Bowen's disease is a cutaneous squamous cell carcinoma (CSCC) in situ. If left untreated, BD may progress to invasive CSCC. CSCC is one of the most common cutaneous carcinoma in the elderly and the advanced, metastasis CSCC usually have a poor outcomes. However, the mechanisms of invasion and metastasis from Bowen’s disease to CSCC is complicated and still unclear.The aim of this study was to explore the biomarkers and molecular alterations in Bowen’s disease development process via analyzing the proteomics changes in tissues of CSCC, Bowen disease and healthy skin.A total of 7 individuals with CSCC , 7 individuals with Bowen disease  and 7 healthy controls  presented to the Department of Dermatology, Yijishan Hospital, the First Affiliated Hospital of Wannan Medical College between January 2021 and December 2021 were enrolled. The proteomics analysis was performed to screen differentially expressed proteins/gens (DEPs/DEGs) in the lesions of CSCC, Bowen disease and healthy skin tissues. The transcriptomic data (GSE32628) of CSCC was selected and downloaded from the GEO database. The common DEGs in our proteomics results and GSE32628 between CSCC and healthy skin tissues were selected. And then, the common DEGs which significantly up or down-regulated between CSCC and Bowen disease in our proteomics results were further screened to identify using Western blot methods in the validation group. CSCC A431 cells were transfected with SERPINB1 small interfering RNA (si-SERPINB1) or small interfering RNA negative control (si-NC). To explore the effect of SERPINB1 silencing on migration and invasion ability of A431 cells.P value < 0.05. These DEPs involved multiple biological functions such as protein binding process, immune, inflammation, ribosome, protein digestion and absorption, ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway and others. A total of 20 common DEGs  between CSCC and healthy skin tissues in GSE32628 and our proteomics results were found. Besides, the proteins of TNC, FSCN1, SERPINB1, ACTN1 and RAB31 in CSCC were significantly up-regulated, while COL3A1, COL1A1 and CD36 were significantly down-regulated relative to Bowen disease in proteomics results. These proteins were mainly involved in multiple pathways, including Focal adhesion, ECM-receptor interaction, Human papillomavirus infection, PI3K-Akt signaling pathway, PPAR signaling pathway, AMPK signaling pathway and others. These eight proteins were selected for further validation. According to the Western blotting analysis, when compared with the Bowen disease and healthy skin tissues, we found that the relative expression levels of TNC, FSCN1, SERPINB1, ACTN1 and RAB31 in the CSCC were significantly increased, while COL1A1 and CD36 were significantly decreased, and the differences were statistically significant (P < 0.05). Furthermore, the relative expression levels of TNC, FSCN1, SERPINB1 in the Bowen disease were also significantly increased, while the COL3A1 were also significantly decreased relative to the healthy control. SERPINB1 siRNA inhibited the expression of SERPINB1 at mRNA and protein levels in the A431 cells. After interfering with the expression of SERPINB1, the migration and invasion ability in the A431 cells were significantly decreased (P < 0.05).A total of 501 proteins were differentially expressed between the CSCC and healthy skin tissues, with 332 up-regulated and 169 down-regulated at least 1.5-fold with a This study highlights that eight proteins, TNC, FSCN1, SERPINB1, ACTN1, RAB31, COL3A1, COL1A1, CD36, were significantly associated with the mechanisms of invasion and metastasis in Bowen’s disease.The online version contains supplementary material available at 10.1186/s12967-022-03622-1. Cutaneous squamous cell carcinoma (CSCC) is one of the most common cutaneous carcinoma in the elderly. With the extension of life expectancy in human, the prevalence of CSCC keeps rasing, with an annual global incidence more than 1 million . PhotodyThe proteomics technology has been successfully used to identify DEPs/DEGs in tumor tissues and to provide insights into the molecular mechanisms underlying and/or potential therapy targets for these complicated diseases . The proA total of 5 inpatients with CSCC (n = 5) and 5 inpatients with Bowen disease (n = 5) between January 2021 and December 2021 were selected in this study according to the postoperative histopathology results. On the other hand, 5 inpatients with pigmented naevi were defined with healthy controls. The CSCC tissues, Bowen disease tissues and healthy skin tissues (more than 2 cm apart from pigmented naevi) were obtained after surgical resection.The 15 enrolled individuals comprised 8 men and 7 women with a male: female ratio of 8:7. The age ranged from 52 to 87 years with a mean age of (67.13 ± 9.20) years. The main lesion site was nose  followed by cheek . There was no significant differences in age, gender and tissues location between the three groups. The clinical characteristics of all individuals are shown in Table g for 5 min at 4 °C, the supernatant were discarded. We collected the sediment and washed with pre-cooled acetone for three times. The protein sample was then diluted by adding 200 mM TEAB and trypsin was added at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight. The dithiothreitol (DTT) was added in each protein sample with a final concentration of 5 mM and stand still at 56 °C for 30 min. Then, iodoacetamide (IAA) was added to make the final concentration at 11 mM, and incubated at room temperature for 15 min in the dark. LC–MS/MS analysis: The tryptic peptides were dissolved in solvent A (0.1% formic acid and 2% acetonitrile) and separated using an NanoElute UHPLC system. The parameters were set as 6–24% solvent B (0.1% formic acid and 90% acetonitrile) for 70 min, 24–32% for 14 min, 32–80% for 3 min and 80% for 3 min at 450 nL/min flow rate. The peptides were separated by UHPLC system and injected into Capillary ion source for ionization and then analyzed by timsTOFTM Pro mass spectrometer.Protein Extraction: Microsections of CSCC (n = 5), Bowen disease (n = 5) and healthy controls (n = 5) from the Department of Pathology in our hospital were collected. Proteins were extracted from the microdissected tissue samples and dewaxed using the method which described previously . TrypsinP < 0.05 threshold. Functional enrichment (GO and KEGG) were performed to identify the relationships among the target proteins associated with CSCC. The Universal Protein Resource  is a well-known protein database, which consists of the UniProt knowledgebase (UniProtKB), the UniProt unique protein identifier archive (UniParc), and the UniProt reference sequence clusters (UniRef). Apart from protein sequence data, the UniProtKB has comprehensive annotations and is the core of the database [P-value < 0.05 was considered significant. On the other hand, the protein–protein interaction (PPI) network among DEPs was analyzed via STRING online software [Bioinformatics analysis mainly covers three domains: Gene Ontology (GO), Kyoto Encyclopedia Genes and Genomes (KEGG) and protein–protein interaction (PPI). The DEPs between CSCC, Bowen disease and healthy control were identified using a 1.5-fold change and the Wilcoxon test database . Besidessoftware . The Cythttps://www.ncbi.nlm.nih.gov/pmc/). In the GSE32628, a total of 13 samples with CSCC and paired 13 samples with normal tissues from 13 inpatients were selected and enrolled. RNA was isolated and gene expression profiles were obtained using HumanWG-6 v2 Expression BeadChips (Illumina). The transcriptomic data were calculated using GEO2R online (https://www.ncbi.nlm.nih.gov/geo/geo2r/). The differentially expressed transcriptomic data between CSCC and normal tissues were further identified using a log2FC > 2/ < -2 and adjusted P values < 0.01. Besides, in our proteomics study, we also identified DEPs between CSCC and healthy skin tissues using a 1.5-fold change and P values < 0.05. Next, a Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) containing two lists of differentially expressed gens was drawn online to identify common gens that were significantly up- or down-regulated in CSCC. And then, the common gens which significantly up- or down-regulated between CSCC and Bowen disease were further filtered to verification using Western blot and Immunohistochemistry methods.The transcriptomic data of CSCC (GSE32628) was selected and downloaded from the GEO database . Equal amounts of protein lysates (20 mg) were loaded into 4–15% precast SDSPAGE gels  and then transferred to polyvinylidene fluoride membranes by the semi-dry transfer method. To block nonspecific protein binding, the membranes were incubated with 3% bovine serum albumin for 2 h. The membranes were washed with TBST for 3 min and then incubated with the corresponding primary antibodies overnight at 4 ℃. The membranes were washed with TBST for 3 min again and incubated with appropriate horseradish peroxidaseconjugated secondary antibodies for 2 h. They were washed again with TBST and visualized using enhanced chemiluminescence substrate kits  in a chemiluminescence imaging system . The band intensity was quantified by Quantity One software . The Western blot examination were performed using primary antibodies to TNC , FSCN1 , SERPINB1 , ACTN1 , RAB31 , COL3A1 , COL1A1  and CD36 . The experimental flow chart are shown in Fig. The Western blot was used to verify the target proteins  expression levels in CSCC tissues relative to Bowen disease, healthy skin tissues. Besides, 2 inpatients with CSCC, 2 inpatients with Bowen disease and 2 inpatients with pigmented naevi were enrolled and defined as independent verification samples. The clinical characteristics of these 6 individuals are shown in Additional file Cell culture: Two human CSCC cell lines (A431 and SCL-1) and a human immortalized keratinocytes cell line Hacat  were obtained from American Tissue Culture Collection . The cells were cultured in Dulbecco modified Eagle medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) and incubated at 37℃ in 5% carbon dioxide.Real-time fluorescence-based quantitative PCR (RT-qPCR) and Western blot analysis were performed to detect the mRNA and protein of A431 and SCL-1 cells respectively, and then A431 cells with highly expressed SERPINB1 served as a research object . The statistical significance was defined as P < 0.05 and the experiments were carried out at least three times.Data are presented as the mean ± standard or frequency and were analyzed by student’s t-test, chi-square test using the SPSS software 20.0 . The differentially expressed microarray between two groups in GSE32628 were further identified using a logIn our study, a total of 501 proteins were differentially expressed between the CSCC and healthy control, with 332 up-regulated and 169 down-regulated. In addition, a total of 252 proteins were up-regulated and 104 proteins were down-regulated in the CSCC relative to the Bowen disease. On the other hand, a total of 246 proteins were up-regulated and 154 proteins were down-regulated in the Bowen disease relative to the healthy control. The total spectra, matched spectra, peptides, unique peptides, and identified and quantified proteins obtained results are shown in Fig. P = 2.17*10–6), actin binding (P = 3.46*10–6) and platelet-derived growth factor binding (P = 1.13*10–5), which were related to protein binding process. The most enriched CC terms, such as extracellular region (P = 1.26*10–13), extracellular matrix (P = 2.47*10–11) and endoplasmic reticulum lumen (P = 2.99*10–9), mainly associated with the protein activation locations. As for BB terms, these DEPs were mainly enriched in nucleobase-containing compound catabolic process (P = 2.88*10–7), cytokine-mediated signaling pathway (P = 9.95*10–7), granulocyte activation (P = 1.14*10–6), neutrophil mediated immunity (P = 1.69*10–6), neutrophil activation (P = 2.33*10–6) and cellular response to cytokine stimulus (P = 3.09*10–6), which were related to immune, inflammation. On the other hand, when compared with Bowen disease, 356 DEPs in CSCC and were also categorized in MF, CC and BP terms. The 8 significantly enriched MF terms, 8 significantly enriched CC terms, and 14 significantly enriched BP terms are shown in Fig. A total of 501 DEPs between CSCC and healthy control were found. GO annotation was made to categorize these DEPs based on 3 biological function types termed including molecular function (MF), cellular component (CC), and biological process (BP). The top 8 significantly enriched MF terms, 8 significantly enriched CC terms, and 14 significantly enriched BP terms are shown in Fig. P = 4.39*10–9); hsa04974, Protein digestion and absorption (P = 7.64*10–6); hsa05171, Coronavirus disease-COVID-19 (P = 1.24*10–5); hsa04512, ECM-receptor interaction (P = 1.43*10–5); hsa04141, Protein processing in endoplasmic reticulum (P = 6.18*10–5); hsa00030, Pentose phosphate pathway (P < 0.001); hsa04510, Focal adhesion (P < 0.001); hsa04151, PI3K-Akt signaling pathway (P = 0.001) and others. In addition, 16 pathways were generated based on the DEPs between the CSCC and Bowen disease. The significantly enriched 8 KEGG pathways as follows: hsa04141, Protein processing in endoplasmic reticulum (P = 3.28*10–7); hsa05130, Pathogenic Escherichia coli infection (P < 0.001); hsa05146, Amoebiasis (P = 0.004); hsa04810, Regulation of actin cytoskeleton (P = 0.004); hsa00500, Starch and sucrose metabolism (P = 0.007); hsa04530, Tight junction (P = 0.008); hsa04144, Endocytosis (P = 0.009) and hsa04666, Fc gamma R-mediated phagocytosis (P = 0.010). The KEGG pathway analysis results are shown in Fig. A total of 22 pathways, 16 pathways and 20 pathways were generated from the KEGG pathway analysis based on the proteins differentially expressed between the CSCC and healthy control, CSCC and Bowen disease, the Bowen disease and healthy control, respectively. When compare with healthy control, these DEPs in CSCC were most enriched in hsa03010, Ribosome  including 499 proteins were detected and showed in Fig. P values < 0.01 and log2FC > 2 and (or) < -2 were set as cut-off criteria for screening out the differentially expressed gens in CSCC relative to normal skin tissues.When compared with normal skin tissues, a total of 115 differentially expressed gens  in CSCC were found. On the other hand, in our proteomics study, a total of 501 proteins were quantified, of which 332 were down-regulated and 169 were up-regulated in the CSCC relative to the healthy skin tissues. Finally, a Venn diagram was generated . Furthermore, the relative expression levels of TNC, FSCN1, SERPINB1 in the Bowen disease were also significantly increased, while the COL3A1 were also significantly decreased relative to the healthy control. The results are shown in Fig. The Western blotting was carried out to verify the relative proteins expression levels of TNC, FSCN1, SERPINB1, ACTN1, RAB31, COL3A1, COL1A1 and CD36 in the independent verification samples . According to the Western blotting analysis, when compared with the Bowen disease and healthy skin tissues, we found that the relative expression levels of TNC, FSCN1, SERPINB1, ACTN1 and RAB31 in the CSCC were significantly increased, while COL1A1 and CD36 were significantly decreased, and the differences were statistically significant (P < 0.05). The results suggested that SERPINB1 knockdown could significantly inhibits CSCC cell migration and invasion.We first confirmed the efficiency of SERPINB1 knockdown, the expression of SERPINB1 mRNA and protein in CSCC cell line A431 were evaluated by qRT-PCR and western blotting, respectively. The SERPINB1 mRNA and protein expression levels in the cells transfected with si-SERPINB1 were decreased relative to the si-NC cells  from the results of Figs. CSCC, a keratinocyte-derived skin neoplasm with malignant potential, represents 20–50% of skin cancers and currently has an increasing incidence in the world . The out+ T cells, macrophage, neutrophil, and dendritic cell). Similar to COL1A1, higher expression of COL3A1 was associated with shorter overall survival in patients with ovarian carcinomas [The extensive family of COL gene products (collagens) is composed of several chain types, including fibril-forming interstitial collagens  and basement membrane collagens (type IV), each type containing multiple isoforms. COL3A1 as well as COL1A1 play a role in cell adhesion process, important for maintaining normal tissue architecture and function . We founrcinomas . CD36 isrcinomas . In the rcinomas , the CD3rcinomas . The tenrcinomas . Higher rcinomas . Besidesrcinomas . In addircinomas  proved Trcinomas .FSCN1, an actin-bindling protein, identifies dendritic cells in the blood and in tissues. ACTN1, one of the alpha actinins, which belong to cytoskeletal proteins and perform important biological functions, such as cell adhesion and migration. Our proteomics results showed expression of FSCN1 and ACTN1 were significant higher in CSCC relative to Bowen disease, and FSCN1 as well as ACTN1 may related to the proliferation, invasion and migration in CSCC progression process. In the previous reported, the higher-regulation of FSCN1 indicates worse prognosis for patients with renal cell carcinoma  and adreP < 0.01) was found by Tseng MY. et al. [RAB31 belongs to the Rab family, mainly localized in the trans-Golgi network and endosomes, which is the largest Ras superfamily of small molecule GTP binding proteins. Accumulating reports has indicated that RAB31 is widely involved in the prognosis of various cancers, including pancreatic cancer , cervica. et al.  NevertheIn conclusion, the proteomics technology provides a new method for the identification of key proteins associated with migration and invasion mechanisms in CSCC. These proteins were mainly involved in multiple pathways, including Focal adhesion, ECM-receptor interaction, Human papillomavirus infection, PI3K-Akt signaling pathway, PPAR signaling pathway, AMPK signaling pathway. Multiple protein factors, such as TNC, FSCN1, SERPINB1, ACTN1, RAB31, COL3A1, COL1A1 and CD36 could be as novel therapeutic target to manage CSCC. The most innovation of this manuscript was to explore the markers of CSCC progression using transcriptomics combined with proteomics methods in Chinese Han population. This also provides a theoretical basis for the targeted therapy of CSCC in the future.The numbers of samples in proteomics are insufficient and a larger cohort could be of benefit however sourcing human samples is a challenge.Additional file 1: Table S1. The clinical characteristics of 6 enrolled individuals for independent verification.Additional file 2: Table S2. The significantly enriched 8 KEGG pathways based on the differentially expressed proteins.Additional file 3: Table S3. The TNC, FSCN1, SERPINB1, ACTN1, RAB31, COL3A1, COL1A1, and CD36 mRNA values in CSCC relative to healthy control in GSE32628.Additional file 4: Figure. S1. a. A total of 246 proteins were up-regulated and 154 proteins were down-regulated in the Bowen disease relative to the healthy control. These differentially expressed proteins were categorized in 3 biological function types terms . The top 8 significantly enriched molecular function (MF) terms, 8 significantly enriched cellular component (CC) terms and 14 significantly enriched biological process (BP) terms are presented. The y-axis denotes the categories of GO terms.Additional file 5: Figure S2. KEGG pathways were generated based on the differentially expressed proteins between Bowen disease and healthy control.Additional file 6: Figure S3. The SERPINB1 protein (a) and mRNA (b) expresison levels in CSCC cell line A431, SCL-1 and a human immortalized keratinocytes cell line Hacat were determined by Western blotting (a) and qRT–PCR (b).Additional file 7. The raw data of 8 proteins in Western blot.Additional file 8. The protocols for biological function experiments.Additional file 9. The raw data of protomics of CSCC, Bowen disease and healthy control tissues.


High rates of new cervical cancer cases and deaths occur in low- and middle-income countries yearly, and one reason was found related to limitation of regular cervical cancer screening in local and low-resource settings. HPV has over 150 types, yet certain 14–20 high-risk and 13–14 low-risk types are common, and, thus, most conventional HPV nucleic acid assays, for examples, Cobas 4800 HPV test  and REBA HPV-ID  were developed to cover these types. We thereby utilized bioinformatics combined with recent isothermal amplification technique at 35–42 °C to firstly describe multiplex recombinase polymerase amplification assay that is specific to these common 20 high-risk and 14 low-risk types, and also L1 and E6/E7 genes that target different stages of cervical cancer development. Multiplex primer concentrations and reaction incubation conditions were optimized to allow simultaneous two gene detections at limit of detection of 1000 copies  for L1 and 100 copies (0.0125 fg) for E6/E7, respectively. The assay was validated against urogenital and other pathogens, normal flora, and human control. In 130 real clinical sample tests, the assay demonstrated 100% specificity, 78% diagnostic accuracy, and 75% sensitivity compared with REBA HPV-ID test, and is much more rapid (15–40 min), less expensive (~ 3–4 USD/reaction) and does not require instrumentation . Hence, the developed novel assay provides alternative screening tool for potential local screening. Furthermore, as this assay uses safe chemical reagents, it is safe for users. The reasons were found primarily related to a smaller rate of access to cervical cancer vaccination as a preventive venue and to regular cervical cancer screening programs  compared with high-income countries2. In Thailand and other Southeast Asia countries, cervical cancer represents the most common cancer among women. The Institute of Oncology/International Agency for Research on Cancer reported that ~ 9158 women were diagnosed with cervical cancer and 4705 died every year, and estimated approximately 3.4% of the Thai women population carried human papillomavirus (HPV) high-risk types 16 or 183. In addition, the cost of radiation therapy for cervical cancer and treatment of side effects, such as gastrointestinal or genitourinary side effects, is as high as ~ 63,103 USD per patient. The 2018 economic burden for merely radiation therapy in Thailand was ~ 131 million USD, and for the diagnosis of these advanced cervical cancer patients was ~ 129 million USD4. Subsequently, an effective, simple, easy-access, and low-price screening method for early prevention is important to minimize the number of cases and the economic burden of advanced cervical cancer patients.Cervical cancer is a major public health problem in developed and developing countries. According to the World Health Organization (WHO), an estimated 604,000 new cases and 342,000 deaths of cervical cancer occurred worldwide in 2020, and 90% of these cases were in low- and middle-income countries7. HPV has over 150 types and is classified into high-risk (HR) or low-risk (LR) for cervical cancer development. Common HPV HR types include 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 58, 59, 68, and 82, given types 16 and 18 generally account for 70% of cervical cancer. Common HPV LR types include 6, 11, 32, 40, 42, 43, 44, 54, 61, 70, 72, 81, 84, and 879.More than 95% of cervical cancer are caused by HPV. HPV is also associated with more than 60–90% of anal cancer, vaginal cancer, oropharyngeal cancer, and penile cancer11. For instance, Spence et al.12 reported a false negative rate of Pap smear to be 35.5% on average with the sensitivity ranging from 30 to 87%11. Furthermore, the Pap smear may require another HPV-specific nucleic acid test, such as Cobas 4800 HPV test  and REBA HPV-ID , as accompaniment and identification of HR types. Therefore, the HPV-specific tests were suggested as an alternative screening tool and some even reported that these tests could allow screening intervals to be extended to ≥ 5 years15. The Cobas 4800 HPV test  utilizes amplification of target DNA by polymerase chain reaction (PCR) and nucleic acid hybridization for detection of HPV HR types 16, 18, and a pool of 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 6815. REBA HPV-ID  is a PCR-based reverse blot hybridization assay for 19 HPV HR probes  and 13 LR probes  for HR and LR genotyping16. Although the Cobas and REBA assays are available commercially and used widely in in vitro clinical laboratories, they require an expensive thermal cycler for PCR amplification of HPV DNA and a special instrument for product-probe detection16.Traditionally, a cytological examination by Papanicolaou test (Pap smear) is simple and established. However, the procedure is inconvenient, cannot be performed in local resource- and clinician-limited settings, and its sensitivity  and specificity to detect precancerous or cancerous changes in cells are relatively low compared with nucleic acid amplification test17. For HPV detection, Ma et al.18 developed RPA for HPV HR types 16 and 18, and Gong et al.19 developed RPA for 13 HPV HR types based on the L1 gene. We, thereby, further developed a multiplex recombinase polymerase amplification (mRPA) assay with attempt to cover broad HPV HR and LR types, as well as normal and precancerous stages of HPV developmental cycle in humans, as potential local use in a single tube for broad and precancerous screenings of cervical cancer. We determined the limit of detection, validated proper specificity on positive controls comprising some HR and LR types and negative controls comprising urogenital bacteria, other viruses, human and mouse, and evaluated the assay efficiency  on real 130 clinical cervical swab samples compared with the Cobas and REBA results. In HPV developmental cycle, a late gene L1 encodes for a major viral capsid protein, while early genes E6 and E7 encode for oncoproteins and their detection suggests an increased risk of cancer development . E6 degrades a human growth suppressor protein p53 and E7 degrades a human retinoblastoma tumor suppressor protein pRb, and, thus, the E6 and E7 induce cancer development by evasion of human growth suppressors, resisting cell death, sustaining proliferative signaling, enabling replicative immortality, angiogenesis induction, and activation of invasion and metastasis20. Consequently, this study designed multiplex degenerate primers based on HPV L1 and E6/E7 genes, covering up to 20 HR and 14 LR types, and the E6/E7 amplification product could suggest an increased risk of cancer.Recently, an isothermal amplification assay like recombinase polymerase amplification is emerged to solve this problem. The recombinase polymerase amplification utilizes 3 enzymes, a recombinase, a single-stranded DNA-binding protein, and strand-displacing polymerase, to allow isothermal amplification of specific primer-binding target(s) at 37–42 °C. This allows the reaction to be simply performed by a heat block or water bath, or even holding a tube in hands, supporting low cost, rapid and point-of-care molecular testswww.megasoftware.net/), and RPA degenerate primers were designed using Primer-BLAST21 or PrimedRPA22 and manual design (Table All GenBank available HPV type L1 (and E6/E7) sequences were aligned using ClustalW , urogenital normal flora (Staphylococcus aureus and Escherichia coli), skin normal flora (Staphylococcus epidermidis), viruses, and mammals ; and the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University , and the Institutional Biosafety Committee (IBC) of the Faculty of Medicine, Chulalongkorn University (MDCU-IBC016/2019), respectively. In brief, cervical swab samples were collected by genecologists from patients who visited the Buddhachinaraj Phitsanulok Hospital during 2013–2014 and the Department of Gynecological Out Patient, King Chulalongkorn Memorial Hospital, Bangkok, during 2019–2020. The samples were lysed in Pathogen Lysis Tubes L  using Tissue Lyser LT (Qiagen), and total nucleic acids were extracted automatically using the Cobas 4800 system (Roche Diagnostics). Then, these genomic DNA were HPV genotyped following the Cobas 4800 HPV test (Roche Diagnostics) and the REBA HPV-ID test (Molecules and Diagnostics); and were also the respective genomic DNA for our mRPA assay. All subjects provided written informed consent, and all data were fully anonymized before assessment. The study was performed in compliance with the Declaration of Helsinki.For HPV positive controls and clinical samples, the respective genomic DNA were provided by the Buddhachinaraj Phitsanulok Hospital, Phitsanulok; and the Faculty of Medicine, Chulalongkorn University, Bangkok® Basic reaction powder , 14.75 μL dehydration buffer (TwistDx), ~ 25 ng DNA (unless specified), and 1.25 μL of 280 mM magnesium acetate . The reaction was incubated at 39 °C (unless specified) for 40 min (unless specified). The optimization of the mRPA reaction condition included the determination of each minimal primer concentration that yielded both L1 and E6/E7 gene amplification products, and the shortest incubation time with the highest limit of detection (LOD) and correct specificity. The reaction was terminated by 80 °C heating for 2 min. The result was analyzed via 3% agarose gel electrophoresis: the amplicon sizes for L1 and E6/7 RPA were ~ 250 bp and ~ 170 bp, respectively.The mRPA reaction (25 μL) comprised 0.12–0.48 µM each of primers (unless specified) in Table ® GT PCR Master Mix , 0.12–0.48 µM each primer, and ~ 25 ng DNA (unless specified). The PCR conditions were 95 °C 5 min followed by 35 cycles of 95 °C 15 s, 56 °C 30 s, and 72 °C 30 s, with a final extension at 72 °C 5 min. The proper size amplicon product was analyzed by 3% agarose gel electrophoresis. The amplicon sizes for L1 and E6/7 PCR were 188–194 bp and 117–121 bp, respectively. Quantitation of the amplicon was additionally performed using Qubit™ dsDNA HS and BR Assay Kits . This kit quantifies the amount of amplicons  at high sensitivity.Single-gene PCR and L1-E6/E7 multiplex PCR (mPCR) were employed as the parallel nucleotide amplification experiments for our mRPA primers and LOD. The PCR reaction (25 µL) comprised 12.5 µL EmeraldAmp23) ÷ (PCR product length (bp) × 6.6 × 1011).For preparation of HPV L1 (and E6/E7) gene as representative HPV HR and LR DNA templates, the PCR primers /e2, given p at an average incidence of 75%, confidence level of 95%, and e of 7.5% for margin of error. This yielded an N of 129. Therefore, we tested 130 random HPV-positive and negative samples by our developed mRPA methods compared with the conventional Cobas and REBA results. The assay efficacy was calculated based on the following equations: sensitivity = true positive ÷ , specificity = true negative ÷ , false positive = 1 − specificity, false negative = 1 − sensitivity, and accuracy = (true positive + true negative) ÷ total samples.To determine the performances of the conventional (Cobas and REBA) and our developed methods, statistically required sample numbers (N)  were calculated according to the equation: N = (p (1-p) z29. Hence, the inclusion of E6/E7 in this study benefited the diagnosis in this HPV DNA integration subjects, and the mPCR and mRPA of the positive HPV HR samples that mostly no E6/E7 gene band appeared might reflect the non-precancerous state of the sample.As sequences of HPV L1 and E6/E7 are highly variable among types, a primer multiplex system containing 8 degenerate primers was required to cover broad HPV L1 and E6/E7 gene types, of 20 HR  and 14 LR  types. We performed bioinformatic analyses by BLASTN on each HPV primer sequence specificity against NCBI GenBank non-redundant database , and the primers were specific to HPV (Table 31. Noted that in our mPCR and mRPA displayed the < 100 bp bands (< ~ 200 bp for mRPA), which were primer dimers and non-specific amplification, and were found in some positive and negative clinical specificity samples, for examples, influenza B, C. trachomatis, N. gonorrhoaea, S. saprophyticus, S. aureaus, S. epidermidis, H. sapiens and M. musculus  in RPA) making the amplicons heavier and we performed a simple one-step clean-up the reaction for low-cost and rapid potential local use, in replace of an amplicon purification step using commercial kits. The shifts of RPA amplicon bands had been reported elsewhere32 to obtain equal amplification efficiency. In brief, each gene primer was individually checked for a single amplification fragment, given that synthetic PCR amplicons of HR-HPV types 18 and 33 and LR-HPV type 11 also supported the validation of the effectiveness of the primers . We found the minimal concentration of multiplex primers for L1 (0.1–0.2 µM each) and E6/E7 (0.2 µM each) RPA to be different . Individual L1 (and E6/E7) primers could successfully amplified the HPV templates Fig. A,C. Cons36. Meanwhile, the Cobas and REBA results exhibited 18.46% diagnostic disagreement between each other. Furthermore, the mRPA result analysis by agarose gel electrophoresis may be replaced by using fluorescent DNA dye, such as SYBR Green I , or DNA probe paper strip for an even more rapid total assay time. We are continuing to improve the mRPA limit of detection by reducing the number of primers to focus on the HPV HR types and the gene expression detection of E6/E7 as reverse transcription-mRPA37.The schematic illustration of RPA and multiplex 2-gene amplification concepts as local HR- and LR-HPV screening tool was summarized in Fig. Our developed HPV mRPA assay for broad-type HPV detection  is simple, rapid, inexpensive, specific, sensitive, and does not require any complicated instrumentation  Fig. . EvaluatSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Power factor calculations resulted in a maximum value of 2.47 × 10−5 W m−1 K−2 at 143 °C. To the best of our knowledge, the observed Seebeck coefficient value is the highest reported for TE thin films fabricated by screen printing. Thus, this study highlights that increased Seebeck coefficients can be obtained by using QD building blocks owing to quantum confinement.Thin-film thermoelectrics (TEs) with a thickness of a few microns present an attractive opportunity to power the internet of things (IoT). Here, we propose screen printing as an industry-relevant technology to fabricate TE thin films from colloidal PbSe quantum dots (QDs). Monodisperse 13 nm-sized PbSe QDs with spherical morphology were synthesized through a straightforward heating-up method. The cubic-phase PbSe QDs with homogeneous chemical composition allowed the formulation of a novel ink to fabricate 2 μm-thick thin films through robust screen printing followed by rapid annealing. A maximum Seebeck coefficient of 561 μV K Thin-film thermoelectric (TE) generators have recently attracted considerable attention in the information and communication technology applications, particularly for the long-term powering of internet-of-things (IoT) devices . ClassicScreen printing relies on a screen with a prepatterned mesh of specific size, where viscous ink is forced to pass through the mesh toward the substrate, with the help of a squeegee. This technique affords few-micrometer-sized TE thin films with compact material packing. Moreover, screen printing allows different substrates and patterns, which widens the range of TE generator geometries . Notably−1 K−1 via phonon scattering at the QD interfaces [From the TE point of view, the intrinsically small dimensions of quantum dots (QDs) provide a natural reduction in thermal conductivity down to 0.9 W mterfaces . Moreoveterfaces ,12,13. Iterfaces ,15. The 2Te3 [2Te3 [2Se [One fundamental challenge in the TE thin film design involves the printed fabrication of the film from TE-relevant QDs. There are several hurdles in this direction during thin-film printing and processing, including the preservation of the QD particle sizes needed to exhibit the quantum confinement effect while avoiding any possible changes in the phase and chemical compositions of constituting QDs. Hence, screen-printed TE thin films from QDs are not common, and most of the reported works are dedicated to the screen printing of TE thin films from non-quantum materials, such as Bi2Te3 ,22,23,24e3 [2Te3 ,26, Ag2STe3 [2Se ,28,29 orTe3 [2Se , obtaine2∙3H2O, ≥99.99%), selenium , trioctylphosphine , oleic acid , 1-octadecene , ethyl cellulose  and terpineol were purchased from Sigma-Aldrich, St. Louis, MO, USA. Analytical reagent grade hexane and toluene (99.8%) were purchased from Fisher Scientific, Waltham, MA, USA. Acetone (≥99.5%), isopropanol (≥99.8%), and ethanol (≥99.8%) were purchased from Honeywell, Charlotte, NC, USA.Lead (II) acetate trihydrate (Pb(ac)2∙3H2O , OA , ODE  and 1 M TOP:Se solution (12 mL) were loaded under ambient conditions into a 250 mL three-neck round-bottom flask, equipped with a magnetic stir bar, thermocouple, condenser, and vacuum adapter. Next, the flask was attached to a Schlenk line, and the mixture was degassed under vacuum at 130 °C for 2 h while stirring to remove low boiling liquids, such as possible water and acetic acid admixtures. Then, the vacuum was switched to Ar, and the water reflux was initiated. Under continuous stirring, the flask was then rapidly heated to 190 °C and kept at this temperature for 10 min. Next, the resultant dark reaction mixture was cooled to room temperature (RT) using a water bath. Afterwards, the as-synthesized PbSe QDs were washed by the addition of 25 mL of ethanol and collected by centrifugation at 9000 rpm for 5 min. The resultant solid was washed with solvent mixture of hexane and isopropanol (1:3) and again collected by centrifugation. Finally, the size of the PbSe QDs was selected by dispersing of centrifuged solid in 15 mL of toluene followed by centrifugation at 3000 rpm for 10 min. The resultant supernatant with colloidal PbSe QDs was stored in closed vial at 4 °C. The concentration of PbSe QDs in toluene was estimated gravimetrically, and adjusted to be 30 mg/mL.To prepare the PbSe QDs, a previously reported colloidal synthesis protocol was adapted and modified . First, A 5% solution of ethyl cellulose in terpineol was prepared by dissolving the appropriate amount of EC in terpineol at 50 °C while stirring. To prepare an ink, 25% weight content of the PbSe QDs in solid form was added to the appropriate amount of 5% EC solution in terpineol. The ink was mixed first with a spatula and then probe sonication was used to improve QDs’ dispersion. The ink was sonicated on 5 min cycles with 5 s of sonication at 40% amplitude and 5 s pause. To achieve high dispersion of the PbSe QDs in the ink, 3 cycles were performed. Finally, the ink was let stir at 50 °C overnight.2) layer was grown by plasma-enhanced chemical vapor deposition on Si (100) substrate. The resultant SiO2/Si substrate was then diced with 0.7 × 2 cm2 and 2 × 2 cm2 dimensions using an automatic dicing saw. The substrates were consecutively cleaned with acetone and isopropanol by ultrasonication for 5 min. Then, the substrates were dried by N2 flow, and finally, subjected to oxygen plasma cleaning for 10 min.A 500 nm-thick silicon dioxide .The heat treatment was performed on a Thermal CVD MicroSys 400 System . The as-printed TE thin films were first annealed at 600 °C for 15 min under vacuum, and then under HKα radiation (λ = 1.541874 Å) and a PIXcel detector. The XRD patterns were matched to International Centre for Diffraction Data (ICDD) PDF-4 database using the HighScore software package .The phase composition of the PbSe QDs and the resultant thin films were analyzed using X-ray diffraction (XRD). The data were collected using an X’Pert PRO diffractometer  with Ni-filtered Cu To investigate the morphology and the chemical composition of the synthesized PbSe QDs, high-angle annular dark-field scanning transmission electron microscopy (HAADF–STEM) and energy-dispersive X-ray spectroscopy in STEM mode (STEM−EDX) were carried out using a JEM-ARM200F electron microscope , operated at 200 kV. QDs’ size distribution was estimated using ImageJ software package. The thickness and the surface morphology of the TE thin films were analyzed by scanning electron microscopy (SEM) using Quanta 650 FEG ESEM .Electrical resistivity and charge mobility were measured by a simultaneous Hall Effect and Van der Paw method at RT. These measurements allowed to determine the surface and volume concentration of carriers, electron mobility and electrical conductivity. The results obtained are an average of five measurements with 5% error.The Seebeck coefficient and electrical conductivity, in the temperature range from RT to 200 °C, were measured using a ZEM-3 system (ULVAC). The scheme of measurement is available in the PbSe QDs were prepared by a facile heating-up colloidal synthesis, relaying on the reaction between organometallic Pb precursor and Se solution in TOP under inert atmosphere. The as-synthesized PbSe QDs adopted a nearly spherical shape with monodisperse size distribution of 13 ± 1 nm, as shown in the representative low-magnification and high-resolution HAADF–STEM images a,c,d. Th2/Si rigid substrates under ambient conditions. Rapid annealing of the as-printed films in vacuum (15 min) and H2 (15 min) at a temperature of 600 °C was then conducted to largely eliminate the organic matter from the printed thin films to improve their electrical conductivity.Having high-quality PbSe QDs in hand, we further formulated a novel ink for screen printing. Since oleate-capped colloidal QDs could be well-dispersed in non-polar organic solvents, the ink was formulated based on terpineol solvent while using ethyl cellulose as a binder . This ne2/Si from the film substrate. Regardless of the conducted heat treatment, the presence of the residual carbon was still detected in the final PbSe QD thin films, as confirmed by Raman spectroscopy  and Seebeck coefficient (S). Electrical properties, measured at RT by the Hall effect, revealed that PbSe QD thin films exhibited a bulk carrier concentration of 3.8 × 1018 cm−3, electron mobility of 7.9 × 10−1 cm2 V−1 s−1, and a quite low σ = 50 S m−1. The σ and S were then measured from RT to 200 °C in air  calculations resulted in a maximum value of PF = 2.47 × 10−5 W m−1 K−2 at 143 °C  above and below the Fermi surface, i.e., by the logarithmic derivative of σ with E. From another point of view, S can also be understood as the average energy per carrier. Outstanding S can be reached by quantum confinement via (i) inducing sharp features in the carrier’s density of electronic states near Fermi energy (high dn(E)/dE), and (ii) introducing energy barriers to selectively scatter charge carriers according to their sign and energy (enhancing dμ(E)/dE).The in films . For inst 350 °C . The obsof 13 nm , which wS > 470 μV K−1, while Tai and co-workers [S = 628 μV K−1 for their thin films, which are a huge enhancement in comparison with S = 265 μV K−1 of bulk PbTe. In another study, Zhou and co-workers [S above 500 μV K−1. Yang and co-workers [2) QDs with S = –420 μV K−1 near 200 °C. A higher Seebeck coefficient value of 580 μV K−1 at room temperature was also reported by Nugraha and co-workers [−1, which is considerably high when compared with the best S = 380 μV K−1 near 100 °C for bulk PbSe [In the literature, thin films produced from different lead chalcogenide QDs showed a higher Seebeck coefficient than the respective bulk material. For example, Yan and co-workers  produced-workers  obtained-workers  fabricat-workers  for PbS ulk PbSe .S after reaching its maximum at 143 °C (gE), despite the fact that some technical issues with the measurement cannot be completely ruled out as a reason for a sharp six-fold decrease in the S value at 200 °C. Interestingly, regardless of the observed drop in thermopower at 200 °C, the electrical conductivity kept increasing with temperature  is larger than the one of bulk PbSe (gE = 0.27 eV) [The observed decreasing trend in the t 143 °C a could bperature a. Such bx occurs . The ban0.27 eV) . The obs0.27 eV) .σ of PbSe QD thin films, near RT, measured by the Hall effect was quite low σ = 50 S m−1. Even at 200 °C, the maximum value obtained was σ = 123 S m−1, which is significantly lower when compared with σ = 1.84 × 104 S m−1 of bulk PbSe [−1, and determined the σ at RT. The σ results listed in σ of the film by nearly twice as much [σ = 165 S m−1 was still far from the 104 S m−1 order when compared with the other screen-printed materials , revealing the high potential of PbSe QDs for TE applications near RT. At the same time, the intrinsic shortcoming of the QD building blocks, namely, the small contact conductance between the spherically shaped QDs, led to very poor electrical conductivity (123 S m−1) of the final PbSe QD thin films. Studies on overcoming this shortcoming with the help of atomic layer deposition of the conductive layer are currently underway in our laboratories.We demonstrate screen printing technology as an easy-to-scale-up approach to fabricate thermoelectric PbSe thin films with a thickness of a few micrometers and potential for near-RT application. This was achieved through (i) a straightforward colloidal synthesis of high-quality PbSe QD building blocks by means of the heat-up method, which allowed obtaining narrow-size and spherical-shape PbSe QDs, (ii) judicious formulation of a novel screen-printable PbSe QD ink obtained from well-dispersed QDs into a polymeric matrix, and (iii) a practical fabrication of the PbSe QD thin films by means of screen printing followed by rapid annealing, applied to eliminate ink organic precursors that could hinder QDs’ charge transport. We propose that owing to the quantum confinement effect from the PbSe QDs’ small size, the resultant thin films exhibit an outstanding Seebeck coefficient (561 μV K


Ubiquitin-specific protease15(USP15), is the 16th identified protease in the USP family and is a key protein in tumorigenesis. However, the predictive value and regulatory mechanism of USP15 in breast cancer are unclear.  The GEPIA, UALCAN, GeneMANIA, and STRING databases were applied to explore the expression of USP15 in breast cancer and associated proteins. In addition, the TIMER database was evaluated for immune infiltration patterns. Moreover, protein immunoblotting assay, cell scratching assay, small compartment invasion assay, 3D stromal gel assay, immunoprecipitation assay, and immunohistochemistry (IHC) were used to USP15 regulatory mechanisms in breast cancer. βRI. In addition, USP15 expression was positively correlated with immune infiltration, including immune score, mesenchymal score, and several tumor-infiltrating lymphocytes (TIL). In addition, IHC results further confirmed the high expression of USP15 in breast cancer and its prognostic potential.  In BRCA, several databases, including GEPIA and UALCAN, describe the upregulation of total protein levels and USP15 phosphorylation. In addition, the expression of USP15 was significantly correlated with gender and clinical stage. Overall survival (OS) was lower in patients with high USP15 expression. Functional network analysis showed that USP15 is involved in tumor-associated pathways, DNA replication, and cell cycle signaling through TGF These findings demonstrate that high USP15 expression indicates poor prognosis in BRCA and reveal potential regulatory networks and the positive relationship with immune infiltration. Thus, USP15 may be an attractive predictor for BRCA. Breast cancer is the most important disease threatening the life and health of women worldwide –3. Amongβ) is a multifunctional cytokine that regulates the cell cycle and affects cell proliferation, differentiation, adhesion, and metastasis in tumor formation and development [β receptor whose involvement in the TGFβ signaling pathway has been confirmed [β, when stimulated by an external signal, first binds to the type II TGFβ receptor to form a complex [β signaling pathway is vital in many tumors such as breast cancer, lung cancer, etc. [Transforming growth factor  and p53. [Epigenetic changes influence tumor development, with protein ubiquitination, a common form of posttranslational modification, playing a crucial role in tumor formation, the reverse process of which is called deubiquitination , 19, whiand p53. . These iand p53. –24.β pathway [β is balanced. When the level of TGFβ expression in cells is too high, smad7 binds to smurf2 downstream of its signaling pathway to form a complex that further binds to TGFβ type I receptors to facilitate ubiquitination and degradation type I TGFβ receptors. The body is stable to induce receptors, thereby reducing the expression level of TGFβ [β in the body is too low, USP15 associates with smad7 and smurf2 to form a complex to deubiquitinate the type I TGFβ receptor and further increase the expression level of the intracellular signaling of TGFβ [β levels to be underexpressed and may tip the balance towards deubiquitination [β and thus promoting tumor proliferation and migration [USP15 acts as a “biological thermostat” in the TGF- pathway . In norm of TGFβ , 27. On  of TGFβ , 29. Howtination , leadingigration , 31, Theigration , 33. Theigration .http://ualcan.path.uab.edu/analysis.html), a comprehensive web resource, provides analyses based on The Cancer Genome Atlas (TCGA) and MET500 cohort data. In our study, expression data for USP15 was obtained using the “Expression Analysis” module of UALCAN Student's t-test was used to generate a p value. The p value cutoff was 0.05. Predictive analysis was performed using a Kaplan–Meier curve [UALCAN (er curve –36.http://gepia.cancer-pku.cn/index.html) is an analysis tool containing RNA sequence expression data of 9736 tumors and 8587 normal tissue samples developed at Peking University. In this study, we performed with the “Multiple Gene Comparison” module of GEPIA. Predictive analysis was performed using a Kaplan–Meier curve. The p value cutoff was 0.05. The student's t-test was used to generate a p value for expression or pathological stage analysis [GEPIA  is a publicly available portal that includes multiomics data from all 32 TCGA Cancer types and 10 Clinical Proteomics Tumor Analysis Consortium (CPTAC) cancer cohorts. The web application has three analytical modules: LinkFinder, LinkInterpreter, and LinkCompare , 39.https://string-db.org/) aims to collect, score, and integrate all publicly available sources of protein-protein interaction (PPI) data, and to complement these with computational predictions of potential functions. We conducted a PPI network analysis of differentially expressed USP15 to explore the interactions among them with STRING [STRING  is a user-friendly website that provides information on protein and genetic interactions, pathways, coexpression, colocalization, and protein domain similarity of submitted genes [GeneMANIA  is a reliable, intuitive tool that systematically evaluates different immune cells' infiltration and their clinical impact. In our study, the “Gene module” was used to assess the correlation between USP15 and the infiltration of immune cells. “Survival module” was used to evaluate the correlation between clinical outcomes and the infiltration of immune cells and USP15 expression [TIMER , and GES-1  from ATCC  purchased or donated to China Normal University by Professor Liu Mingyao's research group. Cells were maintained in DMEM or Death Complete 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cells were maintained at 37°C in a 5% CO2 incubator. All cell lines were regularly checked for mycoplasma contamination.MDA-MB-231, BT549, T47D, SUM159, BT-20, MDA-MB-468 and MCF7 (breast cancer cells), MCF-10A https://www.selleck.cn/No.s7130). Compound stock solutions were prepared in DMSO at a concentration of 100 mM and stored at -20°C.PR619 was purchased from Selleck's official website  precoated with Matrigel or Collagen I. After 12 h, cells on the top side of the filters were wiped by cotton swaps. Cells on the lower side were then fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Images were taken under an inverted microscope (Olympus) [Transwell invasion assay was performed following the manufacturer's instructions with modifications. We selected 1Olympus) , 46.μL per well, with 3 replicate wells and 5 for each group. Drug treatment was given after 24 hours of incubation in a 5% CO2 incubator. The cells were fixed with trichloroacetic acid (TCA) after the drug-addled culture (96 h). Place at 4°C for 1 hour, spin dry, and dry at room temperature. After drying, 50 μL of 0.4% SRB solution was added to each well, and the cells were stained for 10 min at room temperature in the dark. Subsequent 5 washes removed unbound cell dye with 1% acetic acid. Leave the 96-well plate to dry at room temperature. 200μL of 10 mM Trisbase (pH = 10.5) was added, and the plate was shaken until completely dissolved. The OD value of each well at a wavelength of 515 nm was measured on a microplate reader. IC50 values were calculated [Cells with good growth conditions were selected, digested with trypsin, centrifuged, resuspended, adjusted to a cell density of 3000 cells/mL, and inoculated into 96-well plates, 100 lculated , 48.3D culture assay was conducted as described. Briefly, MDA-MB-231 breast cancer cells were seeded on a 48-well plate coated with a thin layer of Matrigel. Thirty minutes postseeding, a medium containing 10% Matrigel and different concentrations of PR619 were added to the plated culture. The culture was maintained for 4 days and the on-top Matrigel–medium mixture with or without PR619 was replaced every 2 days. 3D on-top culture was performed as previously described , 49.Cells were lysed in RIPA buffer  containing protease inhibitors and phosphatase inhibitors, and then analyzed by immunoblotting with the indicated antibodies. The relevant antibody product numbers are as follows: USP15 , PhosphoSmad2 (Ser465/467)/Smad3 (Ser423/425) , SMAD2 , SMAD3 , SMAD4 , SMAD7 , and GAPDH  , 50.μL pipet tip. The cells were washed twice with PBS, and the media in each well was replaced with 1 mL complete RPMI 1640. The compounds at specified concentrations were added to each well, and the cells were incubated at 37°C for 24 h under full conditions. Cell migration was observed and photomicrographed for quantitation and image analysis of each treatment [Cells were seeded onto sterile 6-well plates and incubated at 37°C in complete RPMI 1640 medium to 100% confluence for the experiment and then changed to fresh serum-free media for another 12 h. Wounds were created in cells using a sterile 10.0 reatment , 46.CO-IP kit (ThermoScience) for CO-IP experiments. Four confluent plates of MDA-MB-231 breast cancer cells were selected and cell lysates were prepared in IP buffer . After centrifugation at 14000 g for 20 min, the cell lysate supernatant was incubated with the corresponding antibody overnight. Cell lysates were incubated with A/G beads for 4 hours, then washed after centrifugation, and the eluates were resolved by SDS-PAGE and analyzed by western blotting. All CO-IP experiments were performed at 4°C .t-test was used. For comparison of multiple groups, 1-way ANOVA was used.The data are presented as the mean ± SD unless wise otherwise stated. Statistical tests were performed using GraphPad Prism, version 6.0 (GraphPad Software). For comparisons of 2 groups, an unpaired, 2-tailed Student's p = 0.026) (p = 0.011) and triple-negative breast cancer patients (p = 0.038)  . Then, t= 0.038) . Triple-p = 6.87e − 04) (p = 1.04e − 01), stage 2 (p = 4.05e − 03), and stage 3 (p = 4.11e − 03), respectively. The results showed that the expression of USP15 in stage 2 and stage 3 breast cancer was higher than in stage 1 breast cancer. At the same time, we also found that the expression of USP15 was different in the distribution of breast cancer in different races, including Caucasians (p = 5.12e − 04), African Americans (p = 1.51e − 03), and Asians (p = 5.12e − 04);p = 1.09e − 01). Since age influences tumor development and prognosis, we also performed a statistical analysis of breast cancer patients in different age groups. We found that the expression of USP15 was positively correlated with increasing age, with ages ranging from 21 to 40 years (4.28 e-02), 41-60 years old (4.92e-03), 61-80 years old (8.52e-04), and 81-100 years old (4.10e-03). Analyzing the results, we found that high expression of USP15 was mainly observed in breast cancer patients aged 41-60. To further analyze the influence of USP15 expression on breast cancer prognosis, we also performed a statistical analysis on breast cancer staging. Molecular typing results showed Luminal (p = 2.95e − 0.1), HER2 positive (p = 9.27e − 0.1), and TNBC (p = 6.68e − 01), and pathological typing results showed invasive ductal carcinoma (p = 3.98e − 01), invasive lobular carcinoma (p = 4.43e − 01), and mixed histology (p = 2.85e − 01)). The analysis results indicate that the expression level of USP15 is also closely related to the stage of breast cancer and that the expression of USP15 was higher in invasive breast cancer than in other types of breast cancer. Invasive breast cancer is one of the most common tumor types with a high potential for metastasis and a poor prognosis. Therefore, the above results suggest that USP15 plays an important role in the prognosis and survival of breast cancer patients. At the same time, we selected tumor tissue sections from 20 breast cancer patients from the pathology department of the General Hospital of Ningxia Medical University. By immunohistochemical analysis, we found that USP15 was a high expressed in tumor tissue sections from 3 of these patients.  , the phoatients. . By reviatients. . The resLinkedOmics analyzed 509 BRCA patients with genes coexpressed by USP15, of which 3035 genes were positively correlated with USP15 and 2992 genes were negatively correlated with USP15  . ShowingβRI compared to NPAS4 and CXCL13, where the combined value was 0.918. USP15 was also closely related to smad-related proteins and the combined score of USP15 and Smad7 was 0.976. . The correlation coefficient between USP15 and SMAD7   type ; smurf2 (Cor = 0.569), p = 2.01e − 95); and smad7 ; in the basal BRCA type, USP15 was associated with the expression of TGFβRI ; smurf2 ; and smad7 ; USP15 was BRCA-HER2 type with TGFβRI expression ; smurf2 ; and smad7 . Correlation of USP15 expression with TGFβRI in BRCA luminal type , smurf2 , and smad7    The Smaditation. . TGFβ iselopment . To inve4e − 13) . Based oμM, the half-inhibition rate of MDA -MB-468 was 2.13 ± 0.08 μM, BT549 half-inhibition rate was 6.79 ± 0.31 μM, T47D MCF-7 half-inhibition rate was 4.07 ± 0.15 μM, and the half-inhibition rate of MCF-7 was 3.47 ± 0.01 μM. PR619 can inhibit the cell proliferation of various breast cancer cells in a concentration-dependent manner. Interestingly, we found that PR619 inhibited half of the proliferation of normal mammary epithelial cells at 13.73 ± 0.12 μM, and PR619 interfered with GES-1 with a median inhibition rate of 1.91 ± 0.05 μM, which suggests that PR619 could inhibit proliferation of breast cancer cells with less toxicity. , CD8+ T cells , CD4+ T cells , Macrophage , Neutrophil , and Dendritic cell  were positively correlated; in BRCA-Basal types, USP15 expression was associated with B cells , CD8+ T cells , CD4+ T cells , Macrophage , Neutrophil , and Dendritic cell  were positively correlated; in BRCA-Her2 type, USP15 expression was negatively correlated with B cells , negatively correlated with CD8+ T cells , CD4 + T cells , Macrophage , and Neutrophage ),Neutrophil  and Dendritic cell  were positively correlated; in BRCA-Luminal type In BRCA-Luminal type, USP15 expression was positively correlated with B cells , with CD8+ T cells , CD4+ T cells , Macrophage , Neutrophil , and Dendritic cell  were positively correlated  and histidine (His) . HoweverIn this study, we found that USP15 was highly expressed in breast cancer by searching the TCGA databases , 54. TheIn conclusion, our results suggest that breast cancer metastasis can be blocked by knocking down USP15. The results of this work may reflect the role of USP15 in tumor metastasis, providing an opportunity to assess the clinical relevance of USP15 for metastasis. Our results also offer the opportunity to develop drugs targeting USP15; USP15 may be an attractive predictor of breast cancer prognosis and merits further study in treating breast cancer.


Klebsiella pneumoniae resistance to the last-line antibiotic treatment have prompted a reconsideration of bacteriophage therapy in public health. Biotechnological application of phages and their gene products as an alternative to antibiotics necessitates the understanding of their genomic context. This study sequenced, annotated, characterized, and compared two Klebsiella phages, KP1 and KP12. Physiological validations identified KP1 and KP12 as members of Myoviridae family. Both phages showed that their activities were stable in a wide range of pH and temperature. They exhibit a host specificity toward K. pneumoniae with a broad intraspecies host range. General features of genome size, coding density, percentage GC content, and phylogenetic analyses revealed that these bacteriophages are distantly related. Phage lytic proteins  identified by the local alignment against different databases, were subjected to further bioinformatic analyses including three-dimensional (3D) structure prediction by AlphaFold. AlphaFold models of phage lysis proteins were consistent with the published X-ray crystal structures, suggesting the presence of T4-like and P1/P2-like bacteriophage lysis proteins in KP1 and KP12, respectively. By providing the primary sequence information, this study contributes novel bacteriophages for research and development pipelines of phage therapy that ultimately, cater to the unmet clinical and industrial needs against K. pneumoniae pathogens.Concerns over  Klebsiella pneumoniae is a Gram-negative opportunistic pathogen causing an extensive range of infections in the urinary tract, lungs, liver, abdominal cavity, and surgical sites in humans  K. pneumoniae), # KPN10 strain (ID: K16-KPN-13-022) as the host bacteria was cultured in Tryptic Soy Broth  at 35°C for 18 h. Other bacterial strains tested in this study were cultured in TSB at 35°C for 18 h.To isolate bacteriophages against K. pneumoniae. Plates were incubated at 35°C for 24 h and the lytic activity of bacteriophage was observed.Because sewage is a reservoir of highly abundant enteric pathogens and novel bacteriophages , sewage 4, 50 mM Tris–HCl [pH 7.5]) by 10-fold dilution. Diluted sample (100 μl), host bacteria culture (150 μl), and TSB top agar containing 0.6% agar (3 ml) were mixed and poured onto the TSA containing 2% agar plate followed by a 24 h incubation at 35°C. Single plaques were obtained, resuspended in 400 μl of SM buffer, and layered at room temperature for 4 h. The suspension (100 μl), TSB with 0.6% agar (12 ml), and host bacteria culture (300 μl) were mixed and layered on the TSA containing 2% agar bottom plate (diameter of 150 mm). Plates were incubated at 35°C for 24 h. To extract the bacteriophages, 15 ml of SM buffer was poured onto the cultured plate and stirred slowly at room temperature for 4 h. The suspension was recovered and centrifuged at 4,000 rpm, 4°C for 20 min. Then, the supernatant was filtered through a 0.22 μm pore-size membrane filter to obtain bacteriophage. To concentrate and purify lysates of the bacteriophage, polyethylene glycol 8,000  precipitation and purification via cesium chloride  gradient ultracentrifugation were conducted. DNase 1 and RNase A (1 ug/ml) were added to phage lysate to degrade residual bacterial DNA and RNA during the reaction in 0.5 M NaCl at 40°C for 1 h. The mixture was centrifuged at 6,000 ×g, 4°C for 10 min. Phage-containing supernatant was precipitated by adding PEG 8000 to a final concentration of 10% w/v gradually with stirring at 4°C overnight. After centrifugation at 7,200 ×g, 4°C for 20 min, PEG-bacteriophage pellets were washed twice with 0.1 M ammonium acetate (pH 7.0), resuspended in 0.5 ml of sterile distilled water, and stored or incubated at 4°C overnight. Subsequently, bacteriophage suspension was purified using the CsCl gradient ultracentrifugation. The bacteriophage solution was injected in the CsCl gradient  followed by centrifugation at 78,500 ×g, 4°C for 2 h. The light-gray band was collected and dialyzed three times with membrane  using 500 ml of SM buffer at 4°C. The purified bacteriophage suspension was stored at 4°C until further experiments.Soft agar overlay method  was perfTo obtain the phage titers, soft agar overlay method with 10-fold serial dilutions of the purified phage lysates were performed and plaque forming unit (PFU) was calculated after incubation.K. pneumoniae strains and nine other Gram-negative bacteria. The ESBL (+) K. pneumoniae strains were obtained from the Asian Bacterial Bank (ABB) of Asia Pacific Foundation for Infectious Diseases , which were all isolated from human blood. The other nine Gram-negative bacteria were Acinetobacter baumannii ATCC17978, Citrobacter freundii clinical isolate 15-0628, Cronobacter sakazakii KCTC 2949, Escherichia coli KCTC 1039, ESBL(+) Escherichia coli K01-ECO12-052, Proteus mirabilis KCTC2566, Pseudomonas aeruginosa KCTC2004, Salmonella Typhimurium ATCC14028, and Salmonella Enteritidis KCCM12021. All of these bacteria were handled by Optipharm Inc. using the standard soft agar overlay method for the host specificity test (The host range of the isolated bacteriophages was tested against 11 ESBL(+) ity test . Briefly9 PFU/ml) was applied to glow-discharged carbon-coated copper grids. After allowing the sample to absorb for 2 min, buffer solution was blotted off onto a Whatman paper and the sample on the grids were stained with 2% (w/v) uranyl acetate (UrAc) for 1 min. UrAc was blotted off. These results were recorded with the Tecnai G2 Spirit Twin microscope  at an acceleration voltage of 120 kV.Purified bacteriophage suspension (over 1.0 × 109 PFU/ml) was added to 10 ml of exponential phase culture of K. pneumoniae (1.0 × 108 CFU/ml) to obtain the multiplicity of infection (MOI) of 0.1. The bacteriophage was allowed to adsorb onto the bacterial surface for 5 min at room temperature. Subsequently, the mixture was centrifuged at 10,000 ×g for 1 min and the resultant pellet was resuspended in 20 ml of TSB medium. Aliquots were collected at 5-min intervals, and the bacteriophage titer was measured with the standard double-layer top agar assay  gradient at 26,000 rpm for 4 h. Pellets collected were suspended with SM buffer to a concentration of 1.0 × 10erformed .8 CFU/ml of K. pneumoniae culture was transferred to sterile tubes and infected with bacteriophages at different levels of MOI which were 10, 1, and 0.1. The tubes were incubated at 35°C with shaking for 3 h. Negative control (NC) was incubated without bacteriophage. The cell density (OD600nm) was measured at 1-h intervals using a spectrophotometer .Approximately 108 PFU/mL were incubated at 40°C, 50°C, 60°C, and 70°C for an hour, phage titers were measured using the soft agar overlay method. To determine the phage stability at a range of pH, we used acetic acid and sodium acetate buffer for pH 2–6, phosphate buffer for pH 7–11, and Tris–HCl buffer for pH 8–11. The pH of buffers was adjusted with 1 M HCl or 1 M NaOH. Phage suspension  was added to each pH buffer (990 μl), gently mixed, and incubated at room temperature for 2 h. After treatment, phage titers were measured using the soft agar overlay method.To determine the temperature stability of phages, KP1 and KP12 at a final concentration of 10Bacteriophage suspension was treated with DNase I  to eliminate the host DNA. EDTA (20 mM), proteinase K , and SDS (0.5% w/v) were added to the bacteriophage suspension and allowed to stand at 50°C for an hour. The suspension was mixed with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and centrifuged at room temperature for 10 min. The recovered supernatant was again mixed with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and centrifuged at room temperature for 10 min. Subsequently, the supernatant was mixed with 10% (v/v) of the total volume of 3 M sodium acetate. A double volume of cold 95% ethyl alcohol was added followed by incubation at −20°C for an hour. Centrifugation was performed at 13,000 rpm for 10 min at 0°C. The resultant DNA pellet was washed twice with cold 70% ethyl alcohol . After cGenome sequences of both KP1 and KP12 were annotated using the Rapid Annotation Subsystem Technology toolkit (RASTtk) pipeline . BLASTp Klebsiella endolysin sequences annotated by RefSeq .Subsequent analysis of phage-encoded lytic proteins  was conducted. To identify putative endolysin sequences, protein-encoding genes (PEGs) of KP1 and KP12 were queried against two different curated endolysin databases which were by Klebsiella sp. have been deposited in the NCBI Virus database. These sequences include 5 Ackermannviridae, 45 Autographiviridae, 2 Demerecviridae, 33 Drexlerviridae, 31 Myoviridae, 3 Podoviridae, 4 Schitoviridae, and 10 Siphoviridae. Phylogenetic relationship of 135 bacteriophages including KP1 and KP12 at the amino acid level were evaluated using BPGA pipeline  v6  of bacteriophages infecting iTOL) v6 .Pan-genomic studies determine the core (conserved), accessory (dispensable), and unique (strain-specific) gene pool of a species to uncover species evolution and relationship. Pan-genome analysis was performed concurrently with phylogenetic analysis using BPGA algorithm.Pairwise comparison between KP1 and KP12 was performed using PATRIC Proteome Comparison tool 1 with deUsing the amino acid sequence of putative lytic proteins of KP1 and KP12, their structures were predicted using AlphaFold v2.0. AlphaFold v2.0 was locally installed together with multiple genetic databases which were UniRef90, MGnify, BFD, Uniclust30, PDB70, and PDB with mmCIF format. Structure prediction consisted of five steps which include MSA construction, template search, inference with five models, model ranking based on the average of per-residue confidence metric called predicted local distance difference test (pLDDT) score, and constrained relaxation of the predicted structures. For each target protein sequence, five structural models were generated. Each model was provided with its corresponding pLDDT score, predicted template modeling (pTM) score, and predicted aligned error (PAE) heatmap for the evaluation of model accuracy.E. coli transformants were selected on an LB agar plate containing ampicillin (50 μg/ml). Fragments encoding putative endolysins were recovered from the cloning vector using NdeI and XhoI , and recloned into PET-28a expression vector  to be expressed in Escherichia coli (E. coli) under 0.5-mM isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. After cell disruption using a sonicator , Branson Ultrasonics Corporation, Brookfield, CT, United States), putative endolysins were purified using Ni-NTA Agarose . Purified proteins were analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a Precision Plus Dual Xtra Standard protein marker .Gene encoding putative endolysin in KP1 (KP1.peg.110) was amplified by polymerase chain reaction (PCR) using primers KP1_F (5′-CATATGTTGAAACTTACGCTGGAACAA-3′) and KP1_R (5′-CTCGAGTTATTTTCCTTGATAAGCG-3′). Gene encoding putative endolysin in KP12 (KP12.peg.319) was amplified using primers KP12_F (5′-CATATGTTGAAACTTACGCTGGAACAA-3′) and KP12_R (5′-CTCGAGTTAAGAGGTTAGAACAGAT-3′). PCR was performed under the following conditions: one cycle of 10 min at 95°C, followed by 35 cycles of 90 s at 95°C, 90 s at an annealing temperature between 50°C and 66°C, 90 s at 72°C, and one final cycle of 10 min incubation at 72°C. The PCR products were cloned into the pGEM-T Easy Vector System , and K. pneumoniae (K16-KPN-13-022) was collected, washed once, and resuspended in PBS. The suspension was then autoclaved and centrifuged. The resulting pellet was resuspended in 200 μl of PBS and used as the substrate. The PBS solution contained 0.5 mM EDTA to increase the permeability of Gram-negative bacterial outer membrane according to the previous reference (E. coli BL21(DE3) and E. coli DH5α were prepared via the same process.Spot test was performed as described previously . The oveeference . The solMyoviridae bacteriophages with contractile tails and icosahedral heads. Each phage has a head diameter of approximately 60 nm and a tail with a diameter of 11 nm and length of 90 nm  K. pneumoniae.Subsequently, the host specificities of KP1 and KP12 were determined by their infectivity against various Gram-negative bacteria. Both KP1 and KP12 phages are highly specific for eumoniae . Out of K. pneumoniae, thermostability and pH sensitivity of these phages were further investigated. Overall, the increase in temperature resulted in the decrease of bacteriophage titers , an over 100-fold reduction was observed in both phage titer after 60 min . At 60°C, both KP1 (7.02 ± 0.04 log10 PFU/ml) and KP12 (7.70 ± 0.01 log10 PFU/ml) showed titer reductions but less than those at 70°C. At 40°C and 50°C, phage titers remained relatively stable even after 1 h treatment. Regarding pH stability, the PFU counts (PFU/ml) of phage were over 1.0 × 106 for both KP1 and KP12 which proved to be stable in the range of pH 4–11. At pH 3, there was a decline in both phage titers. However, the decrease observed in viable KP1 . The analysis of KP1 genome showed a total of 295 predicted genes. Of which, 281/295 were identified as PEGs and the remaining 14 genes were identified as tRNA coding genes  defined 179 core genes, 113 accessory genes, and 41 unique genes which account for 53.8%, 33.9%, and 12.3% of the pan-genome, respectively . These sConservation analysis was conducted to identify orthologous genes between KP1 and KP12 bacteriophages. The result indicated that they share only 14 orthologs, which accounted for approximately 5.0% of PEGs in KP1 (14/281) and 3.5% of PEGs in KP12 14/397; . Their rKlebsiella phage-derived endolysins revealed a vast diversity exists even among bacteriophages that target the same host. The highly conserved endolysins of KP1 (KP1.peg.110) and Mineola (YP_010096097.1) formed a lineage with a distinct sequence organization as compared to other phage clusters, while endolysin sequence of KP12 (KP12.peg.319) and of vB_KpnM_KBV79 phage are closely related , with a TM-align score of 0.70975. Nevertheless, KP12.peg.319 structure was highly similar to the Myoviridae P1 phage endolysin Lyz with a TM-align score of 0.83277  when aligned with coliphage endolysins . Instead 0.83277 . Moreove 0.83277  were con 0.83277 . This pr 0.83277 . In KP12 0.83277 .Myoviridae P2 lysis cassette including the overlapped form of i-spanin (KP12.peg.317) and o-spanin . T1–like lysis cassette of KP12 (KP12.peg.278–280) showed the representative arrangement in the order of holin, endolysin, and spanin. However, the putative endolysin (KP12.peg.279) was predicted to have two domains which is not consistent with the small single-domain globular structure of Gram-negative bacteria endolysin. Moreover, its overall pLDDT score distribution at the N–terminus (residue 1–60) was low hence, the predicted structure was not reliable . In addiE. coli expression system with IPTG induction, followed by Ni-NTA based purification. SDS-PAGE showed that both purified proteins were of expected molecular weight (Klebsiella pneumoniae (K16-KPN-13-022), which demonstrated their lytic properties toward K. pneumoniae (E. coli strains BL21(DE3) and DH5α (data not shown).To verify the bacteriolytic capacity of putative endolysins in KP1 (KP1.peg.110) and KP12 (KP12.peg.319), the genes were cloned and expressed using eumoniae  but not K. pneumoniae, the historical practice of bacteriophage therapy emerges as a promising alternative to the traditional antibiotics, due to their remarkable host specificity, hence minimizing the perturbance of microbiota in patients. In the present work, Klebsiella phages KP1 and KP12 were characterized and assigned as members of two distantly related Myoviridae clusters based on their TEMs and phylogenetic analyses. The lifestyle of the bacteriophages were additionally investigated by searching the presence of lysogeny-associated genes such as integrase and using several published prediction tools  K. pneumoniae strains isolated from clinical environment with high morbidity and mortality rates associated (Klebsiella phages (K. pneumoniae 77 capsular serotype reference strains (wzc and wzy sequencing for the tested ESBL (+) K. pneumoniae strains  with its full length mainly covered by enzymatically active domains whereas the lytic proteins from a Gram-positive background are separated into two domains for cell wall binding and enzymatically active site  targeting ESBL(+) K. pneumoniae. Indeed, AlphaFold model prediction has facilitated study of phage structures including adhesion devices of phage  K. pneumoniae. A look into endolysin homologous sequences among Klebsiella phages revealed an interesting find of vast diversity and possible analogous convergence, which challenge our current understanding of this enzyme. Deep learning-based modeling approach followed by a residue-to-residue alignment of predicted 3D structures to experimentally verified X-ray crystals has enabled a comprehensive understanding of not only phage endolysin but also other representative phage lytic proteins such as holin, i-/o-spanin, or anti-holin. An integrated approach of physiological evaluations and comparative genomic analyses supported by rapid structural prediction would certainly expedite functional annotation of phages and their high-value phage-encoded enzymes. With the desired stability and lytic activity against a broad range of K. pneumoniae, KP1 and KP12 themselves and their endolysins have proven their prospects in phage therapy.Taken together, this study characterized two https://github.com/SBML-Kimlab/KP1_KP12.The datasets presented in this study can be found at in vivo bacteriophage experiment and wrote the manuscript. S-ML analyzed data  and wrote the manuscript. LN analyzed data  and wrote the manuscript. JK assisted the data analysis and revised the manuscript. SK discussed the results and supervised the project. DK discussed the results, corrected the manuscript, and supervised the project. All authors contributed to the article and approved the submitted version.YK designed and conducted YK was employed by Optipharm Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction:Cyclocarya paliurus  Iljinsk., a subtropical tree belonging to the family Juglandaceae, is rich in polysaccharides, flavonoids, and terpenoids. It has important pharmacological effects such as lowering blood lipids, blood sugar, and blood pressure. However, little has been discerned regarding anti tumor effects and their potential mechanisms.Method:In vitro cell culture experiments were used to test the effect of C. paliurus total flavonoids (CTFs) extract on apoptosis mechanisms in HepG2 cells. Network pharmacology was applied to further explore the effects of CTFs on liver cancer as well as the mechanisms through which these effects might be achieved. Both 3 hydroxyflavone and luteolin were randomly selected to verify the effect on inducing apoptosis and inhibiting the proliferation of HepG2 cells.Results and Discussion: Network pharmacological analysis was applied to these 62 compounds and their targets, and 13 flavonoids were further screened for their potential anti liver cancer activity. These 13 flavonoids included: tangeretin, baicalein, 7,3′-dihydroxyflavone, velutin, 3-hydroxyflavone, chrysin, kumatakenin, tricin, luteolin, chrysoeriol, apigenin, pinocembrin, and butin. Together, these flavonoids were predicted to interact with AKT1, MAPK3, PIK3CA, EGFR, MAP2K1, SRC, IGF1R, IKBKB, MET, and MAPK14. It was predicted that the inhibitory effect on hepatocellular carcinoma would be accomplished by regulation of core proteins relating to such KEGG pathways as cancer, PI3K-Akt, proteoglycans in cancer, microRNAs in cancer, and endocrine resistance via core target proteins. Both 3-hydroxyflavone and luteolin were demonstrated to induce apoptosis and inhibit the proliferation of HepG2 cells. Our study provides scientific evidence supporting the use of CTFs for the treatment of liver cancer. Brucea javanica seeds was shown to eliminate sphere formation in cancer stem cells and inhibit tumor-initiating behavior in hepatocellular carcinoma cells  signaling pathway. Another study by Network pharmacology, a new branch of pharmacology based on systems and network biology, focuses on revealing the complex network of relationships between drug, target, and disease, systematically observing the intervention and influence of drugs on the disease network and providing insight into the molecular mechanism of drug therapies . Recentlin vitro and in vivo anti-tumor activities in such contexts as acute myeloid leukemia , are widely distributed in plants, including fruits and vegetables . Flavonoleukemia , lung caleukemia , melanomleukemia , and othleukemia . For exaem cells . Recentlem cells .Cyclocarya paliurus  Iljinsk. is endemic to China. The leaves of C. paliurus have long been used in traditional Chinese medicine for their anti-diabetic, anti-bacterial, and anti-cancer properties  have been shown to significantly lower blood sugar, blood fat, and blood pressure, as well as to improve immunity . A portion of leaves were frozen in liquid nitrogen immediately after harvest and transferred to a −80°C freezer until metabolomic analysis. The remaining leaves were dried at 70°C to a constant weight and used for the extraction of total flavonoids.Leaves of 2O and the solution was extracted with 250 mL ethyl acetate for six times. The ethyl acetate extracts were further subjected to polyamide macroporous adsorption resin and the CFTs were collected for downstream in vitro experiments. The total flavonoids content were determined by AlCl3 colorimetry according to the method described by Dried leaves were ground and sieved through a 40-mesh sieve. Powders were ultrasonic soaked in 70% ethanol  for 30 min and filtered with filter paper. This process was repeated a total of three times, and supernatants were combined and concentrated in a vacuum rotary evaporator at 70°C. About 400 g extract was suspended in 1 L H2 at 37°C in a cell culture incubator . Media was replaced at 2-day intervals.HepG2 cells were purchased from the National Collection of Authenticated Cell Cultures, Shanghai, China and STR DNA typing  was useds–OD690s)/(OD5700–OD6900) × 100%, where OD570s and OD690s were absorbance of the tested sample at 570 nm and 690 nm, respectively, and OD5700 and OD6900 were absorbance of the negative control at 570 nm and 690 nm, respectively. Inhibition Rate = (1- Relative Cell Activity) × 100%.Cellular proliferation was assessed using the MTT Cell Proliferation and Cytotoxicity Assay Kit . HepG2 cells were cultured in 96-well plates for 24 h and treated with 0 mg/L, 1.51 mg/L, 3.02 mg/L, 7.55 mg/L, 15.1 mg/L, 30.2 mg/L, 45.3 mg/L, and 90.6 mg/L of CTFs (resolved in DMSO) and incubated for another 48 h. All treatment concentrations, including the control 0 mg/L, contained equal amounts of DMSO. Media was then replaced with fresh media that had been mixed with 10% (V/V) MTT (5 mg/mL). Wells were incubated with formazan solvent for an hour at 37°C before absorbance was detected at OD570 and OD690 using a Multimode Microplate Reader . Relative Cell Activity and the Inhibition Rate of CTFs were calculated as follows. Relative Cell Activity = , 7-AAD (2 μL), and CytoCalcein Violet 450 (2 μL) at room temperature for 20 min in the dark, according to the manufacture’s instructions. Events in the upper left corner of the figures, marked Q1, represent dead cells and naked nuclei. Events in the upper right corner, marked Q2, represent late-stage apoptotic and necrotic cells. Events in the lower left corner, marked Q3, represent normal cells. Events in the lower right corner, marked Q4, represent early-stage apoptotic cells. Apoptotic rate was calculated by summing the percentages of Q2 + Q4. As for Confocal Laser Scanning Microscope (CLSM) analysis , HepG2 cells were cultured in 4-well glass bottom plates specially designed for CLSM applications . Treatment with CTFs and cell staining with the Apoptotic and Necrotic Detection Kit, which was performed as described above.Cellular apoptosis was assessed using the Apoptotic and Necrotic Detection Kit Triple Fluorescence Color . As for flow cytometric analysis , HepG2 cells were cultured in 12-well plates for 48 h and then incubated with 0 mg/L, 7.55 mg/L, 15.1 mg/L, and 30.2 mg/L of CTFs (resolved in DMSO) for 24 h. Next, cells were collected, centrifuged at 300 × g for 5 min, and washed twice with PBS at room temperature. Cells were resuspended with 400 μL 1 × assay buffer at the concentration of 1 × 10C. paliurus were freeze-dried and extracted for secondary metabolite analysis by HPLC/MS/MS and its accompanying self-built database by Metware Biotechnology Co., Ltd. . Among 188 flaovnoids in the leaves of C. paliurus, a total of 114 CTFs were verified to have clear chemical structures which were obtained from Pubchem databases (https://pubchem.ncbi.nlm.nih.gov/). If chemical structures could not be found there, original research articles were reviewed and the structures were drawn by ChemDraw 16.0 software (https://www.chemdraw.com.cn/xiazai html).The flavonoids used for network pharmacology analysis in this study were collected from our previous work . Brieflyhttp://www.swissadme.ch/) to screen compounds used in the present work based on Lipinski’s Rule of Five. Targets of these filtered compounds were then predicted using SwissTargetPrediction (http://www.swisstargetprediction.ch/). Duplicates were deleted after combining the targets of each component, and potential targets were obtained through network topology analysis.CTF structures were saved in SMILES format and searched in SwissADME database  with the following keywords: “Hepatocellular Carcinoma.” The results were summarized and sorted according to the gene names. Common targets between the disease database and the compound database were indicated as potential targets of CTFs responsible for their therapeutic effects in the context of liver cancer.Relevant targets for the inhibition of liver cancer proliferation were identified by searching the MalaCards database (https://david.ncifcrf.gov/) to perform GO and KEGG pathway enrichment analysis. “OFFICE_GENE_SYMBOL” and “Homo sapiens” were selected. The top 10 enriched GO entries and top 30 enriched KEGG pathways that met the criteria of p < 0.01 were selected and uploaded to the OmicShare (http://www.omicshare.com/tools/) cloud platform for data visualization, where the size of the bubble represents the number of genes in the pathway, and the color of the bubble represents the significance of enrichment.Potential targets were imported into the DAVID 6.8 database  was applied to measure the significance of comparisons between groups after homogeneity of variance was confirmed. Tukey’s The total flavonoids in CTFs was 81.82% ± 0.36%. The results of the MTT experiment showed that the cell activity of HepG2 cells decreased as CTFs concentration increased. When the concentration of CTFs reached 90.6 mg/L, the cell activity of HepG2 cells was only 4.94% relative to the DMSO-only control . The IC5After 24, 48, and 72 h of treatment with CTFs, the inhibition rate of 15.1 mg/L CTFs on HepG2 cells was 28.03%, 57.46%, and 93.46%, respectively , indicatvia induction of cellular apoptosis.After 48 h of treatment with 0 mg/L, 7.55 mg/Ll, 15.1 mg/L, and 30.2 mg/L of CTFs, the apoptosis rate of HepG2 cells was 11.2% ± 1.4%, 26.1% ± 1.9%, 38.4% ± 3.5%, and 76.6% ± 2.8%, respectively, indicating that CTFs could promote the apoptosis of HepG2 cells in a dose-dependent manner . Based oAfter screening, a total of 62 compounds met the Lipinski rules , and a tGO enrichment analysis of target proteins indicate that the numbers of target proteins involved in the biological processes (BP), molecular functions (MF) and cellular components (CC) categories are 203 (74.09%), 49 (17.88%), and 22 (8.03%) . The topvia core target proteins.According to the results of enrichment analysis based on KEGG pathway analysis , the topThe component-target-pathway network was constructed to visualize all interactions between target proteins and anti-liver cancer-related pathways. Based on the previous KEGG enrichment analysis, a component-target-pathway network was generated by connecting compounds, targets and pathways . This neThe network analysis showed that the median value of betweenness centrality and closeness centrality was greater than the median with high degree for a total of 10 targets, including AKT1, MAPK3, PIK3CA, EGFR, MAP2K1, SRC, IGF1R, IKBKB, MET, and MAPK14 . These tExcept for MAPK3, for which the corresponding protein structure cannot be found, the remaining top 10 targets were analyzed by molecular docking to assess their binding capabilities. The docking results showed that all binding energies were lower than −5 kcal/mol, indicating the top 10 active compounds have reasonably good binding abilities to the key targets of liver cancer . The aveMolecular docking results showed that there were six active compounds with binding energies of lower than −9 kcal/mol to one of the top targets. These included luteolin (pme0088) modeled with MAP2K1 and 3-hydroxyflavone (pme3134) modeled with PIK3CA. Three amino acid residues—namely MET-146, ASP-208 and LYS-97—in MAP2K1 were modeled to form hydrogen bonds with luteolin . Similarin vitro. With increasing concentration of both 3-hydroxyflavone and luteolin, the inhibitory effect increased in a dose-dependent manner. When the concentration reached 100 μM, the inhibition rate of 3-hydroxyflavone and luteolin reached 46.71% and 81.49%, respectively . The degree of top 13 active flavonoids were ranged from 133 to 144 in present work. Except for 3-Hydroxyflavone (pme3134), the other top 12 active ingredients had hydroxyl or methoxy on their seven position of the diphenylpropane skeleton, indicating the diphenylpropane skeleton was the main active group of flavonoids for anti-tumor effects were also influenced by substituent groups on the diphenylpropane skeleton. Similar studies focused on the anti-liver cancer effects of the flavonoids were performed in traditional Chinese medicines, such as Peperomia dindygulensis  and MAPK (hsa04010) signaling pathways as key mechanisms for the potential therapeutic effect of CTFs on liver cancer. The PI3K/AKT/mTOR pathway, which has been previously implicated in hepatocellular carcinoma carcinogenesis . InhibitThe second group of these proteins, including EGFR, IGF1R and MET, represent membrane receptors of various growth factors. EGFR is a rational target for cancer therapy because it is commonly expressed at a high level in a variety of solid tumors and it has been implicated in the control of cell survival, proliferation, metastasis, and angiogenesis . A signiThe third group of these proteins, including SRC and IKBKB, represent proteins related with kinases. SRC tyrosine kinases regulate many important mechanisms in both normal and cancerous cells and are overexpressed in a broad range of cancers . IKBKB iin vitro with HepG2 cells. Both 3-hydroxyflavone and luteolin were verified to inhibit the proliferation of HepG2 cells in a dose-dependent manner. When the concentration reached 100 μM, the inhibition rate of 3-hydroxyflavone and luteolin reached 46.71% and 81.49%, respectively, while the apoptosis rate of HepG2 cells was 63.5 ± 2.4 and 86 ± 4.9, respectively. For the first time, both of these compounds were shown here to possess anti-liver cancer activity. Little is currently known about 3-hydroxyflavone, while Luteolin  is one of the most common flavonoids present in edible plants and TCMs , PI3K-Akt signaling pathway (hsa04151), proteoglycans in cancer pathway (hsa05205), microRNAs in cancer pathway (hsa05206), and endocrine resistance pathway (hsa01522) via core target proteins. Both 3-hydroxyflavone and luteolin were verified to inhibit the proliferation of HepG2 cells in vitro by inducing apoptosis. Although our results were obtained by network pharmacology and more experiments should be conducted to make it more confidence, our study provides scientific evidence supporting the use of CTFs for the treatment of liver cancer. Furthermore, we should realized that the low water-solubility of the flavonoids might limit the application of flavonoids in in-vivo cancer treatment, however, it may be overcome by different pharmaceutical methods, such as self-nanoemulsion drug delivery system and phospholipid complex.CTFs have significant inhibitory effects on HepG2