Thrombotic microangiopathy (TMA) refers to phenotypically similar disorders, including hemolytic uremic syndromes (HUS) and thrombotic thrombocytopenic purpura (TTP). This review explores the role of the influenza virus as trigger of HUS or TTP. We conducted a literature survey in PubMed and Google Scholar using HUS, TTP, TMA, and influenza as keywords, and extracted and analyzed reported epidemiological and clinical data. We identified 25 cases of influenza-associated TMA. Five additional cases were linked to influenza vaccination and analyzed separately. Influenza A was found in 83%, 10 out of 25 during the 2009 A(H1N1) pandemic. Two patients had bona fide TTP with ADAMTS13 activity <10%. Median age was 15 years (range 0.5–68 years), two thirds were male. Oligoanuria was documented in 81% and neurological involvement in 40% of patients. Serum C3 was reduced in 5 out of 14 patients (36%); Coombs test was negative in 7 out of 7 and elevated fibrin/fibrinogen degradation products were documented in 6 out of 8 patients. Pathogenic complement gene mutations were found in 7 out of 8 patients tested . Twenty out of 24 patients recovered completely, but 3 died (12%). Ten of the surviving patients underwent plasma exchange (PLEX) therapy, 5 plasma infusions. Influenza-mediated HUS or TTP is rare. A sizable proportion of tested patients demonstrated mutations associated with alternative pathway of complement dysregulation that was uncovered by this infection. Further research is warranted targeting the roles of viral neuraminidase, enhanced virus-induced complement activation and/or ADAMTS13 antibodies, and rational treatment approaches.The online version of this article (10.1007/s00467-017-3783-4) contains supplementary material, which is available to authorized users The term thrombotic microangiopathy (TMA) is used to describe a spectrum of phenotypically similar diseases characterized by intravascular (microangiopathic) hemolytic anemia (MAHA), thrombocytopenia, and in most instances, acute kidney injury (AKI). Other organ systems can be affected. Best-known examples are the hemolytic uremic syndromes (HUS) and thrombotic thrombocytopenic purpura (TTP). Although endothelial injury is a triggering event in many instances of HUS, this may not apply to the TTP and some forms of “atypical” HUS (aHUS).Escherichia coli (STEC or Stx HUS) are the most common cause of TMA (HUS) in children. Less frequently, HUS has been linked to infections by other bacteria, such as Shigella dysenteriae type 1, Clostridium perfringens or Streptococcus pneumoniae, and by HIV, coxsackie-, Epstein–Barr (EBV), varicella or influenza viruses [Infections by Shiga toxin-producing  viruses .Thrombotic thrombocytopenic purpura, first described as a clinical entity by Moschcowitz in 1924, is now etiologically defined by the lack of plasmatic ADAMTS13 activity . Most TTTHBD (thrombomodulin), DGKE (diacylglycerol kinase-epsilon), VWF (von Willebrand factor), factor XII, and PLG (plasminogen) [Dysregulation of the alternative pathway of complement (APC) or the coagulation system due to genetic mutations or acquired antibodies, primarily to complement factor H (CFH), increases the risk of HUS, commonly referred to as “atypical” (aHUS) , 8. Geneminogen) . Other fminogen) . Finallyminogen) , 2, 8.This review summarizes current evidence on the link between influenza virus infection and TMA (HUS or TTP) and discusses the overlap between influenza TMA and other forms of aHUS, in addition to the diagnostic workup and management of these conditions.Using PubMed and Google Scholar, the following key words were used alone or in combination: hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, thrombotic microangiopathy, influenza, influenza vaccine/vaccination. Clinical, epidemiological, and demographic features, laboratory results, treatment modalities and outcome were extracted and tabulated. English, French, and German language publications were reviewed.Influenza viruses can cause seasonal infections and epidemics with significant morbidity and mortality. The influenza A(H1N1) pandemic in 1918–1919 was responsible for the death of an estimated 50 million people . The 200S. pneumoniae. Influenza virus belongs to the genus Orthomyxovirus of the Orthomyxoviridae family. Influenza A and B viruses contain eight antisense strand RNA segments and express at least 17 proteins, among them three membrane (glyco)proteins in the lipid envelope: hemagglutinin (HA), neuraminidase (NA), and proton channel matrix protein 2 (M2) [Influenza virus targets the respiratory tract and causes fever, often with acute laryngitis, tracheitis, and pneumonia, and occasionally myocarditis, meningoencephalitis, or rhabdomyolysis, among other symptoms . Infantsn 2 (M2) . Sialic n 2 (M2) . Viral nn 2 (M2) . It facin 2 (M2) . NA inhin 2 (M2) .Hemolytic uremic syndrome triggered by influenza virus (iHUS) is rare. In almost all instances, it is associated with influenza A virus, mainly AH3N2) and AH1N1). Only recently have a few cases of HUS associated with influenza B virus infection been published (Table N1. Only  and AH1NThe first description of iHUS from 1971 is that of a 20-year-old kidney transplant recipient (#1) . The patA typical scenario of HUS due to influenza A(H1N1) infection is a previously healthy, 7-year-old boy with febrile pneumonitis and transient respiratory failure who developed severe AKI, profound MAHA, and thrombocytopenia associated with hypertensive encephalopathy 5 days after the onset of respiratory symptoms (#11). Coagulation profile, plasma fibrinogen, Coombs test, and C3 concentration were normal, as was MCP expression, plasma ADAMTS13 activity and serum CFB, CFH and CFI concentrations. He recovered completely after 2 weeks of peritoneal dialysis. No genetic studies of APC or coagulation factors were reported by the authors .Relevant demographic, clinical and laboratory parameters of all patients identified with influenza A- and B-associated TMA are summarized in Table 9 platelets/L). Peripheral platelet counts recovered after a median of 9.5 days  developed in all 25 patients. Hypertension was present in 10 out of 23 patients (43%). Serum creatinine concentrations were already increased at first measurement in 19 out of 21 patients  and peaked at 327 μM (3.7 mg/dL). Oliguria or anuria was documented in 13 out of 16 cases (81%). Ten patients (40%) initiated renal replacement therapy, mostly in the form of hemodialysis  complications, including drowsiness and mental confusion, focal neurological signs, seizures, and hemiplegia, in addition to Magnetic resonance imaging (MRI) changes and petechiae  HUS cases revealed evidence for variable abnormalities of complement and fibrinolysis, similar to the remainder of influenza A HUS cases Table . It remaThe reported clinical and laboratory features of the children with influenza B-associated HUS , 23 reseInfluenza A virus, including A(H1N1) has been invoked as a cause of TTP in at least four published reports , 39, 40.S. pneumoniae the ability to produce neuraminidase. However, in vivo NA shedding by influenza virus is minimal  compared with S. pneumonia [There is an established link between influenza virus infection and HUS, but the underlying mechanism is speculative . Influenneumonia , 50. ItsAutopsy studies during the 2009 A(H1N1) pandemic revealed viral antigen in endothelial cells . In vitrComplement regulates influenza virus-induced inflammation in the lung, enhances viral clearance, and protects against severe influenza infection . ConsideViral activation of the APC in the respiratory tract leads to ciliary dysfunction in vitro and increased levels of C3a and C5a in bronchial lavage fluid and serum of patients with severe influenza , 67. ComInfluenza-associated acute lung injury (ALI) in A(H5N1)-infected mice has been linked to excessive complement activation with deposition of C3 and C5b-9, and increased expression of complement receptors C3aR and C5aR. Treatment with a C3aR antagonist alleviated pulmonary inflammation in this model . In anotCHF mutation or CFHR1 deletion . Interestingly, all four children with influenza B-associated HUS, published in 2017, carried one or more mutations of complement-related proteins [In our literature survey, plasma C3 levels were reported in 14 patients with iHUS; they were reduced in 5 (36%) and C4 was normal in 11 cases Table . Eight pproteins , 23. AltTen of the 25 reported patients with influenza-related TMA in our survey (40%) were involved in the 2009 AH1N1) pandemic  and risk haplotypes (combined mutations or risk polymorphisms) , 71, whin = 2) and (presumed) TTP  [Thrombotic microangiopathy has been linked to influenza vaccines in a few adults since at least 1973 . Analysis of five accessible reports showed a median interval of 2 weeks (4 days to 3 months) between immunization and onset of HUS  , 72–75. ; n = 3) , 72. In ; n = 3) , 75, or The pathomechanism linking TMA with influenza vaccines is poorly understood. The clinical phenotype and spectrum or TMA  following natural influenza infections and post-vaccination are comparable. However, none of the 5 patients required renal replacement therapy. As with iHUS and idiopathic TTP (iTTP), complement studies and screens for APC and related gene mutations are necessary for a rational treatment strategy. Similar to natural infections, flu vaccines may induce anti-ADAMTS13 antibodies  and actiStreptococcus pneumoniae, a Gram-positive, α-hemolytic, facultative anaerobic bacterium commonly colonizes the human nasopharynx. Commensal strains form biofilms without causing disease [ disease . Pathoge disease , 78.S. pneumonia infection [N-acetyl-neuraminic acid (Neu5Ac) [Influenza virus is known to increase host susceptibility to (severe) nfection . There infection , 79. Neunfection . Ubiquit(Neu5Ac) . Viral a(Neu5Ac) , 82.The risk of pneumococcal pneumonia rises transiently by an estimated 100-fold following influenza , 83. ImpS. pneumoniae infection was first described by Fischer et al. in 1971 [S. pneumoniae-derived circulating Nan cleaves membrane sialic acid residues, unmasking a core disaccharide structure, Galβ1-3GalNAcα1, known as Thomsen–Friedenreich (TF) antigen, on red blood cells, platelets, and glomerular endothelial cells. One hypothesis states that preformed IgM binds to TF antigen and induces a cascade of events leading to HUS [HUS caused by  in 1971 . It pred in 1971 . S. pneug to HUS . Alternag to HUS .pnHUS patients are commonly Coombs test-positive, a feature that has been related to Nan-mediated desialylation , 84, 85.H. influenzae and S. aureus respectively, in addition to NA-producing parainfluenza virus [S. pneumoniae infection and associated with unmasking of the TF antigen , circulating MAC (sC5b-9) and anti-CFH antibodies, in addition to genetic studies targeting HUS-associated complement and coagulation factors Fig.  29, 32,, 38, 39.Patients with influenza-associated TMA benefit from best supportive care, similar to other forms of HUS , 84. TheS. pneumoniae- and influenza-associated HUS series are confounded by small numbers and an unknown proportion of patients with complement regulator defects [In comparison, STEC HUS is associated with mortality rates in children of <4% during the acute illness, irrespective of the infecting STEC serotype , 95, and defects , 87.The efficacy, tolerability, and safety of NA inhibitors for the prevention and management of influenza infections have been demonstrated in large clinical trials that also included infants . Flu vacIndividual and population immunity against endemic or epidemic influenza strains is expected to reduce the occurrence of iTMA. However, the immunization history is rarely mentioned in the available case reports. Furthermore, the notorious variability of the predicted antigen changes hampers the efficacy of influenza A vaccines . AlthougOur survey covers a period of 46 years representing different eras, seasonal and epidemic influenza strains Table , and theTreatment of iHUS hinges on the direct effects of the influenza infection, complicating bacterial pneumonia, “best” supportive care, including dialysis and blood products, if needed, and plasma or anti-complement therapy , particularly in the presence of complement regulator deficiencies Table . RecommeInfluenza-associated HUS or TMA is rare. It has been linked to influenza A and influenza B infections and, in several instances, pathogenic complement gene mutations. The outcome is generally favorable, but depends on underlying complement gene deficiencies and/or the presence of CFH or ADAMTS13 autoantibodies. Identification of the etiology and differentiation between HUS due to complement dysregulation, where the Influenza virus may act as a potent trigger, and influenza-associated HUS without identifiable complement abnormalities, are critical for acute and long-term management.The term “atypical” HUS has been originally coined to describe a heterogeneous group of infrequent forms of HUS not associated with STEC colitis. The current, interchangeable use of the epithet “atypical” that may or may not include HUS owing to pneumococcal infection and various metabolic and other conditions, including “secondary” forms of HUS , 7 and HAs detailed in this review, the pathogenesis of influenza virus-induced TMA is varied. It is intriguing to hypothesize that influenza-derived NA plays a causative role. Membrane glycan desialylation by functionally active, virus membrane-bound NA may cause transient loss of resistance to APC activation on epithelial and/or vascular endothelial cells. Alternatively, virus-specific, alternative mechanisms of (microvascular) endothelial injury resulting in an HUS phenotype need be explored. Both warrant further studies.The diagnostic workup in most of the reviewed cases is incomplete from today’s perspective. Some influenza strains appear to be potent alternative pathway activators in vivo  and therAt present, general treatment recommendations for iHUS and iTTP are lacking. In view of the high proportion of patients with APC dysregulation, PLEX or anti-complement agents constitute a reasonable therapeutic approach, while striving for a rapid and comprehensive etiological diagnosis.ESM 1(DOCX 151 kb)


Grain quality improvement is a key target for rice breeders, along with yield. It is a multigenic trait that is simultaneously influenced by many factors. Over the past few decades, breeding for semi-dwarf cultivars and hybrids has significantly contributed to the attainment of high yield demands but reduced grain quality, which thus needs the attention of researchers. The availability of rice genome sequences has facilitated gene discovery, targeted mutagenesis, and revealed functional aspects of rice grain quality attributes. Some success has been achieved through the application of molecular markers to understand the genetic mechanisms for better rice grain quality; however, researchers have opted for novel strategies. Genomic alteration employing genome editing technologies (GETs) like clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for reverse genetics has opened new avenues of research in the life sciences, including for rice grain quality improvement. Currently, CRISPR/Cas9 technology is widely used by researchers for genome editing to achieve the desired biological objectives, because of its simple targeting. Over the past few years many genes that are related to various aspects of rice grain quality have been successfully edited via CRISPR/Cas9 technology. Interestingly, studies on functional genomics at larger scales have become possible because of the availability of GETs. In this review, we discuss the progress made in rice by employing the CRISPR/Cas9 editing system and its eminent applications. We also elaborate possible future avenues of research with this system, and our understanding regarding the biological mechanism of rice grain quality improvement. Oryza sativa L.) feeds more than 3.5 billion people worldwide [Rice , while the RucV-like domain cleaves the opposite strand of the double-stranded DNA. The sgRNA is a synthetic RNA with a length of about 100 nt. Its 5’-end has a 20-nt sequence that acts as a guide sequence to identify the target sequence accompanied by a protospacer adjacent motif (PAM) sequence, which is often the consensus NGG . The loop structure at the 3’-end of the sgRNA can anchor the target sequence by the guide sequence and form a complex with Cas9, which cleaves the double stranded DNA and forms a double-strand break (DSB) at this site. Once a DSB is generated, nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) DNA repair mechanisms are initiated. A DSB is usually repaired by NHEJ in most situations, and it is a simple way to create mismatches and gene insertion/deletions (indel), leading to gene knockout. When an oligo-template is present, HDR induces specific gene replacement or foreign DNA knock-ins [The functions of various components of the rice genome ultimately enable the production of higher yielding varieties with better quality rice grains ,8. Currerosporus , Staphyls aureus , Streptopyogenes , Streptomophilus , and Strnock-ins ,16. ThesRecent reviews have described the genetics and biotechnologies , integraqBRR3, qBRR5), were mapped to chromosomes five and three, and these influence brown rice recovery [qBRR3 and qBRR5 were also found to regulate grain width and length [qHRR3, located on chromosome three, and it is associated with head rice recovery and grain length [GW2 [GW5 [GW8 [GS5 [Waxy (Wx) gene, on chromosome six, is an important gene which controls amylose content (AC), gel consistency (GC), and rapid visco analyzer pasting viscosity [Wx, tWx, 1Wxg, 2Wxg, and 3Wxg, respectively [Milling quality is based on the recovery of brown, milled, and head rice. It is a complex grain characteristic whose genetics are not fully understood. Over recent years, high throughput mapping technologies have enabled the identification of several major QTLs associated with rice quality. Two major QTLs  in fragrant rice. These mutations render BADH2 non-functional, resulting in increased 2AP [qPC1, has been cloned and functionally characterized [qCFC5, is located on chromosome five [Gelatinization temperature (GT) and amylopectin structure are controlled by a major QTL, SSIIa, which is located on chromosome six ,40,41. H quality ,43. Star quality  or exon  quality  of the Based 2AP . Howevercterized . A majorome five . QTLs coome five  revealedome five ,50. In aome five . Based oome five .GL7 [GS3 [gw5/qSW5 [qPGWC-7 [Traditionally studies on improving rice grain quality through genetic control have been conducted using biparental mapping populations, whilst the latest techniques of genome-wide association studies (GWASs) have allowed the understanding of the genetic basis of complex traits, i.e., grain quality. Although a sufficient number of studies ,55,56,57GL7 , GS3 [60gw5/qSW5 ,62, and [qPGWC-7 . Besides[qPGWC-7 . The int[qPGWC-7 , which dO. sativa L. . Both forward and reverse genetics approaches can identify the genes that are responsible for phenotypic variation . Conventativa L. .GS3, responsible for grain length, and Gn1a, controlling the number of grains, have been successfully edited in four rice varieties [1 plants showed longer grain lengths and increased thousand grain weights in comparison to the wild type. Similarly, three other important genes GW2, GW5, and TGW6, negative regulators of grain weight, were targeted through CRISPR/Cas9-mediated multiplex genome editing. The obtained results indicated that the genome editing of these genes significantly increased grain size and thousand grain weight [OsSPL13, OsSPL16/GW8, GW7, and Chalk5, through knock-in/out using CRISPR/Cas9, and the assessment of their interactions among genes, can greatly improve our understanding of rice grain appearance and milling quality.Many genes responsible for rice grain appearance quality have been identified, and have the potential to be tapped with CRISPR/Cas9 technology. Grain appearance is the primary quality attribute that influences the market acceptability of rice . Howeverarieties . The tran weight . There aWx gene in the endosperm [ALK/SSIIa and RSR1 control GT [Wx gene. Wu et al. [Wx, and they characterized a tetratricopeptide domain-containing flo2 protein regulating the expression of Wx. Similarly, transcription factors like rsr1, OsBP-5, OsEBP-89, OsbZIP58, and OsMADS7 modified Wx expression [Wx gene in the japonica background, leading to reduced AC. Moreover, transgenic Taichung 65 rice lines containing a Wx antisense construct had lower AC, and hybrids obtained from these transgenic lines also showed reduced AC [Wx gene in two widely grown japonica cultivars, “Xiushui134” and “Wuyunjing 7”. The Wx gene mutation led to reduced AC, and offered an effective strategy to improve elite cultivars without any penalty in other desirable agronomic traits. To understand the fine structure and physical properties of starch SBEI and SBEII, they were mutated via targeted mutagenesis. The results demonstrated that SBEIIb plays an important role in creating high-amylose rice. The fragrant gene Badh2 in Zhonghua 11 was edited by the CRISPR/Cas9 mutagenesis system. The mutated line contained an additional T base in the first exon of Badh2, and resulted in an increased amount of 2AP, and enhanced fragrance in rice [Generally, three important physicochemical indices comprising amylose content (AC), gel consistency, and the gelatinization temperature (GT), altogether determine the cooking and eating quality of rice grain. All three indices define (~90%) the starch properties of hulled rice. Rice with good cooking and eating quality determines the cooking ease, along with the firmness and the stickiness features . AC is rndosperm , and ALKntrol GT . Severalu et al.  reportedpression ,85. The pression  successfduced AC . In compduced AC ,84 intro in rice . The sucOsROS1, OsROS1b, OsROS1c, OsROS1d, OsDML3a, and OsDML3b, which can play an important role in enhancing nutritional grain quality. The number of aleurone cell layers was increased by ta2-1, which is a weak mutant allele of OsROS1 [Glu-1D × 5 [Food nutritional quality improvement has great importance worldwide, especially in developing countries where many people rely on rice as their staple food. Additionally, approximately 24,000 people die daily globally, owing to malnutrition ,90. Peopf OsROS1 . Approacf OsROS1 , the ovef OsROS1 , the traf OsROS1 , the prof OsROS1 , the trau-1D × 5 , and theu-1D × 5 .ferritin gene from soybean into rice, and recorded Fe concentrations. Interestingly, it was increased by three- and two-fold in milled and rough rice, respectively. Similarly, Khalekuzzaman et al. [ferritin gene driven by an endosperm-specific glutelin promoter, and found increased Fe concentration in brown and polished seeds of T1 and T2 populations of the cultivar, BRRl Dhan 29 (BR29), respectively, in comparison with controls. Thus, the Fe content was increased by over two-fold in transgenic lines. Subsequently, many researchers have attempted to increase Fe content in rice endosperm using different methods such as: (1) overexpressing the genes involved in Fe uptake from the soil; (2) moving Fe into the grain from the root, shoot and flag leaf; and (3) increasing the efficiency of Fe storage proteins [nicotianamine synthase (NAS) gene from Hordeum vulgare L. has been successfully transferred into rice, which significantly improved the contents of Fe and Zn by two- or three-fold in polished rice grain. In another study, Zheng et al. [NAS genes  increased Fe accumulation by five-fold in polished rice grain. In addition, Johnson et al. [OsNAS genes. β-carotene, which is the precursor of vitamin A, cannot be produced by rice. Therefore, researchers developed golden rice, which rich in β-carotene, by the introgression of two genes, including phytoene synthase and phytoene desaturase, to overcome night blindness caused by vitamin A deficiency in many developing countries [The SSPs include four different categories in rice, albumin, globulin, prolamin, and glutelin, separated by their solubility . In ricen et al.  introducproteins ,106,107.g et al.  observedn et al.  recordedountries . BrieflyOsCYP97A4, OsDSM2, OsCCD4a, OsCCD4b, and OsCCD7), and to increase β-carotene accumulation in rice endosperm [OsWaxy, which caused an amylose content reduction (up to 14%). Multiplex genome editing was also testified with the help of endogenous transfer RNA (tRNA) processing system in rice, wherever each sgRNA was flanked by tRNA and processed into single sgRNAs, which caused large deletions in the genomic sequences of the T0 generation [However, these approaches have some limitations, such as being time consuming, involving the introduction of foreign DNA, off-target genome modifications, the association of undesirable traits with target attributes, and the lower efficiency makes them a hard choice for researchers. However, improving rice grain nutritional quality using the CRISPR/Cas9 system can overcome these issues. The CRISPR/Cas9 system was used to knock out five rice carotenoid catabolic genes  led to the development of rice hybrid lines with low Cd accumulation. The mutant osnramp5 showed less accumulation of Cd in roots, shoots, and seeds [Over the last few years, GETs have revolutionized crop improvement programs. Newly developed crop varieties have improved traits including high yield, resistance against different diseases, and biotic and abiotic stresses. Firstly, the knockout of genes that have a great influence on grain yield, such as enotypes ,114. Theficantly . Moreovent lines ,116. Mulnfection . A knockng yield . Moreoveng yield . ALS1 is and Xia  carried u et al.  targetednd seeds ,119. Hencis-regulatory elements, small RNAs, and epigenetic modifications) can facilitate the engineering of regulatory pathways for different elements of rice grain quality. The rapid shift of research toward the utilization of CRISPR/Cas9 systems for targeted mutagenesis could be a promising approach for overcome barriers to breeding improved quality rice.Premium quality rice grain is the demand of a growing population with better living standards. Presently, the CRISPR/Cas9 system has all genome editing capabilities, e.g., knock-in, knockout, knockdown, and expression activation. This system has tremendous untapped potential, has formed an ever-expanding genetic toolbox for plant biologists to investigate functional genomics, and is a helping hand for breeders to integrate important genes into the genomes of important crops. The successful application of CRISPR/Cas9 for tissue engineering and human stem cell modification has led to further developments in the field of precise genome editing. The ability to target multiple genes via multiplexed genome editing strategies can facilitate pathway-level research to engineer complex multigenic rice grain quality attributes. Previously, few studies have been conducted that are related to targeted mutagenesis for rice grain quality improvement. The pathways of rice grain quality are not well understood, and they can be investigated for the genetic mechanisms controlling quality attributes. The development of novel regulatory components from naturally existing peripherals (genes, promoters,


KEGG enrichment analysis revealed significant enrichment of five pathways: protein processing in ER, protein export, RNA transport, glycosylphosphatidylinositol-anchor biosynthesis, and pyrimidine metabolism. Further, PPI network analysis identified a densely connected sub-module, comprising eight hub molecules: SPCS2, RPL23, RPL26, RPN1, SEC11C, SEC11A, RPS25, and SEC61G. These findings may be helpful in further analysis of the mechanisms underlying parathyroid adenoma development.Parathyroid adenoma is the main cause of primary hyperparathyroidism, which is characterized by enlarged parathyroid glands and excessive parathyroid hormone secretion. Here, we performed transcriptome analysis, comparing parathyroid adenomas with normal parathyroid gland tissue. RNA extracted from ten parathyroid adenoma and five normal parathyroid samples was sequenced, and differentially expressed genes (DEGs) were identified using strict cut-off criteria. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using DEGs as the input, and protein-protein interaction (PPI) networks were constructed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and visualized in Cytoscape. Among DEGs identified in parathyroid adenomas ( PTH) and consequent hypercalcemia [Primary hyperparathyroidism (PHPT) is a common endocrine disease characterized by inappropriate excessive secretion of parathyroid hormone  are the most solidly established molecular drivers of sporadic parathyroid adenoma. Loss of heterozygosity at the MEN1 locus on chromosome 11q13 is the most common genetic aberration in parathyroid adenoma, occurring in 26.2–50.0% of cases [MEN1 and CCND1, other genes that participate in the development of parathyroid adenoma include those encoding cyclin-dependent kinase inhibitors, along with CTNNB1, EZH2, ZFX, GCM2, and CASR [POT1 and RAP1B [To date, of cases ,13,14. Iand CASR . Moreovend RAP1B . AdditioIn this study, we performed transcriptome analysis to compare parathyroid adenomas and normal parathyroid glands, with the aim of identifying differentially expressed genes (DEGs). We subsequently used the Database for Annotation, Visualization and Integrated Discovery (DAVID), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases to systematically extract meaningful biological information from the DEGs identified. We also constructed a protein-protein interaction (PPI) network to ascertain functional relationships between proteins in parathyroid adenoma.PTH levels, in the absence of other possible causes of hypercalcemia. Ultrasound and Sestamibi scans were performed routinely in all patients to localize the tumors and surgical pathology was used to determine whether or not they were parathyroid adenomas. All the PHPT patients were female and the mean age was 53 ± 4.8. The mean levels of serum calcium and intact PTH were 12.7 ± 1.0 mg/dL and 460.4 ± 492.9 mmol/L, respectively. Serum magnesium level was not routinely checked. The mean size of a parathyroid adenoma was 2.3 ± 1.1 cm. None of the patients had evidence of familial disease, a history of neck irradiation, renal insufficiency, or cardiovascular disorders. Fresh frozen parathyroid adenoma tissue was obtained during parathyroidectomy at Seoul National University Hospital, between July 2014 and September 2016.All patients with PHPT had a single parathyroid adenoma, and PHPT was diagnosed based on serum calcium and intact PTH were 8.7 ± 0.5 mg/dL and 34.7 ± 17.0 mmol/L, respectively. The normal parathyroid tissues were collected after intraoperative frozen biopsy for microscopic tissue confirmation, due to macroscopic ambiguity, as previously described in the literature [Normal parathyroid glands were obtained from thyroid carcinoma patients who underwent thyroidectomy at Seoul National University-Seoul Metropolitan Government (SNU-SMG) Boramae Medical Center, between July 2016 and January 2017. All the patients who underwent thyroidectomy were female and the mean age was 50 ± 7.4. The mean levels of serum calcium and intact Total RNA samples were extracted using an RNeasy mini kit , according to the manufacturer’s recommendations. RNA quality was assessed by analysis of rRNA band integrity, using an Agilent RNA 6000 Nano kit . RNA libraries were constructed using a TruSeq RNA Access Library Prep kit , according to the manufacturer’s protocol. The size and quality of libraries were evaluated by electrophoresis using an Agilent High Sensitivity DNA kit (Agilent Technologies); fragments were 350–450 bp. Subsequently, libraries were sequenced using an Illumina HiSeq2500 sequencer (Illumina). Sequencing data were deposited in NCBI’s Sequence Read Archive (SRA) and are accessible through BioProject accession number PRJNA516535.https://github.com/alexdobin/STAR) mapper [p values < 0.001.Quality control of FASTQ data was performed using FastQC . Paired-) mapper . Gene ex) mapper , with LN) mapper ,22. All http://david.abcc.ncifcrf.gov/), a functional annotation tool that allows investigators to unravel the biological meaning behind an input gene list [p < 0.05 was regarded as significant.Functional analyses were performed using DAVID  database, based on identified DEGs (medium confidence score < 0.5) . For furMEN1, CASR, AP2S1, GNA11, CDC73, CDKN1A, CDKN1B, CDKN2C, RET, PTH, CCND1, AIP, CTNNB1, EZH2, ZFX, CDC73, and FGF23, were not detected in either parathyroid adenoma or normal parathyroid tissues.Ten parathyroid adenomas and five normal parathyroid tissue samples were analyzed. The characteristics of the study subjects are presented in The mRNA expression values for genes evaluated in the ten parathyroid adenoma and five normal parathyroid tissue samples are shown in KL) and PTH mRNA were significantly lower in parathyroid adenoma than in normal parathyroid tissue, while those of CASR, FGFR1, FGFR2, and VDR did not differ significantly.In addition, we investigated the expression levels of selected genes that have been implicated in parathyroid function . Levels p < 0.05) were identified , protein export (5 genes), RNA transport (8 genes), glycosylphosphatidylinositol-anchor biosynthesis (3 genes), and pyrimidine metabolism (5 genes) .SPCS2, RPL23, RPL26, RPN1, SEC11C, SEC11A, RPS25, SEC61G), which suggests these hub molecules might play crucial roles in development of parathyroid adenoma. Of them, RPL23 had the highest score, indicating that it was the most strongly interconnected with its neighbor proteins.The PPI network of genes up-regulated in parathyroid adenoma, consisting of 13 nodes and 8 edges  is illustrated in MED12, KMT5A, BMP2K, and ATAD2, are implicated in cell proliferation and transcriptional regulation, supporting the notion that they potentially contribute to tumorigenesis.To better understand the genetics involved in parathyroid adenoma development, we characterized the global expression profiles in parathyroid adenomas and normal parathyroid tissues by transcriptome analysis. A total of 247 DEGs were identified by comparison of ten sporadic parathyroid adenoma and five normal parathyroid tissue samples. Among those genes up-regulated in parathyroid adenomas, some, such as MED12 protein encoded by MED12 is a component of the CDK8 subcomplex, which is essential for CDK8 kinase activation [MED12, exon 2, in both benign and malignant tumors [KMT5A is a protein-lysine N-methyltransferase that can monomethylate Lys-20 of histone H4 (H4K20 methyltransferase) to repress transcription of various genes. This enzyme is an important regulator of the cell cycle [KMT5A expression is elevated in different types of cancer tissues and cancer cell lines, including those of bladder cancer, non-small cell and small cell lung carcinoma, chronic myelogenous leukemia, hepatocellular carcinoma, papillary thyroid cancer, and pancreatic cancer [KMT5A-mediated methylation of proliferation cell nuclear antigen (PCNA), a widely recognized cell proliferation marker of tumor progression including that of parathyroid adenoma [KMT5A-dependent PCNA methylation might promote the development of parathyroid adenoma. Therefore, KMT5A should be further validated as a possible biomarker for parathyroid adenoma progression or development.The tivation . In addit tumors ,31. Furtll cycle , and KMTc cancer ,34. A re adenoma , suggest2D3), is a potent regulator of parathyroid proliferation. A recent study revealed that 1,25(OH)2D3 treatment induces miR-1228, a BMP2K targeting factor, in a dose-dependent manner in human osteoblasts [2D3 alters the cell cycle, ultimately affecting pRB proteins and the E2F family of transcription factors [ATAD2, an E2F target gene, binds to the MYC oncogene and stimulates its transcriptional activity [ATAD2 could link E2F and MYC pathways and contribute to parathyroid tumor development.The active form of vitamin D, 1,25-dihydroxyvitamin D eoblasts . Though  factors . ATAD2, activity . We specKL and PTH genes, which play significant roles in calcium regulation, were down-regulated in parathyroid adenoma compared with normal parathyroid tissue samples. KL encodes the Klotho protein, which is a type I transmembrane protein, and FGF23 co-receptor. Our results are consistent with previously published data, demonstrating decreased expression of Klotho mRNA and protein in parathyroid adenomas [KL is strongly expressed in tissues that require abundant calcium transport, including distal convoluted tubule cells in kidneys, choroid plexus, and parathyroid glands [KL functions in parathyroid gland hyperplasia, this gene may also contribute to the development of parathyroid adenoma [FGF23 a Klotho ligand, was not identified as a DEG in this study.The adenomas ,40. KL id glands . As dele adenoma . By contPTH gene exhibited reduced expression, despite the higher serum PTH levels in patients with parathyroid adenomas. This suggests that parathyroid adenoma cells are less efficient at producing PTH relative to normal parathyroid cells; hence the higher levels of serum PTH in individuals with parathyroid adenoma may simply reflect that these tumors contain more cells that produce PTH than normal parathyroid tissue. This is consistent with previous studies, which reported that parathyroid adenomas have weaker PTH mRNA intensity relative to normal parathyroid tissue, and that disease severity is dependent on tumor weight [Interestingly, the r weight ,44.SPCS2), ribosomal proteins , and the ER membrane .Proteins function through interaction with other proteins; thus investigation of PPI networks is essential for understanding biological processes. Hub nodes, identified by computational measurement of their degree of relationship to neighbor nodes, are considered to have pivotal roles in networks. Hub molecules identified in this study were associated with signal peptidase complex subunit  proteins in PPI analysis.ng cells ,46. PrepThis study has limitations. First, the analysis included a relatively small number of samples; hence there is potential for the results to have been greatly influenced by each sample or individual patient characteristics. To minimize the effects of sex and age, we selected both parathyroid adenoma and normal parathyroid samples from middle-aged female patients. PHPT is observed predominantly in women, although the reason for this is still unclear. It would be interesting to check the influence of gender using a linear regression-based statistical model in a further study with a larger sample size . MoreoveWe identified DEGs in parathyroid adenoma compared with normal parathyroid tissue and GO terms, which may contribute to the development or progression of parathyroid adenoma. Further, PPI network analysis highlighted a densely connected PPI sub-module, consisting of eight hub molecules. Additional validation of these findings, in a larger number of samples and using functional experiments, is required to confirm the results of this study.


Spinacia oleracea L.) is a cold tolerant but heat sensitive green leafy vegetable. In this study, heat adaptation mechanisms in a spinach sibling inbred heat-tolerant line Sp75 were investigated using physiological, proteomic, and phosphoproteomic approaches. The abundance patterns of 911 heat stress-responsive proteins, and phosphorylation level changes of 45 phosphoproteins indicated heat-induced calcium-mediated signaling, ROS homeostasis, endomembrane trafficking, and cross-membrane transport pathways, as well as more than 15 transcription regulation factors. Although photosynthesis was inhibited, diverse primary and secondary metabolic pathways were employed for defense against heat stress, such as glycolysis, pentose phosphate pathway, amino acid metabolism, fatty acid metabolism, nucleotide metabolism, vitamin metabolism, and isoprenoid biosynthesis. These data constitute a heat stress-responsive metabolic atlas in spinach, which will springboard further investigations into the sophisticated molecular mechanisms of plant heat adaptation and inform spinach molecular breeding initiatives.Elevated temperatures limit plant growth and reproduction and pose a growing threat to agriculture. Plant heat stress response is highly conserved and fine-tuned in multiple pathways. Spinach ( Heat stress has become a major deleterious factor affecting crop production worldwide due to global climate change . Heat stArabidopsis thaliana (Oryza sativa) (Zea mays) (Spinacia oleracea) (Solanum tuberosum) (Vitis vinifera) (Lolium perenne) (Rhazya stricta (Dianthus caryophyllus)  , maize (ea mays) , spinachleracea) , potato berosum) , grape (inifera) , perenniperenne) , Rhazya  stricta , carnatiphyllus) , and Sacjaponica . These hMedicago sativa)  (Triticum aestivum) (Hordeum spontaneum) (Glycine max) (Apium graveolens)  and elucidate pathways that are crucial for heat tolerance . Proteom sativa) , rice (L sativa) , wild riionalis) , wheat (estivum) , wild bantaneum) , maize (ntaneum) , soybeanine max) , celery veolens) , grape (veolens) , Pinelli ternata , Carissaspinarum , Portulaoleracea , Agrostilonifera , Populusphratica , and Popnanensis . Numerouresponse . These rcholine monooxygenase, and betaine aldehyde dehydrogenase. The plants overexpressing spinach choline monooxygenase and betaine aldehyde dehydrogenase genes enhanced heat tolerance , Sp75, was grown in a growth chamber with a temperature regime of 22/18°C, 10/14 h day/night cycle, and a relative humidity of 60%. Plants were watered daily to avoid the occurrence of water deficit. When performing heat treatments, plants of the treatment groups were moved to another growth chamber with the same growth condition as the control, except for temperature (37/32°C day/night), and watered daily on a regular schedule as well. The 72 h of heat treatment was started at 10:00 a.m. on the 50th day after seeds were sown, and 48 and 24 h of heat treatments were conducted at 10:00 a.m. on the 51th day and 52th day, respectively. After heat treatments, fully expanded true leaves were collected, morphological changes were recorded, and photosynthetic characteristics were measured for both control and heat-treated plants at 10:00 a.m. on the 53rd day to avoid the differences caused by plant growth, development, and circadian rhythms /(Turgid weight-Dry weight)] × 100%.2 concentration (Ci), and transpiration rate (Tr) were determined at 10:00 a.m. using a portable photosynthesis system LICOR 6400 . The chlorophyll fluorescence parameters were recorded by using a portable modulated chlorophyll fluorometer .Net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular COProline and total soluble sugar contents were determined using ninhydrin reaction and a sulfuric acid-anthrone reagent according to previous methods, respectively . MDA con2O2 measurement, leaf tissue was ground with 0.1% trichloroacetic acid. After centrifugation at 15,000 g for 15 min at 4°C, the supernatant was collected for H2O2 measurement. H2O2 content was determined spectrophotometrically at 390 nm after reacting with potassium iodide as described in a previous study  at 45°C for 1 h, and then alkylated using 125 mM iodoacetamide in 7 M guanidine-HCl (pH 8.0) for 30 min in the dark at room temperature followed by centrifugation. The resulting concentrate was washed three times with 0.5 M triethylammonium bicarbonate at pH 8.5 to remove interfering compounds including DTT, iodoacetamide, and NH4HCO3. Trypsin was added to the samples at 1:50  and incubated at 37°C for 16 h. The tryptic peptides in the filter device were collected by centrifugation and labeled with iTRAQ 8-plex reagents according to manufacturer’s instructions . The samples of 0 hour of heat treatment (HHT) were labeled with iTRAQ tags 113 and 117, 24 HHT with tags 114 and 118, 48 HHT with tags 115 and 119, and 72 HHT with tags 116 and 121. Two independent iTRAQ experiments were carried out, thus each sample has four biological replicates  workflow  by usingplicates . After lThe peptides were dissolved in solvent A  and fractionated on a Shimadzu LC-20A HPLC system  with a XBridge C18 column . Peptides were eluted at a flow rate of 800 μl/min with a linear gradient of 5–55% solvent B  over 42 min followed by ramping up to 90% solvent B in 1 min and holding for 4 min. The fractions were lyophilized and desalted using 3M Empore C18 solid-phase extraction disks .2 beads were added and the mixture was incubated for 30 min at room temperature. After centrifugation, the mixture was washed twice using wash buffer I (65% acetonitrile/0.5% trifluoroacetic acid) and wash buffer II (65% acetonitrile/ 0.1% trifluoroacetic acid), respectively. The phosphopeptides were then eluted with elution buffer I (0.3 M NH4OH/50% acetonitrile) and then elution buffer II (0.5 M NH4OH/60% acetonitrile). The eluted peptides were lyophilized and stored in -20°C.The labeled peptides were resuspended in 500 μl loading buffer (1% glutamic acid/65% acetonitrile/2% trifluoroacetic acid). TiOSamples were reconstituted in 10 μl of 0.1% formic acid and analyzed by online nanoAcquity ultraperformance LC  coupled with an Orbitrap Fusion Tribrid mass spectrometer  according to a previously described method .1)  , 2017 usnsortium  via the 2 using PSI and PHI-BLAST programs3. Protein functional classification was performed manually on the basis of combination of information from KEGG pathway database4, UniProt database5, and the Gene Ontology protein database6, as well as literature. Hierarchical clustering analysis of the protein abundances in leaves was performed on the log (base 2) transformed protein fold change values using Cluster 3.0 available on the Internet7.Proteins without detailed annotation were annotated by searching against the NCBI non-redundant protein database8 , and three biological replicates were used. For proteomics data, four biological replicates were analyzed by Student Figures 1A,B). The number of withered leaves increased, and the withering was more serious at 48 and 72 HHT . The RWC decreased by 11, 21, and 23% after heat treatment for 24, 48, and 72 HHT, respectively . This implied that cellular osmotic potential changed along with the heat-reduced water potential caused by the decrease of RWC. Here, it should be noted that FW was used to normalize the contents of MDA, proline, soluble sugar, H2O2, AsA, and GSH in some previous studies, which might cause a bias given that the stress-caused decrease of RWC. Although we also used FW in this study, DW might be a better denominator to use in the future.The morphology and RWC of spinach leaves were obviously affected by heat stress treatment. Heat stress-treated plants exhibited slight withering at 24 HHT compared to control plants . Gs also decreased in response to the heat stress. There was no significant difference between plants at 24 and 48 HHT, and a 27% decrease in Gs compared to control plants was observed at 72 HHT . Besides these, Ci and Tr increased significantly under heat stress . The parameters Fv/Fm and Y(II) represent the maximal and effective quantum yield of PS II photochemistry, respectively. Fv/Fm decreased gradually during the treatment , while Y(II) was significantly reduced at 72 HHT .Photosynthesis of spinach is very sensitive to heat stress. The Pn decreased gradually in the course of the heat treatment . In addition, MDA and REL were considered as two indexes of heat-induced cell membrane damage. Our data showed that MDA content significantly increased by 1.2-, 1.3-, and 1.6-fold, and REL increased by 1.3-, 1.3-, and 1.5-fold at 24, 48, and 72 HHT, respectively , indicating the loss of membrane integrity in heat-stressed spinach leaves.Proline and soluble sugars are key osmolytes facilitating plant stress adaptation. In spinach leaves, the proline content gradually increased by 1.7-, 1.8-, and 2.5-fold at 24, 48, and 72 HHT, respectively. Similarly, the soluble sugar content also increased by 1.2-, 1.5-, and 1.5-fold, respectively, under the above conditions . The activities of 10 antioxidant enzymes were also affected by heat stress. SOD activity significantly increased by 1.3-, 1.2-, and 1.3-fold at 24, 48, and 72 HHT, respectively . In contrast, POD activity decreased by 0.8-, 0.7-, and 0.9-fold along with the increase of heat treatment duration . GOX activity showed a significant increase of 1.2-fold at 72 HHT, while CAT activity increased by 1.1-fold at both 24 and 48 HHT, and changed to the same level as the control sample at 72 HHT . In the AsA-GSH cycle, APX activity decreased by 0.9-fold, and DHAR activity decreased by 0.8-, 0.9-, and 0.9-fold under three heat stress conditions, respectively. However, MDHAR activity was not altered, and GR activity significantly increased by 1.4-, 1.3-, and 1.3-fold at 24, 48, and 72 HHT, respectively . In addition, GST activity was gradually enhanced by 2.3-, 2.8-, and 2.8-fold in the course of the heat stress treatment . GPX activity was not affected at 24 HHT, and increased by 1.1- and 1.7-fold at 48 and 72 HHT, respectively . Heat stress also led to the changes of antioxidant content, including AsA, DHA, GSH, and GSSG. AsA content decreased by 0.9-, 0.8-, and 0.9-fold, while DHA content increased by 1.9-, 2.1-, and 2.2-fold at 24, 48, and 72 HHT, respectively, leading to the significant decrease of AsA/GSH ratio . In addition, the contents of GSH and GSSG showed a similar heat stress-increased trend . GSH content increased by 1.9-, 1.9-, and 2.6-fold, and GSSG content increased by 1.8-, 2.1-, and 3.1-fold under the three heat stress conditions. However, the GSH/GSSG ratio did not change at 24 and 48 HHT, and decreased by 0.9-fold at 72 HHT .The Hp < 0.05, and with a fold change >1.2 in protein abundance. Among them, 618 protein species increased and 281 decreased under at least one of the heat stress conditions, while the 12 remaining showed differently changed patterns in response to the heat stress treatments . For example, 17 out of 25 (68%) HRPs involved in diverse signaling pathways were heat stress-increased, and they mainly functioned in lipid signaling, calcium signal transmitting, phosphorylation cascades, and 14-3-3-mediated signaling pathways. Importantly, 84% proteins in membrane and transport pathways were heat stress-increased, and they include proteins for endomembrane trafficking, Ran GTPase-mediated nuclear trafficking, as well as water, ion, and metabolite transporters. Also, 52% heat stress-increased proteins were involved in ROS scavenging pathway, e.g., SOD, CAT, GST, Prx, Grx, and Trx, and 57% stress and defense-related proteins  were heat stress-increased in leaves. Furthermore, the majority of HRPs in each group were heat stress-increased, including HRPs in transcription (85%), protein synthesis (90%), protein processing (81%), and protein degradation (64%). This suggested that gene expression and protein turnover were enhanced in response to heat stress. Also, heat stress-increased proteins took a major part in various metabolic pathways, including those of carbohydrate/energy (68%), amino acids (81%), fatty acids (64%), vitamins biosynthesis (63%), isoprenoids biosynthesis (71%), nucleotides (94%), as well as cell cycle, differentiation and development (80%). In contrast, most proteins involved in photosynthesis (65%), hormone metabolism (67%), and cell structure (63%) were heat stress-decreased, indicating these pathways were probably inhibited under heat stress  were detected with more than 1.2-fold changes (p < 0.05) of phosphorylation level, including 25 heat stress-increased and 23 heat stress-decreased phosphopeptides . The phosphorylation levels of seven phosphopeptides involved in six processes in Cluster I increased under two or three heat stress conditions. Cluster II included 18 phosphopeptides, whose phosphorylation levels increased under one heat stress condition. The corresponding proteins were mainly involved in signaling, membrane and transport, transcription, protein synthesis, and other metabolisms. Additionally, in Cluster III, 12 phosphopeptides showed heat stress-decreased phosphorylation levels under two or three heat stress conditions, and they were mainly responsible for membrane and transport, transcription, and other metabolisms. Furthermore, cluster IV included 11 phosphopeptides with heat stress-decreased phosphorylation levels under one of the three treatment conditions, and membrane and transport proteins were most abundant in this cluster .In spinach leaves, 111 phosphopeptides representing 103 phosphoproteins were identified using a TiOFigure 7), while models for the rest 11 phosphoproteins did not contain the corresponding phosphorylation sites. The phosphorylation sites and numbers of helices and beta sheets were presented in the three-dimensional models . However, the phosphorylation levels of ferredoxin-NADP reductase and pyruvate orthophosphate dikinase decreased under heat stress. Their phosphorylation sites, Thr176 and Thr535, were located in the ferredoxin reductase-type FAD-binding domain and phosphoenolpyruvate-utilizing enzyme mobile domain, respectively . Also, the phosphorylation level of heterogeneous nuclear ribonucleoprotein 1 was heat stress-increased. However, the phosphorylation site Ser43 was not located in its functional domain . Similarly, the phosphorylation sites of BAG family molecular chaperone regulator 4 and GDP-mannose 3,5-epimerase 1, Ser30 and Ser443 were heat stress-decreased and not localized in their functional domains .The heat stress-increased phosphorylation site of pyruvate dehydrogenase E1 component subunit alpha occurred on Ser299, which was located in the dehydrogenase E1 component domain  was correlated with the increase of plant freezing tolerance (GF14λ confers plant tolerance to drought stress (High temperature increases plasma membrane (PM) fluidity, leading to a transient calcium influx through calcium channels on PM . High tepathways . We foune leaves , and PP2e leaves . Thus, te leaves . For exaolerance . Overexpt stress . How theFigure 8 and Supplementary Table Figure 8 and Supplementary Table Figure 8 and Supplementary Table Figure 8 and Supplementary Table In spinach leaves, the heat stress-altered abundances and heat-induced phosphorylation of CBPs implied that the increased calcium enhanced the activities of CBPs  and Rho GTPase, and the phosphorylation level of SEC10 isoform 2 were heat stress-increased  and membrane scission of nascent endocytic vesicles   transporters ] of spinach were obviously reduced with the increasing of heat treatment duration , indicating the photosynthetic apparatus was sensitive and impaired. The reduction of Gs may be attributed to low leaf water content caused by high Tr   . It has 120 HHT) . Therefo120 HHT) , our pro2O2 , which causes the autocatalytic peroxidation of membrane lipids thus leading to the impairment of membrane semi-permeability indicated by the increased MDA content and REL, respectively . In order to protect cells from oxidative damage caused by the heat stress, spinach has developed both enzymatic and non-enzymatic detoxification systems to counteract ROS toxicity.In spinach leaves, heat stress led to the accumulation of H2- to O2 and H2O2 , which is in agreement with the heat stress-increased SOD activity in leaves of maize . The POD pathway was inhibited in spinach leaves as evidenced by the heat stress-decreased POD activity  and abundances of all the isoenzymes of POD , glutaredoxin (Grx), and thioredoxin (Trx) were activated for ROS removal under heat stress. SOD acts as the first line of defense by catalyzing the dismutation of Oand H2O2 . Heat stof maize , poplar of maize , and wheof maize . Our proable 2O2 , as welloxisomes . Also, toxisomes . Intereson donor . Meanwhion donor . Additio network , the abuoleracea , A. scablonifera , and poplonifera . Moreovelonifera , carnatilonifera , S. japojaponica , and perjaponica . All thelonifera , spinachlonifera , and perlonifera .Figure 8 and Supplementary Table Non-enzymatic antioxidants  can be employed to scavenge free-radical in heat-stressed spinach . Heat-inFigure 8 and Supplementary Table It is well-known that a number of transcription factors  are heat-inducible . In thisP. oleracea  HRPs were involved in protein synthesis, including various ribosomal proteins, aminoacyl-tRNA biosynthesis enzymes, translation initiation factors, translation elongation factors, and peptide chain release factors , three acetyl-CoA carboxylases, and 3R-hydroxymyristoyl ACP dehydrase isoform 1 involved in saturated fatty acid biosynthesis , vitamin B6 and vitamin E (tocopherol) was induced in spinach, and these metabolites function as antioxidants in response to oxidative stress (Secondary metabolisms were induced by the heat stress in spinach. Vitamin metabolism was altered in spinach leaves, as shown by the increases of five out of eight HRPs in vitamin metabolism under heat stress (Supplementary Table ormation . It has t stress . In addie stress . Moreovee stress .Most spinach varieties are heat stress sensitive, while spinach Sp75 used in this study is a heat tolerant sibling inbred line. Our physiological and proteomics results indicated that Sp75 can accumulate multiple osmolytes/antioxidants and trigger diverse ROS scavenging pathways for heat stress response, although the photosynthesis was inhibited under prolonged heat stress. Importantly, the quantitative proteomics and phosphoproteomics results implied that calcium-mediated signaling, vesicular transporting, transcriptional regulation, protein processing, as well as primary and secondary metabolisms were induced for facilitating heat sensing, transduction, and adaptation. Moreover, we identified 6 candidates of heat-responsive receptors and 24 heat-responsive endomembrane trafficking-related proteins. Their abundance patterns implied that signal peptide recognition, vesicle formation, exocyst-mediated secretory, vesicle fusion, clathrin-mediated endocytosis, endosomal sorting and recycling, as well as endosomal-mediated degradation were active in plant heat response. Importantly, 7 proteins involved in Ran GTPase-mediated nuclear trafficking, as well as 15 heat-increased transcriptional regulation-related proteins were proposed to be novel heat-responsive factors in spinach (Supplementary Table SD conceived and designed the experiments. QZ, WC, JB, CX, JM, XC, XW, YL, and QW performed the experiments. ZZ and YS contributed analysis tools. QZ, HX, JC, and SG analyzed the data. QZ wrote the manuscript with suggestions by SD, ZZ, and SC. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As one of the important cancer hallmarks, metabolism reprogramming, including lipid metabolism alterations, occurs in tumor cells and the tumor microenvironment (TME). It plays an important role in tumorigenesis, progression, and metastasis. Lipids, and several lysophospholipids in particular, are elevated in the blood, ascites, and/or epithelial ovarian cancer (EOC) tissues, making them not only useful biomarkers, but also potential therapeutic targets. While the roles and signaling of these lipids in tumor cells are extensively studied, there is a significant gap in our understanding of their regulations and functions in the context of the microenvironment. This review focuses on the recent study development in several oncolipids, including lysophosphatidic acid and sphingosine-1-phosphate, with emphasis on TME in ovarian cancer. The tumor microenvironment (TME) for epithelial ovarian cancer (EOC) is rather unique. It is mainly confined within the peritoneal cavity and frequently associated with ascetic fluid ,3,4,5,6.The presence of ascetic fluid provides a mobile, easy access, and more dynamic environment for tumor–stromal interactions. In addition to tumor and stromal cells, EOC ascites is rich in cell-free inflammatory cytokines, chemokines, matrix metalloproteinases, integrins, and other secreted molecules, including bioactive lipid factors. These factors are generated by and mutually function in both tumor and stromal cells via autocrine/paracrine mechanisms. They may exist in either extracellular vesicles (EVs) or in “free” forms, including bond forms to proteins or other molecules . EVs areMetabolic reprogramming is one of the major cancer hallmarks  that is 2O with ring, chain, and branched structures), lipids are very diverse in both their respective structures and functions. These diverse compounds are grouped into classes; glycerophospholipids (PLs) (including lysophospholipids (LPLs)) sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides tymidine incorporation [Firstly, LPA is a confirmed mitogen in many cell types. However, MTT dye reduction is not a good method to measure this effect. LPA affected MTT dye reduction with an unknown mechanism in EOC cells , making poration ,26,29. i and G12/13 [Secondly, the most potent roles of LPA in EOC and other cancer cells are likely to be cell migration and invasion. This action is mediated by LPARs and Gd G12/13 ,95,104. d G12/13 ,95. In cd G12/13 ,90,93,951. ATX is highly secreted from ovarian CSCs. Inhibition or knockdown of ATX markedly attenuates the LPA-producing, tumorigenic, and drug resistance potentials of CSCs. In addition, clinicopathological analysis shows a significant survival disadvantage of patients with positive staining of ATX. In addition, LPA is involved in the crosstalk between CSC in TME. EOC cells secrete LPA that activates the expression and secretion of CXCL12 by mesenchymal stem cells (MSCs), enhancing the resistance of OVCA cells to hyperthermia [Thirdly, LPA has been recently shown to be involved in cancer stem cells (CSC) in EOC ,110. Seorthermia .1–3 belong to the endothelial differentiation gene (Edg) family of GPCR and LPAR4–6 belong to the purinergic P2Y family of GPCRs [1–3 in general mediate LPA’s tumor promoting activities [1 may represent a negative regulatory LPA receptor inducing apoptosis in ovarian cancer cells [1–3, LPAR4–6 are less studied. Both pro- and anticancer effects mediated by LPA4–6 in various cancers have been reported, with the majority of them reporting anti-cancer effects [Fourthly, the majority of LPA signaling is mediated by its six G protein couples receptors (GPCRs) ,111. Amoof GPCRs . While Ltivities ,113,114,er cells . At leaser cells . Compare effects ,117,118.Finally, LPA has also been identified as a ligand for the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) ,120. How+ T cell activation [1–3, LPAR6 is the main LPA receptor on TAM and tumor-associated T cells [The ATX-LPA axis has emerged as a novel regulator of lymphocyte homing and inflammation. LPARs are expressed by T cells and LPA enhances the motility of human and mouse T cells in vitro, although generally not in a direct manner . Cancer tivation . LPA alstivation ,124. Alt T cells .ATX is expressed by lymphoid organ high endothelial venule. LPARs receptors are expressed by NK cells, mast cells, eosinophils, and B cells . In addi2s, LPA receptors, including PPARγ, or through LPA degradation by lipid phosphate phosphatases (LPPs). The targeting strategy should take TME into serious consideration. Drugs for these applications have been and will soon be entering clinical practice [In summary, LPA, a simple molecule that mediates a plethora of biological effects, may be targeted at its levels of production by ATX or PLApractice .2 enzymes, which mainly convert phosphatidylcholine (PC) to LPC have been shown to be functionally involved in EOC and/or as markers for various cancer types [Compared to LPA, plasma LPC levels are usually 10 to 100-fold higher and are in the 100–200 µM range in human subjects  Table 1Table 1. er types ,132,133.LPC is present at the highest concentrations among LPLs. Its role in signing is still debatable. Although both of its tumor-promoting and suppressing activities have been reported in various cancers ,134,135,While phosphatidylinositol (PI) is the substrate of PI3K, one of the most pertinent signaling pathways in cancer , LPI as Although a specific receptor of LPI has been reported , they waS1P is the orthologue of LPA with a different backbone (a sphingoid base vs. a glycerol backbone). The physiological and pathologic concentrations of S1P is approximately one order of magnitude lower than that of LPA and is usually present in sub-µM to low µM range ,46,47,48In contrast to LPA, S1P is mainly produced intracellularly by two sphingosine kinases  or by several members of the ABC transporter family . This auOver the past two decades, increasing evidence demonstrates a strong link between S1P and both normal physiology and progression of different diseases, including cancer and inflammation. S1P may affect survival, proliferation, angiogenesis, and metastatic spread of cancer ,146.1–3 and S1PR1–5 belong to the same edg-receptor family [LPA and S1P share structural similarity. In addition, LPARr family . Moreover family ,149, whir family ,150,151 Firstly, S1P has strong concentration dependent differential effects. As mentioned above, the physiological/pathological concentrations of S1P are in general lower than those of LPA ,47,48. TBased on chemically measured S1P concentrations in biological fluids and the binding affinities of S1P to its receptors (in nM to low µM range ), the efSecondly, SphK1 and SphK2 have distinct cellular locations, regulations and functions. In general, SphK1 is tumor promoting and SphK2 is suppressive; SphK1 is upregulated in cancer, while SphK2 is downregulated ,159,160.1–5  [1/S1PR3 and S1PR2 receptors may mediate opposing effects [1 and S1PR3 mediate S1P’s tumor promoting activities, such as cell migration and invasion via activation of Rac. Blockage of SphK1, but not SphK2, or S1PR1/3 could attenuate ovarian cancer angiogenesis and inhibit angiogenic factor expression in a mouse model [1 is upregulated in ovarian cancer tissues and cell lines, which is negatively regulated by miR-148a in EOC cells [2 generally mediates the inhibitory effect via Rho-mediated inhibition of Rac [2 is also involved in negative regulation of tumor angiogenesis and tumor growth in vivo via RhoC activation [2 inhibition in vitro and in vivo [2 has an inhibitor role in macrophage recruitment during inflammation [2 and S1PR3 are expressed higher in ovarian surface epithelial cells than in ovarian cancer cells [Thirdly, different and opposing effects are mediated by different S1PRs. S1P receptors have been identified so far and named S1PR3, 6, 8) . Followi3, 6, 8) ,165. Amo effects ,159,160.OC cells . On the tivation , althoug in vivo . In addiammation . Goetzl er cells .1 axis plays an important role in the immune regulation. It is involved in the mature vascular system; pathological angiogenesis; immune cell egress from tissue compartments; hematopoietic, vascular, and stem cell survival; and cytokine production. In particular, S1P induces STAT3 activation in tumor-associated myeloid derived suppressing cells (MDSCs) [Finally, S1P may have profound regulator effects on inflammation and in the immune system. The SphKs/S1P/S1PR (MDSCs) . In addi (MDSCs) . S1P and (MDSCs) ,172. On  (MDSCs) ,94,173.SphK1 is highly expressed in the tumor stroma of high grade serous ovarian cancer (HGSOC) and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs) . While iA biospecific monoclonal antibody to S1P (S1P mAb) has been developed and investigated for its role in tumorigenesis. The anti-S1P mAb substantially reduced tumor progression and in some cases eliminated measurable tumors in murine xenograft and allograft models. Tumor growth inhibition was attributed to antiangiogenic and antitumorigenic effects of the antibody . The antIn summary, the role of S1P in the pathogenesis of ovarian cancer remains unclear and controversial and more studies are clearly required. Due to the multi-faceted nature of S1P’s roles and signaling, targeting S1P signaling may be a double-edged sword.SPC is an orthologue of LPC with a different backbone (a sphingoid base vs. a glycerol backbone). The levels of SPC in EOC vs. non-malignant ascites is low: 71.5 ± 50.8 nM vs. 17.9 ± 10.1 nM, respectively . The levThe reciprocal interplay of cancer cells and TME is an indispensable prerequisite for tumor growth and progression. Ovarian cancer, the most lethal of all gynecological malignancies, is characterized by a unique TME that enables specific and efficient metastatic mechanisms/routes, impairs immune surveillance, and mediates therapy resistance. More specifically, detached cancer cells—as well as large numbers of T cells, TAMs, and other host cells—cooperate with resident host cells to support tumor progression and immune evasion. The presence of the peritoneal fluid (ascites) enables more efficient tumor-stromal cell interactions and the transcoelomic spread of tumor cells to other pelvic and peritoneal organs. In particular, this fluid is rich in tumor-promoting soluble factors including elevated LPLs, either in EV or non-EV forms. Several important future directions and unresolved questions include, but are not limited to: development of standard and uniform methods for lipid extraction and analyses; further characterization of LPL regulation (both production and degradation) and their signaling mechanisms; development of strategies for cancer-specific targeting those tumor promoting lipids; and conducting more studies on their extracellular associations in order to better develop markers and targeting. Overall, it is critical to take TME into consideration to develop the next generation of therapeutic strategies.


Breast cancer is the second most common cause of cancer-related deaths among women worldwide. It is a heterogeneous disease with four major molecular subtypes. One of the subtypes, human epidermal growth factor receptor 2 (HER2)-enriched (HER2-positive) is characterized by the absence of estrogen and progesterone receptors and overexpression of HER2 receptor, and accounts for 15–20% of all breast cancers. Despite the anti-HER2 and cytotoxic chemotherapy, HER2 subtype is an aggressive disease with significant mortality. Recent advances in molecular biology techniques, including gene expression profiling, proteomics, and microRNA analysis, have been extensively used to explore the underlying mechanisms behind human breast carcinogenesis and metastasis including HER2-positive breast cancer, paving the way for developing new targeted therapies. This review focuses on recent advances on gene expression and miRNA status in HER2-positive breast cancer. Breast cancer is the most common type of cancer diagnosed in women worldwide with around 2.1 million new cases in 2018 according the World Health Organization. Breast cancer is the leading cause of mortality among women in the majority of countries worldwide [Although early detection of the disease through screening programs, education, and availability of therapeutic agents  have led to an overall improvement, the survival rates are drastically reduced in 20–30% of patients who develop metastases. Metastatic breast cancer still represents an incurable disease with a poor outcome, as the median survival is 2–4 years depending on the breast cancer subtype [Several risk factors can be identified at the onset of breast cancer including age at menarche , null paA recent study has shown that breast cancer heterogeneity extends beyond the histopathological classification; however, patients with breast cancer are still grouped according to clinicopathological criteria in order to decide the appropriate therapy and predict their prognosis. These criteria include patient age, tumor size, histological grade as well as the presence or absence of lymph nodes and distant metastases, in addition to hormone receptor, estrogen (ER), progesterone (PR) and human epidermal growth factor receptor 2 (HER2) status . AlthougThe availability of high-throughput technology for gene expression profiling, such as microarray, quantitative, and differential-display reverse transcription polymerase chain reaction (RT-PCR), as well as next generation sequencing, have shown that tumor cells respond differently to treatment which is not resolved by clinicopathological aspects, rather by intrinsic molecular features that can be explored using molecular technology . Gene exHER2 gene copy number or a HER2/CEP17 ratio of 2 or greater [In this review, we will focus on novel prognostic and predictive markers of HER2-positive breast cancer subtype. HER2-positive breast cancer represents 15–20% of breast cancer cases ,19 and iEGFR are transmembrane receptors formed of three parts: An extracellular ligand binding site, a transmembrane part, and an intracellular tyrosine kinase domain that comprises of HER1, HER2, HER3, and HER4 . The humHER2 oncoprotein plays an important role in the pathogenesis of various solid tumors [HER2 receptor is highly expressed in human tissues including the cell membranes of epithelial cells in the gastrointestinal, respiratory, reproductive, and urinary tract as well as in the skin, breast, and placenta . Amplifid tumors , includid tumors , ovariand tumors , lung cad tumors .Also known as HER2-enriched breast cancer, HER2-positive breast cancer represents 15–20% of all breast cancers ,19 and aHER2 protein (receptor) (HER2 overexpression is the phosphatidylinositol 3-kinase (PI3K), mammalian target of rapamycin (mTOR) axis, which are responsible for the regulation of important cellular functions including cellular metabolism, migration, as well as proliferation and angiogenesis [HER2 overexpression is the Ras/Raf/MEK/ERK pathway  pathway) [The primary mechanism of HER2 activation in breast cancer is its gene amplification on the long arm of chromosome 17 (17q12-21-21.32) ; this coeceptor) b causingogenesis ,36,37. Apathway) .HER2 amplification during cancer progression [It is worth noting that HER2 status of breast cancer can change during the disease progression from negative to positive, one plausible reason being the oncogenic gression ,39,40,41HER2 mutations in a subset of breast cancers as well as other malignancies [HER2 mutations in breast cancer is ~3% with most of these affecting the intracellular (kinase) domain of HER2 receptor. Notably, HER2 mutations predominantly affect HER2-negative breast cancers (only 30% are HER2 positive (amplified)) while 63% of breast cancers are ER+ [HER2 mutations in breast cancer are also associated with poor outcomes [Recent studies have also described gnancies ,43. Thesgnancies . A systegnancies  revealed are ER+ . HER2 muoutcomes .HER2-positive breast cancers are morphologically poorly differentiated with a marked pleomorphism and a high proliferation rate (high grade cancers) a; they aAlthough approximately 50% of all HER2-positive breast cancers express the steroid receptors estrogen (ER) or progesterone (PR) , they are usually resistant to endocrine treatment, particularly tamoxifen . There iHER2-positive breast cancer is targeted by personalized therapy using monoclonal antibodies, such as trastuzumab (Herceptin), this treatment targets the HER2 receptor and blocks the related pathways inhibiting proliferation and survival as well as migration and cell invasion, leading to prolonged patient survival . MoreoveEarly diagnosis of breast cancer, as well as monitoring disease progression and its response to treatment, is critical in the management of this disease . HoweverCCND1) and ribosomal protein S6 Kinase B1 (RPS6KB1), groups C and D are ER− and close to the HER2-positive intrinsic subtype with high TP53 expression [DKK1), matrix metalloproteinase 15 (MMP15), baculoviral IAP repeat-containing 5 (BIRC5), CCND1, origin recognition complex subunit 6 homolog-like (ORC6L), MKi67, cyclin E1 (CCNE1), TP53, HER1/EGFR, ATPase H+ transporting V0 subunit a4 (ATP6V0A4), prolyl endopeptidase (PREP), reticulon 4 interacting protein 1 (RTN4IP1), kinesin-like protein (KIF18A)) are upregulated in HER2-positive breast cancer cells and correlated with poor survival rates because of their role in promoting proliferation, progression, lymph node, and metastasis , and contributing to tumor aggressive behavior in colony growth. On the contrary, genes including phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphatase and tensin homolog (PTEN), inositol polyphosphate 4-phosphatase type II (INPP4B), phosphatidylinositol 3-kinase regulatory subunit (PIK3R1), TP63 are downregulated and resulted in poor prognosis and survival. In addition, several genes are studied as targets of recently developed drugs (PGAP3) [GRB7) [HER2 amplified breast cancers. While, 40–80% HER2-positive breast cancer cases are known to harbor TP53 mutations [Progress in microarray technology led to gene expression profiling of breast cancer with the aim of identifying patients that can benefit from adjuvant chemotherapy, and as a prognosis predictor in cancer patients ,60. HER2pression . Moreoveed drugs . Overexp (PGAP3)  and grow) [GRB7) ,14 are autations ,63.PIK3CA mutations, loss of PTEN [CXCR4) upregulation [BRAF); EGFR1; PIK3CA; and proto-oncogene, receptor tyrosine kinase (KIT) are more frequent in trastuzumab-resistant HER2-positive metastatic breast cancer. These mutations may upregulate MAPK/ERK pathway and consequently induce resistance to trastuzumab. Furthermore, use of OncoScantm detected mutations in five genes (catenin beta 1 (CTNNB1); HRas proto-oncogene, GTPase (HRAS); KRAS proto-oncogene, GTPase (KRAS); neurofibromin 2 (NF2); and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1 (SMARCB1) in HER2-positive breast cancer [HER2-positive breast cancer patients generally benefit from neoadjuvant chemotherapy in the form of anthracyclines and taxanes with a higher complete pathological response in comparison with luminal and triple-negative subtypes . Althoug of PTEN  or C-X-Cgulation  can leadgulation . Anothergulation  using Gegulation . They fot cancer . This stt cancer . Anothert cancer . They ust cancer . The rest cancer .Caenorhabditis elegans, they have been shown to play a major role in gene expression modulation and controlling major pathways [MicroRNAs (miRNAs) are a class of small regulatory, noncoding RNA molecules measuring approximately 18–25 nucleotides in length . Since tpathways .miRNAs regulate the expression of 30–60% of human genes and are important modulators of cell proliferation, cell differentiation, cell development, cell-cycle progression, angiogenesis, epithelial-mesenchymal transition, stem cell renewal, apoptosis as well as cell migration, invasion, and metastasis . DysreguRecent studies showed that miRNAs can be detected not only in tissues but also in body fluids such as blood, serum, and urine, thus indicating miRNAs to be easily accessible without the need for invasive procedures . Since mIn the last decade, healthcare systems have greatly evolved, they have integrated point of care (POC) diagnostics as an integral part of their transformation . POC allNumerous miRNAs have been reported to be dysregulated in human malignancies including lung, breast, ovarian, bladder, colon, and other types of carcinoma. Therefore, it is reasonable to assume that dysregulation in the miRNA machinery is a plausible reason for the onset and progression of human cancers . AccumulPrevious studies reported that different breast cancer subtypes display various molecular miRNA signatures ,100,102.To date, only few studies have described the regulation of HER family receptors by miRNAs. Therefore, diagnostic and prognostic roles of miRNAs are still nascent and require further investigations .HER2 gene [A few studies have shown that miRNAs have a diagnostic role in HER2-positive breast cancer. miRNAs have been involved in the expression of 26 proteins within the EGFR1 signaling pathway. Among these, miR-147, miR-124, and miR-193-3p have been shown to act as tumor suppressors for the EGFR1 pathway, inhibiting the proliferation of HER2-positive breast cancers . AnotherER2 gene . In compER2 gene . AnotherER2 gene . A signaER2 gene . This stER2 gene .DICER1 is significantly lower in the HER2-positive, luminal B, and basal-like subtypes and can be used as a predictive marker for metastases-free survival [DORSH to be associated with HER2 gene amplification and protein overexpression [Furthermore, miRNAs also have a prognostic role in HER2-positive breast cancer. miRNA machinery elements, including Dicer, Drosha, DGCR8, Argonaut, and TRBP, and are reported to be involved in the progress of several cancer subtypes . Thus, asurvival ,112. Anosurvival , reportepression .ERBB2/Her2 gene, which is upregulated in HER2-positive breast cancer patients [MicroRNA profiling has helped in enhancing breast cancer classification and in stratifying patients according to their response to therapy . Du et apatients .Recent studies using relative real-time PCR analyzed differential expression of potential miRNAs to U6 RNA  in trasTMEM49, however it was reported that miR-21 mostly has its own transcriptional system and is regulated independently [One of the best characterized miRNAs in HER2-positive breast cancer is miR-21 . It is lendently . The miRendently . Fujito endently , implyinendently .PDCD4 (programmed cell death 4), TPM-1 (tropomycin 1), and PTEN (phosphatase and tensin homolog) [PDCD4 expression is lost by the presence of miR-21 [PDCD4, a target of miR-21, is a regulator of AP1 and also induces the expression of cyclin dependent inhibitor (cdk) 21 [Previous studies have demonstrated that miR-21 acts as an oncogene by targeting tumor suppressor genes homolog) ,126,127 homolog) . In addif miR-21  in HER2-f miR-21 . PDCD4, (cdk) 21 .TGF-β in upregulating miR-21 expression in vascular smooth muscles (VSMCs), as well as in MDA-MB-468 breast cancer cells, through the SMAD signal pathway [TGF-β, is associated with chemoresistance and cell invasion in vitro. In addition, inhibition of PTEN expression was found to be mediated by TGF-β inducing increased expression of miR-21 in breast cancer cells; however, treatment of these cells with miR-21 inhibitor, restored PTEN expression [miR-21 also enhances epithelial-mesenchymal transition, thus promoting progression of primary HER2-positive breast cancer . Loss of pathway . Another pathway , demonstpression . miR-21 pression .ID-1 expression, enhanced by miR-21 upregulation, promote cell invasion as well as enhance oncogene signaling pathways associated with MEK-ERK pathway activation [miR-21 expression is enhanced via MAPK (ERK1/2) pathway upon activation of HER2/neu signaling in breast cancer cells . A studytivation . Moreovetivation ,136, it MiR-21 was also found to significantly affect the response to neoadjuvant therapy in HER2-positive breast cancers . DNA-damAnother important miRNA, miR-210, is located on chromosome 11p15.5 . miR-210Shp2 in hypopharyngeal carcinoma [SMAD3 in breast cancer [AKT3 in ovarian cancer [Dek in ovarian cancer, as well as muscle stem cells [Shp2 was seen to enhance the RAS-MAPK pathway regulating breast cancer proliferation [HER2 mRNA downregulating its expression [Smad3 expression activating EMT-like properties, thus conferring chemo-resistance [Smad3 [miR-489, located on chromosome 7q21.3  has beenarcinoma , SMAD3 it cancer , AKT3 inn cancer , and Dekem cells ,148. Furferation . miR-489pression . miR-488pression . Furtherpression ; indicatsistance . Targetie [Smad3  can be oe [Smad3 . UnregulAs mentioned above, miRNAs have been involved in the onset and progression of breast cancer and have the ability to reverse resistance to drugs like tamoxifen; as studies have shown that re-expression of miR-375 , miR-342WIP1 and oncomiR, miR-21; both of which have been involved in promoting breast tumorigenesis as well as resistance to anti-HER2 therapies [WIP1 and miR-21, reduced proliferation, survival, and tumorigenic potential of HER2-positive breast cancer cells harboring 17q23 amplification, thus, providing an effective therapeutic strategy for HER2-positive breast cancers [WIP1 and miR-21 inhibitors will aid in paving the way for effective future therapeutic approaches against trastuzumab-resistant breast cancers [On the other hand, amplification in the 17q23 region (amplicon) leads to dysregulated expression of oncogene herapies . Researcherapies  showed t cancers . Develop cancers .miRNAs are usually present in microvesicles or bound to lipoproteins in the blood ; while sHER2-positive breast cancer is an aggressive subtype of breast cancer with a poor outcome despite available targeted treatment modalities. There is an increasing body of evidence that various gene and miRNA signatures are specific for HER2-positive breast cancer. None of these has become fully clinically applicable and therefore, further studies are necessary for their validation. More specifically, there is a huge gap regarding the miRNA profile of HER2-positive breast cancer, therefore, it is necessary to conduct more in-depth studies, which may lead to specific miRNA markers or targets for HER2-positive breast cancers.On the other hand, it is important to develop an animal model for HER2-positive breast cancer, which can be used to study the miRNA and gene profile of the main metastatic sites of HER2-positive breast cancer metastasis, more specifically, brain and lung. Such a study may allow to develop specific markers for metastatic HER2-positive breast cancers.