PSEN1, PSEN2, APP, MAPT, GRN, CHCHD10, TBK1, VCP, HTRA1, OPTN, SQSTM1, SIGMAR1, and abnormal repeat expansions in C9orf72 and HTT. Overall, 33.3% (13/39) of the variants were novel, the identified P/LP variants were seen in 2.2% (35/1592) and 10.9% (12/110) of AD and FTD cases, respectively. The overall molecular diagnostic rate was 2.6%. Among them, PSEN1 was the most frequently mutated gene , followed by PSEN2 and APP. Additionally, the age at onset of patients with P/LP variants (51.4 years), ranging from 30 to 83 years, was ~10 years earlier than those without P/LP variants (p < 0.05). This study sheds insight into the genetic spectrum and clinical manifestations of neurodegenerative dementias in South China, further expands the existing repertoire of P/LP variants involved in known dementia-associated genes. It provides a new perspective for basic research on genetic pathogenesis and novel guiding for clinical practice of neurodegenerative dementia.Neurodegenerative dementias are a group of diseases with highly heterogeneous pathology and complicated etiology. There exist potential genetic component overlaps between different neurodegenerative dementias. Here, 1795 patients with neurodegenerative dementias from South China were enrolled, including 1592 with Alzheimer’s disease (AD), 110 with frontotemporal dementia (FTD), and 93 with dementia with Lewy bodies (DLB). Genes targeted sequencing analysis were performed. According to the American College of Medical Genetics (ACMG) guidelines, 39 pathogenic/likely pathogenic (P/LP) variants were identified in 47 unrelated patients in 14 different genes, including  Alzheimer’s disease (AD) is the most common dementia worldwide, accounting for 60–80% of all dementia cases2. Frontotemporal dementia (FTD) is the second most common cause of neurodegenerative dementia after AD in patients younger than 65 years, responsible for 10.2% of cases3, and dementia with Lewy bodies (DLB) has been reported as being the second most common dementia subtype in older people following AD, accounting for 7.5% of all dementia cases4. However, the etiology of neurodegenerative dementias is still obscure, which is thought to be caused by a combination of ageing, environmental, and genetic factors.Neurodegenerative dementias are a group of clinically heterogeneous diseases with frequently overlapping symptoms, such as multi-cognitive impairments, behavioral changes, and movement deficitsPSEN1, PSEN2, and APP are recognized as three causative genes for familial AD (FAD), which explains the genetic background of 5–10% of early onset AD . The estimated mutation frequencies of PSEN1, APP, and PSEN2 in EOAD, are 80%, 15%, and 5%, respectively5. Likewise, FTD is a genetically and pathologically heterogeneous disorder with a higher incidence of familial cases than AD. Genetic etiology has been revealed in ~30–50% of FTD patients with a positive family history7. At present, more than 10 genes are related to FTD, and MAPT, GRN, and C9orf72 are the most common, accounting for ~60% of all cases of inherited FTD3. In contrast, the genetic architecture of DLB remains largely elusive8. To date, only three genes have been confirmed to be related to DLB, including APOE, GBA, and SNCA. However, growing evidence supports that DLB has a strong and unique genetic component9.Recently, substantial progress has been made regarding the molecular genetics of neurodegenerative dementias. PSEN1, the most common etiology of EOAD, has also been found in patients with FTD and DLB13. Similarly, mutations in MAPT, GRN, and C9orf72 have also been detected at lower frequencies in AD and DLB patients16. Homozygosity for APOE4, the strongest genetic risk factor for AD, has also been reported in several studies to increase the risk of FTD and DLB18. In addition, mutations in SNCA have been shown to result in a wide phenotypic spectrum of DLB, Parkinson’s disease (PD), multiple system atrophy (MSA), and FTD21.Interestingly, previous studies have suggested a potential genetic overlap between AD, FTD, and DLB. Notably, PSEN1, PSEN2, APP, MAPT, GRN, C9orf72, CHCHD10, HTRA1, TBK1, OPTN, SQSTM1, VCP, SIGMAR1, and HTT, attempting to summarize the relationship between gene mutations and clinical phenotypes. Then, we compared the age at onset (AAO) of patients with and without P/LP variants and patients carried different genes separately, to depict the AAO spectrum for these dementia-associated genes in our population. Finally, we analyzed APOE genotypes (non-carriers or carriers of APOE4) in AD cohort and conclude the difference between APOE genotypes and different AD subgroups. Our studies provide a new perspective for further basic research of neurodegenerative dementia, especially genetic-associated pathogenesis and facilitated the clinical prediction, diagnosis, and genetic counseling.In this study, we comprehensively analyzed the mutational spectrum of known dementia-associated genes from patients with neurodegenerative dementias in the South Chinese population using integrated targeted gene sequencing analysis. First, we systematically identified pathogenic and likely pathogenic (P/LP) variants of known dementia-associated genes, including known and novel variants, summarized and compared the mutation frequency among patients with different clinical diagnosis. Second, we generalized the clinical manifestation of neurodegenerative dementia patients carried P/LP variants in this study, including PSEN1 , PSEN2 (c.T716C and c.1180delG), GRN (c.20G>A), CHCHD10 (c.121C>T and c.283C>T), OPTN (c.1402_1407del), SQSTM1 (c.558_559insC), SIGMAR1 (c.26G>A), and TBK1 (c.973dup). All identified P/LP variants were responsible for 2.2% of AD (35/1592) and 10.9% of FTD (12/110), which led to an overall molecular diagnostic yield of 2.6%  and one frameshift mutation. Two were novel, including c.T716C, p.239M>T and c.1180delG, p.A394Pfs*8. All patients with the variants had a positive family history except for one who carried PSEN2 p.239M>T. Meanwhile, two APP missense mutations were identified in four FAD probands, including c.2143G>A, p.V715M, and c.2149G>A, p.V717I  were defined as early-onset AD (EOAD) (AAO < 65 years)), 45.5% were APOE4-negative, 48.5% had one APOE4, and 6.0% had two copies of APOE4.In patients with PSEN1/PSEN2/APP variants carriers initially presented with memory decline. Then, language impairment and behavior change were common symptoms in these variants, 52.63% (10/19) PSEN1, 25% (1/4) PSEN2, and 50% (1/2) APP P/LP variants showed language impairment, respectively, such as naming difficulty, repetitive speech, fluency disorder, and speech reduction, while the frequency of mental and behavior change of PSEN1, PSEN2, APP variants were 57.6% (11/19), 75% (3/4), and 100% (2/2), respectively. Meanwhile, 26.3% (5/19) PSEN1, 25% (1/4) PSEN2 P/LP variants presented sensory and movement disorders, such as hallucination, delusion, weakness, involuntary movement, and abnormal gait. Interestingly, the clinical manifestation of patients with PSEN2 mutations at amino acid residue M239 showed high heterogeneity, including memory decline, language impairment, mental and behavior change, and sensory and movement disorders.About clinical phenotypes, all CHCHD10  and HTRA1 , respectively. The patient who carried CHCHD10 p.Q95* showed memory decline at 52 years and gradually developed language dysfunction, behavioral changes, bradykinesia, and depression. Brain MRI showed bilateral atrophy of temporal parietal lobe and hippocampus, and cerebrospinal fluid (CSF) examination showed the level of Aβ42 and Aβ42/Aβ40 ratio decreased, while the phospho-tau (p-tau) and total tau (t-tau) increased. In addition, the Pittsburgh compound B (PiB)-PET showed diffuse amyloid deposition in the whole brain cortex. The patient who carried HTRA1, p.R197*, mainly presented typical forgetfulness of recent events and daily living ability declined at 49 years. Brain MRI showed multiple spot-like hyperintensities in the deep bilateral frontotemporal lobes and paraventricular region, while no microbleeds on susceptibility-weighted images sequence. The level of Aβ42 in CSF decreased, and p-tau increased which supported the diagnosis of AD.Additionally, we also found two female AD patients who carried a nonsense mutation in MAPT, GRN, C9orf72, CHCHD10, TBK1, OPTN, SQSTM1, VCP, SIGMAR1, and HTT, summarized in Table GRN: c. 20G>A, p.W7*; CHCHD10: c. 121C>T, p.Q41*; and SIGMAR1: c.26G>A, p.W9*), two frameshift mutations , and one deletion mutation . Interestingly, only two P/LP variants carriers had a positive family history. The mean ± SD AAO of P/LP variants carriers was 59.2 ± 10.5 years, which was significantly older than P/LP variants carriers in AD cohort . Meanwhile, only three patients carried APOE4 , which tended to be lower than P/LP variant carriers in the AD cohort .A total of 12 P/LP variants in 10 genes were identified in the FTD cohort, including HTT, which supported the diagnosis of Huntington’s disease (HD).As for clinical characteristics, all patients carried P/LP variants in FTD cohort showed memory decline, 58.33% (7/12) patients had language impairment, mental, and behavior changes, and 25% (4/12) P/LP variants carriers accompanied by sensory and movement disorders. Interestingly, one showed personality changes and language impairment, as well as abnormal emotional responses at baseline. In the fifth year of onset, she suffered from memory decline. Brain MRI showed bilateral frontal lobe atrophy, and bvFTD was initially considered. However, molecular testing revealed that she carried heterozygous CAG expanded repeats in p < 0.001), among them, 89.4% were younger than 65 years.Moreover, the mean AAO were significantly younger in patients with P/LP variants in the AD cohort and entire cohort, while no difference was found in the FTD cohort Fig. . SpecifiPSEN1, PSEN2, and APP  were significantly lower than those of non-carriers (p < 0.05) . The results showed that the mean AAO of subjects with P/LP variants of 05) Fig. .APOE4 to compare the difference in AAO between the two groups respectively, which showed that the AAO of FAD patients was significantly younger than that of SAD , while no significant between APOE4 carriers and non-carriers (p = 0.953)  Fig. .APOE genotypes, dementia family history, MMSE scores, mutation status, and clinical diagnosis, the model showed that MMSE scores , disease duration , and status of mutation carried .To analyze the confounding factors affecting AAO, we conducted further multiple linear regression analysis with AAO as the dependent variable. After controlling independent variables, including gender, disease duration, educational attainment, APOE genotypes, there was no significant difference across AD, FTD, and DLB cohorts . Furthermore, APOE4 as the strongest genetic risk factor for AD, we further compared the distribution difference between EOAD and LOAD patients, FAD and SAD. We found no significant difference in APOE4 frequency . Further subgroup analysis in AD cohort showed that the proportion of APOE4-negative patients was higher than that of APOE4-positive patients  . Meanwhile, a higher percentage of APOE4 (+) patients was found in P/LP variants than in patients without P/LP variant in AD cohort (p < 0.001)  Fig. . In addi01) Fig. .Fig. 4ThIn this study, we determined the mutational spectrum of 36 known dementia-associated genes in patients clinically diagnosed with neurodegenerative dementia patients, including AD, FTD, and DLB in a South China population sample using integrated targeted gene sequencing analysis. This is the first report of distributions of gene mutations in patients with neurodegenerative dementias from South China. We observed that the use of an integrated gene analysis could be an effective tool for detecting potential genetic causes in neurodegenerative dementias with high genetic heterogeneity or overlapping phenotypic features, gaining further insight in genetic pathogenesis of neurodegenerative dementia, clinical diagnosis, and genetic counseling.PSEN1 are the most common cause of EOAD, meanwhile, PSEN1 was the most frequently mutated gene in patients with FAD. To date, more than 300 mutations in PSEN1 have been identified to be associated with FAD. In this study, four novel variants were identified, which expanded the mutational spectrum of PSEN1. The AAO of PSEN1 mutation carriers in our study (47.5 years), was older than Ryan et al. reported (43.6 years) in 168 AD patients with PSEN1 mutations22, but younger than Jia et al. reported in a large FAD cohort from China (50.59 years)23. Of interest, in addition to the PSEN1 mutations mentioned above, we found an older female (83 years) carrying a novel mutation (M270L), and the APOE genotype was 3/4 in the DLB cohort. Several algorithms predicted the variant was not disease damaging, whereas the nearby mutations  have been reported to be associated with FAD26. Whether the clinical phenotype of the patient is caused by the novel mutation or the contribution of APOE genotype is unclear; we will perform functional research to further clarify the variant. Meanwhile, regarding clinical phenotypes, PSEN1 mutation carriers often present with atypical cognitive symptoms and additional neurological features22. However, in this study, patients mainly presented with amnesia, language impairment, mental and behavioral changes, and movement disorders, which is one limitation of this study. This might have two explanations. First, some atypical symptoms might not occur at an early stage of the disease, and follow-up is necessary. Second, the mutation locations may lead to distinguishing phenotypes; for example, atypical cognitive presentations and pyramidal signs were seen more frequently in association with PSEN1 mutations involving exon 822, suggesting that multiple factors could contribute to the phenotypic heterogeneity of PSEN1-related AD.Mutations in PSEN1, only 18 pathogenic mutations within PSEN2 have been reported, most of which occurred in European and African populations. In this study, seven P/LP variants were identified, including six FAD cases. Previous studies showed that the AAO of PSEN2-associated cases vary widely, from 45 to 88 years; that is more than 10 years later than the mean AAO for PSEN1-related cases29, which was consistent with our results. Interestingly, six patients with a substitution at PSEN2 amino acid residue 239 were identified, including M239V, M239I, and M239T. Among them, M239V has been reported in European populations to elevate Aβ42 levels and Aβ42/Aβ40, and to exhibit a partial loss of function with respect to the C-terminal fragment-γ as well as a substantial decrease in Aβ40 levels32, but it has been absent from Asian cohorts so far. Our findings suggest that this residue may be a common causative variant in the South Chinese population. In addition, the clinical phenotypes of carriers of the M239V mutation varied widely. Our findings, together with previous reports, further suggest that phenotypic heterogeneity exists even at the same codon site because of different amino acid transversions33.In contrast to APP were residues 715 and 717. Taken together with our previous reports, our team have found four families carrying mutations at this site34. Amino acid residues 715 and 717 are located near the γ-secretase cleavage site, and mutation at this site may increase the hydrophobicity of the APP TM domain to anchor the protein within the membrane and elevate the Aβ42/Aβ40 ratio36. Interestingly, patients with mutations at this site often have non-memory symptoms, which can be misdiagnosed as FTD, because the behavioral problems occur earlier than the memory deficits. Totally, in all PSEN1/PSEN2/APP P/LP variants in LOAD, PSEN1 M457V, and PSEN2 A394Pfs*8 were novel variants, further functional validation was necessary and warranted.In accordance with other populations, the common mutation site of CHCHD10 and HTRA1 mutations in the AD cohort. CHCHD10 has been identified to be associated with a large spectrum of diseases, including FTD, ALS, AD, cerebellar ataxia, mitochondrial myopathy, late-onset spinal motor neuronopathy, and Charcot-Marie-Tooth disease type 240. Previously, we have reported a late-onset AD patient with the CHCHD10 mutation41. A homozygous HTRA1 mutation was known to be causative for CARASIL42, while evidence was also showed that heterozygous HTRA1 mutation, which might result in an impaired HTRA1 activation cascade or be unable to form stable trimers, is related to autosomal dominant hereditary cerebral small vessel disease with delayed onset45. In this study, the female AD patients presented with typical amnesia symptoms at 49 years without any other neurological or extra-neurological symptoms. Meanwhile, the Fazekas score of periventricular white matter hyperintensities was 1, the APOE genotype was 3/3 and the core biomarkers of CSF showed A+T+N-. However, the effect of the heterozygous mutation in the pathophysiologic process of AD remains elusive; further functional studies are still needed. These results further indicate that mutations not only in PSEN1, PSEN2, and APP can cause the AD phenotype, but that variants in other genes might also cause AD-like symptoms. Further follow-up is necessary.In addition, we identified PSEN2 p.M239I and MAPT p.R5H), but her daily living ability remained intact, and the double mutation did not accelerate the cognitive decline, further expanding the phenotype spectrum of the mutation and supporting the phenotypic heterogeneity among subjects carrying the same MAPT mutations. Further in vivo and in vitro studies are needed to determine the effect of MAPT and PSEN2 mutations on the pathology and pathogenesis of AD.Notably, we identified double mutations in a 52-year-old female , followed by pre-capture PCR amplification. After PCR amplification, the DNA fragments were captured by the targeted panel, followed by sequencing using the Illumina NovaSeq 6000 platform. The reads were mapped to the human genome reference (hg19) using the Burrows Wheeler Aligner software (http://bio-bwa.sourceforge.net)57, and duplicate sequence reads were removed using Picard (http://broadinstitute.github.io/picard/). Variant calling was performed using the Genome Analysis Toolkit (https://software.broadinstitute.org/gatk/)58. The variants were annotated using ANNOVAR (https://hpc.nih.gov/apps/ANNOVAR.html)59 and named according to the guidelines of the Human Genome Variation Society (http://www.hgvs.org/)60. Pathogenic or likely pathogenic (P/LP) variants were assessed according to the guidelines issued by the American College of Medical Genetics (ACMG).Genomic DNA was extracted from peripheral blood leukocytes of each participant using the QIAGEN kit according to the manufacturer’s instructions. We designed a dementia-related gene panel containing a total of 36 genes associated with cognitive impairment phenotypes, including C9orf72 and (CAG)n repeats in HTT were performed in all individuals using previously reported repeat-primed polymerase chain reaction and capillary electrophoresis62.Moreover, the (GGGGCC)n repeats in All P/LP variants were estimated by PCR amplification and Sanger sequencing using a Big Dye Terminator V3.1 on an ABI 3730xl DNA analyzer . The DNA sequences were then analyzed using Sequencher software version 4.2. All primers were designed using Primer 5, and the primer sequences and PCR reaction conditions are listed in Supplementary Table t test or the Mann–Whitney U test. The χ2 test and Fisher exact test were used to analyze categorical data, such as the proportion of female patients, family history, the percentage of APOE4 positive or negative, and proportion of EOAD, LOAD or P/LP variants carriers and non-carriers patients. Multiple linear regression analysis was performed to correct the confounding factors and explore the factors affecting the AAO. All tests were two-tailed, and p < 0.05 was considered statistically significant. All analyses were performed using SPSS v.26 (IBM). Data were visualized using Prism 8 (GraphPad).Quantitative variables such as age at onset, age at diagnosis, disease duration, education attainment, and cognitive assessment score are expressed as the mean ± SD. All data were tested for normality and homogeneity of variance using the Shapiro-Wilk test and Levene variance equality test. Two independent samples were conducted using the Further information on research design is available in the Supplementary InformationReporting Summary


All researched ephrins seemed to be upregulated in gynecological cancer, whereas EPHs showed either significant overexpression or extensive loss of expression in gynecological tumors, depending on the particular receptor. EPHA2, the most extensively studied EPH in ovarian cancer, exhibited overexpression both in ovarian carcinoma cell lines and patient tissue samples, while EPHB4 was found to be upregulated in endometrial cancer in a series of studies. EPHs/ephrins were shown to exert their role in different stages of gynecological cancer and to influence various clinicopathological parameters. The analysis of patients’ gynecological cancer tissue samples, most importantly, revealed the significant role of the EPH/ephrin system in the development and progression of gynecological cancer, as well as overall patient survival. In conclusion, the EPH/ephrin system represents a large family of biomolecules with promising applications in the fields of diagnosis, prognosis, disease monitoring, and treatment of gynecological cancer, with an established important clinical impact.Gynecological cancers represent some of the most common types of malignancy worldwide. Erythropoietin-producing hepatocellular receptors (EPHs) comprise the largest subfamily of receptor tyrosine kinases, binding membrane-bound proteins called ephrins. EPHs/ephrins exhibit widespread expression in different cell types, playing an important role in carcinogenesis. The aim of the current review was to examine the dysregulation of the EPH/ephrin system in gynecological cancer, clarifying its role in ovarian, endometrial, and cervical carcinogenesis. In order to identify relevant studies, a literature review was conducted using the MEDLINE and LIVIVO databases. The search terms  Erythropoietin-producing hepatocellular receptors (EPHs) constitute the largest subfamily of receptor tyrosine kinases (RTK) that bind membrane-bound proteins called ephrins . EPHs arephrin, ephrin receptor, ovarian cancer, endometrial cancer, and cervical cancer were employed and we were able to identify 57 studies focused on gynecological cancer and published between 2001 and 2021.Pergaris et al., recently reviewed the clinical impact of the EPH/ephrin family’s expression in solid tumors, reporting their key role in the development and progression of head and neck, thoracic, skin, gastrointestinal tract, urinary tract, gynecological, and pediatric cancer . OvarianOvarian cancer (OC) is the leading cause of death from gynecological malignancies and the fifth most common cause of tumor-related deaths in women in the United States. For 2021, the American Cancer Society estimates the incidence of OC at 21,410 cases and OC-related deaths at 13,770 in the USA . OC inclA number of EPH/ephrin system members have been shown to play an important role in OC, including EPHA1, EPHA2, EPHA4, EPHA5, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, ephrin-A1, ephrin-A3, ephrin-A5, ephrin-B1, and ephrin-B2.EPHA1 gene using the CRISPR/CAS9 technique. The inactivation of EPHA1 suppressed many aggressive properties and resulted in G0/G1 cell cycle arrest, reduction of the cellular adhesion capacity, and inhibition of the migration capacity, proliferation, transwell invasion, and activity of the matrix metalloproteinase (MMP)-2 and c-MYC signaling pathways in SKOV3 and COV504 OC cells [Many studies have investigated the role of the EPH/ephrin system in OC cell lines  and humaOC cells . On the OC cells .EPHA2 has been described to promote the growth of OVK-18 cells . Thaker ephrin-B2 and EPHB4 were upregulated in mutant TP53 cells; ephrin-B2 was upregulated in the A2780 line; and EPHB4 was overexpressed in the larger pool of mutant TP53 lines. In contrast, EPHB2 showed high expression levels in wild-type TP53 cell lines, while overexpression of ephrin-A3 was induced by hypoxia [EPHB2 demonstrated only small variations in RNA expression across OC cell lines. Promoter hypermethylation of EPHB2, EPHB3, and EPHB4 did not, however, seem to play an important role in ovarian tumors, as reported by Wu et al. . Davidso hypoxia . EPHB4 w hypoxia . Ma et a hypoxia .Ephrin-A1 expression was shown to be induced in OC cells by the pleotropic transcription nuclear factor kappa B (NFκB) after stimulation with tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) . Ephrin-Ephrin-B2 was found to be overexpressed in OC cell lines .The upregulation of EPHA1 in OC tissues reveals its implication in disease onset and progression . EPHA1 eSimilarly, EPHA2 has led to overexpression in OC tissues in a series of studies ,26,35,37EPHA4 expression in OC patients correlated with early relapse and showed an adverse clinical association when expressed in both OC tumor cells and tumor-associated macrophages [rophages .On the contrary, loss of EPHA5 immunohistochemistry (IHC) expression in OC cases was shown to be associated with tumor grade, International Federation of Gynecology and Obstetrics (FIGO) stage, and poor patient outcome .Liu et al., employed a tissue microarray IHC analysis and detected high EPHA8 protein expression in 44.80% of OC tissues, but only 6.67–15% of normal or benign ovarian tissues. A high EPHA8 protein level correlated with older age at diagnosis, higher FIGO stage, positive lymph nodes (LNs), the presence of metastasis, positive ascitic fluid, and higher serum CA-125 levels .Loss of EPHB1 protein expression in OC tissue specimens was associated with higher tumor grade, the presence of metastasis, a high proliferative index (assessed by Ki67 expression), and significantly worse overall survival (OS) . OC patiThe expression of EPHB6 was associated with grade and TNM stage, and negatively correlated with Ki67 expression levels in OC samples, while patients with low EPHB6 protein expression exhibited a poorer outcome .Elevated levels of ephrin-A1 expression led to a more aggressive tumor phenotype and were associated with poor survival in OC specimens . High epephrin-B1 as one of the best discriminators more highly expressed in OC [High-grade OCs showed the greatest ephrin-B expression, with a strong correlation found between ephrin-B expression and MVD, disease recurrence, and a decrease in OS . Schanered in OC . Ephrin-ed in OC . Ephrin-ed in OC .With an estimate of approximately 66,570 newly diagnosed cases of cancer of the uterine body and about 12,940 deaths from cancers of the body of the uterus in 2021, endometrial cancer (EC) represents the most common malignant tumor of the female genital tract in the USA . DependiEPHs/ephrins that have been shown to play an important role in EC include EPHA2, EPHB3, EPHB4, ephrin-A1, and ephrin-B2.EPHA2 protein expression was reported to be upregulated in EC cell lines . EPHA2 hEPHB3 and EPHB4 were shown to be overexpressed in EC cell lines through genome-wide microarray-based comparative genomic hybridization (aCGH) technology .Ephrin-A1 induced intercellular dissociation, stimulated cell attachment, and inhibited cell aggregation through the EPHA receptor pathway in human EC-derived Ishikawa cells ,57.EPHA2 protein expression was reported to be upregulated in EC tissue samples, while its overexpression was significantly associated with high tumor stage and grade, increased depth of myometrial invasion, low estrogen receptor (ER) and progesterone receptor (PR) expression, high Ki67 index, high vascular endothelial growth factor (VEGF) expression, high MVD counts, and shorter disease-specific survival ,59.High EPHB4 expression positively correlated with histological tumor grade, adverse clinical disease stage, post-menopausal stage of the patient, dedifferentiation, myometrial invasion, ER expression, and poor survival rates in EC tissue samples ,62,63,64Ephrin-B2 has been reported to be overexpressed in EC cells and to correlate with higher histological grade, adverse clinical disease stage, dedifferentiation, myometrial invasion, and poor survival rates ,62,63,64Cervical cancer (CC) used to be one of the most common causes of cancer death in female patients, but the death rate dropped significantly due to vaccination against HPV, as well as increased use of screening technology such as PAP and HPV tests. For 2021, the American Cancer Society estimates the CC incidence at 14,480 cases and CC-related deaths at 4290 . Even thEPHA2, EPHB2, EPHB4, ephrin-A1, and ephrin-B2 have been shown to play an important role in CC.EPHA2 protein expression exhibited upregulation in CC cell lines, leading to increased cell proliferation and migration of CC. EPHA2 knockdown increased the cellular susceptibility to epirubicin in CC cells and suppressed CC growth in a xenograft mouse model .EPHB2 was also highly expressed in CC cell lines and promoted CC progression by inducing cell migration and invasion as well as the epithelial–mesenchymal transition (EMT) through R-Ras activation . EPHB2 wEPHA2 protein expression exhibited upregulation in CC tissue samples, while its overexpression was associated with cyclin-dependent kinase 6 protein expression, invasion depth, LN metastasis, clinicopathological disease stage, and OS ,72,73.EPHB2 was highly expressed in CC tissue specimens and positively correlated with R-Ras expression ,74.Two large studies investigated the expression of EPHB4 through IHC and real-time RT-PCR in CC tissue samples and reported that high EPHB4 expression correlated with adverse clinical disease stage, larger tumor size, LN metastasis, high MVD, and poor patient OS ,76.Ephrin-A1 seems to be overexpressed in CC tissue samples and associated with poorer OS ,73,77.High ephrin-B2 expression correlated with adverse clinical disease stage, larger tumor size, LN metastasis, high MVD, and poor survival in CC tissue samples ,76.As studies continue to prove the tumorigenic properties of various EPH/ephrin system members in GC, several researchers have focused on the development of novel therapeutic strategies targeting their carcinogenic actions over the past decades. In terms of OC treatment, restoration of miR-520d-3p decreased EPHA2 protein levels, thus suppressing tumor growth and migration both in vitro and in vivo, while dual inhibition of EPHA2 in vivo using 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes loaded with miR-520d-3p and EPHA2-siRNA had synergistic antitumor effects and greater therapeutic efficacy than either monotherapy alone . The comIn vivo therapy experiments in mouse orthotopic models with monoclonal antibodies targeting EPHA2 resulted in significant MVD reduction, decreased proliferation, increased apoptosis, and a lower incidence of distant metastasis in EC ,85.It is established that members of the EPH/ephrin system are implicated in a multitude of molecular procedures, exerting their role in different stages of GC. Consequently, up- or downregulation of each individual EPH/ephrin in gynecological tract tumors influences numerous clinicopathological parameters such as tumor grade, invasion depth, tumor cells’ proliferation rate (assessed by Ki67 index), FIGO stage, hormone receptor status, patient survival, and response to chemotherapy. Therefore, the aforementioned family of biomolecules emerges as potent biomarkers, useful in the fields of diagnosis, prognosis, and disease monitoring in GC. All investigated ephrins exhibited upregulation in OC, EC, and CC, whereas EPHs showed either significant overexpression  or extensive loss (OC) depending on the particular receptor. However, discrepancies between the results reported by different research groups do exist, with studies sometimes reaching contradictory conclusions on the role of some EPHs/ephrins in certain types of GC. EPHA1 expression, for instance, has been reported to be upregulated in OC by several authors ,34,35,36


Cervical cancer is the second most common gynecological malignancy globally; it seriously endangers women’s health because of its high morbidity and mortality. Conventional treatments are prone to drug resistance, recurrence and metastasis. Therefore, there is an urgent need to develop new drugs with high efficacy and low side effects to prevent and treat cervical cancer. In recent years, plant-derived natural products have been evaluated as potential anticancer drugs that preferentially kill tumor cells without severe adverse effects. A growing number of studies have shown that natural products can achieve practical anti-cervical-cancer effects through multiple mechanisms, including inhibition of tumor-cell proliferation, induction of apoptosis, suppression of angiogenesis and telomerase activity, enhancement of immunity and reversal of multidrug resistance. This paper reviews the therapeutic effects and mechanisms of plant-derived natural products on cervical cancer and provides references for developing anti-cervical-cancer drugs with high efficacy and low side effects. Cervical cancer is one of the second most common gynecologic malignancies in the world, with more than 570,000 women diagnosed with cervical cancer and 311,000 deaths annually worldwide ,3. CurreIn recent years, plant-derived natural products have been considered as the most promising candidates for oncology therapies. They can preferentially kill tumor cells with low toxic effect on normal cells. This is attributed to their chemical diversity, structural complexity, inherent biological activity and low side effects ,7. A varhttps://pubmed.ncbi.nlm.nih.gov/ (accessed on 10 June 2021).We collected the relevant experimental works in the literature published in the last five years that elucidate the anticancer effects of natural products on cervical cancer, using PUBMED (including Medline) and the Google Scholar database. Upon searching for the appropriate studies, we used “natural product, cervical cancer” as keywords. After completing the initial search, we removed duplicate works from the literature. Chemical structures of compounds derived from natural products were cited from the NCBI PubChem website, available online: 6-C3-C6 skeletons labeled with A, B and C rings, which can be classified into flavones, flavanones, flavonols, flavanols, isoflavones and anthocyanidins based on their structural diversity [Flavonoids are phenolic phytochemicals that are commonly found in fruits, vegetables and plant-based beverages  . More thiversity ,13. Manyiversity ,15,16. IScutellaria baicalensis is one of the versatile herbs traditionally and has been used in China to treat inflammatory diseases, hypertension, cardiovascular diseases, bacterial and viral infections. A large amount of evidence suggests that S. baicalensis also has potent anticancer activity, and its main bioactive components are baicalein, Wogonin, and baicalin [S. baicalensis and has a variety of pharmacological activities, which can inhibit cell proliferation and migration; induce apoptosis and cell-cycle arrest [baicalin ,18. Baice arrest . Cyclin e arrest ,21. Accoe arrest ,23,24. Me arrest . Anothere arrest . It is sWogonin is a natural monoflavonoid with the potential for selective tumor therapy in vitro and in vivo . Wogonin50 value at 48 h was 47.26 μmol/L. It induced apoptosis in HeLa cells through the death receptor pathway and the mitochondrial pathway, which effectively increased the expression of ROS, Fas, FasL, TNF-α, TNF-r1, Fas-associated death domain (FADD), TNF receptor-associated death domain (TRADD), caspase 3 and caspase 9 and decreased the expression of pro-caspase 8, caspase 10, Bcl-2 and Bcl-2 extra-large protein (Bcl-xl). Additionally, apigenin 7-glucoside treatment increased p16 INK4A expression and reduced Cyclin  and CDK2/6 expression. Meanwhile, it inhibited HeLa cell migration by targeting the phosphatase and tensin homolog (PTEN)/PI3K/AKT pathway [50 values of 10, 68, 76 and 40 μmol/L, respectively. Apigenin also induced mitochondrial redox damage, decreased MMP and lipid peroxidation and inhibited the migration and invasion of cervical cancer cells [Apigenin is an edible natural flavonoid found in various dietary plant foods, such as vegetables and fruits . It has  pathway . Some ster cells . Zhang eer cells . Consequ50 value of 21.8 μmol/L. Luteolin induced apoptosis and G2/M phase cell-cycle arrest in HeLa cells by downregulating UHRF1 and DNA methyltransferase 1 (DNMT1) with a reduction of overall DNA methylation and upregulating p16 INK4A expression [Luteolin is a common plant flavonoid found in various plants, including fruits, vegetables and medicinal herbs. Luteolin-rich plants have been used to treat multiple diseases, such as hypertension, cancer and inflammation ,37. Lutepression . On the pression . It was pression . In addipression . These d50 values of naringin for HeLa, SiHa and C33A cells after 24-h treatment were 793, 764 and 745 μmol/L, respectively, and showed a dose-dependent relationship. Naringin increased the level of endoplasmic reticulum (ER) stress sensors, phosphorylated eIF2α and activated the apoptosis-related protein CHOP and other associated pro-apoptotic proteins (PARP1). Importantly, naringin also blocked the β-catenin signaling pathway by decreasing β-catenin (Ser576) and GSK-3β (Ser9) protein expression and phosphorylation and induced cell-cycle arrest at G0/G1 phase by increasing the expression of cell-cycle checkpoint proteins p21/cip and p27/kip to trigger apoptosis in cervical cancer cells [Citrus fruits are rich in flavonoids and are known for their health-promoting and chemopreventive properties. Naringin, a flavonoid glycoside, can be isolated from citrus fruits, such as orange, tangerine, lemon and lime, and has several pharmacological activities . Naringier cells . Anotherer cells . The res50 values of 650 μmol/L. Hesperetin induced cell-cycle arrest at G2/M phase and apoptosis in SiHa cells by death receptor and mitochondrial pathways, characterized by depolarizing MMP and increasing the expression of caspase 3, caspase 8, caspase 9, p53, Bax, Fas and FADD [Hesperidin is a citrus flavonoid and has various functions, including antioxidation, antitumor and anti-angiogenesis functions, which can protect mitochondrial membranes from free radical attack and inhibit tumor-cell migration and invasion ,48. Hespand FADD . In addiSilibinin is a bioactive polyphenolic flavonoid isolated from the fruits and seeds of silybum marianum. It has been used to treat various diseases, especially the liver, gallbladder and kidney [Silibinin inhibited the growth of Hela and SiHa cells in a dose- and time-dependent manner, with IC50 values of 332 and 275 μmol/L for HeLa cells, and 250 and 195 μmol/L for SiHa cells at 48 h and 72 h, respectively. It reduced ATP content, mtDNA copy number and MMP, activated the dynamin-related protein 1(Drp1)-mediated mitochondrial fission pathway, induced G2/M cell-cycle arrest through reducing the expression of CDK1, cyclin B1 and Cdc25C [ilibinin also suppressed angiogenesis and promoted apoptosis in HeLa cells through inhibiting the mTOR/p70S6K/4E-BP1 signaling pathway and decreasing the accumulation and transcriptional activity of hypoxia-inducible factor-1α (HIF-1α) and the release of VEGF. Moreover, the anti-angiogenic ability of silibinin was enhanced by blocking AKT activation through PI3K/AKT inhibitor LY294002 [ilibinin induced apoptosis in HeLa cells through the mitochondrial pathway and the death-inducing pathway, resulting in decreased expression of CDK1 and CDK2 proteins and increased ratio of Bax/Bcl-2, followed by Cyt c release and activation of caspase 9, as well as increased expression of Fas and FasL and activation of caspase 8 [ilibinin-induced ROS [ilibinin inhibits HeLa cell growth by activating apoptotic vesicles and caspase 3, and promoting the phosphorylation of p53 and JNK in a dose-dependent manner. p53 subsequently interferes with mitochondrial function through the p53-upregulated modulator of apoptosis (PUMA) pathway, which upregulates the Bax/Bcl-2 ratio, reduces MMP and increases ROS production. Then ROS induces autophagy and apoptosis in HeLa cells. Furthermore, p53-mediated glutathione (GSH) depletion significantly enhanced the cytotoxicity of NO in HeLa cells [d kidney ,52. Silid Cdc25C . SilibinLY294002 . At the aspase 8 . The eviuced ROS . SilibinLa cells ,58. It m2+ concentration, which caused shrinkage of spindle-shaped SiHa cells and damage of their microtubule networks [50 value was 10.48 μmol/L at 72 h, while it had weak toxicity for normal cells with IC50 value of 707 μmol/L at 72 h. It induced apoptosis and senescence in HeLa cells by inhibiting PI3K/AKT pathway and human telomerase reverse transcriptase (hTERT) expression and promoting the p53 pathway [Kaempferol is a natural flavonol and widely distributes in many plant families. The growing evidence has shown that kaempferol is a potential cancer therapeutic agent with potent antitumor, anti-inflammatory and antioxidant properties . It inhinetworks . Another pathway . Telomer pathway . It was  pathway . Kaempfe50 of 30 μmol/L for HeLa cells at 24 h and 50 μmol/L for SiHa cells at 48 h. Treatment with cisplatin or quercetin alone did not reduce the expression of MMP2 protein in cervical cancer cells but their combination significantly decreased MMP2 expression, which inhibited the migration and invasion of cervical cancer cells. Meanwhile, quercetin enhanced the chemosensitivity of cervical cancer cells by downregulating the expression of P-gp and methyltransferase-like 3 (METTL3), which mediated HeLa cell proliferation and apoptosis [Quercetin is a dietary polyphenolic compound with wide distribution in vegetables and fruits and has various activities, including anti-allergic, anti-inflammatory and antitumor activities ,65. Querpoptosis . Quercetpoptosis . These sFisetin is a natural flavonols found in vegetables, fruits and nuts and has antitumor, anti-invasive, anti-angiogenic, antidiabetic, cardioprotective and neuroprotective activities ,73. FiseCamellia sinensis) is one of the most commonly used herbs globally and is widely known for its effectiveness in preventing chronic diseases and tumors. This is mainly attributed to the biologically active catechin compounds in green tea, such as (−)-epigallocatechin gallate (EGCG), (−)epigallocatechin 3-gallate (ECG), (−)epigallocatechin (EGC) and (+) catechin. Among them, EGCG is the main component of catechins and has substantial antioxidative, antitumor and anti-angiogenic effects [50 value of 196.07 μg/mL. In addition, catechin inhibited the proliferation and induced apoptosis of SiHa cells partly by regulating TP53 and caspase 3, caspase 8 and caspase 9 [50 value of 20 µg/mL. EGCG inhibited the activation of AKT and NF-κB by blocking the phosphorylation and degradation of the inhibitory κBα and κBβ subunits to result in the downregulation of COX-2 expression. Furthermore, EGCG treatment led to mitochondrial dysfunction through increasing ROS production, p53 and Bax/Bcl-2 ratios to promote Cyt c release and activation of caspase cascade, which induced apoptosis in HeLa cells [Green tea  and downregulation of Bcl-2. In a BALB/c mouse model inoculated with HeLa cells or Caski cells, intraperitoneal injection of dihydroartemisinin for 3 weeks significantly inhibited tumor growth with inhibition rates among 70–80% [In recent years, artemisinin and its derivatives  have been recognized as effective antimalarial agents . They alg 70–80% . Moreoveg 70–80% . In addig 70–80% . Zhang eg 70–80% .50 values of 5.47 and 6.34 μmol/L, respectively. Interestingly, artesunate in vitro and in vivo increased the radiosensitivity of HeLa cells by promoting apoptosis and G2/M phase transition induced by X-ray irradiation, which was related with the increased expression of cytosolic cyclin B1. It was suggested that it could be applied as an effective radiosensitizer in cancer therapy [+CD25+Foxp3+ T cells [Artesunate effectively enhanced TRAIL-mediated cytotoxicity by reducing the expression of pro-survival proteins  via inhibiting the activation of NF-κB and AKT, and increased apoptosis induced by TRAIL . Besides therapy . Meanwhi T cells . It was  T cells . The resSalvia miltiorrhiza. It has anti-inflammatory and antioxidant activities and has been commonly used in prevention and treatment of cardiovascular disease [50 values of 6.97, 14.47, 5.51 and 9.89 μmol/L respectively, and showed a higher anti-proliferative effect than paclitaxel (18.45 μmol/L). Furthermore, tanshinone IIA treatment activated caspase 9 and 3 to cleave PARP, increased the ratio of Bax/Bcl-2 and JNK, released Cyt c that interacted with Apaf-1, and phosphorylated p38 to trigger ER stress-mediated apoptosis through IRE1 and PERK-related pathways. Notably, tanshinone IIA could improve paclitaxel chemosensitivity, suggesting that it may be a potential strategy to overcome paclitaxel resistance [Tanshinone IIA is a diterpenoid naphthoquinone found in traditional Chinese medicine  disease ,132. Rad disease . In addi disease . Anothersistance . Importasistance . It can Rabdosia rubescens with various pharmacological and physiological effects, including antibacterial, anti-inflammatory and antitumor [50 value of 4.13 μmol/L (48 h) [Oridonin is an active diterpene isolated from ntitumor . Oridonintitumor . Hu et aL (48 h) . SeveralL (48 h) ,141. AddL (48 h) . OridoniL (48 h) . It may Ginsenoside Rh2 is a biologically active compound derived from ginseng and has various health effects, including stimulation of immune function, enhancement of cardiovascular health and anti-stress capacity . Ginseno50 value of 30.42 ± 2.39 μmol/L at 48 h of treatment, the phosphorylation of AKT at Thr308 and Ser473, activated Bad, caspase 9 and cell-cycle regulatory factors p27Kip and p21Waf1/Cip1, and induced cell-cycle arrest at G0/G1 phase [Betulinic acid, a natural pentacyclic triterpene found in white birch bark, is one of the most promising cancer therapeutic compound with protective effects against tumor progression, inflammation, metabolic diseases and cardiovascular diseases ,149. BetG1 phase . BetulinG1 phase . FurtherG1 phase . It has G1 phase . BetulinThe chemical structures of terpenoids are summarized in Piperine is a nitrogenous stimulant found in the fruits of black pepper and long pepper. It has various pharmacological properties in vitro and in vivo, such as anticancer, antibacterial, antiulcer hepatoprotective and immunomodulatory properties ,155. AsiSophora flavescens is a kind of Chinese medicine and contains active ingredient matrine. Because of its wide range of pharmacological effects, it has been considered as anticancer drug in China. It has been used clinically to treat viral hepatitis, neuropathic pain, heart disease, skin diseases and other disorders for a long time [S. flavescens inhibits cervical-cancer-cell proliferation and metastasis and induces apoptosis by inhibiting the AKT/mTOR signaling pathway; reducing the expression of Bcl-2, cyclin A and MMP2; blocking the G2/M phase cell cycle; and stimulating Bax and E-calmodulin. Matrine reduces vasodilator-stimulated phosphoprotein (VASP) phosphorylation by inhibiting protein kinase A (PKA) activity, thereby inhibiting HeLa cell adhesion and migration [ong time ,160. S. igration . Besidesigration . Matrine50 value of berberine was 300 μmol/L after the 48-h treatment of HeLa cells. Berberine activated the death receptor pathway in HeLa cells by upregulating Fas, FasL, TNF-α and TRAF-1. Simultaneously, berberine induced apoptosis in HeLa cells by activating the mitochondrial pathway, releasing Cyt c and significantly increasing the ratio of Bax/Bcl-2, which blocked the cell cycle at the S phase. Moreover, berberine activated the MAPK pathway and increased p53 expression, suggesting that p53 may be a drug target in the treatment of cervical cancer [Berberine, an isoquinoline alkaloid extracted from the herb, has various biological activities, such as antibacterial, anti-inflammatory, antidiabetic, anti-angiogenic and anticancer effects. It is widely used as an antibacterial agent in clinical practice ,164. Berl cancer . In addil cancer . It suggThe chemical structures of alkaloids are summarized in 50 values of drug-loaded (curcumin) micelles on HeLa, SiHa and C33a cell lines were 4.7, 3.6 and 12.2 times lower than those of free curcumin . Compared with free curcumin, the number of micelles carrying curcumin is significantly increased by 6-fold. It is worth noting that the percentage of early apoptosis of HeLa, SiHa and C33a cells is increased by curcumin micelles to 30–55% [Curcumin is a phenol compound extracted from the rhizome of the perennial herb turmeric in Zingiberaceae, and has been commonly used as a spice and food coloring. Its safety and efficacy as a traditional herbal medicine have been shown in previous studies. It also exerts anticancer effects through various mechanisms, including inhibition of cell proliferation, invasion and metastasis, and epigenetic alterations ,170. Curo 30–55% . These d50 value of 48.7 ± 2.5 μmol/L. Ellagic acid promoted SiHa cell apoptosis by upregulating p53, Bcl-2 and caspase 3/9; downregulating Bax; and inducing the G2-phase cell-cycle arrest [50 values of 19.47, 16.52 and 10.9 μmol/L, which revealed the synergistic anticancer effect of ellagic acid and curcumin. Their combination synergistically restored p53 protein expression, induced ROS formation and increased DNA damage, accompanied by increased expression of the downstream effectors p21 and Bax, which induced apoptosis in HeLa cells [Ellagic acid is a natural phenol compound primarily found in fruits and nuts, including raspberries, pomegranates and walnuts, with strong anti-proliferative, anti-metastatic and anti-angiogenic activities ,178. Elle arrest . Li et ae arrest . Moreovee arrest . A recenLa cells . Ellagic50 values of 40.06 and 59.07 μmol/L at 72 h. Resveratrol inhibited HeLa and CaSki cell growth and development by inhibiting the expression of HPV oncoproteins E6 and E7; promoting p53, Bax and p16; and inducing cell-cycle arrest at the G1/S phase [Resveratrol is a dietary polyphenol derived from grapes, berries, peanuts and other plant sources. It has several potential biological effects, including cancer prevention and treatment . Resvera/S phase . A study/S phase . Further/S phase . He et a/S phase . Importa/S phase . Another/S phase . ResveraThe chemical structures of phenols are summarized in The high mortality and morbidity rates of cervical cancer remain a primary challenge for scientific research. Although conventional treatments, such as radiotherapy and chemotherapy, are effective, the five-year survival rate of cervical cancer patients is meager and often ends in failure. Moreover, chemotherapy is prone to drug resistance and toxic side effects, causing great suffering to patients. In recent years, plant-derived natural products have been considered as the most promising candidates for oncology therapies. In 2010, Bar-Sela et al.  reportedPlant-derived natural products, a rich treasure trove of drug resources, have driven the development of novel inhibitors with their unique mode of action and molecular structural diversity. They play an important role in human cancer therapy with high safety and low side effects. However, some unresolved issues limit the translation of plant-derived natural products to clinical applications. First, some have poor water solubility, which seriously affects bio-availability. Second, potential toxicity and adverse effects of plant-derived natural products are lack of sufficient evaluation in vivo and clinical trial data. Again, further research on the mechanism of natural antitumor drugs is still the direction of future efforts. We believe that, with additional research, plant-derived natural products will be indeed applied in clinical medicine.


High mobility group box-1 protein (HMGB1), a member of the high mobility group protein superfamily, is an abundant and ubiquitously expressed nuclear protein. Intracellular HMGB1 is released by immune and necrotic cells and secreted HMGB1 activates a range of immune cells, contributing to the excessive release of inflammatory cytokines and promoting processes such as cell migration and adhesion. Moreover, HMGB1 is a typical damage-associated molecular pattern molecule that participates in various inflammatory and immune responses. In these ways, it plays a critical role in the pathophysiology of inflammatory diseases. Herein, we review the effects of HMGB1 on various immune cell types and describe the molecular mechanisms by which it contributes to the development of inflammatory disorders. Finally, we address the therapeutic potential of targeting HMGB1. Living organisms have evolved alarm systems that can sense cellular stress and damage, facilitating the discharge of several critical endogenous factors such as damage-associated molecular pattern molecules (DAMPs) and alarmins ,2,3,4. THigh mobility group box-1 protein (HMGB1), a prototypical alarmin and DAMP molecule, was first described in 1973 ,14. The Almost a decade later, the receptor for advanced glycation end products (RAGE) was found to act as a special receptor for HMGB1, helping to explain the functions of extracellular HMGB1 ,20,21,22Itis evident that HMGB1 plays a cardinal role in regulating inflammatory responses and in cell fate decisions ,29,30. IThis review provides an update in the HMGB1 field, with focus on the pathologic role of HMGB1 in inflammatory and immune conditions. It also highlights the evidence supporting the potential of HMGB1 as a therapeutic target.HMGB1 gene, which was sequenced in 1996, is located on chromosome 13q12.3. It encodes a nuclear protein of 215 amino acids with a molecular weight of 25 kDa [Chironomidae, Drosophila and yeast [The f 25 kDa ,37,38. Tf 25 kDa . Disulfind yeast ,41. Rodend yeast . The DNAHMGB1 is usually located in the nucleus due to its two unique nuclear localization signals (NLS), NLS1 (residues 28–44) and NLS2 (residues 179–185) ,43. DeacMethylation of HMGB1 dampens the strength of HMGB1-DNA binding, leading to its translocation out of the nucleus. This explains the observed release of HMGB1 from neutrophils under chronic inflammation ,49,50. CIn addition to the ability of stress and posttranslational modifications to promote HMGB1 secretion, the protein can also passively diffuse out of leaking necrotic cells to induce inflammation. In fact, HMGB1 released from necrotic cells can promote inflammation more strongly and persistently than HMGB1 released from apoptotic cells ,57,58.At least 14 receptors have been identified to interact with extracellular HMGB1 , with RARAGE was first described in 1995 as the amphoterin-binding receptor . A membeBoth TLR4 and RAGE are required for HMGB1 to trigger the release of mediators from macrophages. In macrophages lacking RAGE, their activation via TLR4 leads to much lower cytokine secretion . HMGB1 cA second potential binding site for RAGE was identified in HMGB1, at residues 23–50 in the A-box . While tToll-like receptors (TLRs) can recognize various danger signals, such as DAMPs and pathogen-associated molecular patterns (PAMPs) to trigger immune responses against pathogens ,69. BindIn addition to RAGE and TLRs, TIM-3 can bind with HMGB1. This interaction inhibits the recruitment of nucleic acids into dendritic cell endosomes ,60. ThusHMGB1 is produced by various immune cell types, especially neutrophils and monocytes, after stimulation with IL-1, TNF-α, DNA, CpG and LPS ,49,50,58Binding of extracellular HMGB1 to TLRs and RAGE on the surface of immune cells initiates downstream signaling cascades involving extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K)/RAC-α serine/threonine-protein kinase (Akt) and NF-κB p56) ,85,86,87 ,85,86,8Neutrophils are the first responders of innate immunity; they produce reactive oxygen species (ROS), cytokines, antibacterial peptides and other inflammatory molecules. In response to infection, neutrophils generated by innate immune cells are recruited to the site of infection and engage pathogens . Once wiThe interaction between HMGB1 and RAGE stimulates the recruitment and infiltration of neutrophils into necrotic sites, which can exacerbate hepatic injury ,91,92. SThe recent discovery of neutrophil extracellular traps (NETs) has expanded our view of how neutrophils function . NETs arMacrophages normally phagocytose apoptotic neutrophils and NETs in a process termed NETosis and impairment in this NET clearance may lead to persistent inflammatory responses and subsequent organ injury ,101. NETMonocytes are not very abundant in the peripheral circulation. They are produced in the bone marrow and released into the peripheral circulation, where they circulate for only about a day before settling permanently within tissues . Once seOn exposure to LPS, macrophages produced hydrogen peroxide that promoted the formation of intramolecular disulfide between C45 and C23 . This oxMacrophages are implicated in initiating, maintaining and resolving inflammatory processes in host defense and during sepsis ,116. C1qHMGB1 has been implicated in various forms of programmed cell death. For example, HMGB1 induces apoptosis of mouse macrophages in a dose- and time-dependent manner ,121. In lowCD45RNhigh DCs prevented acute inflammatory responses by inhibiting the formation of pro-inflammatory mediators [Dendritic cells (DCs) serve as professional antigen-presenting cells that capture antigen in one location, then migrate to lymph nodes, where they present it to native T cells, initiating the adaptive immune response . In addiediators .HMGB1 finely tunes the maturation, differentiation and immune functions of DCs, so it influences the shift of helper T (Th) 1 cells and Th17 polarization necessary for T cell-mediated immunity ,129,130.lowCD45RNhigh subtype, leading to a reduction in T cell-activated immune responses [Several studies suggest that HMGB1 acts as a diphasic immune regulator of DC function. Our own studies revealed that HMGB1 can stimulate DC apoptosis in a time- and dose-dependent manner ,134. Conesponses ,134.In DCs, HMGB1 upregulates PI3K, Akt and mTOR and phosphorylated proteins . The HMGHMGB1-dependent DCs activation induces Th17-type responses and this activation can be blocked using a soluble form of RAGE (sRAGE) , which bHMGB1 upregulates CD86 and CD80 levels on mucosal DCs, whereas the HMGB1 inhibitor glycyrrhizin inhibits induction of DCs and activation of CD8+ cytotoxic lymphocytes that specifically recognize intestinal ovalbumin ,138. The pivotal role of HMGB1 in DC activities may make it a target against human immunodeficiency virus (HIV) infection. HIV can reside within DCs and thereby spread and evade host immunity ,140. NeuT lymphocytes are the principal players in cell-mediated adaptive immunity. T lymphocytes are divided into two major cell types: Th cells and T cytotoxic cells . Th1 celHMGB1 potently modulates T cell immunological reactions, but the underlying molecular mechanisms remain unknown. We observed that serum HMGB1 was markedly increased in rats following thermal injury and it was able to activate T lymphocytes at relatively low concentrations . HMGB1 aIL-2 potently stimulates T lymphocytes and regulates the balance between Th1 and Th2 cells. HMGB1 exerts its immunosuppressive effect on T lymphocytes via p53, p38 MAPK and ERK 1/2 ,148,149.The proinflammatory B-box of HMGB1 is required for soluble CD52 to be able to suppress T cells. CD52 is a glycophosphatidylinositol (GPI)-anchored glycoprotein expressed by T cells, B cells, DCs, natural killer cells, macrophages and eosinophils ,144,150.HMGB1 can directly evoke the differentiation of Th17 and Th2 cells, through which the protein promotes mucus production and airway inflammation in asthmatic mice . After i+ T cells that dampen the proliferation and activity of antigen-presenting cells, T cells, natural killer cells and B cells [Regulatory T cells are a subset of CD4 B cells ,156. The B cells ,156.In regulatory T cells from mouse spleen, we noted that HMGB1 markedly down-regulated expressions of Foxp3 and CTLA-4, which was accompanied by the production of IL-10 ,158. AnoTLRs are sensors that recognize invading pathogens and mediate signaling cascades that trigger and regulate immune responses. The binding of TLR4 with HMGB1 regulates the function of regulatory T cells. We found that stimulation of regulatory T cells with HMGB1 led to a massive elevation of cytoplasmic TLR4 but a steep decline in membrane TLR4 . These cRegulatory T cells are critical for resolving lung damage. Inhibiting HMGB1 dampens pro-inflammatory cytokine production, increases TGF-β release and attenuates lung injury . An HMGBThe activities of HMGB1 depend greatly on its cellular localization. Nuclear HMGB1 acts as a pro-inflammatory molecule in sterile tissue injury or during infection, which in turn affects immune responses. Extracellular HMGB1 engages in various activities affecting inflammation, oxidation, migration, invasion, proliferation, differentiation and tissue regeneration. For example, HMGB1 induces cultured human monocytes to produce a plethora of proinflammatory mediators such as macrophage inflammatory protein (MIP)-1, IL-1, IL-6, IL-8 and TNF ,31. In vHMGB1 appears much later than other proinflammatory cytokines after onset of sepsis: LPS stimulation of mouse macrophages upregulated HMGB1 within 8 h, followed by a massive increase at 16–32 h ,78.HMGB1 can inspect and carry immunogenic nucleic acids, upregulating IL-6 and type 1 interferons in immune cells . BindingThe role of HMGB1 in sepsis has been extensively explored in rodent models with cecal ligation and puncture (CLP). The fact that HMGB1 acts late in sepsis opens a therapeutic window for medical treatment ,170,171.Autoimmune diseases are attributed to dysregulation of both immune and inflammatory responses. Recent findings suggest that HMGB1 is critical for the development of autoimmune pathologies .Rheumatoid arthritis (RA) involves destructive synovitis at the cartilage-bone interface, along with synovial tissue hypoxia ,174. HMGSystemic lupus erythematosus (SLE) is a systemic inflammatory and autoimmune disorder characterized by the deposition of immune complexes in multiple organs . High leIn addition, extracellular HMGB1 was shown to be a source of NET as evidence that HMGB1 levels was high to greater certain among lupus nephritis . HMGB1 pAcute liver injury is a common condition caused by, for example, hepatic viral infection, drug-induced liver dysfunction, or ischemia/reperfusion injury . PatientAcetaminophen is the most common cause of drug-induced liver injury. A hepatotoxic intracellular acetaminophen metabolite causes hepatocyte necrotic death, release of non-acetylated HMGB1 and leukocyte activation. Both the release of HMGB1 and the accompanying activation of macrophages contribute to the inflammatory environment of acetaminophen-induced hepatotoxicity ,195,196.Serum HMGB1 is also recognized as an early clinical marker of hepatic ischemia/reperfusion injury. HMGB1-associated partners such as TLR4, RAGE and CXCR4 are abundantly expressed in various liver and immune cells, including sinusoidal endothelial cells, stellate cells, hepatocytes, Kupffer cells and dendritic cells . ReducinExtracellular HMGB1 has been postulated to act as a danger signal that evokes an inflammatory storm, prevents the phagocytosis of apoptotic cells (necessary to stop inflammation) and changes vascular remodeling in various lung diseases ,198.Asthma is a clinical syndrome characterized by three distinct components: airway inflammation, exaggerated bronchoconstrictor responses and recurrent episodes of airway obstruction ,199. HMGTargeting HMGB1 may a useful treatment for asthma. HMGB1 antagonists repressed leukocyte infiltration, levels of collagen, cell counts and pro-inflammatory cytokines in animal models, which attenuated airway remodeling and airway hyperresponsiveness ,205,206.Acute lung injury (ALI) is characterized by sudden-onset, severe impairment of pulmonary gas exchange and sustained lung inflammation and it is most frequently caused by infections, sepsis, trauma, or inhalation of toxic substances . The rolIntratracheal administration of anti-HMGB1 antibodies alleviate lung inflammation, reduce bacterial burden and improve mortality in mouse models of ALI ,214,215.HMGB1 performs numerous functions during cardiac injury, cardiac remodeling and regeneration ,218. It HMGB1 is released by immune cells or stressed cardiomyocytes and it recruits Ly6C+ monocytes to the damaged cardiac tissue, thereby reprogramming the monocytes into pro-inflammatory M1 macrophages . M1 macrAt the same time, HMGB1 may drive lymphocyte apoptosis, lymphocyte homing, inflammatory resolution and heart remodeling ,149. CarEncephalopathy is a severe condition caused by sepsis, metabolic diseases and liver dysfunction. HMGB1 can trigger inflammatory cascades in the central nerve system (CNS) and cause astrocyte-mediated cerebral swelling .Up to 25% of septic patients display cognitive decline and animal models of sepsis show significant chronic impairments in memory and learning, which are associated with anatomical changes in the hippocampus . AdminisSevere trauma is characterized by extreme cellular stress and excess release of alarmins, especially HMGB1. Indeed, experimental and clinical studies have implicated HMGB1 in systemic inflammation and multiple organ dysfunction as a result of severe trauma ,225. In HMGB1 plays a critical role in bacterial translocation in the gut and in systemic inflammation during severe acute pancreatitis (SAP). Serum HMGB1 levels are elevated in patients with SAP . In thesHMGB1 has been implicated in tumorigenesis, progression, metastasis and chemotherapy resistance of various cancers ,229,230.HMGB1 is a critical endogenous nucleoprotein with multiple biological functions. The properties of HMGB1 depend on its subcellular location, interaction partners and post-translational modifications . Normally, HMGB1 acts as an architectural factor that modulates nucleosomes, DNA and gene transcription by binding DNA in the nucleus. Under stress conditions, HMGB1 can shuttle to the cytoplasm, where it is actively or passively released from cells. HMGB1 is actively released by various types of immune cells, such as neutrophils, macrophages and DCs and it is also secreted during cell injury and death. Once outside the cell, the protein participates in a broad range of biological behaviors, such as autophagy, apoptosis and pyroptosis.HMGB1 has metabolic, immune, chemokine and cytokine activities. HMGB1 induces and maintains potent inflammatory and immune responses by influencing various immune cells involved in the pathology of inflammatory conditions. HMGB1 can recruit and activate neutrophils, promote neutrophil infiltration and promote NET formation. It modulates maturation, differentiation, activation and apoptosis of DCs. It induces apoptosis and pyroptosis in macrophages while impairing their ability to phagocytose apoptotic cells. HMGB1 promotes the migration and survival of regulatory T cells, while suppressing their activity. HMGB1 can modulate the immune functions of T lymphocytes in a dose-dependent fashion.Accumulating evidence indicates that HMGB1 represents a potential biomarker and an appealing target for innovative therapeutic approaches in numerous inflammatory disorders, such as sepsis, lung diseases, autoimmune diseases, acute liver injury, cardiac injury, encephalopathy and other inflammatory conditions. Targeting HMGB1 with pharmacological inhibitors and monoclonal antibodies has shown promise for repressing HMGB1-mediated inflammatory responses in preclinical studies. Blocking HMGB1 can mitigate the cytokine storm and thereby alleviate tissue injury and reduce mortality in various animal models of multiple inflammatory disorders.Targeting HMGB1 has yet to be translated to the clinic. We need further exploration into how HMGB1 drives the pathogenesis of several inflammatory conditions. Considering the dose-dependent effects of HMGB1 in certain contexts, standardized, accurate measurement of HMGB1 levels is key to explore therapeutic strategies based on HMGB1 antagonists. Most preclinical studies have focused on the significance of extracellular HMGB1, so more work is needed on the functions of intracellular HMGB1 and its targetability for the treatment of inflammatory diseases.


The regulator of G-protein signaling 14 (RGS14) is a multifunctional signaling protein that regulates post synaptic plasticity in neurons. RGS14 is expressed in the brain regions essential for learning, memory, emotion, and stimulus-induced behaviors, including the basal ganglia, limbic system, and cortex. Behaviorally, RGS14 regulates spatial and object memory, female-specific responses to cued fear conditioning, and environmental- and psychostimulant-induced locomotion. At the cellular level, RGS14 acts as a scaffolding protein that integrates G protein, Ras/ERK, and calcium/calmodulin signaling pathways essential for spine plasticity and cell signaling, allowing RGS14 to naturally suppress long-term potentiation (LTP) and structural plasticity in hippocampal area CA2 pyramidal cells. Recent proteomics findings indicate that RGS14 also engages the actomyosin system in the brain, perhaps to impact spine morphogenesis. Of note, RGS14 is also a nucleocytoplasmic shuttling protein, where its role in the nucleus remains uncertain. Balanced nuclear import/export and dendritic spine localization are likely essential for RGS14 neuronal functions as a regulator of synaptic plasticity. Supporting this idea, human genetic variants disrupting RGS14 localization also disrupt RGS14’s effects on plasticity. This review will focus on the known and unexplored roles of RGS14 in cell signaling, physiology, disease and behavior. The family of the regulators of G-protein signaling (RGS) proteins regulate G-protein-coupled receptors (GPCRs) and G-protein signaling events ,2. GPCRsffectors . Gα subuproteins  to act aproteins ,6.2+/CaM) and Ca2+/CaM-dependent kinase II (CaMKII) within the R1/2 RBD [While many RGS proteins are dedicated GAPs for target G proteins, some are more complex multifunctional proteins. One such complex RGS protein is RGS14, a ~62 kDa multi-domain scaffolding protein that is a member of the D/R12 subfamily of RGS proteins ,8. RGS14R1/2 RBD  and bindR1/2 RBD . Unlike R1/2 RBD ,20,21. TThe RGS domain is evolutionarily conserved among vertebrate and invertebrate species . RGS14 sWithin the rodent brain, the RGS14 protein is localized entirely to neurons and across cortical and limbic areas, with varying degrees of expression ,27, althRGS14 exists in peripheral tissues as well, where it plays an important role in physiology. RGS14 protein expression can be found in the ventricular tissue of healthy and hypertrophic mice . In humaAnother emerging area of RGS14 peripheral action is the kidney. Recent genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) within the RGS14 gene locus associated with kidney dysfunction ,34,35. SMuch less is known about RGS14’s role in the immune system. RGS14 RNA and protein are highly expressed in mouse lymphoid tissue and cells, including the thymus, spleen, lymph node, peritoneal cells, white blood cells, naïve B cells, and bone marrow ,45,46,47Recent studies reported RGS14 expression in mouse subcutaneous/inguinal white adipose tissue ,25. SurpWhile RGS14 clearly has roles outside of the brain, this review will focus on the physiological actions of RGS14 within the brain. The following discussions will summarize our current understanding of RGS14’s roles in postsynaptic signaling and plasticity at the cellular level, and RGS14’s roles in animal behavior.We have characterized the postnatal developmental expression of RGS14 in the mouse brain . RGS14 mAdult rodents and primates share pronounced RGS14 expression in the hippocampus, striatum, and amygdala ,27,56,57RGS14 is strongly expressed in specific subregions of the hippocampus, notably in pyramidal cell bodies, axons, and dendrites throughout the entire rostro-caudal range of hippocampal area CA2 and in the fasciola cinerea. ,27,57. ROutside of the hippocampus, RGS14 is highly expressed in the basal ganglia, striatum, and amygdala in studies of primate tissue (macaque and human) . The strIn addition to the striatum, several other basal ganglia structures exhibit RGS14 immunoreactivity, including both the internal and external segments of the globus pallidus and the substantia nigra pars reticulata . RGS14 lRGS14 has been studied most extensively in the hippocampus, which is necessary for many types of memory, including spatial, episodic, and social ,59. The Much of hippocampal research has focused on components in the classical tri-synaptic pathway (DG > CA3 > CA1), where excitatory granule cells in the dentate gyrus project onto the excitatory pyramidal cells of area CA3, which then project onto excitatory pyramidal cells of CA1 . ImportaArea CA2, similarly to its neighboring areas CA1 and CA3, contains a high density of glutamatergic pyramidal cells as well as several different types of GABAergic inhibitory neurons . CA2 alsUnlike neighboring CA1 and CA3, CA2 is unique in that pyramidal cells in this region do not support synaptic plasticity ,65,77. URGS14 has been identified as a key actor in area CA2 to suppress synaptic plasticity ,79. In h2+/CaM, and CaMKII  that are essential for spine remodeling during LTP . Actin s2+/CaM and CaMKII [RGS14 also engages calcium signaling pathways in dendritic spines. RGS14 inhibits calcium influx from both voltage-gated calcium channels (VGCCs)  and ionod CaMKII ,120,121,2+/CaM and CaMKII are essential mediators of synaptic plasticity. CaM, CaMKIIα, and CaMKIIβ were identified as novel binding partners in the mouse brain from the RGS14 interactome [2+/CaM and CaMKII to exert its actions on spine calcium.Caeractome . Cell-baeractome . Similareractome . CaM/CaMeractome . CaMKIIβeractome , and CaMeractome  and direeractome ,125. Theeractome , suggestRGS14 regulates synaptic plasticity measured as LTP and spine remodeling ,57,79. L2+/CaM and CaMKII and is phosphorylated by CaMKII [The active translocation of CaMKII to synapses is necessary and sufficient for E-LTP by phosphorylating a host of downstream targets ,125,128.y CaMKII . Phosphoy CaMKII , and they CaMKII ,131. Endy CaMKII ,132, they CaMKII . CaMKII y CaMKII . RGS14 dy CaMKII ,102,104.y CaMKII .E-LTP transitions to L-LTP, as signals from the dendritic spines are transduced to the nucleus, causing the activation of transcription factors. In turn, newly synthesized, plasticity-related RNAs and proteins are translocated to the activated synapse to maintain potentiation . RGS14 s2+/CaM/CaMKII signaling pathways, and the actomyosin system to regulate spine plasticity.By engaging key signaling proteins and pathways essential for spine signaling and plasticity, RGS14 is well positioned to block E-LTP at the spines where it is present . We haveRGS14 blocks LTP in both CA1 and CA2 after high-frequency stimulation during the early phase of LTP (between 0 and 30 min following stimulation). RGS14 inhibits both synaptic potentiation and structural plasticity during this time period, indicating that RGS14 prevents the expression of E-LTP. Genetic mutations in the GPR motif that prevent RGS14–Gαi binding perturb RGS14 plasma membrane localization, thereby blocking RGS14 suppression of LTP . TherefoRGS14 inhibits calcium transients from NMDAR and L-type VGCCs ,117. Sin2+/CaM/CaMKII complex from target proteins that are essential for E-LTP, such as AMPARs, NMDARs, or plasticity-related scaffold proteins [In addition to inhibiting calcium influx, RGS14 likely interferes with calcium-dependent signaling pathways. RGS14 could block spine signaling by serving as a local sink/buffer for key proteins/pathways. For example, RGS14 could bind to CaM after glutamate-induced calcium influx to prevent CaM from interacting with plasticity-inducing proteins . Indeed,proteins .Another possible mechanism by which RGS14 inhibits E-LTP would be its interactions with active H-Ras. Calcium influx and CaMKII can activate H-Ras and cause downstream ERK activation, which phosphorylates AMPAR to promote the insertion of AMPARs into the PSD ,150. OurThe induction of LTP is directly linked to memory formation , and memA recent report from our lab sheds light on this question . As discRGS14 has been observed in the nuclei of striatal neurons of monkey , though Of interest is the curious fact that induction of LTP markedly differs within distinct dendrites of the same CA2 neuron that express RGS14. In proximal dendrites, which receive CA3–CA2 synapses from the stratum radiatum (SR), LTP is absent, but can be elicited with pharmacological modulation. By contrast, distal dendrites of the stratum lacunosum-moleculare (SLM), which receive strong, excitatory input from entorhinal cortex layers II and III, express robust LTP . Here, wPharmacological stimulation and inhibition of several different GPCRs in CA2 can restore LTP in neurons containing RGS14. The activation of vasopressin 1b receptor (Avp1br), oxytocin receptor (Oxtr), and substance P receptor are sufficient to reverse RGS14 inhibition of LTP ,153,154.Regulation of RGS14 subcellular localization could be a key factor. Mutations altering RGS14 localization out of the spines prevent the inhibition of LTP, specifically those that prevent membrane localization and promote nuclear sequestration . It is pAdditionally, activation of Gαq-coupled oxytocin or vasopressin 1b receptors on CA2 pyramidal cells causes downstream PLC-dependent calcium influx ,153, whiLastly, while proximal dendrites of the SR are resistant to LTP, distal dendrites of the SLM readily undergo LTP after activation of layer II/III EC afferents to CA2 ,95,154. As discussed earlier, RGS14-KO mice exhibit enhanced spatial memory in the MWM task and enhanced novel object recognition , both ofWhy would unregulated CA2 LTP enhance hippocampal-dependent memory? In the MWM task, NMDAR-dependent CA3–CA1 LTP is required for learning , and opt414) has been investigated for its effects on rodent behavior linked to hippocampal memory [414, in layer six of the V2 visual cortex of rats, enhances object recognition memory and permits long-term memory of a novel object months after initial exposure [414 into area CA1 of rats enhanced neurite formation and branching in area CA1 pyramidal cells, which was dependent on increases in BDNF production [414 expression in CA1 also enhanced Y-maze and MWM performance in adult, aged, and AD-model rats.A truncated form of human RGS14  is not observed to be naturally expressed in rodents or at detectable levels in the hippocampus of primates [414 lacks an RGS domain and part of the adjacent linker region, suggesting that a loss of binding to plasticity-relevant G proteins could effectively reverse this process. However, this is unlikely since an RGS-null point mutant of RGS14 suppresses LTP [414 encodes the human sequence containing a PDZ-binding ligand (DSAL), which is absent in the RGS14 sequences of rodents [414 reflect the actions of a protein not normally found in hippocampal neurons, they are useful for revealing insights into the potential mechanisms of RGS14 actions by perturbing molecular systems in those neurons.These findings with an overexpressed truncated form of RGS14 conflict with previous reports showing that a loss of endogenous RGS14 enhances hippocampal-dependent spatial memory in mice. In related findings, expression of full-length rat RGS14 and an RGS-null mutant in area CA1 of mice restricts LTP ,79, whicprimates ,27. Furtsses LTP . RGS414  rodents . TherefoRecent reports indicate that RGS14 is also expressed in specific brain regions outside of the hippocampus ,26,56, wRGS14 is expressed in the amygdala of rodents and primates ,26,56. TRGS14 is expressed in the ventral striatum, a brain structure that sits at the limbic–motor interface where these neurons command motivation, reward processing, and goal-directed behavior . It is aRGS14 is also expressed in striatal projection neurons comprising both the direct and indirect pathways of the basal ganglia, where it demonstrates postsynaptic and nuclear localization in the striatum and presynaptic localization in the globus pallidus and substantia nigra pars reticulata . The basRGS14 inhibits hippocampal-based learning and memory by suppressing LTP in area CA2 by engaging and integrating G protein, H-Ras/ERK and calcium signaling pathways ,79. VariRGS14 is found most prominently in the brain regions comprising the limbic system and the basal ganglia. When considered hierarchically, the expression pattern of RGS14 suggests an overarching role in modulating the degree to which the emotional significance of a stimulus permits the shaping of behavior. For example, a car crash victim may experience panic attacks when driving by the crash site, and thus may deliberately choose driving routes that avoid that location. This has clear implications for the treatment of psychiatric disorders characterized by high emotional reactivity, including post-traumatic stress disorder and anxiety disorders. Multiple intervention points exist in the course of substance use disorder/addiction, in which RGS14 manipulation could be beneficial. The period of withdrawal following cocaine exposure is characterized by LTP induction of specific afferents onto ventral striatal projection neurons, and is thought to contribute to the escalation of drug use ,188,189.Large-scale genome and exome sequencing data have generated new information regarding the link between rare human variants, and their impact on disease predisposition and individual traits . RemarkaAs mentioned above, human RGS14 is also expressed outside the brain, in various tissues including kidney, and differs from the rodent protein by expressing a 22 amino acid tail containing a PDZ-binding ligand (DSAL). Many GWAS report a linkage of the RGS14 gene and chronic kidney disease ,39,41,42Since RGS14 was first identified over 20 years ago as a Rap2-binding protein , much haQuite unusual for a large scaffolding protein that regulates cell surface events, RGS14 is a nucleocytoplasmic shuttling protein. Numerous studies show that RGS14 dynamically shuttles from the cytosol to the nucleus and back ,21,98, a2+/CaMKII, PKC and H-Ras/ERK signaling [Additionally, it is unknown how RGS14 is regulated to engage or disengage in plasticity-related events. RGS14 is post-translationally modified via cAMP–PKA, Caignaling ,157,158.ignaling . Furtherignaling , phosphoAs discussed earlier, human/primate RGS14 contains a C-terminal PDZ ligand that is lacking in the rodent protein . NHERF1 Finally, what functions does RGS14 serve in brain outside of the hippocampus? Recent studies show that RGS14 is also expressed in the dorsal striatum, nucleus accumbens, and the central amygdala ,26. As d


This study aimed to explore and identify key genes and signaling pathways that contribute to the progression of cervical cancer to improve prognosis.Three gene expression profiles  were screened and downloaded from the Gene Expression Omnibus database (GEO). Differentially expressed genes (DEGs) were screened using the GEO2R and Venn diagram tools. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Gene set enrichment analysis (GSEA) was performed to analyze the three gene expression profiles. Moreover, a protein–protein interaction (PPI) network of the DEGs was constructed, and functional enrichment analysis was performed. On this basis, hub genes from critical PPI subnetworks were explored with Cytoscape software. The expression of these genes in tumors was verified, and survival analysis of potential prognostic genes from critical subnetworks was conducted. Functional annotation, multiple gene comparison and dimensionality reduction in candidate genes indicated the clinical significance of potential targets.A total of 476 DEGs were screened: 253 upregulated genes and 223 downregulated genes. DEGs were enriched in 22 biological processes, 16 cellular components and 9 molecular functions in precancerous lesions and cervical cancer. DEGs were mainly enriched in 10 KEGG pathways. Through intersection analysis and data mining, 3 key KEGG pathways and related core genes were revealed by GSEA. Moreover, a PPI network of 476 DEGs was constructed, hub genes from 12 critical subnetworks were explored, and a total of 14 potential molecular targets were obtained.These findings promote the understanding of the molecular mechanism of and clinically related molecular targets for cervical cancer.The online version contains supplementary material available at 10.1186/s12885-021-08412-4. Human papillomavirus (HPV) infection is a primary cause of cervical cancer and led to 311,365 deaths in 2018 [Homo sapiens” [porgn] AND (“Expression profiling by array” [Filter] AND (“0001/01/01” [PDAT]: “2020/11/27” [PDAT])). Three expression profile microarray datasets  were selected and downloaded from the GEO database for analysis.CIN is an important process in the development of cervical cancer. The gene expression profiles related to CIN progression were retrieved and downloaded from the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI). The retrieval formula was as follows:  AND “GSE63514 [P-value (adj. P) < 0.05. Then, the Venn diagram tool (http://bioinformatics.psb.ugent.be/webtools/Venn/) was used to compare and analyze the results of the intersection analysis. Based on the intersection of datasets, the final DEGs were obtained. In this study, we selected DEGs according to the intersection of at least two expression profile datasets to avoid the disadvantages of a single dataset and then integrated the results for further biological function analysis.According to the progression of cervical cancer, in the subsequent analysis, samples of each dataset were divided into three groups: N-CIN, CIN-CC, and N-CC. The GEO2R tool was used to analyze the three expression datasets , 7. Normhttps://david.ncifcrf.gov/). Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway [P < 0.05 was set as the cutoff criterion for significant enrichment.DAVID Bioinformatics Resources 6.8 was utilized to distinguish and enrich the biological attributes, such as biological processes, cellular components, molecular functions and pathways, of important DEGs [ pathway  and GeneP < 0.01 were chosen for further analysis. The results of the GSEA of different expression profile datasets were intersected to obtain the common significant KEGG pathways, and core gene sets were analyzed.Gene set enrichment analysis (GSEA) was used to determine the key pathways and core genes during the development of cervical cancer , 11. Enrhttps://string-db.org/). Molecular interactions and PPI networks can promote the exploration of molecular targets, signaling and metabolic pathways, and network functions involved in the progression of cervical lesions. Therefore, the STRING database was used to construct the PPI network.The STRING database is an online search tool used to analyze known proteins and predict PPI networks, including direct and indirect interactions between proteins and their functional correlations   was used to discover the key targets or subnetworks of complex networks –15. The http://gepia2.cancer-pku.cn/) was used to analyze the expression of multiple hub genes and the prognostic value of the selected hub genes in CESC [As cervical cancer is a complex disease, its etiopathogenesis involves compound gene expression and multiple interactions. GEPIA2 : 253 genes with upregulated expression and 223 genes with downregulated expression.Volcano plots Fig.  showed tCDKN2A , 18 was nd CENPF , were shA total of 476 DEGs were uploaded to DAVID for GO/KEGG analyses. The terms of each GO category are provided in Additional file p < 0.01), namely, DNA mismatch repair , the cell cycle , DNA replication , cysteine and methionine metabolism (LDHC), and nucleoside exception repair . In addition, there were 6 common KEGG pathways in the CIN-CC group (p < 0.01), namely, the adipocytokine signaling pathway, small cell lung cancer , namely, the cell cycle , nucleotide excision repair, the Toll-like receptor signaling pathway, prion diseases, spliceosome, DNA replication, proteasome, colorectal cancer, and pancreatic cancer. Moreover, GSEA showed that DNA mismatch repair (N-CIN), small cell lung cancer (CIN-CC), and the cell cycle (N-CC) were the most significantly enriched pathways , and snapshots of the enrichment analysis are shown in Figs. Expression datasets  were subjected to GSEA (version 4.1.0). Then, key pathways and core related genes were obtained. The results showed 5 common KEGG pathways in the N-CIN group , pathwand LAMC2 . There wM5, CDC6 , GSK3B, 1, CDC20 , PTTG1, 3, CCNB1 , RBL1, CL1, CDC7 , WEE1, CE1, CDK2 , CCNA2, P < 1.0E-16). The functional enrichment analysis in the PPI network included 387 GO terms, 5 KEGG pathways, 64 Reactome pathways, and 111 protein domains. Significantly enriched functions of the network are shown in Fig. A total of 476 DEGs were uploaded to the STRING database to construct the PPI network . The PPI network is shown in Fig. NUSAP1, TOP2A, KIF2C, NDC80, ASPM, KIF20A, CDK1, KIF11, BIRC5, MCM2, and CHEK1. Then, the MCODE plug-in was also used to analyze the PPI network, and 12 regions (subnetworks) closely related to the PPI network were found and separated. These regions might represent the molecular complex, and the results from the MCODE analysis included these high scoring hub genes and supported the results of the regional analysis  and enrichment analysis were performed on 14 target genes  were downloaded from the GEO database. According to the development of cervical cancer, the samples were divided into three groups: N-CIN, CIN-CC and N-CC. Intersection analysis of these groups made the screening and validation of DEGs more reliable. A total of 476 DEGs were screened: 253 upregulated genes and 223 downregulated genes. These genes also regulate a number of biological pathways in the progression of cervical lesions, such as the cell cycle, DNA replication, rheumatoid arthritis, the p53 signaling pathway, bladder cancer, complement and coagulation cascades, amoebiasis, cytokine-cytokine receptor interaction, oocyte meiosis, and arachidonic acid metabolism. These pathways need to be further studied.Furthermore, GSEA suggested that the DNA mismatch repair pathway plays an important role in the N-CIN process. Most importantly, the small cell lung cancer pathway might play an important role in the CIN-CC process. The cell cycle pathway might play an important role in the N-CC process. These results provide a better understanding of the molecular pathways associated with the development of the disease from normal cervical epithelium to CIN to cervical cancer. In the N-CIN stage, the enriched molecular pathways were DNA replication, nucleoside exception repair, DNA mismatch repair and specific amino acid metabolism (cysteine and methionine). With the development of cervical epithelial neoplasia, the enriched molecular pathways in the CIN-CC stage were small cell lung cancer, the adipocytokine signaling pathway, pathways in cancer, the Toll-like receptor signaling pathway, graft versus host disease and the TGF-beta signaling pathway, which play important roles in the occurrence and development of cancer. In the whole process of cervical lesions, we may need to pay close attention to pathways such as the cell cycle, DNA replication, DNA repair and pathways in cancer. Our results indicate the importance of these pathways in the occurrence and development of cervical cancer. We need to pay attention to the roles of these pathways in cervical carcinogenesis and further study their interactions.NUSAP1 [, TOP2A, KIF2C [, NDC80 [, ASPM [, KIF20A [, CDK1 [, KIF11 [, BIRC5 [, MCM2 and CHEK1 [P < 0.01). By analyzing the prognostic values of the hub genes from the 12 subnetworks, a total of 14 potential molecular targets  were obtained and annotated.Notably, the PPI network related to cervical lesions was composed of functional proteins that interacted with each other to participate in biological signal transmission, gene expression regulation, energy and material metabolism, cell cycle regulation and cell division. Eleven genes were identified as hub genes from 12 critical subnetworks of cervical cancer. Critical subnetworks might strongly contribute to the occurrence and development of cervical cancer and have high diagnostic value. Further analysis showed that NUSAP1 , TOP2A, A, KIF2C , NDC80 [[, NDC80 , ASPM [2 [, ASPM , 38, KIF, KIF20A , CDK1 [1 [, CDK1 , 38, KIF[, KIF11 , 41, BIR[, BIRC5 , MCM2 annd CHEK1 , 41, 43 ts . Table S1. GO enrichment analysis of DEGs in Biological Processes (DAVID). Table S2. GO enrichment analysis of DEGs in Cellular Components (DAVID). Table S3. GO enrichment analysis of DEGs in Molecular Functions (DAVID).