UMOD) is a major risk gene for monogenic and complex forms of kidney disease. The encoded kidney-specific protein uromodulin is highly abundant in urine and related to chronic kidney disease, hypertension, and pathogen defense. To gain insights into potential systemic roles, we performed genome-wide screens of circulating uromodulin using complementary antibody-based and aptamer-based assays. We detected 3 and 10 distinct significant loci, respectively. Integration of antibody-based results at the UMOD locus with functional genomics data  of primary human kidney tissue highlighted an upstream variant with differential accessibility and transcription in uromodulin-synthesizing kidney cells as underlying the observed cis effect. Shared association patterns with complex traits, including chronic kidney disease and blood pressure, placed the PRKAG2 locus in the same pathway as UMOD. Experimental validation of the third antibody-based locus, B4GALNT2, showed that the p.Cys466Arg variant of the encoded N-acetylgalactosaminyltransferase had a loss-of-function effect leading to higher serum uromodulin levels. Aptamer-based results pointed to enzymes writing glycan marks present on uromodulin and to their receptors in the circulation, suggesting that this assay permits investigating uromodulin’s complex glycosylation rather than its quantitative levels. Overall, our study provides insights into circulating uromodulin and its emerging functions.Uromodulin (  UMOD) locus (Chronic kidney disease (CKD) can progress to kidney failure, is a major risk factor for cardiovascular morbidity and mortality, and is a leading cause of death –3. CKD aD) locus –7. The eD) locus . It is eD) locus . UrinaryD) locus . GlycosyD) locus .UMOD are also associated with higher risk of hypertension, hyperuricemia, and gout and lower risk of kidney stone disease  placed the PRKAG2 locus in the same pathway as UMOD with respect to its disease associations; and (iii) showed that p.Cys466Arg in the uromodulin-glycosylating enzyme B4GALNT2 was a loss-of-function allele leading to higher serum uromodulin levels. Using an aptamer-based assay, we identified non-overlapping loci that pointed to enzymes writing glycan marks present on uromodulin and to their receptors in the circulation. Together, our study based on human genetic evidence provides insights into circulating uromodulin and its emerging functions.Here, we performed meta-analyses of GWAS of circulating uromodulin to obtain insights into factors that may be relevant to CKD pathophysiology and into any systemic functions of this kidney-specific protein. Using an antibody-based assay, we (i) identified an upstream variant at the https://doi.org/10.1172/jci.insight.157035DS1 There were 29,439 participants of European ancestry (EA), 400 African American (AA) participants, and 2216 Hispanic (HIS) participants. GWAS of age-, sex-, and eGFR-adjusted and rank-based inverse normal transformed circulating uromodulin measurements were carried out in each of the 7 studies using densely imputed genotypes , including distributions of age, sex, and eGFR, are shown in enotypes , 26  revealed 3 genomic loci with at least 1 significantly associated (P < 5 × 10–8) genetic variant , B4GALNT2 , and PRKAG2 . The genomic control parameter was 0.99, consistent with the absence of undetected population stratification  more than 1% across 5 studies with antibody-based uromodulin quantification  showed no evidence of inflation . ST3GAL6 codes for ST3 beta-galactoside alpha-2,3-sialyltransferase (ASGR2), with each minor allele associated with 1 standard deviation higher age- and sex-adjusted plasma aptamer uromodulin readout. The estimated SNP-based heritability was 0.177 (95% CI –0.032–0.386). For each of the 2 assays, regional association plots for all 13 loci that achieved genome-wide significance are shown in The GWAS meta-analysis of 8,815,558 genetic variants across 2 studies with plasma aptamer uromodulin readout . Moreover, genes identified with both assays can be connected to uromodulin through different sources of external evidence (see below). This indicates that the antibody- and aptamer-based assays for circulating uromodulin deliver reproducible measurements but assess different properties of their respective targets, such as protein amount and glycosylation pattern, respectively.The 2 meta-analyses of antibody- and aptamer-based uromodulin measurements identified different genetic loci. A Genome-wide discovery screens without adjustment for eGFR showed virtually identical results , A and BWe next evaluated the presence of sex-specific genetic effects, motivated by the observation that women have higher serum uromodulin levels than men , 29, 30.UMOD locus (UMOD/PDILT (P = 5.3 × 10–97), which explained 1.4% of the urine uromodulin variance, none of the other SNPs showed significant (P < 3.8 × 10–3 = 0.05/13) associations , B4GALNT2 (n = 3), ST3GAL6 (n = 4), and ASGR1/ASGR2 . Conditional analyses supported the presence of more than 1 independent signal at 2 n = 2; . For eac2 n = 2; .PDILT, the gene upstream of UMOD. Another independent set of 8 variants in the locus mapped to UMOD  and gene expression (RNA-Seq) from cortex and medulla of native human kidney tissue (Methods) that showed transcription of UMOD, more strongly in medulla than in cortex, but not of PDILT. Both independent variants, rs77924615 in PDILT and rs4293393 in the UMOD promoter, mapped into regions of open chromatin in medulla, where UMOD transcript levels in human kidney are highest (GTEx Project V8)  . These rject V8) . Both vaB4GALNT2 locus, rs7224888, was identified with the antibody assay and is a missense variant. Its minor C allele encodes a cysteine-to-arginine substitution  in the encoded enzyme beta-1,4-N-acetyl-galactosaminyltransferase 2. The C allele was associated with higher circulating uromodulin in our study and has been linked to the absence of the Sda antigen  in micro-dissected kidney tubules , the major G allele at the most likely causal variant rs2075803 leads to a lysine-to-glutamine substitution in SIGLEC9 . It was associated with lower aptamer signal in our study (P = 3.8 × 10–100) and with lower circulating SIGLEC9 protein (P = 6 × 10–2142) (P = 5 × 10–10)   and seru× 10–10)  in previ× 10–10) , indicatUMOD to monogenic autosomal-dominant tubulointerstitial kidney disease , diastolic blood pressure (DBP), and uromodulin levels in urine (Methods). Interestingly, genetic associations with antibody-based circulating uromodulin at the PRKAG2 and UMOD/PDILT loci shared a very similar pattern of colocalization with numerous kidney-related traits . These 2 genes are indeed functionally related, as ST3GAL6 is involved in the synthesis of sialic acid moiety that is bound by SIGLEC9.There were several other examples of positive colocalizations supported by biological knowledge: first, genetic associations at the  in mice   were identified between carrier status of rare UMOD variants and anemia of chronic disease , hypertensive CKD , and CKD , potentially deleterious variants in the genes prioritized at each of the 13 uromodulin-associated genetic loci on 770 complex diseases (Methods). Using data from whole-exome sequencing of 173,688 UK Biobank participants, significant associations (cis based on transcriptome-wide RNA-sequencing of 36 non-brain tissues (GTEx Project V8) .Colocalization analyses of the uromodulin association signals at all 13 significant loci were also performed with the expression of genes in ject V8) , tubuloiject V8) , as wellject V8)  in orderject V8) , 43, 44.ject V8) . For exaPRKAG2, B4GALNT2, and UMOD/PDILT as the genes most likely causing the association with antibody-based uromodulin, and CFH, MGAT5, ST3GAL6, HLA-DRB1, B4GALT1, ABO, DPP7/MAN1B1, ST3GAL4, ASGR1/ASGR2, and SIGLEC9 for the aptamer-based readout  implicated  readout .cis association at UMOD/PDILT, the aptamer-based assay may identify differences related to glycan marks known to be present on uromodulin and their receptors, for example, because such modifications may lead to differential aptamer binding. In order to detect any shared functions, processes, and pathways among the respective genes identified by each assay, enrichment analyses were performed (Methods). Terms and pathways related to protein glycosylation were highly enriched for genes identified via aptamer plasma uromodulin readout  and the Sda antigen , likely as a consequence of the higher steric hindrance of arginine . For thi antigen , confirm antigen .Western blot analysis on cell lysates showed that Arg406 B4GALNT2 had a slightly reduced molecular weight compared with WT protein. Such difference is related to different glycosylation, due to retention of Arg406 B4GALNT2 in the endoplasmic reticulum (ER). Indeed, the lower band observed for the Arg406 isoform was fully sensitive to treatment with Endo H, a deglycosylating enzyme that is specific for high-mannose, ER-type N-glycans, while only a minor fraction of WT B4GALNT2 was cleaved by Endo H . To subsd-galactosaminyltransferase activity present in microsomal preparations of guinea pig kidney transfers N-[14C]-acetylgalactosamine to N-linked glycans of UMOD for the synthesis of Sda antigen (B4GALNT2 transcript in TAL and DCT segments where uromodulin is expressed (+) and a low but consistent signal in UMOD-positive cells  and aptamer-based  assays has 4 principal findings. First, it identifies an upstream variant at the UMOD/PDILT locus for which integration with functional genomics data from primary human kidney tissue supports differential chromatin accessibility and transcription in cells synthesizing uromodulin as underlying its strong association with circulating and urine uromodulin, as well as CKD and hypertension. Second, shared association patterns of uromodulin-associated genes with complex traits and diseases are plentiful and place the PRKAG2 and UMOD loci into the same context with respect to their associations with CKD, hypertension, and kidney stone disease. Third, the missense variant p.Cys466Arg in the uromodulin-glycosylating enzyme B4GALNT2 is a loss-of-function allele leading to higher levels of circulating uromodulin. Fourth, our study reveals enzymes that write glycan marks found on uromodulin and their receptors that may be related to uromodulin’s complex glycosylation pattern, function, and clearance.This GWAS meta-analysis of circulating uromodulin using complementary antibody-based  activity . AlternaPRKAG2 locus showed a striking similarity to those at UMOD with respect to shared disease association patterns. Considering that we tested for colocalization with hundreds of human traits and diseases, these mirroring patterns are extremely unlikely to result from chance. Our results imply that genetic variants at PRKAG2 are associated with higher risk for CKD and hypertension, as well as lower risk for kidney stone disease, through the same biological context as UMOD. PRKAG2 codes for the regulatory γ subunit of the AMP-activated protein kinase (AMPK), an enzyme with a key role in regulating multiple processes related to cellular energy metabolism. AMPK has been described to phosphorylate the kidney-specific Na+-K+-2Cl– cotransporter sialoprotein receptors may affect circulating uromodulin, possibly modulating crosstalk between the kidney and the innate and adaptive immune system.Our study also provides insights of potential clinical relevance. It indicates that a common genetic basis of urine uromodulin levels with higher risk of CKD and hypertension extends to circulating uromodulin levels and identifies kidney cell type–specific regulation of uromodulin expression as a mechanism. Interventions aimed at reducing uromodulin synthesis can therefore be expected to have concomitant effects on both urine and serum levels of the protein, which may be of importance given its emerging systemic relevance. The association of genetic variants at In conclusion, our study provides human genetic evidence of pathway members of uromodulin and delivers insights into its determinants and systemic role in the circulation.Seven prospective studies participated in the genome-wide analyses of serum/plasma uromodulin levels : the CHSDetails about genotyping and imputation in each of the 7 studies are provided in The CHS, GCKD, KORA, and LURIC studies quantified uromodulin from serum using a commercial ELISA , 67, 68 Each study performed 4 sets of GWAS according to a prespecified analysis plan: a primary analysis, in which rank-based inverse normal transformed age-, sex-, and eGFR-adjusted residuals of uromodulin levels were used as the dependent variable and regressed on genotypes as the predictor, controlling for principal components. In addition, 3 secondary analyses were carried out: the primary analysis was repeated separately for men and women (without adjustment for sex and only when at least 50 participants were available), and a sex-combined analysis was performed in which the residuals were not adjusted for eGFR. Studies with participants of different ancestries performed separate GWAS for each ancestry group. Sex-stratified GWAS of chromosome X markers, assuming X inactivation and an additive model, were only available from the CHS and GCKD studies. Genome-wide summary statistics were collected in a prespecified format and uploaded to a central server for meta-analysis.P < 5 × 10–8. EA-specific meta-analyses were also performed, as EA participants were the largest subsample. SNP-based heritability was estimated using LDSC v1.0.1 with the option --h2. Precomputed LD scores from 1000 Genomes European data were used as reference. Input files for LDSC were GWAS summary statistics from the primary association analyses filtered for MAF > 0.01 (antibody-based assay) or MAF > 0.005 (aptamer-based assay).Prior to meta-analysis, GWAS summary statistics from individual studies were subjected to thorough quality control using GWAtoolbox . The varSeveral complementary approaches to characterize genetic loci identified through genome-wide screens of circulating uromodulin were employed, with detailed methods described in Supplemental Methods. These include (i) associations with urine uromodulin levels; (ii) annotation, enrichment analyses, and functional genomics; (iii) independent SNP selection, statistical fine mapping, and credible set annotation; (iv) colocalization with gene expression, plasma protein levels, biomarkers, and diseases; (v) phenome-wide association studies of serum uromodulin levels; as well as (vi) gene-by-gene interaction analyses.http://pfam.xfam.org) to map functional domains. The region 254–464 containing the glycosyltransferase domain was analyzed in iTasser (The sequence of B4GALNT2 isoform 2 (Uniprot Q8NHY0-2) was analyzed in PFAM  are reported in Supplemental Methods. Protein and RNA extracts, Western blot, RT-qPCR, and immunofluorescence analyses were carried out as previously described , 75.https://nxc-1453.imbi.uni-freiburg.de/s/gReQNkMJtkYYLxa Publicly available single-nucleus ATAC-seq data were downloaded from https://susztaklab.com/human_kidney/igv/ and generated as described previously  and the legislation of animal care and experimentation of Canton Zurich .YL, YC, FC, LR, and AK conceived and designed the study; YL, YC, MRC, M Pietzner, NQHN, NS, MLB, MEK, BG, P Sekula, IS, P Schlosser, MW, SA, LR, and AK performed statistical analysis; MEK, M Pigeyre, JAB, MEG, CH, UTS, BKK, FK, AP, JS, DS, CT, KUE, CG, EB, BMP, JC, CL, BY, and AK managed an individual study; JS, DS, CT, WM, GP, and JES measured uromodulin; YL, YC, MEK, NS, SH, CBJ, and MW integrated bioinformatic data; YL, YC, FC, GS, PJO, SA, OD, LR, and AK interpreted results; FC, GS, SA, OD, and LR performed experimental studies; YL, YC, FC, GS, SA, LR, and AK wrote the initial manuscript draft; and all authors critically reviewed the manuscript. The order of the co–first authors was determined based on the overall analytical and conceptual contribution to the project.


Diosgenin (DIO) is a dietary and phytochemical steroidal saponin representing multiple activities. The present study investigated the protective effect of DIO on type II diabetes-associated nonalcoholic fatty liver disease (D-NAFLD). The rat model was established by high-fat diet and streptozotocin injection and then administered DIO for 8 weeks. The results showed that DIO reduced insulin resistance index, improved dyslipidemia, and relieved pancreatic damage. DIO decreased hepatic injury markers, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). H&E staining showed that DIO relieved hepatic lipid deposition. Mechanistically, DIO inhibited hepatic de novo lipogenesis (DNL) and increased fatty acid β-oxidation (FAO) through regulation of the AMPK-ACC/SREBP1 pathway. Endoplasmic reticulum (ER) stress was inhibited by DIO through regulation of PERK and IRE1 arms, which may then inhibit DNL. DIO also decreased reactive oxygen species (ROS) and enhanced the antioxidant capacity via an increase in Superoxide dismutase (SOD), Catalase (CAT), and Glutathione peroxidase (GPx) activities. The mitochondria are the site for FAO, and ROS can damage mitochondrial function. DIO relieved mitochondrial fission and fusion disorder by inhibiting DRP1 and increasing MFN1/MFN2 expressions. Mitochondrial apoptosis was then inhibited by DIO. In conclusion, the present study suggests that DIO protects against D-NAFLD by inhibiting DNL and improving FAO and mitochondrial function. NAFLDide (TG) ,11. Steride (TG) . FAO is ide (TG) .Since the bulk of lipid synthesis takes place in the smooth endoplasmic reticulum (ER), ER stress involves in the development of hepatic steatosis. ER stress affects the process of NAFLD via regulating lip-stasis, modulating hepatic insulin sensitivity, and regulating hepatic autophagic flux. Growing evidence has suggested that chronic or acute ER stress will aggravate hepatic steatosis and hepatocyte death ,15. ThusThe primary route of lipid depletion is FAO, which is carried by mitochondria. However, persistent FAO will produce massive ROS and then damage mitochondria due to the increase of FFA. Subsequently, the defective FAO appears . At the 2DM has attracted increasing attention. Diosgenin (DIO) is a dietary and phytochemical steroidal saponin that displayed efficacy against various life-threatening diseases such as diabetes and its complications, hyperlipidemia, and cardiovascular diseases [Severe side effects have been reported to accompany long-term administration of drugs for lowering blood glucose and regulating lipid metabolism. Seeking natural active substances with low toxicity to prevent and treat NAFLD and Tdiseases ,22. It idiseases . Howevern = 8) and the T2DM group (n = 32). T2DM rats were established by high-fat diet  and streptozotocin  injection. Firstly, the rats were administrated with HFD for 4 weeks. The formula of HFD refers to our previous article [2DM rats (random blood glucose > 300 mg/dL) were distributed into 4 groups (n = 8): D-NAFLD group, D-NAFLD+DIO  group, D-NAFLD+DIO (20 mg/kg) group and D-NAFLD+Metformin   group, positive control. Rats, except for the control and D-NAFLD group, were given DIO and Met by gavage daily for 8 weeks. Except for the control group, rats in the other groups were given HFD ad libitum during the intervention period. The animal experimental process is shown in Sprague Dawley male rats (200 ± 20 g) were obtained from Dossy Experimental Animals Co., Ltd. . After adaptation for 1 week, the rats were distributed into the control group . The levels of TG, total cholesterol (TC), FFA, low-density lipoprotein (LDL), high-density lipoprotein (HDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured by corresponding assay kits purchased from Jiancheng Bioengineering Institute .At the end of the experiment, the rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg). The blood was drawn from the heart and then centrifugated to collect serum  was used to measure the fasting insulin level. Briefly, 10 uL serum was added to the detection holes containing 40 uL sample diluent and then incubated at 37 °C for 30 min. Then the holes were washed with scrubbing solution for 30 s and then patted dry, repeated 5 times. Subsequently, 50 uL enzyme labeled reagent was added and incubated at 37 °C for 30 min. After washing, the chromogenic solution was added and incubated at 37 °C for 15 min in the dark. After reaction termination, the absorbance at 450 nm was measured using a multifunctional microplate reader . The insulin resistance index was calculated with the formula: Fasting blood glucose × fasting insulin/22.5.After euthanasia, the liver and pancreas were processed, including 10% neutral formaldehyde fixing, paraffin embedding, 5 µm thickness sectioning, and H&E staining . The pat−ΔΔCt method.The total RNA of liver tissue was extracted using Ultrapure RNA Kit . The RNA was reverse-transcribed into cDNA using HiFiScript cDNA Synthesis Kit . The mRNA expression was analyzed by the semiquantitative real-time PCR system  with SYBR reagent (CWBIO). The primers are shown in ROS was measured by DHE probe . In detail, the liver was quickly taken out, embedded in an optimal cutting temperature compound, rapidly frozen in liquid nitrogen, and then sectioned at 5 µm. The sections were incubated with the DHE probe (37 °C for 30 min), washed with PBS, and photographed under an inverted fluorescence microscope .Liver antioxidant enzyme activities, including Superoxide dismutase (SOD), Catalase (CAT), and Glutathione peroxidase (GPx) were measured by corresponding commercial kits from Beyotime. Protein level was measured using bicinchoninic acid (BCA) protein assay kit (CWBIO). The activities of SOD, CAT, and GPx are presented as units/mg protein.The detection of lipid peroxidation was performed by Lipid Peroxidation MDA Assay Kit (Beyotime). Protein content was also detected. The MDA level is presented as nmol/mg pro.79, 1:1000, Proteintech), AMPK , p-AMPK , Protein kinase RNA-like endoplasmic reticulum kinase , p-PERK , Phosphorylated eukaryotic initiation factor 2 , IRE1 , p-IRE1 , X-box binding protein 1 spliced , Activating transcription factor 4 , C/EBP homologous protein , p-CHOP , B-cell lymphoma-2 , Bcl2-associated X , CytC , Apoptotic protease activating factor-1 , caspase 9 , caspase 3 , DRP1 , p-DRP1 , MFN1 , MFN2 , Fission 1 , ACTB  and GAPDH .The protein extraction and western blot were performed according to the methods previously described . The prip < 0.05 indicates significant difference and ** p < 0.01 indicates a highly significant difference.All data were expressed as the mean ± SD. Significant differences between different groups were determined by one-way factorial analysis of variance (ANOVA), followed by Duncan’s for multiple-range test using SPSS 20.0. * Insulin resistance and elevated blood lipid are two main pathogenic factors of D-NAFLD. Thus, insulin resistance index and blood lipid were measured. As shown in 2DM. As shown in Prolonged insulin resistance will impair islet function, mainly through damaging islet β cells. In D-NAFLD rats, the Langerhans cells are irregular and partially absent. DIO treatment improved the structure of islets A. β cellInsulin resistance could increase FFA and exacerbate hepatic lipid deposition. To investigate whether DIO relieves lipid deposition and liver injury in D-NAFLD rats, liver/body ratio, serum ALT, serum AST, liver TG, and H&E staining were measured. As shown in SREBP1c, FASN, and CD36 molecule (thrombospondin receptor) (CD36), but decreased FAO-associated genes, including Peroxisome proliferator activated receptor α (PPARα) and Carnitine palmitoyltransferase-1 (CPT1). DIO administration decreased SREBP1c, FASN, and CD36 expressions, and increased PPARα and CPT1 expressions. ACC serves as an important precursor for the biosynthesis of fatty acid and an effective inhibitor of long-chain fatty acyl-CoA transporting into mitochondria. SREBP1 is a key transcription factor for DNL through upregulating genes coding FASN. As shown in As shown in Saturated fatty acids can alter phospholipids’ composition in the ER membrane and directly activate the sensors of IRE1 and PERK to induce ER stress, which could exacerbate hepatic steatosis through regulating DNL. Thus, the protein expressions of PERK, p-PERK, IRE1, p-IRE1, and their downstream were measured. It is shown that proteins in the PERK pathway including PERK, p-PERK, and p-EIF2α were upregulated in the liver of D-NAFLD rats . SimilarMCP-1, IL-1β, and TNF-α were upregulated significantly in the liver of D-NAFLD rats, while IL-4 and IL-10 were downregulated. DIO treatment downregulated MCP-1, IL-1β and TNF-α expressions, and increased IL-10 expression, but had no significant effect on IL-4 expression . It is documented that insulin resistance could lead to hepatic steatosis and the elevated serum ALT and AST are indicators of liver damage. In the present study, the levels of serum ALT and AST increased significantly in D-NAFLD rats; after DIO and Met treatment, they decreased greatly. The results of H&E staining and liver triglyceride consistently showed that DIO and Met decreased hepatic steatosis. In addition, administration with DIO and Met to D-NAFLD rats reduced liver/body weight. The effects of DIO and Met on the above results were equivalent and there was no statistical significance between DIO (10 mg/kg) and DIO (20 mg/kg) treatments, except for pancreatic cell apoptosis (20 mg/kg DIO is better).Elevated blood lipid and insulin resistance are two main pathogenic factors of D-NAFLD. In this study, dyslipidemia was ameliorated by DIO and Met through decreasing TG, TC, FFA, and LDL levels, and increasing HDL content. In addition, DIO and Met decreased the insulin resistance index and improved pancreatic structure. Lipotoxicity and glucotoxicity of T79 and then its inactivation. ACC serves as an important precursor for the biosynthesis of fatty acid and an effective inhibitor of long-chain fatty acyl-CoA transporting into mitochondria [372, suppressing the proteolytic cleavage of precursor SREBP1 into mature SREBP1, which then reduces FASN expression and inhibits lipid synthesis [79 was also upregulated greatly after DIO administration. Moreover, DIO inhibited the over-expression of SREBP1 in D-NAFLD rats. In addition, the mRNA expressions of lipogenic genes including SREBP1, FASN, and CD36 decreased greatly. The suppression of genes involving in β-oxidation such as PPARα and CPT1 impairs the metabolic function of mitochondria [PPARα and CPT1 decreased greatly in the liver of D-NAFLD rats, which was enhanced by DIO. These results indicated that DIO can reduce lipid synthesis and enhance FAO. In general, there was no significant difference between the two concentrations of DIO.In this study, Met is used as a positive control. Since our aim is to explore the protective effect of DIO on D-NAFLD, mechanistically, we only focused on DIO. One of the pathogeneses of hepatic steatosis is the disorder between hepatic lipid uptake, DNL, and FAO. Emerging evidence has indicated that AMPK plays a crucial role in NAFLD by stimulating FAO and inhibiting lipogenesis. Once activated, AMPK induces the phosphorylation of ACC at Serchondria ,31. In aynthesis . We showchondria ,33. In tER stress is also a therapeutic target for NAFLD. It can be directly activated by glucolipotoxicity. Saturated fatty acids alter phospholipids’ composition in the ER membrane and then trigger ER stress through the sensors of IRE1α and PERK even in the absence of unfolded proteins ,35. StudMitochondria is the main site for FAO in cells. Per hepatocyte is thought to contain 500 to 4000 mitochondria, and a decrease in their capacity to oxidize fatty acids contributes to the development of hepatic steatosis . As an a616) expressions. To the contrary, MFN1 and MFN2 expressions were upregulated greatly after the DIO application. It is worth noting that the changes of p-DRP1, MFN1, and MFN2 were more significant in the D-NAFLD+DIO (20 mg/kg) group.Mitochondria continuously undergo fusion and fission. Various studies have suggested that excessive mitochondrial fission is involved in hepatic steatosis and liver inflammation, which results in hepatocyte death ,42,43,442DM rats induced by HFD and STZ injection and that DIO (20 mg/kg) is the preferred option. Firstly, DIO reduced the insulin resistance index and relieved pancreatic damage and apoptosis. Then, DIO improved dyslipidemia and hepatic steatosis, mainly through suppressing DNL and enhancing FAO via AMPK-ACC/SREBP1 and AMPK-ACC pathways. DIO also relieved ER stress, which may then inhibit DNL and improve mitochondrial function, a site for FAO. Thus, the dietary intervention of DIO has great potential in relieving D-NAFLD, which can help or even substitute for medication, and it is valuable to study the synergistic effect of DIO with drugs such as Met.In conclusion, the current study revealed that DIO can protect against NAFLD in T


IDH2 and FLT3 mutations, whereas non-responders were associated with PTPN11 mutations. The combination of PI3K/mTOR dual pathway inhibition with bimiralisib and BCL2 inhibition with venetoclax has emerged as a candidate treatment in IDH2- and FLT3-mutated AML.In October 2020, the FDA granted regular approval to venetoclax (ABT-199) in combination with hypomethylating agents for newly-diagnosed acute myeloid leukemia (AML) in adults 75 years or older, or in patients with comorbidities precluding intensive chemotherapy. The treatment response to venetoclax combination treatment, however, may be short-lived, and leukemia relapse is the major cause of treatment failure. Multiple studies have confirmed the upregulation of the anti-apoptotic proteins of the B-cell lymphoma 2 (BCL2) family and the activation of intracellular signaling pathways associated with resistance to venetoclax. To improve treatment outcome, compounds targeting anti-apoptotic proteins and signaling pathways have been evaluated in combination with venetoclax. In this study, the BCL-XL inhibitor A1331852, MCL1-inhibitor S63845, dual PI3K-mTOR inhibitor bimiralisib (PQR309), BMI-1 inhibitor unesbulin (PTC596), MEK-inhibitor trametinib (GSK1120212), and STAT3 inhibitor C-188-9 were assessed as single agents and in combination with venetoclax, for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Enhanced cytotoxic effects were present in all combination treatments with venetoclax in AML cell lines and AML patient samples. Elevated in vitro efficacies were observed for the combination treatment of venetoclax with A1331852, S63845 and bimiralisib, with differing response markers for each combination. For the venetoclax and bimiralisib combination treatment, responders were enriched for  The combination of venetoclax and the HMA azacitidine results in a remission rate of approximately 70% [With a median age of 68 years at diagnosis, acute myeloid leukemia (AML) is predominantly a disease of the elderly. The majority of these patients are not eligible for intensive chemotherapy with curative intent, and the standard of care has been treatment with hypomethylating agents (HMA) ,2. The ately 70% . HoweverNPM1, DNMT3A, or IDH2 mutations, while resistance was associated with mutations in FLT3, RAS, or TP53 genes [TET2, IDH1, and IDH2 mutations, while non-responders were associated with FLT3 and RAS mutations [RUNX1 and SRSF2 mutations may also be associated with venetoclax response [High response rates and durable remissions to venetoclax treatment were found to be associated with 53 genes ,5,6. In utations . RUNX1 aresponse ,9. Differesponse .RAS-mutated AML [FLT3- or TET2-mutated AML [Hematological cells of various origins, including AML, exhibit specific dependencies on either BCL-2, BCL-XL, or MCL-1 for survival ,12. Thisated AML , combinaated AML . In vivoated AML , and a pated AML , while vated AML ,21.FLT3 gene mutations lead to abnormal activation of the pathway in AML [In myeloid cells, FLT3 is a growth factor receptor signaling via PI3K-AKT-mTOR, and y in AML ,23. Abouy in AML . Pan PI3y in AML . PQR309 y in AML . Bimiraly in AML  and in ay in AML . Unesbuly in AML . The comy in AML . Chemothy in AML .In the current study, the BCL-XL inhibitor A1331852, the MCL1-inhibitor S63845, the PI3K inhibitor bimiralisib (PQR309), the BMI-1 inhibitor unesbulin (PTC596), the MEK-inhibitor trametinib (GSK1120212), and the STAT3 inhibitor C-188-9 were assessed as single agents and in combination with venetoclax, for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Several combinations were found to be effective in vitro: venetoclax and S63845, venetoclax and A1331825, and venetoclax and PQR308.FLT3-ITD and FLT3 wild type, NPM1 mutant and wild type, as well as TP53 wild type, mutant, hemizygous, and null cells , trametinib (p = 0.003), S63845 (p = 0.005), A1331825 (p = 0.01), PTC596 (p = 0.01), or C-188-9 (p = 0.04), Combination indexes were calculated according to Chou Talalay [Cell viability was determined in seven AML cell lines and one BCL2-driven DLBCL cell line treated with increasing dosages of single compounds and in combination using a variety of targeted therapies including the BCL-XL inhibitor A1331825, the PI3K inhibitor bimiralisib (PCR309), the STAT3 inhibitor C-188-9, the BMI-1 inhibitor PTC596, the MCL1 inhibitor S63845, and the MEK inhibitor trametinib. Drug concentrations in the combination studies were chosen to correspond to minimally effective concentrations in single compound assays determined in initial titration. Five AML cell lines and OCI-Ly1 were susceptible to 100 nM venetoclax and multiple combination treatments, while PL-21 and MOLM-16 cells were resistant to 100 nM venetoclax and most combination treatments, with the exception of venetoclax and A1331825 . Across To investigate the efficacy of venetoclax combination treatments in the bone marrow environment, cell viability was determined in the susceptible AML cell lines MOLM-13, ML-2, SKM-1, and OCI-AML3, grown in the absence or presence of bone marrow stroma cells. Stroma cells secrete granulocyte and macrophage colony-stimulating factors  and a variety of cytokines, which can induce STAT signaling in leukemic cells ,40,41,42Effects of venetoclax combination treatments on cell cycle and apoptosis were studied in the susceptible AML cell lines MOLM-13, ML-2, SKM-1, and OCI-AML3. Treatment with bimiralisib and venetoclax lead to the induction of apoptosis, cell cycle arrest, and cell death in MOLM-13 cells , as wellAfter initial studies in AML cell lines, the treatment combinations of venetoclax with A1331825, S63845, bimiralisib, trametinib, C-188-9, or PTC596 were applied to patient-derived mononuclear cells isolated from peripheral blood (PBMC) or bone marrow (BMMC) . A totalIDH2 mutation, as well as elevated blast cell percentage in peripheral blood or bone marrow associated with response, presence of PTPN11, TET2 or ASXL1 mutation indicated lack of response, while FLT3, NPM1, RUNX1, and TP53 status as well as CD34 levels were not associated with treatment response  family and the activation of intracellular signaling pathways were the major factors leading to resistance to venetoclax ,5,7. AccIn this study, we describe a panel of venetoclax combination treatments with enhanced cytotoxic effects on AML cells grown in the absence or presence of bone marrow stroma, including the BCL-XL inhibitor A133825, the MCL1 inhibitor S63845, the BMI1 inhibitor PTC596, the dual PI3K-mTOR inhibitor bimiralisib, the STAT3 inhibitor C-188-9, and the MEK inhibitor trametinib. The in vitro IC50 concentrations of the tested inhibitors were determined to be in the range of physiologically relevant concentrations for all compounds, except C-188-9, where in vitro IC50 levels exceeded the plasma concentrations present in PDX mice. AML cells grown in the presence of bone marrow stroma were generally less affected by the combination treatments than AML cells grown in the absence of stroma, indicating a protective effect of the bone marrow environment on AML cells. MOLM-13 cells were protected toward venetoclax and combination treatments when grown on stroma, ML-2 cells were protected toward trametinib, OCI-AML3 cells were protected toward all venetoclax combination treatments, and SKM-1 cells were more susceptible to venetoclax and A1331825, and protected toward bimiralisib and trametinib, when grown on stroma. SKM-1 cells have been found to be more susceptible to venetoclax and to the STAT5 inhibitor AC-4-130 when grown on stroma . A cell-IDH2, NPM1, FLT3, DNMT3A, PTPN11, ASXL1, TET2, KRAS, RUNX1, and TP53 genes have been described as response markers to venetoclax treatment [IDH2 mutation was the single most significant and consistent biomarker associated with response to venetoclax and multiple venetoclax combination treatments, while TP53 mutation was not associated with response. Notably, the TP53 double mutant cell line OCI-Ly1 was most sensitive to venetoclax, while the TP53 double mutant cell line MOLM-16 was resistant.To validate the findings in a translational setting, venetoclax combination treatments were applied to mononuclear cells isolated from the peripheral blood or bone marrow of primary AML patients. The addition of the BCL-XL inhibitor A133825, the MCL1 inhibitor S63845 or the PI3K inhibitor bimiralisib to venetoclax induced substantial reductions of cell viability in 50–70% of the tested AML samples, while the addition of the MEK inhibitor trametinib, the BMI-1 inhibitor PTC596 or the STAT3 inhibitor C-188-9 to venetoclax was less effective, with substantial reductions of cell viability in 20–40% of tested AML samples. In order to define the patient subgroups who may profit from novel targeted combination therapies, potential response markers were deduced from the correlation analysis of cell viabilities grouped according to diagnostic parameters, including gene mutation status of prevalent tumor suppressors and oncogenes, peripheral blood and bone marrow blast cell percentage, and levels of CD34 positive cells. A significant association between venetoclax response and elevated blast cell percentage has been previously described , and wasFLT3 mutations were associated with response to venetoclax and bimiralisib, but not to venetoclax monotherapy or in combination with A1331825 or S63845, indicating that FLT3-mutated cells may be specifically susceptible to the combined inhibition of BCL2, PI3K, and mTOR. The TET2 gene mutation has been associated with response to venetoclax and HMA combination treatment [TET2 mutation apparently associated with resistance to venetoclax in combination with A1331825 or bimiralisib, possibly due to presence of concurrent mutations in KRAS, PTPN11, or TP53 genes in the primary AML samples. PTPN11 mutations were associated with lack of response to venetoclax and A1331825 or bimiralisib, but not to venetoclax and S63845, indicating differential target cell specificity and differential mechanisms of action for S63845 in combination with venetoclax. Activating mutations of the SHP2 protein, encoded by the PTPN11 gene, leads to hyper-activation of the downstream RAS-MAPK signaling pathway and confer resistance to venetoclax and multiple venetoclax combinations. The combination of venetoclax and the MCL1 inhibitor AZD5991 was proposed to overcome this resistance [PTPN11 status. ASXL1 mutations have been associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine [ASXL1 mutation apparently associated with resistance to venetoclax in combination with A1331825, but not in combination with S63845 or bimiralisib, possibly due to the presence of concurrent mutations in KRAS, PTPN11, RUNX1, or TP53 genes in the primary AML samples. Further studies in larger cohorts may be required to validate the relevance of TET2, TP53, and ASXL1 mutation status as response markers to venetoclax combination treatments. Additional biomarkers of response to venetoclax combination treatments may arise from studies on expression levels of pro-apoptotic BCL-2 family proteins and anti-apoptotic proteins  as well as the components of the upstream signaling pathways in larger cohorts.The mutation status of other genes was relevant to response, however, with differential indicators in specific venetoclax combination treatments. sistance . In accocytidine . In our IDH2-mutated AML in the absence of PTPN11 mutations, while the combination of venetoclax and S63845 may be effective in AML in the presence of PTPN11 mutations. The combination treatment of venetoclax and bimiralisib may be effective in AML with IDH2, NPM1, and FLT3 mutations. We propose the combination of PI3K/mTOR dual pathway inhibition with bimiralisib and BCL2 inhibition with venetoclax as a candidate treatment in clinical trials for IDH2- or FLT3-mutated AML.In conclusion, different combinations of targeted therapies emerge that are suitable in the treatment of specific subsets of AML patients. In this study, elevated in vitro efficacies were detected in the combination treatments of venetoclax with BH3 mimetics A1331852 and S63845 and the dual PI3K-mTOR inhibitor bimiralisib, in AML with elevated blast cell percentage, at drug concentrations that can be reached in vivo in the plasma, with different response markers in each combination. The combination treatment of venetoclax and A1331825 may be effective in the treatment of FLT3, NPM1, TP53, and conventional karyotype analysis of at least 20 metaphases were performed in all samples. In addition, all samples were analyzed by NGS sequencing of the myeloid panel genes. The genes tested within the NGS panel can be categorized into several major functional categories, including the spliceosome , epigenetic modifiers , cohesions , transcription factors , signaling molecules , and chromatin modifiers (EZH2 and ASXL1).Mononuclear cells of AML patients diagnosed and treated at the University Hospital, Bern, Switzerland, between 2018 and 2022, were included in this study. Informed consent from all patients was obtained according to the Declaration of Helsinki, and the studies were approved by decisions of the local ethics committee of Bern, Switzerland, decision number 221/15. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were collected at the time of diagnosis before initiation of treatment. The AML cells were analyzed at the central hematology laboratory of the University Hospital Bern according to state of the art techniques . Mutatio2. Human bone marrow stroma cell line HS-5 (ATCC® CRL-11882™) was supplied by the American tissue culture collection . HS-5 cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  in standard tissue culture flasks . HS-5 cells secrete granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), macrophage-CSF (M-CSF), Kit ligand (KL), macrophage-inhibitory protein-1 alpha, interleukin-1 alpha , IL-1beta, IL-1RA, IL-6, IL-8, IL-11, and leukemia inhibitory factor (LIF) [Human AML cells lines OCI-AML3 , MOLM-13 , FLT3-ITD, and TP53wt), MOLM-16 , ML-2 , FLT3wt, TP53mut) and HL-60  as well as B-cell lymphoma cell line OCI-Ly1 , PTEN del, TP53 mut) were supplied by the German Collection of Micro-organisms and Cell Cultures . AML cells were grown in RPMI-1640 medium , OCI-Ly1 in Iscove’s modified Dulbecco medium , supplemented with 20% fetal bovine serum , in tissue-culture flasks  in a standard cell culture incubator at 37 °C with 5% COor (LIF) ,42. For 5/mL on tissue culture plates suspension 96 well , and treated with targeted compounds. For assays with patient-derived mononuclear cells, the cells were cultured for 24 h prior to treatment. The BMI1 inhibitor PTC596 (HY-112041), the MCL1 inhibitor S63845 (HY-100741), the MEK inhibitor trametinib (HY-10999), the STAT3 inhibitor C-188-9 (HY-112288) and the PI3K inhibitor PQR309 (HY-12868) were purchased at MedChemExpress . PQR309  is a novel brain-penetrant dual PI3K/mTOR inhibitor with in vitro and in vivo anti-lymphoma activity as single agent and in combination. A stock solution of Venetoclax was prepared by dissolving a tablet in DMSO . Cell viability was determined 20 h after the start of treatment using the MTT-based cell proliferation kit I . This time point was selected because the cellular responses were effectual for the calculation of combination indexes after 20 h of treatment with two compounds in leukemic cells. For AML cell lines, four independent assays  with four measurements  per dosage were performed. For hematological patient samples, two independent assays with three technical replicates per dosage were performed. For the calculation of combination indexes, two dosages of venetoclax and two dosages of the other compounds were applied alone and in combination. Combination indexes were calculated on Compusyn software . Data are depicted as scatter plots with median values and SD. In grouped analysis, significance of differences in median values was calculated by Mann–Whitney test.For assays with AML cell lines, cells were plated at a density of 5 × 10Imaging cytometry was carried out on the NC-3000 cell analyzer  with reagents supplied by ChemoMetec. To determine the induction of cell death apoptotic cells were stained with AnnexinV-CF488A conjugate  in AnnexinV buffer and Hoechst 33,342 (10 μg/mL) for 15 min at 37 °C, followed by several washes. Propidium iodide was added shortly before imaging. For cell cycle analysis, cells were incubated in lysis buffer with DAPI (10 μg/mL) for 5 min at 37 °C before imaging on the NC-3000 cell analyzer.


Type 2 diabetes (T2D) is a chronic metabolic disease defined by insulin insensitivity corresponding to impaired insulin sensitivity, decreased insulin production, and eventually failure of beta cells in the pancreas. There is a 30–40 percent higher risk of developing T2D in active smokers. Moreover, T2D patients with active smoking may gradually develop many complications. However, there is still no significant research conducted to solve the issue. Hence, we have proposed a highthroughput network-based quantitative pipeline employing statistical methods. Transcriptomic and GWAS data were analysed and obtained from type 2 diabetes patients and active smokers. Differentially Expressed Genes (DEGs) resulted by comparing T2D patients’ and smokers’ tissue samples to those of healthy controls of gene expression transcriptomic datasets. We have found 55 dysregulated genes shared in people with type 2 diabetes and those who smoked, 27 of which were upregulated and 28 of which were downregulated. These identified DEGs were functionally annotated to reveal the involvement of cell-associated molecular pathways and GO terms. Moreover, protein–protein interaction analysis was conducted to discover hub proteins in the pathways. We have also identified transcriptional and post-transcriptional regulators associated with T2D and smoking. Moreover, we have analysed GWAS data and found 57 common biomarker genes between T2D and smokers. Then, Transcriptomic and GWAS analyses are compared for more robust outcomes and identified 1 significant common gene, 19 shared significant pathways and 12 shared significant GOs. Finally, we have discovered protein–drug interactions for our identified biomarkers. Type 2 diabetes(T2D) is a chronic metabolic disease defined by insulin insensitivity corresponding to impaired insulin sensitivity, decreased insulin production, and eventually, failed beta cells in the pancreas ,2. So, TIt is clinically statistically shown that professional smokers are more likely to develop T2D with a 30–40% higher chance than nonsmokers . AnotherThe development of T2D can be amplified by regular smoking, as well as many complications which can be developed in T2D patients with a smoking history. Hence, we have tried to retrieve common molecular mechanisms that exist between T2D and smokers. Molecular mechanisms and relationships underlying the T2D-smoker are yet unclear in medical endocrinology , but theWe used data from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI) . DatasetAccession numbers of the datasets, which is human gene expression, are GSE106177  and GSE4p-value ≤ 0.05, the significant DEGs were filtered. By utilising the match function, we were able to find shared DEGs between T2D and smoking and create a Venn diagram (Shown in The GREIN Gene Expression Omnibus(GEO) provided us with gene expression RNA-sequence datasets. The DESeq2, an R package, was used to identify differentially expressed genes (DEGs) of T2D and Smoking related RNA-Seq count data . In DESeShown in .Many bioinformatic techniques often focus on measuring the relevance of gene set similarity with previously annotated gene sets in order to find functional pathways connected to a particular disease . We usedp-value that is less than 1.0 × 10−8. We also identified the GOs and pathways associated with the identified common significant genes from GWAS analysis. Finally, we have compared the transcriptomic analysis result with the GWAS analysis to better understand and relate the common biological outcomes by both studies.Researchers use genome-wide association studies (GWAS) to examine a group of genetic mutants which are linked with a certain illness or condition. During this procedure, the genomic sequences of a large number of patients were collected and evaluated so that we could determine the DNA polymorphisms that are present in the genomes of the patients. We have collected GWAS data from both T2D patients and smokers. We have collected the GWAS data from two well-known databases such as PheGenI  and GWASWe have collected RNA-Seq data from Grein or NCBI to examine the impact of gene expression on T2D patients and Smokers. As shown in TMEM178B, TAPBP, ZNF469, SPP1, ARHGAP27, XIST, HCN2, TDRD12, METRN, MEGF6, KIT, IDUA, DUSP8, ABR, C11orf96, ZBTB22, GLIS2, EPPK1, CPXM2, PRRC2A, NTRK1, HLA-C, LOC101928994, VGF, ICAM5, FSCN2 and KCNJ12. Moreover, the most significant shared down regulated genes between T2D and Smoking: LRRCC1, EPHA7, ARHGAP20, SELL, FLOT1, PCDH18, CFI, ARHGAP11A, CENPH, KRBOX1, MYB, PRIM1, MICA, HMSD, CDCA7, KIFC1, HLA-DPA1, ZNF385D, GPR158, SMN1, HLA-DPB1, MGP, CERS6-AS1, PSMB8, PCYT1B, E2F2, TCF19 and RNVU1-4.Then, we have compared the upregulated genes of T2D with the upregulated genes of smokers. Similarly, we have compared the downregulated genes of both conditions. Between T2D and Smoking, the number of upregulated common genes are 27 and the number of downregulated common genes are 28 Shown in . The mosp-values below 0.05 are considered. Therefore, we got 169 most significant signalling pathways. Finally, we sorted the pathways in ascending order depending on p-value. The top 20 pathways were represented in In pathway analysis, we have used five different databases named BioCarta, Elsevier Pathway, KEEG, Reactome and WikiPathway. Then, we have looked for highly expressed pathways by utilizing shared DEGs between T2D and smoking. These pathways were then divided into functional groups for analysis. Initially, we got 573 signalling pathways linked with both disease and condition. Manual curation was then utilised to abate the number of pathways. Pathways with p-value less than 0.05 were included as the most significant GO terms. A total of 137 GO terminologies are found as the mostly enriched GO terms between the conditions. The GO approach uses the biological process (BP), cellular component (CC), and molecular function (MF) to categorise them into functional categories, but we only consider the BP database of GO terminologies. At first, 489 GO terms found common between T2d patients and smokers. After that, the terms with a STRING , a databARHGAP27, E2F2, KIT, EPHA7, TAPBP, KIFC1, ARHGAP11A, SPI1, POU5F1, NANOG, SOX2, METRN, TCF19, SMN1, TDRD12, MGP, E2F1, GATA2, NFKB1, NFIC, SRF, YY1, TFAP2A, FOXL1, TP53, POU2F2, FOXC1, PRRC2A, VGF, CDCA7, SPP1, FLOT1, ABR and MYB. For TFs, protein–drug interactions, and gene–microRNA interactions, we have used Net-workAnalyst tools . Transcrp-value which is less than 1.0 × 10−8. After that, we found 595 significant genes for T2D and 535 significant genes for smokers. Then, we compared both significant genes to obtain the candidate genes associated with both conditions. There are 57 candidate genes  to distinguish the most significant pathways and GOs. Thus, the number of pathways reduced to 196; similarly, GOs reduced to 300. A comparison performed between transcriptomic and GWAS analysis where we have found only one biomarker gene (HLA-C) common between two studies. We have also compared significant pathways from both transcriptomic and GWAS analysis and found 19 significant pathways are shared by both studies: Genes with Mutations Associated with Psoriasis, Proteins Involved in Ulcerative Colitis, Proteins Involved in Psoriatic Arthritis, Proteins Involved in Neuroblastoma, Proteins Involved in Spontaneous Abortion, Proteins with Altered Expression in Osteoarthritis, NTRK FOXO/MYCN Signalling, NTRK1/2/3 Acetylcholine Production, Receptors and Adaptor Proteins Activated in Cancer, Proteins Involved in Glioma, Kaposi sarcoma-associated herpesvirus infection, MAPK signalling pathway, PI3K-Akt signalling pathway, Endosomal/Vacuolar pathway, VEGFR2 mediated cell proliferation, PI3K-Akt signalling pathway WP4172, Allograft Rejection WP2328, Pathways Regulating Hippo Signalling WP4540 and NOTCH1 regulation of endothelial cell calcification WP3413. Similarly, 12 significant GOs are shared by both studies: regulation of neuron apoptotic process (GO:0043523), positive regulation of phosphorylation (GO:0042327), antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway (GO:0002484), nerve growth factor signalling pathway (GO:0038180), negative regulation of sprouting angiogenesis (GO:1903671), positive regulation of bone resorption (GO:0045780), positive regulation of DNA-binding transcription factor activity (GO:0051091), ovarian follicle development (GO:0001541), neurotrophin TRK receptor signalling pathway (GO:0048011), positive regulation of histone H3-K4 methylation (GO:0051571), positive regulation of cell projection organization (GO:0031346) and sympathetic nervous system development (GO:0048485).GWAS analysis was performed to find the most significant genes for both T2D and smokers. We employed a threshold Shown in  that areWe investigated the previously published literature to verify the presence of biomarker genes that are found both in smokers and T2D Shown in . The expThere is no doubt that T2D is a major threat to humanity. People who smoke cigarettes have a 30–40% increased chance of acquiring T2D in comparison with nonsmokers . MoreoveTo investigate if there was any significant dysregulation, we have performed Differential Expression Analysis (DEA) followed by identification of common genes including up or downregulated genes shown in , pathwayFirst of all, we have focused on four pathways, namely PI3K/AKT signalling pathway, MAPK signalling pathway, Endosomal/Vacuolar pathways and Allograft Rejection pathway, which are found by the shared profile of T2D and smoker. These pathways were resulted from both transcriptomic and GWAS analysis.T2D development is significantly influenced by the MAPK signalling pathway , where cFor significant common genes, we have focused on certain genes named SPP1, PSMB8, PRRC2A, and PRIM1 that are found from transcriptomic analysis. These genes are highly connected with each other, which also resulted in hub–protein analysis  are validated using previous studies. They are also repeated in multiple analyses such as GWAS analysis, protein–protein interaction, protein–drug interactions, and transcriptional and post-transcriptional Regulators. Signalling pathways such as PI3K/AKT signalling pathway, AMPK signalling pathway, Endosomal/Vacuolar pathways and Allograft Rejection are also verified using published literature. However, still, there are many novel findings that make way for further analysis by the researchers. Finally, our study suggests that people should quit smoking as soon as possible in order to reduce the risk of being diagnosed with type 2 diabetes. Moreover, type 2 diabetes patients must quit smoking to avoid other complications/diseases and take medication accordingly.


Bushen Huoxue Huatan Decoction (BHHD) is a classic prescription for treating polycystic ovary syndrome (PCOS). This study aims to explore the effects and possible mechanisms of BHHD on PCOS by integrating network pharmacology and clinical study.  The components and potential drug targets of BHHD were analysed using the TCMSP platform, and the potential pathogenesis targets for PCOS were analysed using the GeneCards and OMIM databases. Subsequently, a disease-compound-target network diagram was established to identify the targets of BHHD treatment on PCOS. In addition, protein-protein interaction analysis, KEGG pathway analysis, and Gene Ontology biological analysis were carried out to reveal the mechanisms of BHHD. To further validate the analysis, a clinical trial involving 62 PCOS patients was conducted. All patients were treated with BHHD for 3 months and the ovulation rate, anthropometric indicators, clinical symptoms, and serological indicators were measured and compared before and after treatment. β, IL-8, macrophage migration inhibitory factor, and human leukocyte differentiation antigen CD40 ligand were greatly reduced.  The network pharmacology analysis showed that quercetin, luteolin, and kaempferol are the most significant active components in BHHD; STAT3, Jun, AKT1, MAPK3, MAPK1, and TP53 are the most critical drug targets; regulating hormones, reversing insulin (INS) resistance, exerting anti-inflammatory effects, and improving fertility might be the most important mechanisms of BHHD in the treatment of PCOS. After BHHD administration, the cyclic ovulation rate and the clinical symptoms including acanthosis nigricans and acne of patients were obviously improved. The serum endocrine levels of luteinising hormone (LH), LH/follicle-stimulating hormone, testosterone, dehydroepiandrosterone sulphate, insulin (INS), and area under the INS curve were evidently reversed, and the serum inflammatory factors levels including human interleukin (IL)-18, IL-16, IL-1 BHHD has a good therapeutic effect on PCOS, and its mechanisms may be related to regulating hormone levels, improving insulin resistance, alleviating inflammation, and promoting pregnancy. Curcuma aromatica. Good clinical effects have been achieved in the application of BHHD in long-term clinical practice; however, its mechanism remains unclear.Polycystic ovary syndrome (PCOS) is the one of the most prevalent endocrine and metabolic disorders in premenopausal women; it is characterised by hyperandrogenism and chronic anovulation . PCOS afNetwork pharmacology, based on systems biology, multidirectional pharmacology, computational biology, network analysis, and other disciplines, fully combines pharmacology and informatics to explore the overall correlation between drugs and diseases, discover drug targets, and guide the development of new drugs . Networkhttp://lsp.nwu.edu.cn/tcmsp.php, 2021-4-7), a database containing the chemical compositions of Chinese medicines  and waist-hip ratio  were calculated. In addition, the clinical symptoms including acne (based on the Rosenfield scoring system), hirsutism  and acanthosis nigricans  were comprehensively evaluated by two or more professional clinicians.Endocrine and Metabolic Parameters. The serum levels of luteinising hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), estradiol (E2), dehydroepiandrosterone sulphate (DHEAS), and prolactin (PRL) were detected by chemiluminescence immunoassay, the serum level of fasting blood glucose (FBG) was detected by the hexokinase method, and the serum INS concentrations at 0 min, 30 min, 60 min, 90 min, 120 min, and 180 min were detected by electrochemiluminescence. In addition, the area under the INS curve (IAUC) calculated according to the formula [IAUC = 0.5 × (INS_0 min + INS_180 min) + 0.75 × INS_60 min + INS_30 min + INS_120 min], INS resistance index (HOMA-IR) calculated according to the formula [HOMA-IR = fasting INS  × FBG (mmol/L)/22.5], and INS sensitivity index (ISI) calculated according to the formula [ISI = 1/(FBG × INS_0 min)].Serum Inflammatory Cytokines. The levels of serum inflammatory cytokines of PCOS patients  were tested by Proteome Profiler Human Cytokine Array Kit  and analysed by Shanghai Uninvest Biotechnology Company; the significant inflammatory factors were verified by ELISA assay (Beyotime Systems).The cyclic ovulation rate, clinical symptoms, endocrine and inflammatory levels of patients were compared before and after treatment. The observation indexes and test methods are as follows:A total of 1033 ingredients were obtained from BHHD based on the TCMSP platform, including 85 BS, 202 DG, 125 DS, 143 HQ, 121 MG, 105 SCP, 30 ZJC, and 222 YJ. According to OB ≥ 30% and DL ≥ 0.18, 142 potential active ingredients were screened out. The same ingredients were then removed from different herbs, resulting in 129 potential active ingredients.A total of 292 genes corresponding to potential active components of BHHD were screened using the targets information analysis function of TCMSP platform and 175 target genes were obtained after de-duplication. All these 175 target genes were annotated using UniProt data. In addition, a total of 4797 PCOS disease-related genes were gained from the GeneCards and OMIM database. Moreover, a total of 189 cross-targets genes for BHHD treatment of PCOS were captured through intersection analysis of drug components and PCOS disease-related genes. β-sitosterol, and baicalein, as shown in For further explanation, a component-target network was constructed, as shown in Homo sapiens with a confidence level >0.900. There were 173 nodes and 1838 edges in It is important to accurately reveal and annotate all functional interactions between proteins in cells. We used PPI networks to explore the relationships between different target genes and to find key genes in the network. 189 cross-targets were imported into STRING and the species selected was P value . Amongst them, several KEGG pathways were closely related to PCOS, including TNF signalling pathway (hsa04668), HIF-1 signalling pathway (hsa04066), FoxO signalling pathway (hsa04068), PI3K-Akt signalling pathway (hsa04151), Toll-like receptor signalling pathway (hsa04620), T-cell receptor signalling pathway (hsa04660), VEGF signalling pathway (hsa04370), p53 signalling pathway (hsa04115), INS resistance (hsa04931), ErbB signalling pathway (hsa04012), and oestrogen signalling pathway (hsa04915). The 30 pathways with the highest significance associated with PCOS are shown in A total of 130 pathways were revealed by KEGG enrichment analysis with statistically significant differences (CHIECRCT-20160050).Based on the inclusion and exclusion criteria, 62 PCOS were actually included in this study. The mean age of patients was (26.21 ± 4.67) years, and the mean course of disease was (4.89 ± 5.02) years. This study was approved by the Ethics Review Committee of Chinese Registered Clinical Trials . According to the calculation results, the ovulation rate of patients with PCOS after BHHD treatment was 61.2%, which was significantly increased compared with that before treatment .2), central obesity (WHR >0.8), hirsutism (F-G scoring >9), acne (Rosenfield scoring >0), and acanthosis nigricans (positive) were 28.5%, 61.3%, 45.2%, 77.4%, and 38.7%, respectively. After BHHD treatment, the clinical symptoms of acne and acanthosis nigricans were obviously improved (P < 0.05); however, there were no significant differences in BMI, WHR, and hirsutism before and after treatment (P > 0.05)   Tables –8, whereα, CXCL12/SDF-1, IL-1ra/IL-1F3, IL-8, IL-13, IL-16, IL-18/IL-1F4, MIF, and serpin E1/PAI-1 were obviously increased in PCOS compared with the healthy women (P < 0.05)   . As ELIS < 0.05) .PCOS is the most common endocrine and metabolic disorder that disturbs the physical and reproductive health of women of childbearing age . Therefoβ-sitosterol, and baicalein. Quercetin is a flavonol compound with multiple biological activities and is widely distributed in the plant kingdom. Pourteymour et al. reported [According to the “compound-target-disease” network diagram, the core components of BHHD were obtained in our study, and the top 10 ingredients were quercetin, luteolin, kaempferol, fisetin, wogonin, tanshinone IIA, naringenin, dihydrotanshinlactone, reported  that Quereported  found threported  shown thS. baicalensis, has shown biological activities against several androgen-associated disorders, such as prostate cancer, androgenetic, and acne. Interestingly, our previous research revealed that baicalin would potentially be an effective therapeutic agent for hyperandrogenism in PCOS by inhibiting the recruitment of GATA1 to the HSD3B2 promoter in ovarian tissue [Baicalein, a flavonoid glycoside isolated from n tissue . In addiIt is known that about two-thirds of PCOS patients ovulate sparsely or even anovulate, which is the main cause of infertility in PCOS. In recent years, assisted reproductive technology has greatly improved the pregnancy rate of PCOS; however, there are several problems, such as high incidence of ovarian hyperstimulation syndrome, low endometrial receptivity, and increased risk of abortion , so it mβ-hydroxysteroid dehydrogenase, 17-steroid reductase, and aromatase P450. The lack of these enzymes leads to impaired ovarian steroid synthesis, failure of ovarian androgen conversion to oestrogen, and increased androgen levels, which prevent ovulation [Hormone disorder is an important feature of PCOS. Gonadotropin-releasing hormone, androgen, estrogen, growth hormone, cortisol, parathyroid hormone, calcitonin, and other hormones are out of balance in PCOS patients . Androgevulation . MultiplOur network pharmacology analysis results showed that estrogen signalling pathway, GnRH signalling pathway, ovarian steroid production pathway, and other hormone-related signalling pathways were significantly expressed, suggesting that BHHD may have a certain regulatory effect on various hormones. The effects of natural compounds in regulating hormones have always been a research hotspot. Quercetin can inhibit the proliferation and apoptosis of bovine ovarian cells and the release of IGF-I, progesterone, and testosterone; reverse the effect of xylene on ovarian function; and protect ovarian function . KaempfeInsulin resistance (IR) and compensatory hyperinsulinemia are also important endocrine features in PCOS patients, with an incidence of about 70%. INS regulates the activity of enzymes in the ovaries and liver and its signals are interconnected through a variety of intracellular pathways; at present, IRSS, PI3K/AKT, and MAPK are generally considered to be key nodes of INS function. These nodes are also active in the ovary and their interaction with FSH and LH signalling pathways allows INS to directly regulate ovarian function , 39. Preα levels are higher in PCOS patients and are directly related to body fat percentage levels [α system triggers ovarian apoptosis and reduces oocyte capacity, affecting follicle maturation and elimination, thereby reducing fertility in patients with PCOS [α can directly reduce the capacity of endometrial tissue glucose uptake, affect endometrial function, and lead to infertility [β and IL-18, further enhancing the inflammatory environment [Recent studies show that chronic low-grade inflammation is closely associated with PCOS. Inflammatory processes are involved in ovulation and play a key role in follicular dynamics. Visceral adipose tissue induces inflammatory responses and maintains the inflammatory state of adipose cells by increasing the recruitment of inflammatory cytokines, monocyte chemotactic proteins, and immune cells. The TNF signalling pathway and NOD-like signalling pathway are classical inflammation-related pathways. TNF-e levels . The actith PCOS . At the ertility . In termironment .α, CXCL12/SDF-1, IL-1ra/IL-1F3, IL-8, IL-13, IL-16, IL-18/IL-1F4, MIF, and serpin E1/PAI-1. These findings confirm the presence of chronic inflammation in PCOS patients. While, after BHHD intervention, the serum levels of inflammatory cytokines IL-1ra/IL-1F3, IL-8, IL-16, IL-18, MIF, and CD40L in PCOS patients were significantly reduced. According to previous studies, IL-1ra/IL-1F3 is an endogenous inhibitor of IL-1; it can bind to IL-1RI and IL-1RII, but it does not initiate signal transduction and thus functions as a competitive inhibitor of the activity of IL-1 through IL-1R. A large number of animal studies have shown that IL-1ra can reduce the occurrence of low blood sugar levels generated by colony-stimulating factor due to endotoxemia, as well as strengthen organic tolerance [κB pathway in the ovary and promotes the expression of IL-1β, IL-6, iNOS, and TNF-α [α-induced apoptosis and inflammation by blocking the nuclear factor-κB (NF-κB) and AP-1 signalling pathways. Luteolin inhibits proinflammatory mediators such as IL-1β, IL-6, IL-8, IL-17, IL-22, TNF-α, and COX-2. It also regulates various signalling pathways, such as NF-κB, JAK-STAT, and TLR [Our protein chip analysis showed that compared with healthy women, PCOS patients had an inflammatory state with significantly increased serum levels of inflammatory cytokines CCL5/RANTES, CD40 ligand/TNFSF5, complement component C5/C5a, CXCL1/GROolerance . IL-8, aolerance , and eleolerance . IL-16 iolerance . Il-18 iolerance . MIF is olerance . Elevatend TNF-α . CD40 isnd TNF-α . Serum Cnd TNF-α . The cor and TLR . All of This study is the first to identify the potential of BHHD in the treatment of PCOS from a network pharmacology perspective and reveal the mechanism of BHHD action in four aspects, namely, regulating hormone levels, reversing insulin resistance, anti-inflammatory immunity, and promoting pregnancy in patients. It is noteworthy that these aspects have been confirmed in our subsequent clinical trial. In addition, this study also found that monomer compounds such as quercetin and kaempferol have excellent performance in all aspects of PCOS treatment and may become new hot drugs for clinical treatment of PCOS if more rigorous pharmacological and clinical studies are carried out.


Restricting ribosome biosynthesis and assembly in response to nutrient starvation is a universal phenomenon that enables cells to survive with limited intracellular resources. When cells experience starvation, nutrient signaling pathways, such as the target of rapamycin (TOR) and protein kinase A (PKA), become quiescent, leading to several transcription factors and histone modification enzymes cooperatively and rapidly repressing ribosomal genes. Fission yeast has factors for heterochromatin formation similar to mammalian cells, such as H3K9 methyltransferase and HP1 protein, which are absent in budding yeast. However, limited studies on heterochromatinization in ribosomal genes have been conducted on fission yeast. Herein, we shed light on and compare the regulatory mechanisms of ribosomal gene transcription in two species with the latest insights. Saccharomyces cerevisiae, and each cell contains 100,000–200,000 ribosomes in the fission yeast Schizosaccharomyces pombe [S. cerevisiae [S. cerevisiae and S. pombe [The ribosome is massive intracellular machinery translating mRNA, transcribed from DNA, into proteins in living organisms. This machinery is constructed by a small 40S subunit composed of 18S ribosomal RNA (rRNA) and 33 ribosomal proteins (RPs) and a large 60S subunit composed of three rRNAs  and 46 (in yeast) or 47 (in human) RPs [S. cerevisiae carries approximately 150 rDNA tandem repeats on chromosome XII, S. pombe has 100–150 repeats on both ends of chromosome III, and humans carry a total of ~350 repeats on five different chromosomes [S. cerevisiae [S. pombe [S. cerevisiae [To achieve rapid and robust rRNA transcription, multiple copies of ribosomal DNA (rDNA) containing 18S, 5.8S, and 25S/28S regions exist tandemly in the genome . For exaomosomes ,7. The 5S. pombe . FurtherS. cerevisiae and more than 500 assembly factors and 300 snoRNAs in humans [Transcription of ribosome-associated genes requires three RNA polymerases. That is, consecutive sequences of 18S, 5.8S, and 25S/28S rRNA are transcribed by RNA polymerase I in the nucleolus as the precursor rRNA from 5′ETS  to 3′ETS, including ITS  1 and ITS 2 . 5S rRNAIn summary, numerous factors contribute to ribosome biogenesis, and these genes are actively transcribed. Accordingly, intracellular resources are substantially devoted to ribosome synthesis. Therefore, ribosome biosynthesis must be promptly halted to reduce intracellular energy consumption when the cells encounter nutrient depletion.One way to cease ribosome biogenesis is epigenetic gene regulation through modifying histones and methylation of cytosine residues on DNA without altering the DNA sequences. DNA methylation does not occur in budding and fission yeasts ,14. HistThis review summarizes the findings on the transcriptional regulation mechanisms of ribosomal genes and compares the difference between budding and fission yeasts.S. cerevisiae revealed that the concentration of active rDNA is reduced in the stationary phase  compared to that in the logarithmic growth phase (or when cells are cultured in a nutrient-rich medium) [When the nutrient supply decreases, ribosome biosynthesis must be halted immediately to avoid the consumption of intracellular resources. Early studies using  medium) . In addi medium) , and tyr medium) , indicat medium) . Jorgens medium) .S. pombe, the depletion of sulfur, an indispensable component for many coenzymes such as coenzyme A and biotin, diminishes the expression of ribosome-related genes [In ed genes . A compred genes . We alsoed genes ,27.S. cerevisiae [S. pombe, oxidative, cadmium, heat, osmotic, and DNA damage stress commonly repress ribosomal genes [Intriguingly, ribosomal genes can also be repressed by stresses other than nutritional starvation. For example, mild heat shock leads to quick but transient repression of RP genes in revisiae ,28. In Sal genes . Since nS. cerevisiae. Two types of TOR proteins, Tor1 and Tor2, are found in budding yeast, forming TOR complex 1 (TORC1) and TOR complex 2 (TORC2), respectively [TOR was initially identified as a target gene for the macrolide immunosuppressant drug rapamycin by genetic screening using ectively ,31. Notaectively ,33. Withectively ,35,36  strongly accumulates on 35S rDNA through the DNA binding domain box A and box B, forming a fragile chromatin structure ,54,55,56cription ,58. An icription . These fMO1 gene ,60  in the rDNA region rather than by repressing transcription of 35S rRNA [S. cerevisiae) by SIRT7 (Sir2 in S. cerevisiae) enhances the association of histone and RNA polymerase I on rDNA, leading to an upregulation of rRNA transcription [Finally, we refer to a case where rDNA transcription is epigenetically regulated in ng yeast . Hence,  repeats , it does35S rRNA . Interescription ,80.S. cerevisiae COMPASS complex involving Set1 promotes histone H3K4 methylation and represses transcription from rDNA during nutrient-rich conditions, suggesting that the Set1 complex silences the dormant rDNA unit [S. cerevisiae rDNA silencing during starvation remains to be determined, and whether it is regulated by TORC1.Instead, the DNA unit ,82  by methyltransferase Nop1 and is found to accumulate specifically in the nucleolus. In addition, the ChIP-seq profile indicates that H2AQ105me is enriched within the 35S rDNA regions. Since a mutant with an alanine substitution at Q105 mimicking methylation exhibits increased rRNA transcription, the enrichment in H2AQ105me is assumed to facilitate rRNA transcription [Remarkably, cription . This idcription   carry a UAS (Upstream Activating Sequence), a T-rich element, and a TATA box as upstream regulatory elements. The transcriptional activator Rap1 (repressor/activator protein 1) recognizes the UAS and associates with ~90% of the RP genes [lacZ gene, which is fused downstream of the RPL16 gene, revealed that transcription levels in cells lacking the Rap1 binding site at the RPL16 gene promoter were reduced to 10% of that in wild-type cells, confirming that Rap1 activates RP genes [S. cerevisiae cells are exposed to heat, osmotic stress, or rapamycin, the localization pattern of Rap1 is not altered despite the observed repression of RP gene transcription, suggesting that Rap1 is just a scaffold for other transcription factors governing their expression [The repressive mechanisms of RP genes ,86,87,88RP genes . Howeverpression   pathway during nutritious conditions. In contrast, it is increased during amino acid starvation [As described above, RP gene expression relies on complicated mechanisms, but the behavior of these factors is dynamically altered when cells shift into nutrient-poor conditions. Starvation and rapamycin treatment triggers the dissociation of Ifh1, Sfp1, and Esa1 from the RP gene. The dissociation of Esa1 was studied by Joo and coworkers . A transarvation   are involved in ogenesis . The upsogenesis ,111,112.ogenesis . It is kogenesis . Additioogenesis   demonstrated that the Sfp1-binding motif is concentrated in the promoter of ribosome biosynthetic genes repressed during nitrogen starvation [S. pombe ribosomal gene expression. A homolog of the transcriptional activator Sfp1 is also present in arvation , indicatS. pombe. However, Fhl1 predominantly contributes to the regulation of genes related to the transport process and mating/sporulation, with little or no influence on the ribosome-related genes [The forkhead protein Fhl1 is also regulated by TORC1 in ed genes .S. cerevisiae Crf1 and Ifh1 are annotated with the S. pombe homolog Crf1, it has not been determined whether Crf1 regulates ribosome-related genes. A homolog of Stb3 is also present in S. pombe, but its function has yet to be discovered. Finally, there is no annotation and homolog information for Dot6 and Tod6 on PomBase (https://www.pombase.org/ accessed on 31 January 2023). Altogether, the analysis of transcription factors equivalent to those revealed in S. cerevisiae is poor in S. pombe.Although S. pombe ribosomal gene repressions. Yet, histone deacetylase (class I) Clr6, homologs of Rpd3 repressing rRNA, RP, and Ribi genes in S. cerevisiae, do not contribute to the repression of ribosome-related genes. In contrast, class II deacetylase Clr3, a homolog of Hda1, represses at least rRNA gene expression [S. pombe, accumulate at rDNA regions to repress transcription [S. pombe as in S. cerevisiae  positively regulates rRNA gene expression . Previouctivator . Additioctivator , which mctivator  , also contributes [D. melanogaster or mice as model organisms.In HEK293 cells and β-TC6 cells, a mouse pancreatic beta cell, mTOR binds to the rDNA promoter, and this binding is dissociated upon rapamycin treatments and amino acid starvation . This sutributes . In the


TWIST1 and the involved genes in the process of CSCs self-renewal in human esophageal squamous cell carcinoma (ESCC) line KYSE-30. Methods: TWIST1 overexpression was enforced in the ESCC KYSE-30 cells using retroviral vector containing the specific pruf-IRES-GFP-hTWIST1 sequence. Following RNA extraction and cDNA synthesis, the mRNA expression profile of TWIST1 and the stem cell markers, including BMI1, CRIPTO1, DPPA2, KLF4, SOX2, NANOG, and MSI1, were assessed using relative comparative real-time PCR. Results: Ectopic expression of TWIST1 in KYSE-30 cells resulted in an increased expression of TWIST1 compared to control GFP cells by nearly 9-fold. Transduction of TWIST1-retroviral particles caused a significant enhancement in BMI1, CRIPTO1, DPPA2, KLF4, and SOX2 mRNA expression, approximately 4.5-, 3.2-, 5.5-, 3.5-, and 3.7-folds, respectively, whereas this increased TWIST1 expression caused no change in the mRNA expression of NANOG and MSI1 genes. Conclusions: TWIST1 gene ectopic expression in KYSE-30 cells enhanced the level of cancer stem cell markers’ mRNA expression. These results may emphasize the role of TWIST1 in the self-renewal process and may corroborate the involvement of TWIST1 in the stemness state capacity of ESCC cell line KYSE-30, as well as its potential as a therapeutic target.Background: Stemness markers play critical roles in the maintenance of key properties of embryonic stem cells (ESCs), including the pluripotency, stemness state, and self-renewal capacities, as well as cell fate decision. Some of these features are present in cancer stem cells (CSCs). TWIST1, as a bHLH transcription factor oncogene, is involved in the epithelial–mesenchymal transition (EMT) process in both embryonic and cancer development. Our aim in this study was to investigate the functional correlation between  Tumor progression and metastasis is a complicated process, during which the primary tumor cell population is able to escape and survive in the circulation and invade and proliferate in another microenvironment . EpithelTWIST1, a basic helix-loop-helix transcription factor, is a key mediator of the mesenchymal/CSC state and can link EMT to self-renewal ,12. The TWIST1 ectopic expression on the expression pattern of stemness and self-renewal-associated genes, including BMI1, CRIPTO1, DPPA2, KLF4, SOX2, NANOG, and MSI1 in ESCC cell line KYSE-30, to elucidate possible crosstalk between TWIST1 and the self-renewal state of the cells.Hence, the evaluation of the EMT mechanism in CSCs regulation is an essential need to identify new approaches for therapeutic strategies preventing cancer progression and improving the prognosis of the disease . Our aimBMI1, CRIPTO1, DPPA2, KLF4, and SOX2 mRNAs and gene sequences were obtained from Genebank . The sequence analysis was performed by using CLC Main Workbench version 20 (CLC bio).http://en.pasteur.ac.ir/). These cells were cultured in Dulbecco’s modified Eagle’s medium  and RPMI 1640 medium (Biosera), respectively, supplemented with 100 U/mL penicillin–streptomycin  and 10 % heat-inactivated fetal bovine serum  in a humidified atmosphere at 37 °C with 5% CO2.GP293T  and the human ESCC (KYSE-30) cell line were obtained from the Pasteur Institute Cell Bank of Iran (5 cells/6-well plate). Retroviral transduction was performed twice and evaluated through inverted fluorescence microscopy.The Pruf-IRES-GFP-hTWIST1 and control Pruf-IRES-GFP plasmids were obtained for generating retroviral particles, as described before ,14. BrieBMI1, CRIPTO1, DPPA2, KLF4, SOX2, NANOG, and MSI1, were evaluated by specific primer sets demonstrated in The RNA extraction from the Pruf-IRES-GFP- and Pruf-IRES-GFP-hTWIST1-transduced ESCC cell line, DNase I treatment, cDNA synthesis, and quantitative RT-PCR expression analysis have been described in detail previously ,15,16. TBMI1, CRIPTO1, DPPA2, KLF4, and SOX2 genes were evaluated to check and find the probable E-boxes. Several different E-boxes were found in the −2 kb upstream region of these genes before the starting site. Interestingly, some of the E-boxes were located close to the transcription start site in position −70 for BMI1; −46, −97 and −203 for CRIPTO1; in −348 and −437 for DPPA2; in− 412 and −422 for KLF4; and finally in −1 and −6 for SOX2. Other E-boxes were highlighted in CSCs gene transcription units located in both exon and intron regions. , while TWIST1 overexpression had no effect on the mRNA expression of NANOG and MSI1 genes. Data are represented in The stable enforced high expression of The EMT process and stemness state are tightly linked together. The common shared regulators and signaling pathways in these events can assist in a better understanding of the connection between EMT and stemness states, as well as the identification of novel and effective targets to develop new therapeutic strategies and preclude relapse for patients . CSCs prTWIST1, as a known EMT marker in cancer, can trigger the generation of a CSC status through the overexpression of stemness markers in different types of cancers [TWIST1 ectopic expression on different involved genes in stemness state and self-renewal capacities and determined the increased levels of BMI1, CRIPTO1, DPPA2, KLF4, and SOX2 expression following TWIST1-enforced expression in KYSE-30 cells. These results may highlight the potential of TWIST1 to support self-renewal capacity through modulating stem cell genes’ expression pattern in ESCC line KYSE-30.  cancers . In currTWIST1 can expand CSCs with self-renewal potential for the growth of secondary tumors [TWIST1 induced the expression of stemness markers and enhanced self-renewal in human head and neck squamous cell carcinoma, ESCC, as well as cervical and breast cancers [TWIST1, CRIPTO1, SOX2, and MSI1 were evaluated in ESCC patients, indicating their role in tumorigenesis and tumor cell aggressiveness [TWIST1 gene expression in ESCC cell lines KYSE-30 and YM-1 resulted in the significant overexpression of OCT4 (stem cell-associate transcription factor), MAGEA4, and NY-ESO1 (testicular cancer antigens), N-cadherin, Occluding, ZEB2, Fibronectin, and Vimentin (EMT markers), suggesting a key relation between EMT and CSC formation in esophageal tumor cells [SNAIL gene was observed following the enforced expression of TWIST1 in KYSE-30 cells, suggesting a negative regulatory effect of TWIST1 on SNAIL expression [TWIST1 expression, it may be extrapolated that the ectopic expression of TWIST1 induced cells to undergo the EMT process for the acquisition of a CSC population and the self-renewal ability of stem-like cells. TWIST1 has been demonstrated to be involved in self-renewal through the regulation of stemness markers [TWIST1 overexpression can stimulate EMT-induced CSC state and metastasis in the tumor.TWIST1, as a regulator of EMT in embryogenesis, is involved in the tumorigenesis of different cancers, including sarcomas, carcinomas, and hematological malignancies . It has y tumors . It has  cancers . The expsiveness ,22,23,24or cells ,13,16. Mpression . Based oBMI1 by TWIST1 can enhance the expression of SOX2, KLF4, NANOG, NF-κB, MRP1, and TERT [NANOG is also able to regulate the expression of BMI1, TWIST1, and SNAIL1 to promote EMT, invasion, migration, the induction of stemness markers, metastasis, and tumor-initiating ability in breast, colon, non-small cell lung cancer (NSCLS), and head and neck squamous cell carcinoma (HNSCC) via promoter occupancy [BMI1 and TWIST1 in the E-cadherin promoter can lead to EMT activation and develop the CSCs subpopulation [BMI1 via the enforced expression of TWIST1 can suppress the expression of E-cadherin and induce proliferation, self-renewal, and chemoresistance [BMI1 and TWIST1 in hypoxia can induce the expression of OCT4 and CD44 [BMI1 can facilitate tumor invasion and progression [BMI1 and TWIST1 in cancer initiation, differentiation, self-renewal, stemness, EMT, and CSC-mediated metastasis [The induced expression of ccupancy ,26. The sistance . The corand CD44 . The actgression ,30. ConsTWIST1 increased the expression of stemness transcription factors, including KLF4 and SOX2 in KYSE-30 cells, while no change in NANOG expression was observed. The roles of these stemness genes in tumorigenicity state have been reported previously. Numerous studies demonstrated that KLF4, SOX2, and NANOG constitute CSC markers and have key roles in sphere formation, stem cell self-renewal, cell motility, the formation and maintenance of CSC phenotype, clonogenicity and tumor regenerative ability, long-term proliferative potential of CSCs, repression of differentiation, cell cycle, migration, invasion, EMT, metastasis, and cancer progression in various malignancies [KLF4, SOX2, and NANOG were found in several malignancies, and their expression levels were correlated with poor prognosis, advanced-stage cancer, and shorter patient survival [NANOG gene expression after TWIST1 overexpression in this study, consisting of involved transcription machinery and transcription factors, DNA-binding proteins that compete with TWIST1, and other regulatory proteins in the cell. Describing this situation needs further investigation.Our study indicated that the induction of gnancies ,32. Moresurvival . DiffereKLF4 and BMI1 reduced the number and size of tumor sphere formation and tumor-initiating ability, suggesting that both KLF4 and BMI1 may contribute to inducing stem-like property and metastasis by TWIST1-JAGGED1-NOTCH-KLF4 signaling in HNC [KLF4 in triggering EMT in non-small cell lung and endometrial cancers and human nasopharyngeal carcinoma [TWIST1 may induce and promote EMT through KLF4 upregulation and its interaction with SOX2/NANOG/BMI1 in KYSE-30 cells. Similar to KLF4, the biological impact of SOX2 in tumor cells is dependent on tumor type [SOX2 expression can enhance malignancy by inducing stemness properties and EMT in ESCC. It has been indicated that the ectopic expression of SOX2 and TWIST1 in breast cell lines MCF7 and ZR751 resulted in SOX2-mediated invasion/EMT via the TWIST1-dependent mechanism [SOX2 and TWIST1 in glioblastoma cells, suggesting their interaction to maintain stemness and EMT ability [SOX2 and OCT4 can suppress E-cadherin expression during the reprogramming of fibroblasts and activate SNAIL1 expression and the EMT process through the suppression of TGF-β signaling [TWIST1 and SOX2 in ESCs and breast cancer cells can be modulated through their binding to the promoter region [TWIST1 led to the increased expression of SOX2 in KYSE-30 cells, which may promote the invasiveness of the cells.KLF4, as an anti-proliferative factor in differentiated epithelia, play a dual function depending on tumor type, tissue, and cancer stage. While it performs as an oncogene in HNSCC, breast, skin, advanced-stage of ESCC and pancreatic cancers, it functions as a tumor suppressor gene (TSG) in lung, liver, colorectal, prostate, bladder, gastric malignancies, and high-grade dysplasia, as well as early-stage ESCCs ,34. Knocg in HNC . Evidencarcinoma . Based omor type . Due to mor type , SOX2 exechanism . There i ability . Interesignaling . Since tr region , we hypoCRIPTO1, as a pluripotential ES marker, is a linker between EMT and tumor-initiating cells (or CSCs) and regulates self-renewal and tumorigenicity in cancers [CRIPTO1 was significantly associated with poor prognosis of patients, self-renewal, aggressive phenotypes, and tumorigenesis in esophageal and renal malignancies [CRIPTO1 expression significantly reduced the expression of NANOG, SOX2, and OCT4 and consequently reduced the stemness properties of ESCC cells, while its high expression was correlated with EMT, invasion, and metastasis of ESCC [TWIST1 and CRIPTO1 co-expression was correlated with larger tumor size, advanced stages of tumor progression, poorly differentiated state of tumors, presence of distant metastases, poor 3-year survival rates, and disease progression in non-small cell lung carcinoma [TWIST1 and CRIPTO1 in KYSE-30 cells. This report is in line with previous studies describing the regulatory role for CRIPTO1 in EMT, as an early step of invasion and metastasis, in epithelial cancer cells, including KYSE-30, EC109, and TE-1 ESCC cell lines. cancers . The uprgnancies ,41,42,43arcinoma . Herein DPPA2, as a pluripotency-related oncogene, with the pluripotency genes of NANOG, SOX2, OCT4, and SALL4, leading to the rearrangement of the epigenome during reprogramming [MAGE-A4), LAGE, and NY-ESO1, are particularly expressed in DPPA2-positive NSCLC tumors [MAGEA4 and TWIST1 in ESCC [DPPA2 and TWIST1 in ESCC. Herein, we elucidated that the enforced expression of TWIST1 led to the upregulation of DPPA2. Consistent with the reports [Cancer cells can resemble the undifferentiated state with stem cell properties, including self-renewal ability, invasiveness, unrestricted proliferation through stem cell-associate transcription factors and germ cell or cancer/testis genes . There igramming ,47. MoreC tumors . There i in ESCC . Therefo reports ,50,51,52TWIST1 and other EMT transcription factors, as well as stemness state regulators, which are involved in tumor resistance against traditional therapeutic modalities, are key to the future study of cancer diagnosis and therapy. Therefore, novel therapeutic strategies focused on targeting CSCs and their molecular regulators can eradicate the CSCs and therapy resistance. Our results confirmed the potential of TWIST1 as a proper therapeutic target for cancer treatment to inhibit EMT progress and the cancer stem-cell-like phenotype through its regulatory role of CSC markers’ gene expression, expanding insight into the TWIST1 biology in ESCC and paving the road to an efficient targeted therapy. A combination of traditional therapeutic modalities with TWIST1-targeted therapy may target the whole tumor mass, including its cancer cells, as well as its CSCs subpopulations, and offer a promising strategy for cancer cure.Since traditional cancer treatment modalities, including surgical resection, chemo- and radiotherapies, are not sufficient to inhibit tumor relapse, BMI1, CRIPTO1, DPPA2, KLF4, and SOX2, in ESCC line KYSE-30. These results may suggest a role for TWIST1 in the stemness and self-renewal maintenance of ESCC cells and provide clues to the molecular pathway controlling the EMT-induced stemness state in cancer cells.In summary, we showed that the enforced expression of TWIST1 can upregulate stem cell markers