. In this study, the myofiber density and sarcomere length of common carp in D2 and D3 treatments were significantly increased, while the fiber diameter was significantly decreased. These results are in line with the finding of the study of blunt snout bream, which indicated that high dietary DHA levels (0.8%) enhance the fiber density and sarcomere lengths . In the ontents) . These rontents) .Dicentrarchus labrax L.), the cultured fish species has low muscle DHA levels, and its myofiber density and textural firmness are significantly inferior to those of wild fish species [Previous studies found that fiber density is correlated with muscle hardness, springiness, chewiness, and gumminess ,34. In bmyog and mrf4 expression and increases the myofiber development of blunt snout bream. In our study, the expressions of myod, myf5, mrf4, and myog were up-regulated in the muscle of DHA dietary treatments (D2 and D3 groups), while the negative factor mstna was decreased. In mammals, the effects of DHA treatment on the differentiated C2C12 myoblasts also showed an up-regulation of mrf4, myog, and myod mRNA expression levels, and an increase in myogenic differentiation [Myoblast proliferation and differentiation contribute to the hyperplastic growth and hypertrophic growth of fish, which is regulated by a series of positive factors  and negative factors (Mstn) ,7. Myod ntiation . The resmyod and its promoter activity, and increased Mstn expression [foxo1 was decreased in the muscle of common carp fed diets supplemented with DHA. Accordingly, high myogenic factor expression and low mstna expression were detected in dietary DHA groups (D2-D3). In line with these results, the diets supplemented with DHA had a decrease in FoxO1 expression in the adipose and liver of weaned cross-bred pigs, and DHA treatments also reduced the activity of FoxO1 promoter in humans [foxo1 mRNA abundance, and increased muscle protein mass. These studies suggested that dietary DHA negatively mediates FoxO1 to influence the expression of myogenic regulatory factors.Besides myogenic factors, the proliferation and differentiation of myofibers can be regulated by the upstream transcription factors, such as FoxO1 . A seriepression . In thisn humans . Liu et n humans  reportedpi3k, pdk, and sgk) and MAPK  were evaluated in the common carp fed high-DHA diets (D2 and D3). These discoveries are consistent with a previous mammal study that reported that n-3 LC-PUFA induces airway smooth muscle cell proliferation through PI3K and MAPK pathways [foxo1 expression level by activating the PI3K and MAPK pathways, which supplementation of DHA modified the muscle hyperplastic growth and textural firmness of the common carp fed plant-derived diets. Moreover, FoxO activation is also regulated by a variety of upstream signaling pathways, which includes the inactivation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways ,39,40. Ipathways . The resIn conclusion, given the results of growth performance, muscular fatty acid nutrition, and textural properties, the present study demonstrated that dietary DHA can modify the growth and textural firmness of common carp fed plant-derived diets. Moreover, the dietary DHA is beneficial for the textural firmness of common carp by inducing muscle hyperplastic growth, which could be attributed to the activation of PI3K and MAPK pathways, and the inactivation of the inhibitor of myofiber development—FoxO1. These results provide new insights for understanding the potential mechanisms of DHA supplementation to improve the textural firmness of common carp fed plant-derived diets.


The protein expression of IKK-α and IKK-β, as inhibitors of NF-KB which exhibit a critical function in the regulation of genes involved in chronic inflammatory disorders, were studied in the tested cancerous cell lines. In this study, the anticancer activity of household-processed fenugreek leaves and seeds against HepG2, HCT-116, MCF-7, and VERO cell lines was measured using an MTT assay. DNA fragmentation of both HepG2 and MCF-7 was investigated by using gel electrophoresis. RT-PCR was used to evaluate the relative expression of each p53, caspase-3, Bax, and Bcl-2 genes, whereas ELISA assay determined the expression of caspase-3, TNF-α, and 8-OHDG genes. Western blotting analyzed the protein-expressing levels of IKK-α and IKK-β proteins in each studied cell line. Data showed that at 500 µg mL−1, ADFL had the highest cytotoxicity against the HepG2 and HCT-116 cell lines. Although, each UFS and GFS sample had a more inhibitory effect on MCF-7 cells than ADFL. Gel electrophoresis demonstrated that the IC50 of each ADFL, UFS, and GFS sample induced DNA fragmentation in HepG2 and MCF-7, contrary to untreated cell lines. Gene expression using RT-PCR showed that IC50 doses of each sample induced apoptosis through the up-regulation of the p53, caspase-3, and Bax genes and the down-regulation of the Bcl-2 gene in each studied cell line. The relative expression of TNF-α, 8-OHDG, and caspase-3 genes of each HepG2 and MCF-7 cell line using ELISA assays demonstrated that ADFL, UFS, and GFS samples reduced the expression of TNF-α and 8-OHDG genes but increased the expression of the caspase-3 gene. Protein-expressing levels of IKK-α and IKK-β proteins in each studied cell line, determined using Western blotting, indicated that household treatments decreased IKK-α expression compared to the UFS sample. Moreover, the ADFL and SFS samples had the most activity in the IKK-β expression levels. Among all studied samples, air-dried fenugreek leaves and unprocessed and germinated fenugreek seeds had the most anti-proliferative and apoptotic-inducing properties against human HepG2, MCF-7, and HCT-116 cell lines, as compared to the VERO cell line. So, these crude extracts can be used in the future for developing new effective natural drugs for the treatment of hepatocellular, breast, and colon carcinomas.Household processing of fenugreek seeds and leaves, including soaking, germination, and boiling of the seeds, and air-drying of the leaves, has improved the levels of human consumption of the bitter seeds and increased the shelf life of fresh leaves, respectively. The potential anticancer activity of either unprocessed or processed fenugreek seeds or leaves and the relative expression of pro-apoptotic and anti-apoptotic genes of the studied cancerous cell lines exposed to IC Cancer remains an aggressive cause of death on a global scale despite significant efforts. The chemotherapeutic agents currently in use have negative side effects and have not performed as expected. Medicinal herbs have been used for ages to cure a variety of illnesses and are also known to have anticancer properties .Tumor chemoprevention is a procedure used to decelerate, degenerate, or prevent the cross-mechanism of tumorigenesis, including blocking its important morphogenetic stages, such as preneoplasia, neoplasia, and metastasis. Alternatives such as naturally occurring phytochemicals found in food are increasingly preferred over manufactured medications for a number of reasons, including less (or no) toxicity or side effects, improved availability, and safety. Programmed cell death (PCD)-type I (apoptosis), PCD-type II (autophagic cell death), and necrosis are some of the mechanisms involved in cell death produced by plant products . A complex biological process called apoptosis is crucial for removing damaged cells from living things. A type of controlled cell death known as apoptosis is characterized by distinctive morphological and biochemical alterations in the cells. The apoptotic cascade is fueled by a variety of proteins. They can be assessed utilizing common analytical procedures based on protein detection. Caspases are enzymes , and their activity can be used to determine the stage and rate of apoptosis, particularly when they are cleaving the peptide bonds of the right substrate. In humans, there are eleven isoenzymes of caspases, seven of which  are involved in apoptosis .The I kappa B protein kinase complex, often known as IKK, is a 700 kDa protein that can phosphorylate the conserved N-terminal serine residues of IκB-α and IκB-β and is triggered by inflammatory signals  . The IKKTrigonella foenum-graecum, is a traditional herbal remedy used for the treatment of diabetes, high cholesterol, wounds, inflammation, digestive problems, etc. It is also thought to have anti-tumor characteristics; however, the exact mechanisms for this activity are still unclear [Fenugreek, or  unclear . Numerou unclear .50 doses of ethanolic crude extracts of each tested sample. Finally, a Western blot assay was used to measure the protein-expressing levels of both IKK-α and IKK-β protein kinases in each cancerous and normal cell line exposed to the IC50 doses of the studied samples.The aim of this work is to evaluate the anti-proliferative activity of air-dried fenugreek leaves (ADFL), unprocessed fenugreek seeds (UFS), germinated fenugreek seeds (GFS), soaked fenugreek seeds (SFS), and boiled fenugreek seeds (BFS) against three cancerous cells from hepatic (HepG2), colon (HCT-116), and breast (MCF-7) cancer cell lines in comparison to normal kidney cell lines (VERO) using MTT assays. AGE, comet assay, RT-PCR, and ELISA methods were used to study the ability of each studied sample on DNA damage and regulation of pro- and anti-apoptotic genes, by exposing p53- caspase-3, Bax, and Bcl-2 of HepG2, HCT-116, MCF-7, and VERO cells to ICAll RPMI 1640 culture media, glutamine, FBS, penicillin, streptomycin, trypsin, primers for p53, caspase-3, Bcl-2, Bax, GAPDH, solvents, dyes, and chemicals were purchased from Sigma-Aldrich, St. Louis, MO, USA. Primary antibodies included TNF-α, 8-OHDG, Cas-3, GAPDH, IKK-α, and IKK-β, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, . Western blot kits were purchased from Amersham, Buckinghamshire, UK.All cell lines were obtained from the American Type Culture Collection . Anti-proliferative activity was investigated in vitro against three human cancerous cell lines, including the human hepatic (HepG2), breast (MCF-7), and colon (HCT-116) carcinoma cell lines, and normal human kidney cell line (VERO), and determined by the Bioassay—Cell Culture Laboratory, National Research Centre. All cell lines were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% FBS, and 1% penicillin/streptomycin. Then, sub-confluent cultures (70–80%) were trypsinized (trypsin 0.05%/0.53 mM EDTA) and split depending on the seeding ratio.Trigonella foenum-graecum Linn.) were purchased from the local market in Egypt in the month of December 2019 and identified at the Faculty of Science, Cairo University. The leaves were washed thoroughly with tap water, and the surface water was removed by air-drying under shade for 15 days. Leaves were subsequently dried in a hot air oven at 50 °C for 4 h, homogenized to a fine powder, and then stored at 4 °C.Fresh and healthy leaves of fenugreek ((a)Untreated Fenugreek Seeds (UFS)Two kilograms of fenugreek seeds were purchased from the local market in Egypt, identified at the Faculty of Science, Cairo University, and divided into four groups as follows:Five hundred grams of seeds were cleaned manually to remove dust and foreign particles, crushed into a fine powder flour with the help of Moulinex blender LM 241, and sieved through a 0.5 mm mesh size. The powdered flour was kept at 4 °C to prevent changes until further analysis .(b)Soaked Fenugreek Seeds (SFS)w/v) for 12 h at room temperature. After removing the soaking water, the seeds were air-dried in shade for 5 days, followed by hot air oven-drying at 50 °C for 4 h in a conventional oven, and stored at 4 °C [Five hundred grams of untreated seeds were soaked in tap water at the ratio of 1:5 ((c)Germinated Fenugreek Seeds (GFS)w/v) for 12 h at room temperature. After removing the soaking water, seeds were kept in the dark for germination (tied in a cotton cloth) at 20 °C for 60 h in darkness. After harvesting the sprouts, they were air-dried for 5 days, followed by hot air oven-drying at 50 °C for 4 h in a conventional oven, and stored at 4 °C [Five hundred grams of untreated seeds were soaked in tap water at the ratio of 1:5 ((d)Boiled Fenugreek Seeds (BFS)2O (1:5 w/v). The sample was boiled on a hot plate for 10 min, and then, after the water was discarded, the boiled seeds were air-dried in shade for 5 days, followed by hot air oven-drying at 50 °C for 4 h in a conventional oven, and stored at 4 °C [Five hundred grams of untreated seeds were put in 2 L beaker containing 1250 mL tap H at 4 °C .w/v) 80% ethanol in distilled H2O through sonication for 60 min. Extraction was repeated three times. After filtration, an extract of each sample was condensed until dry (resulting in crude extracts) by use of a rotary evaporator at 40 °C. A known weight of each crude extract was dissolved in appropriate volume of dimethyl sulfoxide (DMSO) and stored at −80 °C until further analysis [One hundred grams of each sample were extracted with 1 L -2,5-diphenyltetrazolium bromide (MTT) assay, according to Mosmann .The qualitative method for assessing cell death by detecting DNA fragments using AGE was performed according to Kasibhatla et al. .50 of each studied sample. The method of Kent et al.  was foll50 doses of the tested samples for 24 h, according to Robert [®cycler software 2.1  to calculate the threshold cycle (Ct) using the second derivative maximum. The fold change value for each gene was determined after normalization to the expression levels of GAPDH as an HK gene, which was calculated using the equation 2−∆∆Ct.Gene expression of pro-apoptotic genes  and Bax, an anti-apoptotic gene, were tested against HepG2, HCT-116, MCF-7, and VERO cells exposed to ICo Robert . Raw dat50 of crude extracts of each studied sample was seeded at a density of 2 × 104/well in a 96-well plate and incubated for 24 h. Absorbance was measured using an ELISA reader . Determination was performed in triplicate, and standard deviation was determined.Quantitative analysis of TNF-α, 8-OHDG, and Cas-3 antibodies using ELISA technique was investigated according to the method of Khalil . Each ceThe Western blot for IKK-α and IKK-β primary antibodies was performed according to the method of Morgia . The exp50 was calculated by using GraphPad prism 7. One-way analysis of variance (ANOVA) was used to analyze the difference between groups by applying the least significant difference (LSD) test with a 5% level of significance (p < 0.05).All assays used in this work were evaluated in triplicate, and the data obtained were represented by the mean ± standard deviation (SD). Statistical Analysis Software (SAS 9.1) was applied for the statistical analysis of data, and IC−1. In contrast, the household processing of fenugreek seeds at 1000 µg mL−1 caused a significant decrease in the cell proliferation of the HepG2 cells, and BFS had the lowest cytotoxicity (56%). The IC50 values were 220 µg mL−1 for ADFL and 320 µg mL−1 for UFS, although these values increased after household treatments. The impacts of household-processed fenugreek leaves and seeds on the cell proliferation of HepG2, HCT-116, MCF-7, and VERO cell lines exposed to serial concentrations of the tested samples are presented in −1 also had the highest anti-proliferative activity (95%) against the HCT-116 cell line, and the germinated (GFS), soaked (SFS), and boiled (BFS) fenugreek seeds reduced the activity compared to the untreated seeds (UFS). The IC50 values were 240 µg mL−1 for ADFL and 275 µg mL−1 for UFS, although these values increased after household treatments. Furthermore, the impact of the tested samples on the human colon cancer cell line (HCT-116) is represented in −1, both the untreated and germinated fenugreek seeds (UFS and GFS) had the highest potential cytotoxicity against the MCF-7 (95%), compared to the other tested samples. ADFL had 92% cytotoxicity at 1000 µg mL−1, SFS had 90% at 2000 µg mL−1, and BFS had 95% at 2000 µg mL−1. The IC50 values of both UFS and GFS were 500 and 620 µg mL−1, respectively. In addition, the soaking and boiling treatments of seeds at 1000 µg mL−1 caused a significant decline  in the cell proliferation of MCF-7. Protodioscin, a steroidal saponin of fenugreek, had an inhibitory impact on the leukemic cell line (HL-60) and a weak inhibitory effect on the growth of the gastric cell line (KATO-III) [On the contrary, as shown in 50 (µg mL−1) values of ADFL, UFS, and GFS were 1660, 1840, and 1940 µg mL−1, respectively. However, the soaking and boiling treatments had no effects on the normal VERO cell line.Finally, the effect of the tested samples on the cell proliferation of the normal kidney cell line (VERO) can be observed in 50 doses of each tested sample can be observed in Agarose gel electrophoresis (AGE) was used to evaluate the DNA fragmentation activity of the tested samples to indicate whether or not the action of the extract is associated with apoptosis. DNA fragmentation of the HepG2 and MCF-7 cancer cell lines treated with the IC50 doses of each studied sample was observed. The treatment of the HepG2 and MCF-7 cell lines with the IC50 doses of the household-processed fenugreek leaves and seeds  caused the fragmentation of DNA in each cancer cell line compared to the untreated control cell lines and the cells exposed to the IC50 of the BFS sample. Therefore, the air-dried fenugreek leaves (ADFL) and unprocessed and germinated fenugreek seeds  induced DNA fragmentation and, thus, apoptosis in both HepG2 and MCF-7 cancer cell lines.As shown in The DNA damage of the HepG2 and MCF-7 cancer cell lines was observed by using single-cell gel electrophoresis (SCGE) (Comet assay), which is able to detect DNA strand breaks (SBs) and oxidative DNA damage in cultured cell lines.50 doses of ADFL, UFS, GFS, SFS, and BFS, which were 13.01, 12.91, 11.41, 10.00, and 9.31%, respectively, for HepG2 cell lines, and were 12.27, 11.10, 9.89, 9.66, and 8.77%, respectively, for MCF-7 cell lines. As presented in 50 doses of the studied samples. The head diameters of the untreated HepG2 and MCF-7 cell lines were 17.61 and 17.15 px, respectively. The values of head diameters are also observable in 50 doses of the tested samples had a decrease in DNA % in the head , except in the BFS sample (84.01%), which was not significantly different compared to the control sample. The values were 76.01, 78.00, 79.00, 81.04, and 81.48% for the MCF-7 cell lines treated with ADFL, UFS, GFS, SFS, and BFS, respectively.The percentages of DNA values in the heads of the untreated HepG2 and MCF-7 cell lines (control) were 84.00 and 81.51%, respectively. In contrast, the HepG2 cell lines exposed to the IC50 of ADFL (4.00 px), UFS (3.94 px), and GFS (3.91 px), but was not significantly changed after treatment with either SFS (3.85) or BFS (3.81) samples. On the other hand, the tail length value of the untreated control cell line of MCF-7 was 3.80 px, and increased to 4.01, 3.98, 3.90, and 3.85 px after exposure to ADFL, UFS, GFS, and SFS, respectively, with the exception of the BFS sample (3.81 px).The data in Furthermore, the percentages of DNA in the head were found to be 22.17% for ADFL, 21.00% for UFS, 19.41% for GFS, 18.12% for SFS, and, lastly, 18.00% for BFS relative to the untreated control HepG2 cell lines. In addition, the untreated MCF-7 cell line (control) had a DNA% in the head of 18.20%, which was also raised to 23.21, 22.11, 20.00, and 18.41% for ADFL, UFS, GFS, and SFS, respectively, but did not vary with the BFS sample (18.21%). 50 of ADFL (1.10), UFS (1.07), GFS (1.01), and SFS (0.96), but not with the BFS sample (0.94). Lastly, the tail moment values of DNA of the untreated (control) HepG2 and MCF-7 cell lines were 1.02 and 0.93, respectively. These values were higher after the exposure of HepG2 cell lines to ADFL (1.16), UFS (1.14), GFS (1.10), and SFS (1.07), but did not differ from the BFS sample (1.01). The untreated control MCF-7 cell lines had a tail moment of 0.93 and increased after treatment with the IC50 doses of the selected sample, is summarized in The gene expression of the pro-apoptotic genes involved either p53, Caspase-3 (Cas-3), or Bax, as well as the anti-apoptotic Bcl-2 gene, against HepG2, HCT-116, MCF-7, and VERO cell lines treated with IC50 doses of ADFL, UFS, GFS, SFS, and BFS are compared to the untreated cell lines (control). The results showed that air-dried fenugreek leaves (ADFL) were the most potent sample, which significantly up-regulated p53, Cas-3, and Bax  and down-regulated Bcl-2 (at a 0.52 fold change) compared to untreated cell lines. However, the standard drug (doxorubicin) caused an up-regulation of the studied pro-apoptotic genes, as follows: 1.87 for p53, 3.82 for Cas-3, and 3.11 fold change for Bax genes, and a down-regulation of the Bcl-2 gene (at a 0.32 fold change). In addition, each of the UFS, GFS, SFS, and BFS samples significantly increased the up-regulation of p53 , Cas-3 , and Bax  genes relative to the untreated control cell lines. According to 50 doses of ADFL, UFS, GFS, SFS, and BFS compared to both the control cell line and the other samples that were treated with doxorubicin were also used to study the apoptosis-inducing property of the tested samples  exposed to IC samples B. Data s50 in each studied sample were compared to the untreated cell lines, and the other samples treated with doxorubicin were also used to evaluate the apoptotic-inducing activity of the tested samples  treated with IC samples C. Findin50 concentration of each studied sample was also used to study the apoptosis-inducing properties of the tested samples  exposed to the IC samples D. The da50 doses of household-processed fenugreek leaves and seeds involved ADFL, UFS, GFS, SFS, and BFS, which enhanced the up-regulation of the studied pro-apoptotic p53, Cas-3, and Bax genes and down-regulation of anti-apoptotic Bcl-2 genes, which induced apoptosis of the studied cell lines, then decreased the tumor growth and cell proliferation.According to these findings, we summarized that the treatment of hepatic, colon, and breast cancer cell lines exposed to IC−1 protein, respectively, for each HepG2 and MCF-7 cell lines treated with IC50 doses of ADFL, UFS, GFS, SFS, and BFS. This caused a significant decline up to 51 and 57%, 61 and 61%, 72 and 81%, 89 and 92%, and finally, 96 and 99%, respectively for the same tested samples. The control (untreated cell line) showed 25.03 and 23.00 pg mg−1 protein for each untreated HepG2 and MCF-7 cell line.Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine which is involved in immune function and is also proposed to play a role in metabolic disorders. The results presented in An 8-Hydroxy-2′-deoxyguanosine (8-OHDG) biomarker was evaluated to measure DNA damage. The results presented in The caspase-3 (Cas-3) factor is usually used to evaluate the apoptotic cascade. This biomarker factor was activated and increased after exposure to the HepG2 and MCF-7 cell lines to the studied samples, with the exception of the boiled fenugreek seeds (BFS). According to the presented data in To further confirm the results of the IKK-α and IKK-β expressions, the signaling pathway was further investigated through a Western blotting assay . 50 doses . Meanwhile, IKK-α expression levels in VERO cells were not detected in all studied samples, except for UFS (60.78%) and GFS (64.83%).As observable in 50 doses . Meanwhile, IKK-β expression levels were been high in HCT-116 cells treated with SFS (46.90%), GFS (45.57%), and ADFL (44.56%), respectively.In contrast, the results shown in The fenugreek plant has had an important role for a long time in the treatment and prevention of diseases. Various studies have also proved many of these conventional applications and have observed the beneficial value of this plant and its capabilities in traditional medicine. However, the scientific information about the evaluation of the mechanism of action of this plant is not enough .Finding naturally occurring chemo-preventive compounds has received more attention in recent years, especially those found in dietary and medicinal plants due to their bioactive components ,23. These findings are in agreement with Raju  and ThirThe majority of these bioactive chemicals inhibit cell cycle progression and cause apoptotic cell death to carry out their cancer-chemotherapeutic activities. As a result, the induction of apoptosis in tumor cells has evolved into a marker of the tumor treatment response when using a bioactive chemical derived from plants to lower and manage cancer-related mortality in humans ,27. TherIn this work, household processes were been involved in the air-drying of fenugreek leaves for preserving the leaves from spoilage resulting from the moisture content of fresh leaves, and soaking, germination, and boiling of raw fenugreek seeds to reduce the bitter taste, due to the presence of saponins, and specific smell, due to the presence of alkaloids and volatile oils, which limit their usability and acceptability in food industries. This study aimed to evaluate the anti-proliferative activity of air-dried fenugreek leaves (ADFL), unprocessed fenugreek seeds (UFS), germinated fenugreek seeds (GFS), soaked fenugreek seeds (SFS), and boiled fenugreek seeds (BFS) against three cancerous cells involved hepatic (HepG2), colon (HCT-116), and breast (MCF-7) cells in comparison to normal kidney cell lines by using an MTT assay.A typical genetic change linked to cancer is the mutation of the tumor suppressor p53, which inactivates 50% of human malignancies . A complDNA damage is one of the most well-known effects of oxidative stress. Biomarkers for nucleic acid oxidation are frequently utilized to evaluate this damage . The mosThrough the inhibition of Akt activity, diosgenin reduced TNF-induced NF-κB activation, IκB-α kinase activation, IκB-α phosphorylation, IκB degradation, p65 (RELA) phosphorylation, and p65 nuclear translocation. Diosgenin also inhibited the TNF-induced production of genes related to cell proliferation , anti-apoptosis , and invasion -9). Additionally, TNF and chemotherapeutic medicines such as paclitaxel and doxorubicin that promote apoptosis were potentiated by diosgenin .The primary public health issue across the globe is cancer. The identification of naturally occurring chemo-preventive compounds has recently received increased interest, particularly those found in dietary and medicinal plants because of their bioactive components. The majority of these bioactive chemicals inhibit cell cycle progression and cause apoptotic cell death to carry out their cancer-chemotherapeutic activities. As a result, the induction of apoptosis in tumor cells has evolved into a marker of the tumor treatment response when using a bioactive substance produced from plants to lower and manage cancer-related mortality in humans. When compared to the VERO cell line, air-dried fenugreek leaves, raw fenugreek seeds, and germinated fenugreek seeds had the most anti-proliferative and apoptosis-inducing effects on each of the human HepG2, MCF-7, and HCT-116 cell lines. So, these crude extracts can be used in the future for developing new effective natural drugs for the treatment of hepatocellular, breast, and colon carcinomas.


Malus hupehensis seedlings. Results showed that treatment with 100 μM exogenous SL analog (GR24) could effectively alleviate salinity-alkalinity stress with higher chlorophyll content and photosynthetic rate than the apple seedlings without GR24 treatment. The mechanism was also explored: First, exogenous GR24 regulated the expression of Na+/K+ transporter genes and decreased the ratio of Na+/K+ in the cytoplasm to maintain ion homeostasis. Second, exogenous GR24 increased the enzyme activities of superoxide, peroxidase and catalase, thereby eliminating reactive oxygen species production. Third, exogenous GR24 alleviated the high pH stress by regulating the expression of H+-ATPase genes and inducing the production of organic acid. Last, exogenous GR24 application increased endogenous acetic acid, abscisic acid, zeatin riboside, and GA3 contents for co-responding to salinity-alkalinity stress indirectly. This study will provide important theoretical basis for analyzing the mechanism of exogenous GR24 in improving salinity-alkalinity tolerance of apple.Salinity-alkalinity stress can remarkably affect the growth and yield of apple. Strigolactone (SL) is a class of carotenoid-derived compounds that functions in stress tolerance. However, the effects and mechanism of exogenous SL on the salinity-alkalinity tolerance of apple seedlings remain unclear. Here, we assessed the effect of SL on the salinity-alkalinity stress response of  Malus domestica) is a highly valued and widely cultivated fruit around the world , hydrogen peroxide (H2O2), and singlet oxygen, result in oxidative stress that can lead to plant cell membrane permeability increasing and ion leakage, which may impede plant development , as a class of carotenoid-derived compounds, is essential in regulating numerous aspects of plant development . The enzMalus hupehensis, one of the important rootstocks in apple culture, under salinity-alkalinity stress. Different concentrations of exogenous GR24 were applied on M. hupehensis seedlings under salinity-alkalinity stress, the positive regulation of GR24 was evaluated in terms of the photosynthetic system, oxidative damage, osmotic balance, and ion homeostasis. The expression levels of ion transporter genes, key SL signaling pathway genes, and SL biosynthesis genes under GR24 treatments were also determined. This study helped clarify the regulatory mechanism of SL in apple plants under salinity-alkalinity stress and provided a new way to improve salinity-alkalinity tolerance in apple production.In the present study, we explored the functions of exogenous GR24 in M. hupehensis (an apple rootstock with apomixis characteristics) were sown in nutrient soil and grown in a greenhouse under controlled temperature (25 ± 2°C), photoperiod (16/8 h day/night), humidity (60–65%), and light intensity (100 μmol/m2/s). After one-month-old, when the seedlings developed into to four leaves, they were transplanted into a plastic pot and irrigated with Hoagland solution every 3 days. Ten days later, seedlings with similar growth status were selected for subsequent saline-alkali stress and exogenous GR24 treatment.After low-temperature vernalization, the seeds of M. hupehensis seedlings were randomly divided into five groups. The seedlings in group I were watered with a complete nutrient solution as the control, group II were treated by 100 mM NaHCO3 and NaCl with concentration ratio of 1:1. On the basic of group II, groups III-V were treated with the 10, 100, and 1,000 μM of exogenous GR24, respectively. GR24  was sprayed every 3 days. After 15 days of treatment, the seedlings were photographed, and the wilting rate, fresh weight, and dry weight were measured. Both the technical and biological duplications of each experiment were repeated thrice.A total of 200 2/s at an approximately 50% humidity, and the temperature was set at 22°C. 2,3,5-triphenyltetrazolium chloride (TTC) method was applied for qualitatively and quantitatively assess the root activity according to After 15 days of saline-alkali stress and exogenous GR24 treatment, 20 apple seedlings from each group were randomly selected to determine the chlorophyll content and basic photosynthetic parameters. Four leaves of each seedlings were measured. Under light condition, the chlorophyll content was measured using SPAD-502 Plus . The photosynthesis rate, transpiration rate, and stomatal conductance were measured using the CIRAS-3 portable photosynthetic system . The light intensity was controlled at 800 μmol/m2O2 and O2–, respectively. The H2O2 level was measured using H2O2 kits . The MDA content of the leaves was measured using a plant MDA extraction kit . Three biological duplications for each experiment were set.Nitroblue tetrazolium and 3,3-diaminobenzidine were used to stain HFresh leaves (0.5 g) were ground in 5 ml of extracted buffer after saline-alkali and exogenous GR24 treatment for 15 days. After centrifugation at 12,000 rpm for 10 min, the supernatants were immediately used for SOD, POD, and CAT content assay. SOD, POD, and CAT kits  were used to detect the activities of antioxidant enzymes according to the manufacturer’s instructions. The malic acid and citric acid content of apple leaves were measured using Malic acid assay Kit and Citric acid assay Kit , respectively. Each experiment was repeated thrice.After saline-alkali and exogenous GR24 treatment for 15 days, fresh leaves (0.5 g) from each group were used for the detection of electrolyte leakage and osmolytes. Electrolyte leakage was measured as described by 3 and HClO4 with the ratio of 5:1. After digestion, the solution was diluted with deionized water to 25 ml. The concentrations of sodium (Na), potassium (K), calcium (Ca), iron (Fe), magnesium (Mg), and phosphorus (P) were determined through inductively coupled plasma-optical emission spectrometry  as described by The apple seedlings were collected and washed with deionized water to remove the excess impurities after 15 days of saline-alkali stress and GR24 treatment. The leaves were dehydrated at 105°C for 30 min and baked at 80°C for 72 h. Afterward, 0.5 g of kiln-dried leaves was ground into powder and added with 12 ml of HNOvia electrospray ionization-high-performance liquid chromatography-tandem mass spectrometry, as described by After saline-alkali and exogenous GR24 treatment for 15 days, the endogenous acetic acid (IAA), gibberellin3 (GA3), zeatin riboside (ZR), and jasmonic acid (JA) concentrations were determined. Fresh leaves (0.5 g) were prepared for phytohormone extractions, and hormonal analysis and quantification were performed ® 480 II system  was used for the qPCR assay, and the primers are listed in M. hupehensis actin gene (GenBank accession number GQ339778.1) was used to normalize gene expression levels. Data were analyzed using the 2–ΔΔCt method , which includes RNase-free DNase treatment. Total RNA was adjusted to the same concentration for cDNA synthesis by using 5 × All-In-One RT MasterMix  according to the manufacturer’s instructions. LightCyclert method . All qRTt-test analysis. Statistically significant differences were indicated by P < 0.05. Statistical computations were conducted by using SPSS software .Data were subjected to ANOVA followed by Fisher’s LSD or Student’s The apple seedlings were seriously damaged by saline-alkali stress, and the leaves became withered and chlorotic after 15 days. After the treatment of low (10 μM) and high (1 mM) concentrations of GR24, the growth vigor of the seedlings was much better than those without GR24 treatment, but the leaves remained withered and chlorotic. At the low (10 μM) and the high (1 mM) concentrations, the wilting rates of the seedlings substantially decreased from 73.3% to 45% and 48.3%, respectively , and the2/s to 4 μmol/m2/s but recovered to 11.5 μmol/m2/s when exogenous GR24 was applied  and H2O2 revealed that the leaves of apple seedlings were seriously damaged by saline-alkali stress  was more than 1.6 times that of the control group (1.6 nmol/g), but was significantly decreased to 2 nmol/g after exogenous GR24 was applied  enzyme family genes to 2.57, 8.36, and 4.36 times, respectively. The expression levels of antioxidant enzyme genes MhGPX6, MhPER65, MhpOXN1, were significantly induced by salinity-alkalinity stress and were substantially decreased after exogenous GR24 treatment. However, the expression levels of MhSOD, MhPOD and MhCAT were significantly decreased under the salinity-alkalinity stress, while exogenous GR24 significantly upregulated the expression levels of MhSOD, MhPOD and MhCAT to 1.36, 1.93 and 2.47 times, respectively. Moreover, the expression of three kinases, namely, MhANP2, MhMAPKKK, and MhGK, and three selected transcription factors, namely, MhMYB39, MhERF019, and MhNAC56, remarkably changed under salinity-alkalinity stress and exogenous GR24 treatment  and three DWARF14 genes . The expression of MhD14-1 was decreased by salinity-alkalinity stress. The transcription levels of MhMAX2 and MhD53 were increased by salinity-alkalinity stress. However, all of the four genes were induced by exogenous GR24 treatment (MhCYP711), two carotenoid cleavage dixoygenase genes (MhCCD7 and MhCCD8), and a 9-cis/all-trans-β-carotene isomerase gene (MhD27), were also quantified by qPCR. The results showed that the expression levels of these four genes were decreased by salinity-alkalinity stress but remarkably induced by exogenous GR24 treatment  under salt stress. Plants have evolved some important protein, which may protect themselves and reduce the poisoning of Na+, such as the cation/H+ exchangers, salt overly sensitive l (SOS1), and cation exchangers, which expel Na+ from cells. Our results showed that MhCHX15, MhSOS1, and MhCAX5 expression levels were increased by exogenous GR24 treatment under salinity-alkalinity stress  is responsible for K+ efflux from the cytoplasm to the outside of the cell (+/H+ antiporters (NHX) in the tonoplast facilitate K+ influx and efflux in the vacuoles  were substantially affected by exogenous GR24, and the tendencies of MhSOD, MhPOD and MhCAT expression levels were correlated with SOD, POD and CAT activities  and cucumber (Cucumis sativus)  . In the t stress . Here, wMhD14-1, MhD14-3, MhMAX2, and MhD53 were substantially induced by exogenous GR24 treatment  signaling pathway enzymes include SL receptor D14, transcriptional repressor protein D53/MXL6/7/8, and F-box protein D3/MAX2 . The trareatment . Similart stress . Our resreatment . Therefo+/K+ transporter genes, eliminate ROS through enhancing the activities of SOD, POD, and CAT, regulate osmotic balance by increasing the proline content, balance root pH through secretion of organic acid, and cooperate with IAA, GA3, ZR, JA responding to saline-alkali stress (Our study explored that strigolactones could effectively improve the tolerance on salinity-alkalinity stress in apple. Exogenous GR24 could affect ion homeostasis by regulating Nai stress . This woThe original contributions presented in this study are included in the article/XZ and CW planned and designed the research. CB, CM, WL, XX, XL, DG, ZS, and YT performed the experiments, conducted the fieldwork, and analyzed the data. XZ and CM wrote the manuscript. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Choledochal cysts (CC) is characterized by extra- and/or intra-hepatic b\ile duct dilations. There are two main theories, “pancreaticobiliary maljunction” and “congenital stenosis of bile ducts” proposed for the pathogenesis of CC. Although family cases or CC associated with other anomalies have been reported, the molecular pathogenesis of CC is still poorly understood. Recent advances in transcriptomics and genomics analysis platforms have unveiled key expression signatures/genes/signaling pathways in the pathogenesis of human diseases including CC. This review summarizes insights from genomics and transcriptomics studies into the pathogenesis of CC, with the aim to improve (i) our understanding of its underlying complex pathomechanisms, and (ii) clinical management of different subtypes of CC, in particular their associated hepatic fibrotic change and their risk of malignancy transformation. Choledochal cysts (CC), also known as congenital biliary dilation, presents as the dilation of extrahepatic bile ducts. It has a worldwide incidence of about 1:100,000 to 1:150,000 but the incidence can be as high as 1 in 1000 in Asians. In addition, the disease incidence shows a female predominance, with a female to male ratio of 3~4:1. CC is often seen in infants, and can be diagnosed incidentally or when it causes biliary pancreatitis with abdominal pain, jaundice, and even liver enlargement. According to Todani’s classification [The etiology of CC remains unclear. There are two main theories, “pancreaticobiliary maljunction” and “congenital stenosis of bile ducts”, proposed for the pathogenesis of CC ,3,4,5. PThere is evidence suggesting a genetic contribution for CC. For example, some CC cases were reported in families ,12,13, sHNF1B gene, and HNF1B mutations or 17q12 microdeletions are usually seen in patients with nephritic cysts, diabetes syndrome, and neurologic and psychiatric symptoms [HNF1B encodes a transcription factor called hepatic nuclear factor-1 β (HNF1B), which is involved in bile duct organogenesis [HNF1B mutations and found 6 patients had CC, which exceeds the overall incidence of CC in the general population, indicating that mutations HNF1B in 17q12 may play a role in the pathogenesis of CC [HNF1B gene also suffered from CC, which is similar to the phenotype as seen in patients with a 17q12 microdeletion. The 17q12 duplication led to an overdosage of HNF1B and was found both in the young boy and his mother, indicating that the autosomal dominant inherence may exist, although his mother was completely within normal range [Chromosomal anomalies have been identified in CC . Chromossymptoms ,25,26,27ogenesis , and itsogenesis . Kettuneis of CC . Later, al range .CCT4, COMMD1, B3GNT2, and EHBP1 genes has been shown to associate with multiple congenital anomalies including renal anomalies, intractable seizures, and a choledochal cyst [EHBP1, the gene-encoding and actin-binding protein for cytophagy, was the only gene deleted in a type II CC patient [APC gene [De novo 2p15p16.1 microdeletion encompassing the hal cyst ,35,36,37 patient . ChromosAPC gene ,39. DeleAPC gene . A cytogAPC gene . Genes lAPC gene ,42. CC hAPC gene ,44,45, aERBB2 and WNT11 were the two hub genes in this same module that distinguished fusiform CC and cystic CC. ERBB2 mutations/amplifications were implicated in extrahepatic cholangiocarcinoma [There have been few microarray or RNA sequencing studies on the etiology of CC. Lv et al. performed transcriptome sequencing analysis on subtypes of Type I CC including cystic CC (Ia) and fusiform CC (Ic), and identified 6463 differentially expressed genes (DEGs) implicated in the biological processes of epithelial cell differentiation or extracellular matrix between these two CC subtypes ,48. An earcinoma ,54,55. APXDN, RTEL1, ANKRD11, MAP2K1, CYLD, and ACAN) with the de novo variants are involved in human developmental diseases, such as sclerocornea, spondyloepimetaphyseal dysplasia, dyskeratosis congenita, and familial multiple trichoepitheliomata [PIK3CA, TLN1 CYLD, and MAP2K1) with the damaging variants are linked to bile duct and liver cancer. De novo variants in significantly mutated regions (SMRs) genes  were over-represented in CC patients, especially PIK3CA with excess mutations implicated in 9 cancer types, indicating that it may play a role in malignancy [DCHS1, EPS15, DNM1, C5orf42, POU2F2, THBS1, POU2F2, BYSL, C5orf42, THBS1, BYSL, TXLNB) with de novo damaging variants recurrently identified in different individuals, forming either protein–protein interactions (PPIs) or compound heterozygotes or in homozygous. PPI pair TRIM28 and ZNF382 were overrepresented in CC group compared to controls in PPI analyses, with 3 and 2 patients carrying rare damaging variants on these two genes, respectively. TRIM28, known for regulation of the endoderm differentiation and involvement in malignancy, may play a role in CC [A trio-based whole exome-sequencing of 31 CC trios was performed to identify de novo variants . A totaleliomata ,63,64,65lignancy ,68,69,70le in CC ,72,73,74le in CC .Type V CC, also called Caroli disease, is a congenital disorder and rare genetic condition characterized by multiple dilations of intrahepatic bile ducts, with an incidence of around 1 in 1,000,000 . When itPKHD1 encodes a protein called fibrocystin that is involved in kidney organogenesis, and mutation of PKHD1 gene is responsible for cyst formation in kidney. PKHD1 gene mutations were identified in Caroli disease associated with autosomal recessive polycystic kidney disease (ARPKD) [PKHD1 causes cilia dysfunction with shortening and deformation of cilia, leading to an abnormal Wnt signaling and ductal plate malformation [PKHD1 were identified in a patient with Caroli syndrome, these mutations led to a replacement of the polar amino acid by the less polar amino acid in the extracellular domains of fibrocystin affecting fibrocystin deposition/function in the cilia [PKHD1 was seen in Caroli syndrome as a paternal inheritance [PKHD1 missense mutation was seen in Caroli syndrome associated with type II Abernethy malformation  [ (ARPKD) ,20,79,84he cilia . Anothereritance . Later, anomaly) .PKD1 or polycystin-2 encoding gene PKD2 were commonly seen in cases of Caroli disease associated with autosomal dominant polycystic kidney disease (ADPKD). Caroli disease has also been identified in families with a young sister, older brother, and father, which suggests that the disease could be inherited in an autosomal dominant mode, although autosomal recessive mode of inheritance was still the main mode of inheritance [PKD1 gene was associated with Caroli disease and PKD1 phenotype in two family members [Mutations in polycystin-1 encoding gene eritance . More faeritance ,90,91,92eritance ,94. A mu members . FolliclWDR19/NPHP13 gene were first reported in Caroli disease associated with nephronophthisis-related ciliopathies (NPHP-RC), an autosomal recessive cystic kidney disorder [GLIS2/NPHP7 recessive missense mutation was also found in NPHP-RC [Pathogenic variants in the disorder . The samdisorder . GLIS2/N NPHP-RC . Homozyg NPHP-RC   with multiple cysts in liver and kidney, by sib mating of a female rat derived from Crj:CD (SD) rats . Unlike TP53 and RBM10 mutations have been identified in those patients, which showed an 8.9% and 1.1% frequency for cholangiocarcinoma from CC, respectively [KRAS amplification was also discovered. KRAS mutation was first seen in a gallbladder adenoma of a patient with FAP, suggesting that KRAS mutation may induce malignancy transformation [Choledochal cysts are associated with an increased risk of cancer, particularly cholangiocarcinoma (70.4%) but also pancreatic and gallbladder cancers (23.5%), and especially those with type I and IV cysts . Althougectively ,108,109.ormation . Immunohormation . Expressormation .Recent genomics and transcriptomics studies have yielded important insights into the complex pathogenesis of CC, but our knowledge of genetic heterogeneity and the mechanisms underlying the causation/progression and malignancy transformation of CC is still in its infancy. To demonstrate the causal links between the genomic and transcriptomic signatures and CC will be a daunting task. Application of CRISPR/Cas9-based genome editing in patients’ liver tissue-derived and human iPSC-derived liver organoids could help in the functional evaluation of genomic variants and dysregulation of genes/pathways in the pathogenesis of CC.Due to the genetic heterogeneity and its underlying complex pathomechanisms, it is likely that CC is caused by the joint contribution of a number of independently acting or interacting polymorphic genes, each displaying different effect sizes that act simultaneously or interactively in the causation and progression of the disease. Therefore, to unravel the pathogenesis of CC, a combined analysis of the huge database of genomic variants and transcriptomic architectures, together with a genotype–phenotype correlation analysis of their clinical manifestations, associated anomalies, and malignant transformation are essential, and such analysis is too large or complex to be efficiently dealt with by traditional data-processing application software.Artificial intelligence (AI) solves complex problems by mimicking human brains. Using supervised, semi-supervised, and unsupervised learning, as well as evolutional manners, AI identifies human genetic patterns and disorders from genetic data ,114. DeeAfter decades of research on choledochal cysts, its causes remain unclear. It is generally believed that mutations in multiple genes with genetic heterogeneity drives this complex disease. Recent advances in transcriptomics and genomics analysis have unveiled key expression signatures/genes/signaling pathways in the pathogenesis of subtypes of CC and their relevance in the malignant transformation of CC. Elucidation of the common and unique genomics and transcriptomic signatures for each CC subtype not only provides insights into their complex molecular pathogenesis but also improves clinical management of the disease.Data for this review were collected from PubMed and Web of Science databases using the Mesh terms “Choledochal Cyst/genetics’’ or keywords “choledochal cyst” or “common biliary dilation” and “genetics”. All article types published between 1992 and 2022 were included. Each relevant text was thoroughly reviewed and examined, including relevant papers in the references list. Articles were excluded from review if the paper was not in English or the full-text was unobtainable .