Aflatoxin B1 (AFB1) is a natural feed and food contaminant classified as a group I carcinogen for humans. In the dairy industry, AFB1 and its derivative, AFM1, are of concern for the related economic losses and their possible presence in milk and dairy food products. Among its toxic effects, AFB1 can cause oxidative stress. Thus, dietary supplementation with natural antioxidants has been considered among the strategies to mitigate AFB1 presence and its toxicity. Here, the protective role of resveratrol (R) has been investigated in a foetal bovine hepatocyte cell line (BFH12) exposed to AFB1, by measuring cytotoxicity, transcriptional changes (RNA sequencing), and targeted post-transcriptional modifications . Resveratrol reversed the AFB1-dependent cytotoxicity. As for gene expression, when administered alone, R induced neglectable changes in BFH12 cells. Conversely, when comparing AFB1-exposed cells with those co-incubated with R+AFB1, greater transcriptional variations were observed . Functional analyses revealed that several significant genes were involved in lipid biosynthesis, response to external stimulus, drug metabolism, and inflammatory response. As for NQO1 and CYP3A activities and lipid peroxidation, R significantly reverted variations induced by AFB1, mostly corroborating and/or completing transcriptional data. Outcomes of the present study provide new knowledge about key molecular mechanisms involved in R antioxidant-mediated protection against AFB1 toxicity. Aflatoxins (AFs) are mycotoxins naturally affecting cereal grains and animal feeds around the world ,2. They The great toxicity of AFB1 is based on a bioactivation process. Indeed, AFB1 is biologically activated by a number of cytochromes P450 (CYPs), namely CYP1A and CYP3A, to an extremely reactive and electrophilic derivative, AFB1-8,9-epoxide (AFBO), that binds DNA and proteins . Such AFBesides AFB1, the most toxic aflatoxin is AFM1. AFB1, following animal intake, can be rapidly hydroxylated to form AFM1, which is efficiently transferred to milk, thus representing a safety concern for humans consuming dairy products . AflatoxRemarkably, species differences in the constitutive expression and catalytic activity of bioactivating and detoxifying enzymes appear crucial in determining the different susceptibility to AFB1 among farmed species . In thisThe animal intake of AFs always accompanies the production of Reactive Oxygen Species (ROS) and the resulting oxidative damage ,18,19, wPolygonum cuspidatum [Resveratrol  is a polyphenolic phytoalexin naturally found in grapes, red wine, berries, peanut skins, and other plant parts and products. It shows a broad range of pharmacological activities, and in plants it serves as a sort of antitoxin. Being an antioxidant polyphenol, R also shows numerous beneficial health effects, such as anticancer, anti-ageing, and anti-inflammatory effects ,36,37,38spidatum ,47,48 leThe molecular mechanisms triggered by R have been investigated in farm animal species both in vitro and in vivo. For instance, a whole transcriptome study conducted in bovine skeletal muscle cells has recently demonstrated how R significantly affects the mRNA expression of genes regulating cholesterol and fatty acid metabolism, cell cycle and proliferation . In the Recently, the potential anti-AFB1 activity of R has been explored in rodents. In alcohol-AFB1-induced rat hepatocellular carcinoma, a two-week treatment with R (100 mg/kg bw) restored the level of key antioxidant enzymes  toward normal levels ,58. In mAs far as farm animals are concerned, while the overall benefits of dietary R have been largely investigated, the ability of this polyphenol to prevent or mitigate AFB1 toxicity and the underneath molecular mechanisms have been scarcely studied, except for broilers in vivo  and cattTo date, the impact of R on AFB1-mediated whole transcriptional changes in an established hepatic cell line isolated from veterinary species has never been investigated. This study will help in developing R-supplemented diets to mitigate toxicity induced by AFB1 in dairy cattle. Such feeding improvements might contribute to reduce the risk of dairy products contamination, ultimately preventing significant economic losses.The bovine SV40 large T-antigen-transduced foetal hepatocyte-derived cell line BFH12  was cultExperimental conditions for the cytotoxicity test were those reported in our previous study . Briefly50) obtained in the present study: the selected concentrations were always below the corresponding IC50.Resveratrol concentrations were chosen based on the half maximal inhibitory concentration  are available in our previously published manuscripts ,63.As far as the RNA quality, all samples had an RNA Integrity Number (RIN) value > 7.A targeted qPCR approach was preliminarily performed to assess the effects of R at the selected sub-cytotoxic concentrations . Only transcripts known to be regulated by AFB1  were considered.The target gene expression analysis was carried out on four biological replicates . A total of four experimental conditions were assessed: CTRL, R , AFB1, R  + AFB1. Reverse transcription, qPCR amplification conditions, target genes, primers, and qPCR data analysis were described elsewhere . Based on the preliminary qPCR results, besides CTRL and AFB1 experimental conditions, only cells exposed to the maximum concentration of R , either alone (R) or in combination with AFB1 (R+AFB1), were subjected to deep transcriptome investigations.For each experimental condition, three independent biological replicates  were assessed. A total of 12 tagged RNA-seq libraries were prepared and sequenced following a 50 bp single-end strategy in an Illumina Hi-Seq 4000 instrument . Libraries were prepared as previously reported . BrieflyNotably, libraries representing CTRL and AFB1 conditions have been previously analysed in a stand-alone study assessing the whole transcriptional effects of PCB126 and AFB1 on BFH12 cells . In the estimateDisp), a linear model was fitted (glmQLFit), and the differential expressed genes (DEGs) were determined (glmQLFTest) by setting the following thresholds of significance: FDR ≤ 0.05 and Fold Change (FC) ≥ 1.5 . The complete R code used for the DE analysis is reported in Differential expression (DE) analysis has been conducted as previously described in  with minClusterProfiler package  [ClusterProfiler package. Gene Ontology terms redundancy was removed by using the simplify function (similarity cutoff = 0.5). Dotplots display the most significant enriched terms , while gene-concept networks highlight which genes were involved in the significant GO terms.A functional interpretation of significant DEGs was obtained through GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) over-representation tests implemented in R environment, and using functions included in the ichKEGG) . Ensemblp-value ≤ 0.05) indicates that the biological activity , characterized by the gene set, is correlated with the supplied ranking. The ranked input was prepared as specified in [gseKEGG function provided by the ClusterProfiler package [A pre-ranked KEGG Gene Set Enrichment Analysis (GSEA)  was alsoified in . The ana package .The detailed R codes used for the bioinformatic analysis are reported in At the end of the experiment, medium and cell samples were collected and total AFB1, AFM1, and AFL were measured by LC-MS/MS in all experimental conditions, following the procedure detailed in .Pro-oxidant effects of AFB1 and the potential benefits of R were assessed by measuring lipid peroxidation (MDA production) in the same experimental groups assessed by RNA-seq. Notably, lipid peroxidation is one of the main manifestations of oxidative damage and it is known to play an important role in AFB1 toxicity. The detailed protocol is provided in .The catalytic activity of two key enzymes involved in the antioxidant response and in AFB1 bioactivation, i.e., NQO1 (NAD(H):quinone oxidoreductase 1) and CYP3A, were investigated, as they were here reported to be transcriptionally modulated by treatments. Enzymatic activities were measured in the same experimental groups considered for RNA-seq analysis.Diaphorase, or NQO1, was selected as it is a multifunctional and stress-inducible protein involved in antioxidant defence and detoxification processes. It reduces quinone to hydroquinone, thus avoiding the formation of semiquinones and species with reactive oxygen radicals that are deleterious to cells . The diaTM CYP3A4 assay with Luciferin-IPA), following the protocol detailed in [TM cell viability assay kit (Promega), assaying ATP as a marker for basal metabolic activity, was used.The cytochrome P450 3A was selected as it plays a pivotal role in the bioactivation of AFB1 and in the formation of the highly reactive epoxide intermediate AFB(1)-8,9-epoxide [ailed in . Six indDose–response curves, box and whiskers plots, bar plots and related statistical analysis were obtained by using GraphPad Prism software .50 values. After 64 h of incubation, the IC50 value was 56.06 μM (R2 = 0.97)  a.The cytotoxicity assessed following the AFB1 and R co-treatment showed that R mitigated AFB1-induced cytotoxicity in a dose-dependent manner. In particular, the highest R dose  significantly reduced the median AFB1 cytotoxicity, dropping from 86.00% to 49.22% b.Bovine foetal hepatocytes metabolized AFB1, producing and releasing into the medium its foremost derivatives, i.e., AFM1 and AFL . ResveraIn cellular pellets, AFM1 and AFL were not detected, while the amount of cellular AFB1 was generally low in all different experimental conditions .Some key genes involved in phase I and II biotransformation, AHR pathways, and antioxidant mechanisms were affected by R alone or co-administered with AFB1 . The obsTo obtain net transcriptional results, the RNA-seq studies have been carried out only in cells treated with the highest R concentration.A total of 291,836,355 raw reads were sequenced and deposited in GeneBank under the BioProject accession PRJNA627332.B. taurus reference genome. In Raw reads of all samples passed quality control measures. After trimming and rRNAs removal, approximately 24 million reads per sample were retained. About 99% of the reads obtained from each RNA-seq library mapped to the In cells incubated with R alone, a total of 5 DEGs were found; compared to controls, 4 genes were overexpressed, while only one gene was downregulated. The EdgeR output of the DE analysis conducted in this study was reported in Gene expression profiles of cells co-treated with R and AFB1 were compared to those of cells treated with AFB1 alone. In BFH12 cells co-treated with R and AFB1, a total of 840 DEGs were identified. The majority of significant genes, i.e., 647, were upregulated, while 193 were downregulated. The EdgeR output of the DE analysis conducted in this study is reported in Among the top-10 downregulated genes, superoxide dismutase 2  showed the highest fold change. A further gene involved in regulating oxidative stress and inhibited in R + AFB1 co-treated cells was mitochondrial calcium uptake 1 . Besides, genes with a role in apoptosis were also found, i.e., MER proto-oncogene, tyrosine kinase , Rap guanine nucleotide exchange factor 1 .The enrichment analysis, carried out on the complete list of DEGs (both up- and downregulated), resulted in 17 significant Biological Processes (BPs) and 7 KEGG pathways . BesidesNotably, a significantly enriched BP was also the “response to external stimulus”  with genes like aquaporin 1 , fos proto-oncogene, AP-1 transcription factor subunit , nuclear receptor subfamily 1 group H member 4 , serpin family G member 1 , and fibronectin type III domain containing 4 . All these genes are known to be involved in anti-inflammatory processes. Furthermore, the expression of some pro-inflammatory cytokines like C-X-C motif chemokine ligand 9 , CXCL10 (logFC = 3.11), CXCL11 (logFC = 2.20), and CXCL16 (logFC = 0.64) was significantly induced. As for downregulated genes, some of them have a pro-carcinogenic activity, like the activated leukocyte cell adhesion molecule , and semaphorin 3C . The OMA1 zinc metallopeptidase  is involved in apoptosis, and DNA damage inducible transcript 3  in cell stress and DNA damage.Differentially expressed genes playing a role in processes involving cell–cell communication were particularly represented, resulting in the significant enrichment of the BP “extracellular matrix organization” , “second-messenger-mediated signalling” , and “cell-cell signalling” .As far as KEGG enrichment, the significance of “steroid biosynthesis”  is consistent with the enriched BP “lipid biosynthetic process” (see above). Among significant KEGGs, “PPAR signalling pathway”  appears particularly interesting.The GSEA conducted on the output of the DE analysis resulted in 35 significant KEGG pathways . Among tAmong the downregulated gene sets, we found some terms related to RNA processing, like “spliceosome” , “RNA degradation” , “RNA transport” , and “aminoacyl-tRNA biosynthesis” . Processes related to protein synthesis and degradation were also suppressed, as demonstrated by the negatively enriched KEGG pathways like “ubiquitin mediated proteolysis” , “proteasome” , and “ribosome” .In cells, oxidative stress may result in lipid peroxidation. To confirm the pro-oxidant effects of AFB1 and the potential benefits of R we measured the amount of MDA, which is a marker of lipid peroxidation. The amount of MDA significantly increased in BFH12 cells exposed to AFB1 compared to control conditions . The co-The activity of NQO1 was selected as a marker of the cell antioxidant status and, additionally, to confirm the trend observed for this specific target at the mRNA level (qPCR and RNA-seq: p = 0.067). However, R alone or in combination with AFB1 significantly induced the NQO1 enzymatic activity, bringing it above that of CTRL cells  experimentally exposed for 48 h  [50 observed at 48 h in six human cancer cell lines, ranging from 70 to 150 μM [50 at 72 h of approximately 150 and 200 μM, respectively [50 in normal human and bovine hepatocytes cannot be established, because BFH12 are foetal-derived cells; moreover, to our knowledge, no study has yet investigated the cytotoxicity of this natural compound in primary bovine hepatocytes. However, the cytotoxicity data here obtained let us suggest that bovine hepatocytes are more sensitive to R than the human counterpart. Worth of note, when comparing IC50 of different natural antioxidant extracts at 64 h in BFH12, R shows a cytotoxicity potential similar to that observed for quercetin , but considerably lower than those reported for curcuminoids [The ICy 50 μM) . This reo 150 μM . Resverauminoids .The significant decrease in AFB1-induced cytotoxicity was crucial in demonstrating the positive impact of R in cattle, and confirmed the extraordinary potential of this natural extract. In BFH12 exposed to AFB1, curcuminoids were already proven to increase cell viability , but sucThe amount of AFM1, the most toxic AFB1 derivative , appeareConversely, the amount of highly toxic AFL increased with R concentration, thus indicating that this natural polyphenol impacts on AFB1 reduction, the reaction forming AFL in the liver . AflatoxOverall, the impact of R on AFM1 and AFL formation seems to be consistent with a beneficial influence of this natural polyphenol.Overall, R administered alone induced neglectable transcriptional changes. Worth of mention, in this same cellular model, curcuminoids had a considerable impact on gene transcription, as they significantly affected the expression of hundreds of genes . NotablyThe most significant DEG was a gene involved in lipid metabolism, the desaturase FADS1, whose transcription was significantly induced by R. This result is consistent with previous literature reporting FADS1 induction in human HepG2 treated with 40 μM R . FADS1 pTwo key players in inflammatory processes, cell proliferation and survival by regulating apoptosis, i.e., HMGA1 and SPP1  ,80,81, wThe sole gene downregulated by R, SCUBE1, a cell surface glycoprotein found in platelet and endothelial cells , was parIf R alone did not deeply interfere with BFH12 transcriptional patterns, the co-treatment with AFB1 greatly modulated the whole transcriptome when compared to that of cells exposed to AFB1 only. This let us think that the potential of this natural polyphenol is maximal in presence of a stressful event, like AFB1. Indeed, R pre-treatment was effective in counteracting molecular processes triggered by AFB1, and promoting cell survival.The functional analysis pointed out some interesting pathways underlying the mechanisms by which R is able to mitigate AFB1-induced toxicity. Among the most significantly enriched BP, we found “lipid biosynthetic process”. This term is mainly associated with the tight connection between cholesterol metabolism and inflammation, and is mostly represented by genes whose upregulation aims at decreasing AFB1-induced inflammatory events. Among these ones we found APOA1, PTGIS, and LDLR. The former codes for the major protein component of high-density lipoprotein (HDL), widely known for regulating cholesterol trafficking. It exhibits tumor-suppressive activity by promoting anti-inflammatory processes and modulating immune responses ,94. The Two out of the 5 downregulated genes representing the lipid biosynthesis term  were most likely implicated in mitigating the inflammatory response, thus reducing cell injury. The former gene (PLA2G4A) encodes for an enzyme that catalyzes the hydrolysis of membrane phospholipids to release arachidonic acid, a hinge molecule in eicosanoids synthetic pathway. Eicosanoids are lipid-based cellular hormones key for the inflammatory response. Notably, in rat mesangial cells pre-treated with R derivatives and stimulated with the pro-inflammatory cytokine TNF-α, Lee et al. reported an inhibition of PLA2G4A protein expression, thus suggesting an anti-inflammatory activity of these natural compounds . LikewisThe enriched BP “response to external stimulus” was represented by some key genes which provide additional information about the molecular mechanisms underlying the R protective role against AFB1-induced toxicity in BFH12. For instance, AQP1 (upregulated by R) might exert a cytoprotective role and help to alleviate the inflammatory reaction induced by AFB1. Its overexpression has been previously suggested to confer survival advantage in LPS-induced acute kidney injury, both in vivo in rats ) and in In the presence of R, CXCR3 ligands , that are pro-inflammatory cytokines, were upregulated. Interestingly, CXCL10 (the IFN-inducible protein-10/IP-10) is a general marker of hepatic inflammation and injury, but it has also shown hepatoprotective effects in mice, as it plays a role in hepatic repair and regeneration ,110. How2O2, and mammalian cells lacking OMA1 showed protection against this mitochondrial stress [Some interesting genes involved in cancer development and invasion, and belonging to the BP “response to external stimulus” were downregulated by R, thus suggesting that R might attenuate the activation of biological processes normally triggered by AFB1 stimulus and responsible of the hepatocarcinogenicity of this mycotoxin. This is the case of ALCAM , SEMA3C, and OMA1. The first gene is a cell surface member of the immunoglobulin superfamily playing a pro-carcinogenic role in liver cancer ; interesl stress . In agrel stress . Again, Careful analysis of the list of up- and downregulated genes pointed out some additional interesting mRNA changes triggered by R that most likely contribute to prevent cell damage and death occurring in cells exposed to AFB1.For instance, CYP1B1 and CYP1A1 were both more expressed in cells co-treated with R and AFB1 compared to those treated with AFB1 only. Notably, if compared to control cells, the expression of these genes was significantly re-pressed by AFB1 . Interes2O2, and it is a target of NRF2 [2O2 in cardiovascular tissue, and it prevents oxidative stress in prostate cancer in vitro [BFH12 pre-treated with R and then exposed to AFB1 exhibited transcriptional changes in some genes implicated in the antioxidant response and detoxification. Transferrin (TF), glutathione peroxidase 7 (GPX7), and peroxidasin (PXDN) were all significantly upregulated by R, while SOD2 was downregulated. Transferrin sequestrates iron, that produces free radicals, in a redox-inactive form, thus possessing an antioxidant capacity and providing a cytoprotective effect in various animal models . Glutath of NRF2 . Notablyin vitro . The dowin vitro ,129,130.in vitro , thus sup-value was minimally below the threshold of significance (FDR = 0.056). This gene targets the same compounds of MGST1, including carcinogens, environmental toxins, and products of oxidative stress, by conjugation with glutathione. Overall, their transcriptional induction by R contribute to AFB1 detoxification and antioxidant mechanisms of response. Interestingly, in human and mice MGST1 and GSTA2 gene expression is regulated by NRF2 [Genes involved in detoxification were also significantly impacted by R. For instance, the microsomal glutathione S-transferase 1 (MGST1) was significantly induced by R and AFB1 co-treatment. This gene has a well-established role in the conjugation of electrophiles and oxidative stress protection . Worth o by NRF2 ,134,135, by NRF2 . ConversAs for the ABC transporters, ABCA1, multidrug-resistant protein 1 (MDR1), and ABCG2, showed a transcriptional pattern similar to that observed after curcumin pre-treatment: ABCA1 and MDR1 were downregulated, while ABCG2 was upregulated by R . The dowTo the best of our knowledge, this is the first study assessing the potential benefits of R in AFB1-exposed bovine cells through an integrated approach encompassing cytotoxicity, whole-transcriptomics, and specific post-transcriptional confirmatory assays. Based on the present results, and the experiments that we have previously performed on this cell model, the following conclusions can be drawn. (1) Resveratrol pre-treatment hampered AFB1-induced cytotoxicity. (2) Resveratrol treatment scarcely affected the BFH12 transcriptome. (3) Resveratrol pre-treatment deeply modified the transcriptome of BFH12 cells exposed to AFB1. (4) The pathways mostly affected by R pre-treatment, and driving protective mechanisms against AFB1 toxicity, are related to lipid biosynthetic processes, inflammation, drug metabolism, and drug transport. (5) Prospective molecular studies are recommended to investigate the role played by specific pathways/genes in AFB1 mechanistic toxicology and, consequently, better characterize the protective role of R. (6) In vivo studies implementing R-supplemented diets are envisaged to assess R’s bioavailability and its beneficial effects on aflatoxicosis. (7) Compared to curcuminoids, R showed a greater potential to mitigate AFB1-induced toxicity; this suggests that R might be a better choice for further in vivo investigations assessing the introduction of this polyphenol in dairy cattle feeding strategies. Indeed, we expect that the beneficial effects, and the underneath molecular mechanisms, might occur also in vivo, yet with slight differences. Thus, in perspective, R-supplemented diets might improve health status of cattle exposed to AFB1-contaminated feed.


Arabidopsis thaliana (Arabidopsis) transformant lines which harbor molecular modifications to the miR396 and miR399 expression modules to phosphate (PO4) starvation. We show that molecular alteration of either miR396 or miR399 abundance afforded the Arabidopsis transformant lines different degrees of tolerance to PO4 starvation. Furthermore, RT-qPCR assessment of PO4-starved miR396 and miR399 transformants revealed that the tolerance displayed by these plant lines to this form of abiotic stress most likely stemmed from the altered expression of the target genes of these two miRNAs. Therefore, this study forms an early step towards the future development of molecularly modified plant lines which possess a degree of tolerance to growth in a PO4 deficient environment.In plant cells, the molecular and metabolic processes of nucleic acid synthesis, phospholipid production, coenzyme activation and the generation of the vast amount of chemical energy required to drive these processes relies on an adequate supply of the essential macronutrient, phosphorous (P). The requirement of an appropriate level of P in plant cells is evidenced by the intricately linked molecular mechanisms of P sensing, signaling and transport. One such mechanism is the posttranscriptional regulation of the P response pathway by the highly conserved plant microRNA (miRNA), miR399. In addition to miR399, numerous other plant miRNAs are also required to respond to environmental stress, including miR396. Here, we exposed  The rnt roots . Due to  tissues ,8. To acres of P ,9.It is predicted that up to 40% of the yield of the world’s most economically significant crop species is limited by poor soil P availability . In modeArabidopsis thaliana (Arabidopsis) for example, the mechanisms of N and P uptake from the soil are reciprocally linked to one another. Therefore, a miRNA with a reduced accumulation profile during N stress, will usually be enhanced in abundance during the cultivation of Arabidopsis in a P-depleted environment [One such molecular approach to provide crops with an enhanced tolerance to cultivation in a P limited environment, or for their growth in other environments where the plant is exposed to other forms of abiotic stress, is the identification of abiotic stress-responsive small non-protein-coding regulatory RNAs (sRNAs) which function at the posttranscriptional level to modulate gene expression ,21,22,23ironment ,29,30.Arabidopsis to regulate PO4 uptake from the soil to maintain P homeostasis [Arabidopsis aerial tissues, the transcriptional activity of the five genomic loci (including MIR399A-MIR399D and MIR399E/F) that encode the precursor transcripts from which the miR399 sRNA is processed, is activated by the PO4 stress-responsive transcription factor, PHOSPHATE RESPONSIVE1 (PHR1) [Arabidopsis miRNA pathway to specifically repress the transcript abundance of its target gene, PHOSPHATE2 (PHO2) [Arabidopsis roots, PHO2 targets the PO4 transporter proteins, PHOSPHATE TRANSPORTER1; 4 , PHT1; 8 and PHT1; 9 for ubiquitin-mediated degradation [PHO2 transcript abundance, promotes root-to-shoot P transport in an attempt by Arabidopsis to maintain P homeostasis in its aerial tissues in P limited conditions [One such miRNA is miR399, with altered miR399 abundance central to the molecular response of eostasis ,20. More1 (PHR1) ,32,33. A2 (PHO2) ,26,31,34radation ,35. The nditions ,36,37.Arabidopsis, miR396-directed posttranscriptional regulation of six members of the GROWTH REGULATING FACTOR (GRF) family of transcription factors has been extensively documented to play a central role in all aspects of both the vegetative and reproductive phases of development [GRF1, GRF2, GRF3, GRF7, GRF8 and GRF9 gene expression is required for root [Arabidopsis [Arabidopsis development, the miR396/GRF expression module has also been associated with the adaptive response of Arabidopsis and of maize (Zea mays), rice (Oryza sativa) and wheat (Triticum aestivum) to the abiotic stressors, abscisic acid (ABA), cold, drought, heat, osmotic, salt, and ultraviolet light [GRF expression module at the posttranscriptional level across this evolutionary diverse group of plant species, and to such an extensive range of environmental stressors, strongly suggests that this miRNA expression module plays an equally important role in the molecular response of plants to abiotic stress as it does in standard plant development.In elopment ,41,42,43for root ,39, leaffor root ,41 and fbidopsis ,41. In aet light ,48,49,50PHO2 expression module has been repeatedly demonstrated to direct a central role in modulating soil PO4 uptake to maintain P homeostasis [Arabidopsis growth and development. Namely, Arabidopsis plants molecularly modified to over-accumulate miR399, and as a direct consequence of this molecular alteration, to have highly reduced PHO2 expression levels, were also shown to have increased stomata numbers due to elevated stomatal density [PHO2 expression module has been associated with the early flowering phenotype expressed by Arabidopsis plants exposed to reduced ambient temperature during the seedling stage of development [PHO2 target transcript, as part of the molecular response of Arabidopsis seedlings exposed to salt stress [PHO2 expression module and the response of Arabidopsis to PO4 stress, miR396-directed alterations to the abundance of its GRF target gene transcripts has been widely documented across an evolutionary diverse group of plant species and environmental stressors. For example, alteration of the abundance of the miR396 sRNA and/or the expression of its GRF target genes has been reported in Arabidopsis, rice, tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), cotton (Gossypium hirsutum L.) and creeping bentgrass (Agrostis stolonifera) following the exposure of these plant species to salt stress [Although the miR399/eostasis ,20,31,35 density . In addielopment . More ret stress . In diret stress ,56,57,58Arabidopsis transformants, including the MIM396, MIR396, MIM399 and MIR399 plant lines, were generated to confirm our initial finding [GRF and miR399/PHO2 expression modules forms part of the molecular response of Arabidopsis to salt stress [MIM396, MIR396, MIM399 and MIR399 seedlings to a 7-day growth period in the absence of PO4 to (1) establish the requirement of the miR396/GRF expression module as part of the molecular response of Arabidopsis to PO4 starvation, and (2) confirm the central requirement of the miR399/PHO2 expression module for Arabidopsis to mount an adaptive response to this form of abiotic stress. This approach clearly demonstrated that during the seedling stage of Arabidopsis development, unmodified Col-0 plants were the most sensitive to cultivation in a growth environment that lacked P. Accordingly, the experimental approach used here also revealed that both sets of transformant lines which harbored molecular modifications to either the miR396 or miR399 expression module were less sensitive to the imposed stress than were Col-0 seedlings. More specifically, molecular alteration of the miR396 expression module afforded MIM396 and MIR396 seedlings a mild degree of tolerance to PO4 starvation, whereas the alteration of miR399 abundance via a molecular approach provided MIM399 and MIR399 seedlings with a further enhancement in tolerance to this form of abiotic stress. Subsequent quantification of GRF and PHO2 target gene expression strongly suggested that the tolerance displayed by the miR396 and miR399 transformant lines to PO4 starvation was the result of altered miRNA target gene expression in MIM396, MIR396, MIM399 and MIR399 seedlings. This study therefore forms an important early step in the future development of molecularly modified plant lines tolerant to cultivation in a growth environment deficient in the level of P required for normal plant growth and development.The  finding  that molt stress ,58. In tArabidopsis whole seedlings following their cultivation for 7 days on Arabidopsis growth medium supplemented with 150 millimolar sodium chloride (150 mM NaCl) [PHO2 expression module has been repeatedly demonstrated to occupy a central role in the molecular response of Arabidopsis to PO4 starvation [Arabidopsis transformant lines with significantly altered miR396 and miR399 abundance. Following experimental verification that miR396 and miR399 abundance was reduced in MIM396 and MIR399 plants, respectively, and that the abundance of these two miRNAs was elevated in the MIR396 and MIR399 transformant lines [MIM396, MIR396, MIM399 and MIR399 seedlings, together with unmodified Col-0 seedlings of the same age, were exposed to a 7-day period of PO4 starvation. This approach was undertaken to attempt to establish a role for the miR396/GRF expression module in the molecular response of Arabidopsis to PO4 starvation, as well as to compare any established requirement of the miR396/GRF expression module to this form of abiotic stress to that of the well documented and central role occupied by the miR399/PHO2 expression module.We have previously demonstrated that the abundance of miRNAs, miR396 and miR399, is significantly altered in 15-day-old arvation ,20,31,35nt lines ,58, 8-daArabidopsis growth medium (referred to as P+ samples from herein), the growth of MIM396/P+ and MIM399/P+ seedlings was promoted (MIR396/P+ and MIR399/P+ transformant lines was mildly repressed (+ whole seedlings (4 deficient Arabidopsis growth medium (referred to as P- samples from herein) was detrimental to the development of Col-0/P- seedlings. More specifically, the elongation of Col-0/P- rosette leaf petioles was repressed, and the distal tips of Col-0/P- rosette leaves curled down towards the growth medium. In addition, the healthy light-green coloration of Col-0/P+ rosette leaves  and 6.6% (30.0 mg), respectively  and 14.2% (24.1 mg), respectively  and 10.4% (21.4 mg), respectively, compared to the fresh weight of a MIM399/P+ and MIR399/P+ seedling.To determine the degree to which altered miR396 or miR399 abundance influenced the phenotypic or physiological response of the assessed . The fre 28.1 mg A. As indeedlings C,G, and ectively A. Similaeedlings E,I, the ectively A. The av 19.1 mg A. AlthouIM396/P- D and MIRIR396/P- F seedlinIR396/P- A. Figure4-starved Col-0, MIM396, MIR396, MIM399 and MIR399 whole seedlings was next assessed  by a similar degree (39.9%) as was determined for the Col-0 sample. The imposed stress induced a milder response in the miR396 and miR399 overexpression lines than it did in either Col-0 or MIM396 seedlings. More specifically, the rosette area of MIR396/P- and MIR399/P- seedlings was moderately, yet significantly reduced by 18.8% (13.6 mm2) and 17.4% (11.3 mm2), from 17.0 and 14.5 mm2 for MIR396/P+ and MIR399/P+ seedlings, respectively  reduced by 47.4% compared to the primary root length of a MIR396/P+ seedling . In 15-day-old control-grown MIM396, MIR396, MIM399 and MIR399 seedlings, anthocyanin accumulated to a level equivalent to its abundance in the aerial tissues of Col-0/P+ seedlings . Similarly, and when compared to a Col-0/P+ seedling, the chlorophyll a content of a control-grown MIR396 seedling was only mildly reduced by 4.4% to 0.70 mg/g FW. In contrast, the chlorophyll a content of MIM396/P+ and MIM399/P+ seedlings was moderately elevated by 10.0% and 8.5%, respectively, and the chlorophyll a content of the MIR399/P+ sample was significantly increased by 12.4% , MIR396/P- (down by 3.5% to 0.67 mg/g FW) and MIR399/P- (down by 3.9% to 0.79 mg/g FW) seedlings. In addition, the chlorophyll a content of MIM399/P- seedlings, when compared to MIM399/P+ seedlings, was significantly reduced by 10.7% to 0.71 mg/g FW , was further mildly elevated by 3.3% to 0.83 mg/FW in MIM396/P- seedlings following the stress treatment period , in the aerial tissues of 15-day-old Col-0/P+ seedlings. In comparison to the value determined for control-grown Col-0 seedlings, spectrophotometry next revealed that the abundance of chlorophyll b was significantly elevated by 12.8%, 13.8% and 16.0% in the aerial tissues of the MIM396/P+, MIM399/P+ and MIR399/P+ transformant lines, respectively  to the already significantly reduced abundance of the miR396 sRNA in MIM396/P- seedlings  mildly decreased the expression level of GRF1, GRF2 and GRF3 by 1.3-, 1.2- and 1.4-fold, respectively  mildly reduce GRF1 expression by 1.1-fold  mo1.6-fold C,E,G, an3.8-fold D,F, respeedlings A–G againMIR396/P- seedlings (down by 2.6-fold), compared to the MIR396/P+ sample (GRF1 (1.3-fold), GRF2 (1.1-fold), GRF3 (1.2-fold) and GRF7 (1.3-fold) target transcripts  transformant lines, and to be significantly elevated by 2.0-fold in the miR396 overexpression line following the cultivation of these four Arabidopsis lines for the entire 15-day experimental period under standard growth conditions. In contrast to elevated PHR1 expression in MIM396/P+, MIR396/P+ and MIM399/P+ seedlings, the expression of PHR1 was mildly repressed by 1.3-fold in the MIR399/P+ sample  in the MIR399/P- sample , MIR396/P+ (1.2-fold) and MIM399/P+ (1.4-fold) seedlings, and to only be decreased in its abundance level (down by 1.4-fold) in the MIR399/P+ sample, when compared to the level of PHT1;9 expression observed in the Col-0/P+ sample  attempt to establish the requirement of the miR396/GRF expression module in the molecular response of Arabidopsis to PO4 starvation, and to (2) compare the miR396 findings to those obtained for Arabidopsis transformant lines which harbored molecular alterations to the expression module of the well characterized PO4 stress responsive miRNA, miR399.Due to the major annual losses of up to 40% in total yield of the world’s most important crop species stemming from their cultivation on soils which lack an appropriate supply of P ,13,14,15t stress ,53,58. Tnditions ,31,34,35tressors ,48,49,50Arabidopsis growth regime for the entire experimental period, both of the eTM transformant lines displayed enhanced vegetative development , PHT1;8 (down 1.4-fold) and PHT1;9 (down 1.4-fold)  E–G. PHT1he xylem ,67,69. Fhe xylem ,69. Ther4-starved Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings  than in the Col-0/P- sample (RE = 2.11 ± 0.03)  than it was in the Col-0/P- sample (RE = 0.15 ± 0.03). Direct comparison of miR399 and PHO2 transcript abundance in PO4-starved Col-0 and MIM399 plants  C. Accordectively D, in resectively C. Again,9 plants C,D, impl4 transporter targets of PHO2-directed ubiquitination, namely PHT1;4, PHT1;8 and PHT1;9, was therefore subsequently assessed via RT-qPCR and revealed that the expression was higher for all three PO4 transporters in Col-0/P- seedlings than it was in MIM399/P- seedlings , is highly detrimental to the later stages of vegetative development of the Arabidopsis lines that harbor such modifications. Perturbed vegetative development in these lines is the result of the over-accumulation of P in the aerial tissues, leading to an overall reduction in rosette area and the development of areas of chlorosis and necrosis in mature rosette leaves [a and b content were elevated to their highest levels in MIR399/P+ seedlings, when all five assessed Arabidopsis lines were cultivated under a standard Arabidopsis regime for the entire 15-day experimental period, the vegetative development of this transformant line was also repressed to the greatest extent  and salt stress [GRF target gene expression [GRF expression module post PO4 starvation ) for the generation of the four transformant lines analyzed in this study has been described in detail previously [AtMIM396, pAtMIR396, pAtMIM399 and pAtMIR399, were produced via the placement of artificially synthesized  DNA fragments behind the Cauliflower mosaic virus (CaMV) 35S promoter of the pBART plant expression vector. More specifically, for the construction of the pAtMIM396 target mimicry transgene, the endogenous non-cleavable miR399 target site of the Arabidopsis non-protein-coding RNA, INDUCED BY PHOSPHATE STARVATION1 , was replaced with a miR396a-specific non-cleavable target site according to the design method outlined in [IPS1 sequence was artificially synthesized without any requirement for further design modification. To generate the miR396 and miR399 overexpression transgenes, pAtMIR396 and pAtMIR399 respectively, DNA fragments were artificially synthesized to exactly match the non-protein-coding RNA sequences of the Arabidopsis miRNA precursor transcripts, PRE-MIR396A (AT2G10606) and PRE-MIR399C (AT5G62162).The construction of the plant expression vectors used to transform wild-type eviously ,58. In blined in ,74. For AtMIM396, pAtMIR396, pAtMIM399 and pAtMIR399 plant expression vectors were subsequently introduced into Agrobacterium tumefaciens (Agrobacterium) strain GV3101 and these Agrobacterium cultures were used to transform wild-type Arabidopsis plants according to the protocol of [2 generation of transformants, the (1) transgene copy number, and the (2) zygosity of each transformant was determined via a standard PCR-based genotyping approach. Of the T2 transformants determined to be homozygous for a single chromosome insertion event, the ‘best performing’ transformant line was selected to represent the MIM396, MIR396, MIM399 and MIR399 transformant populations for subsequent phenotypic, physiological and molecular analyses, with all experimentation reported here conducted on the T3 transformant generation. It is important to note here that the best performing MIM396, MIR396, MIM399 and MIR399 transformant line was selected via RT-qPCR analysis of miRNA abundance in the T2 generation, that is; identification of the (1) MIM396 and MIM399 transformant lines with the greatest degree of reduced miR396 and miR399 abundance, and (2) MIR396 and MIR399 transformant lines with the highest degree of elevated miR396 and miR399 abundance.Post their generation, the resulting ptocol of . In the MIM396, MIR396, MIM399 and MIR399 plants were surface sterilized via incubation in a sealed chamber for 90 min (min) at room temperature with chlorine gas. The sterilized seeds were plated out onto standard Arabidopsis growth medium  salts), and post sealing each plate with gas permeable tape, the plates were transferred to 4 °C and incubated in the dark for 48 h for stratification. Post stratification, the plates were transferred to a temperature-controlled growth cabinet  and cultivated for an 8-day period under a standard Arabidopsis growth regime of 16 h light/8 h dark, and a day/night temperature of 22/18 °C. At 8 days of age, an equal number (n = 48) of Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings were transferred to either (1) fresh standard Arabidopsis growth medium , or (2) Arabidopsis growth medium where the PO4 had been replaced with an equivalent molar amount (1.0 millimolar (mM)) of potassium chloride (KCl) . Post seedling transfer, the control and PO4 starvation plates were sealed with gas permeable tape and returned to the temperature-controlled growth cabinet. The control and PO4 starved Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings were then cultivated for an additional 7-day period under a standard Arabidopsis growth regime.The seeds sourced from Col-0, MIM396, MIR396, MIM399 and MIR399 transformant lines to 15-day-old Col-0 seedlings, each performed phenotypic and physiological assessment was converted to a percentage. Taking this approach, control-grown Col-0 seedlings were assigned a value of 100% for each assessed metric. Rosette area (millimeters squared (mm2)) was calculated via the analysis of photographic images of control-grown and PO4-starved Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings grown on media plates which were orientated horizontally for the entire 15-day experimental period with the freely available software, ImageJ. Similarly, primary root length (millimeters (mm)) was determined via ImageJ analysis of photographic images of control-grown and PO4-starved 15-day-old Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings which were grown on vertically orientated plates for the 7-day treatment period post the transfer of 8-day-old seedlings to fresh Arabidopsis growth medium. The fresh weight of 15-day-old control-grown or PO4-starved Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings was determined via the transfer of 12 pooled seedlings of each assessed plant line into pre-weighed 1.5 mL microfuge tubes. Each sample tube was immediately capped post seedling transfer and then the weight of the sample tube was recalculated. The initial weight was then subtracted from the final weight of each 1.5 mL microfuge tube, and this value was subsequently divided by 12 to give the average fresh weights of 15-day-old control-grown or PO4-starved Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings. This procedure was repeated four times for each plant line, and growth condition, and post calculation of the final weight of each tube, the tubes were immediately submerged in liquid nitrogen (LN2) followed by storage at −80 °C for future use as the four biological replicates for the RT-qPCR analyses reported here in To allow for the direct comparison of the a and b content were also calculated for the MIM396, MIR396, MIM399 and MIR399 transformant lines for their direct comparison to 15-day-old control-grown Col-0 seedlings. To determine the anthocyanin abundance of each Arabidopsis line, 100 mg of freshly harvested rosette leaves were ground into a fine powder in LN2. Once the powder had completely thawed, 1.0 milliliter (mL) of acidic methanol (1.0% (v/v) of 12 N hydrogen chloride (HCl)) was added to each sample. The samples were briefly vortexed to thoroughly mix the ground plant material into solution, and then incubated at 4 °C for 2 h. Any remaining cellular debris was pelleted out of solution via centrifugation at 15,000 × g for 5 min at room temperature. The absorbance (A) at 530 (A530) and 657 (A657) nanometers (nm) of each sample was then measured in a GENESYS 10S spectrophotometer  with acidic methanol used as the blanking solution. The abundance of anthocyanin in each sample, in units of milligrams per gram of fresh weight (mg/g FW), was calculated using the equation; A530 − 0.25 × A657/fresh weight (g), according to [a and b content of control-grown and PO4-starved Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings, 100 mg of freshly harvested rosette leaves was ground into a fine powder in LN2. To this powder, 1.0 mL of 80% (v/v) ice-cold acetone was added and each sample was thoroughly mixed by careful hand inversion. The samples were then incubated in the dark for 24 h at room temperature. Following this incubation period, any cellular debris that remained in solution was removed via centrifugation at 15,000 × g for 5 min at room temperature. The A at wavelengths 646 (A646) and 663 (A663) nm was then determined in a GENESYS 10S spectrophotometer with the resuspension agent, 80% (v/v) acetone, used as the blanking solution. The chlorophyll a and b content (micrograms per gram of fresh weight (μg/g FW)) of each sample was then determined using the Lichtenthaler’s equations exactly as outlined in [The physiological parameters of anthocyanin abundance and chlorophyll rding to . To detelined in .4-starved 15-day-old Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings, TRIzol™ Reagent was used according to the manufacturer’s  protocol. A NanoDrop® spectrophotometer  was used to determine the concentration of each sample and the quality of each total RNA extraction was ensured via the use of a standard electrophoresis approach on a 1.2% (w/v) ethidium bromide-stained agarose gel. Four biological replicates consisting of pools of 12, 15-day-old control-grown and PO4-starved Col-0, MIM396, MIR396, MIM399 and MIR399 seedlings were subsequently used as templates to synthesize complementary DNA (cDNA). The synthesis of a cDNA product specific to either the miR396 or miR399 sRNA was conducted as previously reported in [® qPCR Master Mix  was used as the fluorescent reagent for all performed RT-qPCR experiments. Following the RT-qPCR assessments, miRNA abundance or mRNA expression was quantified using the 2−ΔΔCT method. The small nucleolar RNA, snoR101, and the housekeeping gene, UBIQUITIN10 , were used to normalize miRNA abundance and mRNA expression, respectively, in control-grown or PO4-starved MIM396, MIR396, MIM399 and MIR399 transformants, for comparison to the Col-0/P+ sample. To extract total RNA from control-grown and POorted in  using a orted in , and a corted in ,80. All post hoc test was performed using the SPSS program . The results of the statistical analyses are presented as letters above the columns on the relevant histograms in p > 0.05), whereas a different letter above a histogram column indicates a statistically significant difference (p < 0.05).As stated above, the phenotypic, physiological, and molecular data reported in this study were obtained from the analysis of four biological replicates with each biological replicate consisting of 12 pooled seedlings. Statistical analysis was performed using the one-way analysis of variance  method while the Tukey’s Arabidopsis transformant lines molecularly modified to harbor alterations to the miR396 and miR399 expression modules are less sensitive to growth in a PO4 deficient environment than were unmodified wild-type Arabidopsis plants at the seedling stage of vegetative development. The global reduction in all assessed transcript components of the miR396/GRF expression module identified a previously unreported requirement for this miRNA, and potentially, all six of its GRF target genes as part of the molecular response of Arabidopsis to PO4 starvation. We also confirm that Arabidopsis transformant lines with a molecularly altered miR399/PHO2 expression module are more resistant to cultivation in a growth environment lacking PO4. More specifically, RT-qPCR suggested that the PO4 stress tolerance afforded to the Arabidopsis miR399 transformant lines was likely the result of decreased PHO2 transcript abundance, and therefore, defective PHO2-mediated ubiquitination of the PO4 transporters, PHT1;4, PHT1;8 and PHT1;9. In turn, enhanced activity of these three PO4 transporters would promote root-to-shoot P translocation, and subsequently; an elevated level of available P in Arabidopsis aerial tissues would enable enhanced cellular function. When taken together, the results presented in this study represent an early, yet important step in the use of molecular technologies to develop new Arabidopsis lines that are tolerant to growth in an environment where P is either in limited supply or is completely lacking. Furthermore, due to the high level of conservation of the miR396 and miR399 expression modules in plants, future advances made in Arabidopsis hold a degree of promise for their translation into agronomically important crop species as part of the development of new plant lines that can be cultivated in an environment that lacks an adequate supply of P.Considering that the molecular and metabolic processes of nucleic acid synthesis, phospholipid production, coenzyme activation and the generation of the vast volume of chemical energy required to drive these processes all rely on an adequate supply of the essential macronutrient, phosphorous (P), an alternate approach to develop new plant lines with a decreased reliance upon P is of extreme importance if agriculture is to continue to achieve the outputs required by an ever increasing population under less favorable environmental conditions in the future. One such approach is the use of molecular technologies to alter key steps in nutrient response pathways. At the posttranscriptional level, the highly conserved plant miRNAs, miR396 and miR399, have been repeatedly demonstrated to mediate central roles as part of the molecular response of a diverse range of evolutionarily diverse plant species to an array of environmental stressors. Here we demonstrate that


Assessing the phenotypic diversity underlying tumor progression requires the identification of variations in the respective molecular interaction in the tumor microenvironment (TME). Despite emerging studies focusing on the association between cation-chloride cotransporters (CCCs) and carcinogenesis, direct evidence that CCCs (KCC2 and NKCC1) mediate tumor progression in pan-cancer remains unclear.We conducted a comprehensive assessment of the expression, DNA variation profiles, and prognostic and immunologic implications of CCCs based on a large-scale pan-cancer population, including 10,967 cancer patients from the Cancer Genome Atlas, 9,162 cancer patients from Genomics Expression Omnibus, 48,834 cancer patients from 188 independent studies, and 356 cancer patients from three real-world cohorts.p < 0.05). Besides emphasizing the clinical implications of CNV of CCCs for cancer patients, we found that NKCC1MUT could prominently prolong progression-free survival (p = 2.59e-04), disease-specific survival (p = 0.019), and overall survival (p = 0.034) compared with NKCC1WT cancer patients possibly via regulation of cell proliferation and oncogenic stress pathways. Additionally, KCC2 positively correlated with the levels of tumor-infiltrating macrophages and CD4+ T cells, but NKCC1 showed a significantly widely negative association with tumor-infiltrated lymphocytes, suggesting an immune-excluded TME in cancers. Similarly, expression of KCC2, rather than NKCC1, was positively correlated with the immune checkpoint molecules, indicating its role as an immune regulator in a wide variety of cancers. Finally, to verify our hypothesis and altered expression of CCCs, we performed IHC analysis and revealed the staining distribution in tumor and adjacent normal tissues of glioma, clear cell renal cell carcinoma, papillary cell renal cell carcinoma, and hepatocellular and breast cancer from three real-world cohorts, and validated prominently prognostic implications of CCCs in patients with clear cell renal cell carcinoma.In this study, we first found that CCCs were highly expressed in most tumors, and prominently associated with prognosis. Kaplan–Meier analysis and Cox regression analysis revealed that KCC2 and NKCC1 significantly predicted survival for patients with pan-cancer, suggesting that CCCs have inconsistent tumorigenesis regulatory mechanisms in cancers. Next, we examined the DNA variation landscape of KCC2 and NKCC1 and their prognostic implications in pan-cancer. The results demonstrated that UCEC patients with somatic copy number variation (CNV) of NKCC1 received significantly better outcomes (This study first comprehensively investigated the molecular and clinical role of CCCs, and illustrated the significant association among KCC2/NKCC1 expression, DNA variation profiles prognosis, and TME of pan-cancer. The pan-cancer findings provided an in-depth understanding of potential oncogenic and immunologic of differential expression and DNA alteration of KCC2/NKCC1 cancers. CCCs(KCC1–4) , 6. Stud(KCC1–4) , 8.With the deepening of research, tumor cell-unlimited proliferation and strong invasive and metastasis ability are the main causes of high malignancy degree and aggressive prognosis , 10. TheWith the rapid development of modern technology, bioinformatics and computational biology have recently been utilized for the profiling of tumor tissues to assist the investigators in developing novel insights into anti-tumor studies –19. Manyhttps://portal.gdc.cancer.gov) database , and the clinical pathological information of the participants was fully described in the official GTEx annotation. Besides, 48,834 cancer samples with available DNA variation profiles from 188 independent cohorts were enrolled using cBioPortal for Cancer Genomics  in this study to investigate the genomics alteration role of KCC2 (SLC12A5) and NKCC1 (SLC12A2) in cancers. Genomics Expression Omnibus (GEO) database provided 875 gastric cancer patients, 1,925 lung cancer patients, 1,435 ovarian cancer patients, and 4,929 breast cancer patients with available RNA sequencing data, and follow-up survival information was enrolled in this study. Additionally, three real-world cohorts, People’s Hospital of Zhengzhou University , Affiliated Hospital of Youjiang Medical University for Nationalities , and Fudan University Shanghai Cancer Center , provided 20 samples of LIHC (liver hepatocellular carcinoma), 33 samples of BRCA (breast invasive carcinoma), 40 samples of LGG (brain lower grade glioma), 40 samples of GBM (glioblastoma multiforme), 70 KIRC  samples, 50 KIRP  samples, and 50 normal kidney tissue samples for further experimental validation. All of the study designs and test procedures were performed in accordance with the Helsinki Declaration II.The RNA-seq, copy number variation, mutation, clinical, and survival data of 10,967 pan-cancer patients were obtained from The Cancer Genome Atlas  was implemented with an anti-KCC2  and anti-NKCC1  at 1/500 dilution. IHC staining was performed according to the manufacturer’s instructions as previously described . On the t-test was performed to compare the differences among cancers. The Kaplan–Meier method in 95% confidence interval (95% CI) and log-rank test was implemented to evaluate the significance of disease-specific survival (DSS) and overall survival (OS) benefits in a separate KCC2/NKCC1 expression group and all subgroups classified by tumor microenvironment infiltration characteristics using Kaplan–Meier Plotter . Univariate and multivariate Cox regression analysis was performed to identify the proper terms to build the Nomogram. The forest was used to show the p-value, HR, and 95% CI of each variable through “forestplot” R package  (To further investigate the role of KCC2 (SLC12A5) and NKCC1 (SLC12A2) in pan-cancers, we analyzed mutation abundance and frequency of KCC2/NKCC1 in pan-cancer using cBioportal for Cancer Genomics  . A totalal.org/) .http://timer.cistrome.org/) was utilized to analyze the comprehensive correlation between abundance of tumor-infiltrating immune cells and KCC2 (SLC12A5) and NKCC1 (SLC12A2) expression using Pearson’s test  database was used for functional enrichment analyses of biological processes (BP), molecular functions (MF), and cellular components (CC).p < 0.05 was considered as statistically significant.All statistical analyses were performed using SPSS software  , GraphPaTo investigate differential cation-chloride transportation patterns in tumors, we analyzed the differential expression of KCC2 (SLC12A5) , KIRC , PRAD , and THCA . NKCC1 expression was found as a significant prognostic biomarker in BLCA , CESC , and KIRC . Taken together, cation-chloride cotransporters KCC2 (SLC12A5) and NKCC1 (SLC12A2) have a certain impact on the survival of patients in pan-cancer, especially in KIRC, GBM, PRAD, THCA, BLCA, and CESC.In order to further optimize the prediction model and improve the clinical transformation efficiency, we performed a stepwise analysis of the impact of KCC2 (p = 0.015), PAAD , and breast cancer , the higher the expression of KCC2, the better the prognosis of the patient. However, in some tumors, KCC2 has the opposite predictive prognostic implications, such as UCEC , KIRC , LIHC , gastric cancer , and lung cancer .Furthermore, we conducted a detailed Kaplan–Meier survival analysis of the specific effects of KCC2 (SLC12A5) and NKCC1 (SLC12A2) on patient survival in various cancers. As shown in p = 0.0026), LIHC , sarcoma , and ovarian cancer  from TCGA database. In HSCC , KIRC , LSCC , READ , UCEC , gastric cancer , and breast cancer , the higher the expression of NKCC1, the better the prognosis of the patient. Overall, in nearly 20,000 pan-cancer samples with overall survival information, KCC2 and NKCC1 showed different prognostic values in different cancer types, suggesting that CCCs have inconsistent tumorigenesis regulatory mechanisms in cancers.At the same time, we analyzed the prognostic implications of the expression of NKCC1 in pan-cancer on patients. In MUT were under significantly better PFS (p = 0.02) and OS (p = 0.05).Next, we examined the DNA variation profiles of CCCs in pan-cancer. As shown in p < 0.05). As shown in p < 0.001), while the CNV and expression of NKCC1 had a markedly positive relationship in OV, BRCA, and UCEC (p < 0.001).In order to further explore the effect of DNA alteration on tumorigenesis, we verified the expression of correlation of CNV with transcriptional expression of KCC2 and NKCC1  and NKCC1 (SLC12A2)  and NKCC1 (SLC12A2) have a high frequency of CNV in pan-cancer, and significantly interfere with prognosis for cancer patients  and NKCC1 (SLC12A2) affect the prognosis of cancer patients, we conducted a specific analysis of CCC mutation in cancer samples from TCGA database. In p < 0.05), we found that NKCC1MUT could prominently prolong DSS (p = 0.019), PFS (p = 2.59e-04), and OS (p = 0.034) compared with NKCC1WT cancer patients. Then, we comprehensively studied the main functional contributors associated with the mutation of KCC2 and NKCC1, and found that KCC2MUT and NKCC1MUT were significantly involved in multiply malignant biological processes, such as WNT ligands, SFRP, WNT dual receptor complex, DNA replication stress and cell proliferation, and apoptosis  and NKCC1 (SLC12A2) among 10,967 pan-cancer patients  and NKCC1 (SLC12A2) mutation, and we then queried 48,834 samples in 188 studies for a comprehensive analysis. As shown in WT cancer patients (p = 0.0122). Although there were no statistically significant PFS and OS difference between KCC2 mutation and the WT group (p < 0.05), we found that NKCC1MUT could prominently prolong DFS (p = 0.021), DSS (p = 0.019), and OS (p = 5.74e-04) compared with NKCC1WT cancer patients.Importantly, we analyzed the survival difference between the altered and unaltered group of KCC2 (SLC12A5) and NKCC1 (SLC12A2) among cancer patients from 66 databases  and NKCC1 (SLC12A2) in pan-cancer, and found that the expression of KCC2 was significantly correlated with the increase in the number of immune cells, including B cells, Th2 cells, and M1 macrophages  and NKCC1 (SLC12A2) in pan-cancer and immune checkpoint genes, including SIGLEC15, IDO1, CD274, HAVCR2, PDCD1, CTLA4, LAG3, and PDCD1LG2. Interestingly, both KCC2 and NKCC1 are significantly related to tumor-infiltrating immune cells, including tumor purity and immune checkpoint molecules such as PD-L1, CTLA-4, LAG-3, and TIGIT in ccRCC. Overall, KCC2 and NKCC1 expression positively correlated with the immune checkpoint molecules  and insignificant expression difference of NKCC1 expression (p = 0.752) in liver cancer samples (p = 0.018). Overall, through IHC validation analysis of KCC2/NKCC1 in tumor and normal tissues from multiple cohorts, we found the significantly differential transcriptional expression pattern of cation-chloride cotransporters in pan-cancer and normal tissues, suggesting the potential involvement of channels in the occurrence and progression of cancers.In order to verify our hypothesis and predictive results of bioinformatics, we performed IHC analysis of KCC2 (SLC12A5) and NKCC1 (SLC12A2) to reveal the staining distribution in tumor and adjacent normal tissues of glioma (from AHYMUN cohort), renal cell carcinoma (from FUSCC cohort), and liver and breast cancer (from PHZU cohort). The scatter plot of the IHC scores revealed that KCC2 and NKCC1 expression was significantly elevated in 40 GBM (WHO IV Grade) compared with 40 LGG (WHO II Grade) samples from the AHYMUN  and OS , which is inconsistent with the prognostic value of KCC2 in the public database. Besides, we found that higher NKCC1 expression prominently predicts shorter PFS  and OS  for 232 patients with KIRC.In order to verify our hypothesis and predictive implications of KCC2 (SLC12A5) and NKCC1 (SLC12A2) expression, we enrolled 232 patients with follow-up and protein expression data from the FUSCC cohort .CCCs can catalyze Ks ratios , 31. CCCs ratios , 33. NKCo of 1:1 , 34. In + channels can accelerate the progression of breast cancer in G1 stage , the Henan Province Medical Science and Technology Research Program (No. SB201902029), National Key Research and Development Program of China (No. 2019YFC1316000), and the Natural Science Foundation of Shanghai (No. 20ZR1413100).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Naphthenic acids (NAs) are compounds naturally present in most petroleum sources comprised of complex mixtures with a highly variable composition depending on their origin. Their occurrence in crude oil can cause severe corrosion problems and catalysts deactivation, decreasing oil quality and consequently impacting its productivity and economic value. NAs structures also allow them to behave as surfactants, causing the formation and stabilization of emulsions. In face of the ongoing challenge of treatment of water-in-oil (W/O) or oil-in-water (O/W) emulsions in the oil and gas industry, it is important to understand how NAs act in emulsified systems and which acids are present in the interface. Considering that, this review describes the properties of NAs, their role in the formation and stability of oil emulsions, and the modern analytical methods used for the qualitative analysis of such acids. In petroleum fields, crude oil and produced water (PW) emulsions are formed during oil production when the fluid undergoes flow restrictions. The inevitable emulsification of crude oil and PW may increase fluid viscosity, leading to several problems in flow assurance and primary processing. Emulsion stability is mainly determined by the structural properties of its interfacial layer –3.Being among the most important surfactants naturally found in oil sources , naphtheWhen flowing through pipes, oil and water are subjected to agitation, promoting dispersion from one phase to another and resulting in emulsions . DefinedEmulsions are complex structures characterized according to the liquid that forms the continuous phase : if the In the petroleum industry, W/O is the most common type of emulsion formed during oil production. Such emulsions typically contain ≤50% of water . One mayKinetically stable emulsions are formed in the presence of surfactants, whose hydrophilic-lipophilic balance (HLB) is the most important parameter for predicting emulsion type. For instance, while O/W emulsions are preferably formed with hydrophilic surfactants, W/O emulsions occur in the presence of hydrophobic emulsifiers . Each emThe aqueous phase contains inorganic salts such as chlorides, sulfates, and carbonates of various metals, which may contribute to the process of corrosion, incrustation formation, and catalyst deactivation . Hence, Visually, emulsions are a single-phase system produced by applying shear or sufficient energy to form dispersed droplets, regardless of the oil origin , 18. HowSurfactants enable emulsification by reducing interfacial tension (IT) and forming a film at the water-oil interface, slowing the flocculation and coalescence of the droplets . Resins Mechanisms such as steric hindrance, electrostatic repulsion, and the Gibbs–Marangoni effect may likewise affect emulsions stability –30. SterThe presence of solid particles can also provide kinetic stability to emulsions. Although not part of oil intrinsic composition, inorganic particles can act as barriers to prevent coalescence at the water-oil interface. The efficiency of these solids in stabilizing an emulsion depends on factors such as particle size  and wetInorganic solids commonly found in petroleum are derived from corrosion products , mineral scale, formation sand, clay, drilling muds, and stimulation fluids. When incorporated into petroleum, these particles can aggregate with asphaltenes and resins, stabilizing emulsions , 36. Thewt), and organic compounds containing small amounts of sulfur, nitrogen, oxygen, and organometallics + ions. Derivatization with N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) generates t-butyldimethylsilylated (t-BDMS) acids, which produce stable [M-57]+ ions, where M represents the molecular mass of the derivatized acid zed acid . Such psic acids . Moreoveic acids –112 are +) depending on the analyte nature. For allowing an unequivocal detection of the adduct ion and its corresponding nominal (or monoisotopic) mass and isotopic pattern, PCI is an interesting method for assigning the elemental composition of complex mixtures.Although incipient in such applications, positive chemical ionization (PCI) is an interesting alternative for GC-MS analysis of NAs. PCI uses methane or ammonia as reagent gas, which provides soft ionization and generates the adduct ion ([M+H]Z = −8) − adduct ions [m/z 200) and 120,000 (at m/z 400) are required to resolve isobaric doublets with a mass difference of only 3.4 mDa [Liquid chromatography (LC) is a well-suited technique for analyzing polar analytes in complex samples, complementing the molecular coverage of GC-based methods. LC is considered extremely versatile by enabling multiple separation modes, such as normal phase (NP), reversed phase (RP), and hydrophilic interaction chromatography (HILIC) . The mosuct ions , 138. El 3.4 mDa .2, O3, and O4) in OSPW samples [2-, O3-, and O4 classes accounted for 33.6% of total organic matter extracted. The results indicate that adding oxygen atoms in the molecules increased their pKa values, while double bonds and aromatic components reduced pKa values [N-(3-dimethylaminopropyl)-N′-ethylcarbodimide (EDC). The precursor ion scan was used to monitor the common product ion of the EDC derivatives, regardless of the original structure of the NA [Several studies in the literature have applied LC-MS, thus providing important information on NAs. This technique has been used, for example, for analyzing oxy-NAs  were observed only at very low abundances when compared with O2. Saturated carboxylic acids (Z = 0) were also detected in all studied samples in considerable concentrations [2, O3, O4, O5, O6, S, O2S, O3S, O4S, and O5S classes. Within this sample, Ox (x ≥ 2) was the predominant species, accounting for more than 90% of the total abundance, followed by OxS. Nitrogen-containing species were detected in very low abundance (≤0.4%) [LC-MS was used to characterize NAs in crude oils and refined petroleum products , verifyitrations . In anot (≤0.4%) .in situ OSPW [1-6, N, NO1-4, S, SO1-4, and NO2S. Class distributions were similar in both OSPW samples, but surface mining samples had higher prevalence of O1-6 species, while in situ samples showed relatively more N, NO1-4, S, SO1-4, and NO2S species. MS/MS experiments revealed that the O2 class included nonacidic compounds with dihydroxy, diketo, or ketohydroxy moieties [2 species was performed in ozonated OSPW samples [x and SOx species that indicated that species detected in ESI (+) mode were more recalcitrant to ozonation than those detected in ESI (−) ionization mode. The study highlighted the importance of using more than one ionization mode to improve molecular coverage, particularly for characterizing NAs by LC-MS [A different study used LC-MS to characterize samples of surface mining OSPW and itu OSPW , findingmoieties . In a su samples , obtainiby LC-MS .3 and SO5 species were the major classes detected by APCI (+). O2 class compounds with carbon number from 16 to 18 and DBE equal to 6 were the most prevalent, along with O4 classes, which are mainly bicyclic and tricyclic nonaromatic species, probably comprising mainly unesterified diacids with carbon number from 18 to 24 [A polar fraction of OSPW was used to assess the composition of NAs with LC-MS , whereby18 to 24 .2, O3, O1, O2, NO2, NS, and OS classes. While O2 class showed higher relative abundance in the FT-Orbitrap MS, the other classes were detected in lower proportions compared to FT-ICR MS [Direct MS analysis is possible by high-resolution mass analyzers such as FT-ICR MS and electrostatic ion trap with Fourier transform (FT-Orbitrap MS). Fourier transform-based MS instruments are key for petroleomics research, offering unprecedented mass resolving power and reliable compositional analysis . More spT-ICR MS .The most common ionization methods for direct MS analysis include ESI , APCI 1, and APP15 to C35), while calcium naphthenate deposits consisted mainly of ARN acids (C80), besides including also smaller species of ARN (C60-C70). Another study identified similar trends in crude oil samples from South America [1 and N1 [1O1, N1O2, N1O3, N1O4, N1O5, N2O1, N2O2, and N2O3. In negative mode (−), APPI also detected O5, O6, O7, and S1O4. Regarding O2 class, APPI (−) exhibited the same classes as ESI [Before selecting the ionization method, one must carefully consider the desired classes of analytes to be measured. ESI is highly suitable for studying polar compounds containing heteroatoms, such as NAs . Differe America . Moreove America , thus in America . APPI al1 and N1 . When ap1 and N1 , APPI lis as ESI .n-hexane, and cyclohexane: butyl acetate [x, SOx, and NOx) and enabling the extraction of hydrocarbon species with five or more oxygen atoms. Compared to other solvents, the cyclohexane: butyl acetate mixture allowed the detection of NAs with a wider range of DBE values and carbon number and oxygen-containing classes up to O9, while n-hexane and cyclohexane extracted classes of compounds up to O5. Similar trends were observed for SOx and NOx, suggesting that cyclohexane: butyl acetate was more efficient than n-hexane and cyclohexane for extracting polar compounds [For enabling selective characterization and reducing the effects of ionic suppression, sample preparation methods are vital for the analysis of NAs by direct MS. In this context, FT-ICR MS was used to characterize NAs fractions in OSPW samples extracted with different solvents, namely, cyclohexane, dichloromethane,  acetate . The solompounds .m/z 200-1200), thus reducing the suppression of ions with higher molecular mass (m/z 700-1200 Da) [2 class), nitrogen-containing compounds, and NO2 compounds were also detected [A study conducted with NAs present in a thermally degraded oil evaluated liquid-liquid extraction (LLE) and solid-phase extraction (SPE), finding SPE to allow the detection of a larger range of NAs species (1200 Da) . LLE witdetected .1-6) were the most prevalent in acid extracts, especially O4. When extracted under basic conditions, classical NAs (O2) exhibited the highest intensity (ranging from 30 to 44%). A different study employed FT-Orbitrap MS to evaluate different solvents and SPE phases for the extraction of NAs in OSPW [2 species when compared with other solvents, such as chloroform and ethyl acetate. These findings indicate that, when comparing results from a sample, one must necessarily standardize the analytical method by employing the same extraction procedure.SPE and LLE were also evaluated under acidic and basic conditions to profile OSPW samples by FT-Orbitrap MS . Oxygen- in OSPW , verifyi3S1, O2, and O4 species, as both monocharged and double-charged acids [Isolation of interfacial material requires dedicated sample preparation methods, such as fractionation using a modified aminopropyl silica (MAPS). FT-ICR MS analyses of interfacially active materials isolated by MAPS revealed a high abundance of low molecular weight molecules. All fractions presented Oed acids . A studyed acids . A compoed acids .2 classes, suggesting that O2 compounds were the main contributors to the observed TAN values. Such approaches are particularly interesting for highly complex mixtures, providing important information that may be disregarded in univariate analysis due to the high amount of data generated by high-resolution MS.Lastly, chemometric methods can be used to process compositional data, enabling the recognition of useful patterns and establishment of predictive models. For example, in a study conducted with partial least squares regression to estimate the TAN value of Brazilian crude oils , 161, moThis review showed that the stability of water-oil emulsions is related to the interfacial film formed between the continuous and the dispersed phases. To the best of our knowledge, the literature still lacks reports addressing oil-waters systems. Interface stability is improved by the occurrence of surface-active compounds naturally present in petroleum, where NAs are among the most important classes. NAs play an important role in the stability of oil emulsions by reducing the system interfacial tension according to the nature and distribution of NAs, among other factors. In this context, the development of reliable analytical methods for the qualitative and quantitative analysis of NAs is extremely necessary for improving our understanding of these compounds and their occurrence in crude oils. NAs composition is highly variable and depends on the oil source. To introduce the available solutions, significant developments using hyphenated MS methods and direct MS approaches were presented in this study, which also discussed basic aspects of sample preparation and data processing that accompany GC-MS, LC-MS, and MS.We hope this review will raise awareness as to the existing challenges for improving our understanding on naphthenic acids. Despite the major advances in analytical methods for NAs, the elucidation of all NAs remains a critical research requirement. Such advances and their application to study naphthenic acids may provide a starting point for developing standard quantitative methods for characterizing NAs.


Aspergillus Nidulans; however, the role of NEK family genes for tumor development remains unclear and requires additional study. In the present study, we investigate the prognosis relationships of NEK family genes for breast cancer development, as well as the gene expression signature via the bioinformatics approach. The results of several integrative analyses revealed that most of the NEK family genes are overexpressed in breast cancer. Among these family genes, NEK2/6/8 overexpression had poor prognostic significance in distant metastasis-free survival (DMFS) in breast cancer patients. Meanwhile, NEK2/6 had the highest level of DNA methylation, and the functional enrichment analysis from MetaCore and Gene Set Enrichment Analysis (GSEA) suggested that NEK2 was associated with the cell cycle, G2M checkpoint, DNA repair, E2F, MYC, MTORC1, and interferon-related signaling. Moreover, Tumor Immune Estimation Resource (TIMER) results showed that the transcriptional levels of NEK2 were positively correlated with immune infiltration of B cells and CD4+ T Cell. Collectively, the current study indicated that NEK family genes, especially NEK2 which is involved in immune infiltration, and may serve as prognosis biomarkers for breast cancer progression.Breast cancer remains the most common malignant cancer in women, with a staggering incidence of two million cases annually worldwide; therefore, it is crucial to explore novel biomarkers to assess the diagnosis and prognosis of breast cancer patients. NIMA-related kinase (NEK) protein kinase contains 11 family members named NEK1-NEK11, which were discovered from  Breast cancer is one of the most common cancers that frequently occurs in women. Moreover, it has become one of the significant causes of death in women throughout the world. According to the most recent global cancer burden report, 2.26 million new breast cancer cases were diagnosed globally . FurtherNIMA -Related Kinase (NEK) is a family of protein kinases . NEK con2/M development [The protein kinase family of NEK has been implicated in the development of various cancers . Previouelopment .Various studies looking for novel polymorphisms in carriers of the BRCA type 1/2 susceptibility protein (BRCA1/2) mutation revealed that NEK10 mutations were associated with breast cancer ,27. MoreNEK gene family on breast cancer are lacking in the current stage. Furthermore, analyses with bioinformatics methods have not been widely used to investigate the performance of NEK family members in breast cancer. Cell cycle kinases play an essential role in the development of cancer therapy [NEK family members can regulate cell cycle kinase procedure is still unclear [NEK gene family expression differences between breast cancer and normal tissue. Second, we used Kaplan-Meier plots to reveal the significance of NEK gene family members to the prognosis of breast cancer patients. After that, we used MethSurv to determine the expression and prognostic patterns of single CpG methylation of the NEK gene family in breast cancer. Then, we studied the gene potential of the NEK family in depth through functional enrichment analysis and miRNA-regulated networks. This analysis was used to reveal the biological processes, molecular pathways, potentially regulated miRNAs, and their involvement in cell cycle kinases. In addition, we also used MetaCore to delve deeper into the enrichment pathways of potential NEK family genes in breast cancer development. Finally, we used the TIMER database to uncover the correlation of potential genes from the NEK family with immune cell markers in breast cancer. Comprehensive analyses of the promising effects of the  therapy . However unclear . Therefo unclear ,41,42,43NEK gene family expressions between breast cancer and normal tissues. Through the DiffExp module in the TIMER database , we revealed differential expression patterns between normal and adjacent tumor tissues for each gene of interest in all TCGA tumors [https://portals.broadinstitute.org/ccle, accessed on 12 May 2020) contains 1457 cancer cell lines (n = 1457) with 136,488 unique data ; therefore we used the CCLE to explore the expression of NEK family members in these cancer cell lines. UALCAN  contains omics cancer data  [NEK gene family members in normal and breast cancer tissues, and their clinicopathological significance. Furthermore, we used an independent-sample t-test to confirm the significance of expressions of NEK gene family members by normal and breast cancer tissues, and in terms of clinicopathological parameters. We used p < 0.05 for the threshold of concluding that there was a significant difference [We used the tumor immune estimation resource (TIMER), Cancer Cell Lines Encyclopedia (CCLE), and UALCAN to determine differences in A tumors ,45. Furtd CPTAC) , which wfference ,48,49.http://kmplot.com/analysis/, accessed on 12 May 2020) has a dataset of about 54,000 genes [n = 7830) [NEK gene family members on distant metastasis-free survival (DMFS) in breast cancer patients. The hazard ratio (HR) with 95% confidence interval (CI) and log-rank p values were used to determine the significance of the overexpression of NEK family genes in terms of DMFS in breast cancer patients [The KM plotter  . These dpatients ,53,54.https://biit.cs.ut.ee/methsurv/, accessed on 12 May 2020) to determine the expression and prognostic patterns of single CpG methylation of the NEK gene family in breast cancer [DNA methylation plays a vital role in prognostic assessment and potential biomarker in cancer development . We usedt cancer . In thisp < 0.05 was chosen as the threshold for statistical significance. Next, we used the “fgsea” packages in R Studio software to evaluate enriched pathways in transcriptional data by a GSEA [https://portal.genego.com/, accessed on 12 May 2020) was used to perform functional analyses on various omics data, which calculates the p-value of enrichment in different gene pools of an uploaded dataset [NEK gene family members were collected and in-depth integrated, by applying Venny vers. 2.1 , and results were subsequently uploaded to MetaCore for analysis. Finally, we also investigated the gene potential of the NEK family with miRNAs. We used miRWalk  to investigate the regulatory potential of miRNAs, analyzing regulated pathways and networks by Ingenuity Pathway Analysis (IPA) [We used the Bioconductor “clusterProfiler” and “GOplot 1.0.2” packages in R Studio software ,58 to tey a GSEA ,61. Thisy a GSEA ,63,64. F dataset ,67,68,69is (IPA) ,72,73,74http://timer.cistrome.org/, accessed on 12 May 2020) was used to examine the relationships among immune cells and various types of cancer [NEK gene family members with the quantity of immune infiltrates in breast cancer through this dataset. The main objective was to find potential biomarkers at the level of immune infiltration in breast cancer tissues. We studied potential associations of gene expressions of the NEK family with various levels of infiltration such as purity, B cells, cluster of differentiation-positive (CD8+) T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells (DCs) [p values, and adjusted p values to make statistical decisions.TIMER 2.0  ,76. The NEK family. We determined the distributions of gene expression levels using the TIMER database, which are displayed in a box plot. This study found significant differences between normal and breast cancer tissues, and we determined that differences in NEK1/2/3/5/6/7/8/9/10 expressions were statistically significant at p < 0.001  between normal and breast cancer tissues , NEK2 , NEK4 , NEK6 , NEK8 , NEK9 , NEK10 , and NEK11 .We analyzed a breast cancer database with the KM plotter , to unvep-value < 0.05) in breast cancer.DNA methylation is an epigenetic alteration that plays a role in the development of several cancers . DNA metp values < 0.001, and in the CCLE analysis, we also found overexpression levels of NEK gene family members in breast cancer cell lines. In the UALCAN analysis, we discovered that NEK1/2/3/4/5/6/7/8/9/11 were statistically significantly overexpressed in breast cancer compared to normal tissues. Furthermore, we found that NEK1/2/3/7/9/10 were overexpressed in terms of clinicopathological indicators and had higher statistical significance in more advanced tumors. We found a significant result in the survival analysis, which revealed that NEK2/6/8 had high HRs and overexpression prognostic significance in DMFS in breast cancer patients. DNA methylation analysis also concluded that NEK2/6 had the highest level of DNA methylation and a significant prognostic value (likelihood ratio (LR) test p-value < 0.05) in breast cancer. Therefore, this study further explored the NEK2 gene by investigating MFs, CCs, and BPs using GO and KEGG. Enriched pathways in transcriptional data were evaluated by a GSEA. A functional analysis was conducted on various omics data, which calculated p values of enrichment across different gene pools in datasets uploaded on the MetaCore platform. Then, this study also investigated the relationship of NEK2 with miRNA-regulated networks. Finally, we also studied the correlation of NEK2 transcriptional levels with immune infiltration.Collectively, the results of several integrative analyses such as TIMER, CCLE, UALCAN, survival analysis with the KM plotter, and DNA methylation revealed that several NEK family genes were consistently overexpressed in breast cancer. In the TIMER database, we found that NEK1/2/3/5/6/7/8/9/10 had significant NEK2 from associated METABRIC and TCGA datasets , purity , CD4+ T cells , neutrophils , and DCs  , QUANTISEQ , XCELL , and Class-switched memory XCELL  , activated CIBERSORT-ABS , resting CIBERSORT-ABS , XCELL , Th1 XCELL (r = 0.1474 p = 0.000), and Th2 XCELL   and , respectively  A. Subseqsubtypes ,80,81. T= 0.001) . Further= 0.000) . We alsoectively .NEK family with miRNA. miRNA is a major post-transcriptional gene expression regulator known to play an important role in regulating cancer development [According to previous studies, NEK dysregulation was linked to the occurrence and progression of several cancers ,83. We kelopment ,91,92.NEK gene family reveals that NEK2/6/8 were closely related to the development of breast cancer in humans. We found a significant result in the survival analysis, which revealed that NEK2/6/8 have high HRs and overexpression prognostic significance in DMFS in breast cancer patients. Meanwhile, a previous study revealed that NEK2 has distinctly essential roles as a tumor-suppressor gene in different cancers, such as lung adenocarcinoma [NEK2 and its relationship with other prognostics, we used integrated data analysis with high-throughput technology and several bioinformatics tools.Our comprehensive study of 11 members of the current, exploratory arcinoma , ovarianarcinoma , hepatocarcinoma , and brearcinoma . The higarcinoma . Moreovearcinoma , gastricarcinoma , hepaticarcinoma , and brearcinoma . Furtherarcinoma  and breaarcinoma , while Narcinoma . To reveNEK2 gene was significantly overexpressed in human breast cancer tissues compared to normal tissues through the TIMER and UALCAN exploration. Based on the clinicopathological characteristics of the tumor stage, we discovered that NEK2 mRNA expression tended to be significantly higher in more advanced tumor stages, whereas NEK6/8/11 mRNA expressions were only found in tumor stages 1, 2, and 3. In DNA methylation analysis, this study found that the prognostic value of NEK2/6 in a single CpG was significant in breast cancer development. We found prognostic significance of DNA methylation expression levels in cg17931972 from NEK2 and cg14289738 from NEK6. As a result of our investigation, we found that NEK2 consistently has a poor prognosis in breast cancer. Therefore, we thoroughly investigated the biological processes of NEK2 and the regulation of NEK2 with miRNA.This study found that the NEK gene family members in the development of breast cancer. The GO analysis of genes co-expressed with NEK2 from the METABRIC and TCGA datasets showed that almost all of them were associated with cell division and DNA replication in BPs [Results of the GO analysis revealed a significant presence of n in BPs ,103, ATPn in BPs ,106,107,n in BPs , and celn in BPs ,110,111.n in BPs ,113,114.n in BPs ,116,117.n in BPs ,119,120.n in BPs . Meanwhin in BPs . The resn in BPs .CDCA1, CDC20, MAD2a, PLK1, Aurora-A, BUB1, and BUBR1  and , respectively (Finally, we identified NEK2 as a potential biomarker of immune cells in breast cancer tissues. We discovered the correlation between the NEK2 gene’s expression and immunological levels of B cells, CD4rvention ,140. Preelopment , neutropelopment ,143,144,elopment ,147,148,ectively B. IntereOur study revealed that among all members of the NEK family, NEK2 is overexpressed in breast cancer patients and is associated with a poor prognosis in breast cancer. On the enrichment pathway, we also discovered an important role of NEK2 on the cell cycle and metaphase checkpoint regulation. In conclusion, NEK2 may have potential value as a prognostic and immune infiltration marker for breast cancer development.