The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.
Trends in faculty demographic composition, promotion success, and retention are important considerations in Academic Health Centers (AHC). This paper reviews the design, implementation, and utility of a faculty promotion and tenure (P&T) database (PROMO/TE©) over 12 years in a large southwestern academic health center. Review of the system design, portfolio creation, P&T tracking, interface with other faculty databases, and lessons learned will be offered. PROMO/TE© was developed to improve the P&T packet creation, application, and review process in one College and was expanded to other colleges at the AHC. The PROMO/TE© system is integrated with Workday® and FACFACTS© to track trends in recruitment, attrition, and P&T trends across gender, underrepresented minorities, and other subgroups. PROMO/TE© has several advantages including improving communication, transparency, uniformity, and efficiency in the P&T packet creation, application, and review process. Increased cost savings ($217,198 annually) were noted with elimination of hard copy packets and decreased time spent. The first college reviewed 743 dossiers in the PROMO/TE© system since its creation in 2012 and there has been on average a 10% increase in P&T approvals since its inception. PROMO/TE© facilitates and tracks trends in the P&T process and has many benefits as well as significant cost savings. PROMO/TE© serves as a potential model for other institutions.
Heated tobacco products (HTPs) are readily available at diverse points of sale (POS) in Egypt. This study aims to assess these advertisements and promotions to provide evidence for policymakers on the need for tobacco control law amendments and enforcement in Egypt. A cross-sectional descriptive study was conducted in Cairo and Giza governorates in 2022 through a convenience sample to collect data from 150 POS. The study's data collection tools assessed the availability, display, advertisement, and promotion of HTP at each site. Price promotions were available at 18% of the visited sites, ranging between bundles and promo code discounts; 75% of the points of sale had some type of advertisement, either inside (67.3%) or outside (36%), stating that HTP are less harmful than traditional cigarettes because they do not burn. HTP display was commonly around the cashier area (87.3%), followed by candy and gum (80.7%) or soda, ice cream, or coffee machines (66%). The reported advertisement and promotion of HTP at POS and their sale to minors violate the National Tobacco Control Law 52/1981. These violations risk the health of the youth. We call on policymakers to explicitly ban all sorts of advertisement and promotion of tobacco products at POS, and enforce the ban of sale to minors (under 18 years).
This study was targeted at investigating the biological functions of E74-like ETS transcription factor 1 (ELF1) in pancreatic cancer (PC) and its underlying mechanism. ELF1 expression in PC tissues was detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. Cell counting kit-8 (CCK-8) method, EdU method and flow cytometry were used to detect the cell proliferation and apoptosis of PC cell lines after transfection. A subcutaneous tumorigenesis model was constructed to validate the oncogenic role of ELF1 in vivo. PROMO database was used to predict the binding site of ELF1 on the promoter region of doublecortin-like kinase 1 (DCLK1). Dual-luciferase reporter gene assay, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assay and quantitative real-time PCR were performed to detect the binding of ELF1 to the promoter region of DCLK1. The effect of ELF1 on DCLK1 expression was detected by Western blot assay. It was found that ELF1 expression in PC tissues and cells was up-regulated. ELF1 overexpression promoted the proliferation and inhibited the apoptosis of PC cells, while knocking down ELF1 had the opposite effects. ELF1 could bind to the promoter region of DCLK1 and ELF1 overexpression promoted the expression of DCLK1. Bioinformatics analysis suggested that Janus kinase (JAK) - signal transducer and activator of transcription (STAT) signaling pathway was associated to DCLK1 expression, and overexpression of ELF1 promoted the expression of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). In conclusion, ELF1 promoted the malignant progression of PC via regulating DCLK1/ JAK/STAT signaling pathway.
To explore the biological function of RELA proto-oncogene, NF-kB subunit (RELA) in hepatocellular carcinoma (HCC) progression, and its potential regulatory effects on the regulators of m6A modification. GEPIA, UALCAN and Human Protein Atlas databases were applied to analyze the expression characteristics of RELA in HCC tissues and non-cancer liver tissues, and its relationship with clinicopathologic indicators and prognosis. Quantitative real-time PCR (qRT-PCR) was used to examine the expression level of RELA mRNA in HCC cells. Cell counting kit-8 (CCK-8) assay, EdU assay and flow cytometry were used to examine cell growth and apoptosis. PROMO database was applied to predict the binding sequence between RELA and methyltransferase like protein 3 (METTL3) promoter region, and this prediction was verified by dual luciferase reporter gene experiment and chromatin immunoprecipitation assay. The effect of RELA on METTL3 expression was examined by Western blot and qRT-PCT, and the regulatory effects of RELA on the other m6A regulators were evaluated by qRT-PCR. RELA was highly expressed in HCC tissues and cell lines, and was closely associated with adverse clinicopathologic indicators and poor prognosis of patients. Overexpression of RELA promoted the growth of HCC cells and inhibited apoptosis; Knocking down RELA had the opposite effects. Overexpression of RELA promoted METTL3 transcription. Knockdown or overexpression of METTL3 reversed the effects of overexpression or knockdown of RELA on HCC cell growth and apoptosis, respectively. RELA also promoted the expression of a series of m6A regulators at mRNA expression level in HCC cell lines. RELA promotes the transcription of METTL3 by binding to METTL3 promoter region, thus promoting the malignancy of HCC cells. This study suggests NF-κB signaling contributes the dysregulation of m6A modification in HCC tumorigenesis.
The present work was performed to clarify the role of TATA-binding protein (TBP) in hepatocellular carcinoma (HCC). TBP expression in adjacent liver tissues and HCC tissue sample was detected by immunohistochemistry and qRT-PCR. With CCK-8, BrdU, flow cytometry, and transwell assays, the malignancy of cancer cell lines were evaluated. The binding sites of TBP and AKT serine/threonine kinase 3 (Akt3) promoter region were predicted by PROMO database, and the binding relationship between TBP and AKT3 promoter was verified with dual luciferase reporter gene assay and ChIP-qPCR assay. The effect of TBP on AKT3 expression was examined by immunoblotting. The signaling pathways associated with AKT3 were predicted by gene set enrichment analysis (GSEA) with LinkedOmics database. It was revealed that, TBP expression in HCC tissues and cell lines was up-regulated, which was associated with the short survival time of patients. Up-regulation of TBP promoted the viability and aggressiveness of HCC cells, while knockdown of TBP had opposite effects. TBP could bind with AKT3 promoter region, and TBP overexpression promoted the expression of AKT3, while its knockdown worked oppositely. Additionally, TBP/AKT3 axis modulated mTOR expression in HCC cells. In conclusion, TBP promotes the transcription of AKT3, thus accelerating the malignant progression of HCC.
Elevated evidence suggests that KIF20A plays an important role in hepatocellular carcinoma (HCC) progression. Nevertheless, the underlying mechanism by which KIF20A promotes HCC cell growth are not well understood. Using TCGA-LIHC RNAseq and GEO datasets, we assessed the KIF20A expression and patient survival in HCC and hepatitis B virus (HBV)-related HCC. Mutant and CNV analysis were performed to evaluate the genetic alteration of KIF20A in HCC. PPI network and GSEA enrichment was utilized for analyzing the KIF20A-related genes and involved pathways in HCC. To further explore regulatory mechanism in HBV-related HCC, PROMO prediction and luciferase reporter system was utilized for verifying HBx/GATA2/KIF20A binding sites. CCK-8 and flow cytometry were carried out to determine the regulation of GATA2-KIF20A on HBV-related HCC cell proliferation and apoptosis. KIF20A was significantly upregulated in pan-cancer (including HCC). KIF20A mRNA level was a significant independent predictor of overall survival in HBV-related HCC patients. Genetic alterations analysis revealed the copy number gain and amplification triggered KIF20A upregulation in HCC. In addition, the genes associated with KIF20A expression in HCC was enriched in PLK1 pathway and cell cycle in HCC. HBx might indirectly binds to KIF20A promoter via regulating GATA2. Additionally, transcription factor GATA2 directly binds to the promoter region of KIF20A. Overexpression of GATA2 promotes HepG2.2.15 cell growth and inhibits cell apoptosis via modulating KIF20A. Our findings demonstrated that HBx contributed to cell proliferation by interacting with GATA2 and KIF20A in HBV-related HCC.
This study aimed to describe and evaluate a standardised workflow for cone beam computed tomography (CBCT)-guided, motion-managed intensity-modulated proton therapy (IMPT) in non-small cell lung cancer (NSCLC), integrating four-dimensional (4D) robust optimisation, surface-guided setup verification, and a CBCT-triaged quality assurance CT (QACT) pathway for adaptive replanning (ARP). This retrospective study included 32 consecutive patients with biopsy-proven NSCLC (stages I-III and oligometastatic, oligoprogressive, or recurrent disease treated with curative intent) who underwent CBCT-guided IMPT at our centre. Patients were treated either in free breathing (FB), breath hold (BH), or FB with compression belt (CB), depending on tumour excursion on 4DCT scan. QACTs were obtained when CBCT showed anatomic or beam path changes (triggered QACT [T-QACT]) or at periodic intervals (periodic QACT [P-QACT]). Adverse events were graded per Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Most patients (53%) had stage III disease and were predominantly treated with conventionally fractionated IMPT. With a median follow-up of 28 months (3-70), 2-year local control, progression-free survival, and overall survival were 84.2%, 70.6%, and 73.9%, for the entire cohort and 94.4%, 78.5%, and 83.1% for nonmetastatic cohort, respectively. Local control (LC) was significantly better in the nonmetastatic cohort than in the metastatic/recurrent cohort (P = 0.017). ARP was required in 50% of patients, with 81% initiated within the first 3 weeks. Motion management included BH in 53%, FB in 28%, and FB + CB in 19%. Failures were predominantly distant (97%), with no marginal recurrences. Among 32 T-QACTs, 47% triggered replanning, whereas only 1.8% of P-QACTs led to changes, demonstrating redundancy of routine periodic scans. Acute grade 2 oesophagitis occurred in 12%; one patient (3%) died of neutropenic sepsis after 4 weeks of chemoradiation. Late grade 3 oesophagitis (3%) and airway complications (6%) were infrequent, with no reported grade 3 cardiac adverse events. CBCT-guided, 4D robustly optimised IMPT was feasible, safe, and effective, providing a reproducible ARP workflow for NSCLC.
Globally, gastric cancer (GC) is a major cause of cancer death. This study is aimed at investigating the biological functions of activating transcription factor 2 (ATF2) and the underlying mechanism in GC. In the present work, GEPIA, UALCAN, Human Protein Atlas and StarBase databases were adopted to analyze ATF2 expression characteristics in GC tissues and normal gastric tissues, and its relationships with tumor grade and patients' survival time. Quantitative real-time polymerase chain reaction (qRT-PCR) method was employed to examine ATF2 mRNA expression in normal gastric tissues, GC tissues, and GC cell lines. Cell counting kit-8 (CCK-8) and EdU assays were utilized for detecting GC cell proliferation. Cell apoptosis was detected by flow cytometry. PROMO database was applied to predict the binding site of ATF2 with the METTL3 promoter region. The binding relationship between ATF2 and the METTL3 promoter region was verified through dual-luciferase reporter gene assay and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay. Western blot was performed to evaluate the effect of ATF2 on METTL3 expression. METTL3-related signaling pathways were predicted using Gene Set Enrichment Analysis (GSEA) in the LinkedOmics database. It was found that, ATF2 level was elevated in GC tissues and cell lines in comparison with normal tissues and correlated with short patients' survival time. ATF2 overexpression facilitated GC cell growth and suppressed the apoptosis, whereas ATF2 knockdown suppressed GC cell proliferation and facilitated the apoptosis. ATF2 bound to the METTL3 promoter region, and ATF2 overexpression promoted the transcription of METTL3, and ATF2 knockdown restrained the transcription of METTL3. METTL3 was associated with cell cycle progression, and ATF2 overexpression enhanced cyclin D1 expression, and METTL3 knockdown reduced cyclin D1 expression. In summary, ATF2 facilitates GC cell proliferation and suppresses the apoptosis via activating the METTL3/cyclin D1 signaling pathway, and ATF2 is promising to be an anti-drug target for GC.
As an indigestible component of human breast milk, Human Milk Oligosaccharides (HMOs) play an important role as a substrate for the establishing microbiome of the newborn. They have further been shown to have beneficial effects on the immune system, lung and brain development. For preterm infants HMO composition of human breast milk may be of particular relevance since the establishment of a healthy microbiome is challenged by multiple disruptive factors associated with preterm birth, such as cesarean section, hospital environment and perinatal antibiotic exposure. In a previous study it has been proposed that maternal probiotic supplementation during late stages of pregnancy may change the HMO composition in human milk. However, there is currently no study on pregnancies which are threatened to preterm birth. Furthermore, HMO composition has not been investigated in association with clinically relevant outcomes of vulnerable infants including inflammation-mediated diseases such as sepsis, necrotizing enterocolitis (NEC) or chronic lung disease. A randomized controlled intervention study (PROMO = probiotics for human milk oligosaccharides) has been designed to analyze changes in HMO composition of human breast milk after supplementation of probiotics (Lactobacillus acidophilus, Bifidobacterium lactis and Bifidobacterium infantis) in pregnancies at risk for preterm birth. The primary endpoint is HMO composition of 3-fucosyllactose and 3'-sialyllactose in expressed breast milk. We estimate that probiotic intervention will increase these two HMO levels by 50% according to the standardized mean difference between treatment and control groups. As secondary outcomes we will measure preterm infants' clinical outcomes (preterm birth, sepsis, weight gain growth, gastrointestinal complications) and effects on microbiome composition in the rectovaginal tract of mothers at delivery and in the gut of term and preterm infants by sequencing at high genomic resolution. Therefore, we will longitudinally collect bio samples in the first 4 weeks after birth as well as in follow-up investigations at 3 months, one year, and five years of age. We estimate that probiotic intervention will increase these two HMO levels by 50% according to the standardized mean difference between treatment and control groups. The PROMO study will gain insight into the microbiome-HMO interaction at the fetomaternal interface and its consequences for duration of pregnancy and outcome of infants.
Hallux valgus deformity consists of a lateral deviation of the great toe, metatarsus varus, and pronation of the first metatarsal. Most osteotomies only correct varus, but not the pronation of the metatarsal. Persistent postoperative pronation has been shown to increase deformity recurrence and have worse functional outcomes. The proximal rotational metatarsal osteotomy (PROMO) technique reliably corrects pronation and varus through a stable osteotomy, avoiding fusing any healthy joints. The objective of this research is to show a prospective series of the PROMO technique. Twenty-five patients (30 feet) were operated with the PROMO technique. The sample included 22 women and 3 men, average age 46 years (range 22-59), for a mean prospective follow-up of 1 year (range 9-14 months). Inclusion criteria included symptomatic hallux valgus deformities, absence of severe joint arthritis, or inflammatory arthropathies, with a metatarsal malrotation of 10 degrees or more, with no tarsometatarsal subluxation or arthritis on the anteroposterior or lateral foot radiograph views. The mean preoperative and postoperative Lower Extremity Functional Scale (LEFS) score, metatarsophalangeal angle, intermetatarsal angle, metatarsal malrotation, complications, satisfaction, and recurrence were recorded. The mean preoperative and postoperative LEFS scores were 56 and 73. The median pre-/postoperative metatarsophalangeal angle was 32.5/4 degrees and the intermetatarsal angle 15.5/5 degrees. The metatarsal rotation was satisfactorily corrected in 24 of 25 patients. An Akin osteotomy was needed in 27 of 30 feet. All patients were satisfied with the surgery, and no recurrence or complications were found. PROMO is a reliable technique, with good short-term results in terms of angular correction, satisfaction, and recurrence. Long-term studies are needed to determine if a lower hallux recurrence rate occurs with the correction of metatarsal rotation in comparison with conventional osteotomies. IV, prospective case series.
Diabetic kidney disease (DKD) is a complication caused by end-stage diabetes mellitus and usually results in glomerular podocyte injury. Exosomes are important for intercellular information exchange. However, the effect of podocyte exosomes on DKD has not been elucidated. GEO, PROMO, and GSE1009 databases were used to identify the gene APOC1 and transcription factor HOXD9. qRT-PCR, western blot, and transmission electron microscopy (TEM) were investigated to confirm APOC1 change in high glucose-treated podocytes and exosomes. Flow cytometry, immunofluorescence, qPCR, immunoblotting, wound healing, Transwell invasion assays, dual luciferase assay, and ChIP-PCR assay were performed to detect the effect of APOC1 and HOXD9 on macrophage polarization in high glucose-treated podocytes and exosomes. qRT-PCR and immunoblotting assays were employed to assess the impact of APOC1 knockdown on the M1 polarization of macrophages in response to liraglutide treatment. The results suggested that the expression of APOC1 in human podocytes (HPC) and exosomes was elevated. High glucose-treated HPC exosomes promoted macrophage M1-type polarization, which was reversed by adding sh-APOC1. Afterward, HOXD9 was identified as a potential transcription factor for APOC1. Knockdown of HOXD9 led to macrophage M2 polarization, and overexpression of APOC1 polarized macrophage M1. In addition, enhanced p65 phosphorylation verified that HOXD9/APOC1 induced macrophage M1-type polarization by activating the NF-κB signaling pathway. Knocking down APOC1 enhanced the inhibitory effect of liraglutide on macrophage M1 polarization. Our findings highlighted that HOXD9/APOC1 was a key player in causing podocyte injury in diabetic kidney disease and led to macrophage M1 polarization through the NF-κB signaling pathway.
The human leukocyte antigen (HLA) class Ib molecules HLA-F and HLA-G are implicated in pregnancy success, but how do HLA-G and HLA-F genetic polymorphisms impact recurrent implantation failure (RIF)? Prospective cohort study at a fertility clinic including a cohort of 84 women experiencing RIF and 35 IVF controls to assess the influence of HLA-G haplotypes and diplotypes and HLA-F single nucleotide polymorphisms (SNP) on RIF. Over-representation trends for HLA-F SNP genotypes rs1362126, rs2523405 and rs2523393, previously linked with a short time-to-pregnancy, were detected in female control groups compared with RIF patients with no identified pathology linked to infertility. The HLA-G promoter haplotype PROMO-G010101b/c linked with the HLA-G 3'-untranslated region (3'UTR) haplotype UTR-4, which previously has been associated with positive IVF outcome and pregnancy success, was less frequent in the RIF group. For RIF patients carrying the UTR-4 haplotype, the odds ratio (OR) was 0.27 (95% CI 0.12-0.66; P = 0.0044, Pc = 0.026). The HLA-G PROMO-G010104-UTR-3 haplotype was associated with an increased risk of RIF. For RIF patients carrying the UTR-3 haplotype, the OR was 5.86 (95% CI 1.52-26.23; P = 0.0115, Pc = 0.069). These results show that specific HLA-G haplotypes based on the promoter region and the 3'UTR are either associated with an increased risk of reduced fertility, including the manifestation of RIF, and lower chance of achieving pregnancy, or with a reduced risk of experiencing RIF.
The late diagnosis and easy metastasis of lung adenocarcinoma (LADC) remains a challenge. SGPP2 is reported to modulate cell processes in many cancers. However, the roles and molecular mechanisms of SGPP2 in LADC are unclear. Online bioinformatics tools GEPIA, CPTAC, and K-M plotter were used to analyze the expression of SGPP2 and the prognosis in LADC. JASPAR and PROMO were used to predict the transcription factors of SGPP2. Real-time quantitative reverse transcription PCR and western blot were used to detect the levels of SGPP2 in LADC cell lines and tissues. Cell counting kit-8, colony formation, flow cytometry, and transwell assay were used to detect cell proliferation, apoptosis, and invasion. The anti-cancer effect of SGPP2 silence was evaluated in the LADC xenograft model. It was found that SGPP2 was highly expressed and related to the poor prognosis of LADC patients. Elevated SGPP2 expression was detected in LADC cell lines and tissues. The chi-square test indicated that the expression of SGPP2 was positively related to tumor, node, metastasis grades and lymph node metastasis. Knocking down SGPP2 significantly inhibited LADC cell viability, and invasion, but induced apoptosis. The anti-tumor effects of SGPP2 were verified in vivo. The upstream transcription factor of SGPP2 was predicted to be SP1, which was highly expressed in LADC tissues and cell lines. Overexpression of SP1 partly rescued the inhibition of SGPP2-shRNA in cell growth, colony formation, and invasion capabilities, and decreased apoptotic cell number in LADC cells. This study demonstrated that SGPP2, activated by SP1, promotes LADC cell proliferation and invasion, and suppresses apoptosis in LADC.
Apolipoprotein C3 (APOC3) is known for its important functions in metabolism-related diseases. However, the function and molecular mechanism of APOC3 in polycystic ovarian syndrome (PCOS) have not been reported. Quantitative polymerase chain reaction and western blot assays were used to detect the expression of APOC3 in KGN cells. Small interference APOC3 (siAPOC3) was applied to reduce APOC3 expression, and the proliferation ability of human granulosa cell line (KGN cells) was measured by cell counting kit-8 and colony formation assays. The protein levels of key genes related to apoptosis were detected by western blot assay. The transcriptional regulator of APOC3 was predicted by the UCSC and PROMO website, and verified by dual luciferase assay. siAPOC3 and pcDNA3.1-specific protein 1 (SP1) vector were co-transfected into KGN cells to detect the function of SP1 and APOC3 in KGN cells. APOC3 was overexpressed in KGN cells, and siAPOC3 transfection significantly reduced the growth ability of KGN cells and increased the apoptosis ability of KGN cells. SP1 directly bound to the promoter of APOC3 and transcriptional regulated APOC3 expression. Overexpression of SP1 increased the growth ability of KGN cells and decreased the apoptosis ability of KGN cells, which were reversed after siAPOC3 transfection. The increased levels of toll-like receptor 2 (TLR2) and p65 phosphorylation (p-P65) nuclear factor kappa B (NF-κB) caused by SP1 overexpression were inhibited by siAPOC3 transfection. APOC3, transcriptionally regulated by SP1, promoted the growth of KGN cells, and inhibited the apoptosis by regulating TLR2/NF-κB signalling pathway.
Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignant tumor with unique geographical and ethnic distribution characteristics. NPC is mostly found in south China and Southeast Asia, and its treatment mainly depends on radiotherapy and chemotherapy. However, NPC is usually found in the late stage, and local recurrence and distant metastasis are common, leading to poor prognosis. The receptor tyrosine kinase AXL is up-regulated in various tumors and it is involved in tumor proliferation, migration, invasion, and other processes, which are associated with poor prognosis of tumors. This study aims to detect the expression of AXL in NPC cell lines and tissues, and to investigate its biological function of AXL and the underlying molecular mechanisms in regulation of NPC. The expression levels of AXL in normal nasopharyngeal epithelial tissues and NPC tissues were analyzed by GSE68799, GSE12452, and GSE53819 data sets based on Gene Expression Omnibus (GEO) database. The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between AXL and prognosis of head and neck squamous cell carcinoma (HNSC). The indicators of prognosis included overall survival (OS), disease-free interval (DFI), disease-specific survival (DSS), and progression-free interval (PFI). Western blotting assay was used to detect the AXL protein expression levels in normal nasopharyngeal epithelial cell line and NPC cell lines. Immunohistochemical method was used to detect AXL expression levels in normal nasopharyngeal epithelial tissues and NPC tissues. Cell lines with stable AXL knockdown were established by infecting 5-8F and Fadu cells with lentivirus interference vector, and cell lines with stable AXL overexpression were established by infecting C666-1 and HK-1 cells with lentivirus expression vector. Real-time PCR and Western blotting were used to detect the efficiency of knockdown and overexpression in stable cell lines. The effects of AXL knockdown or overexpression on proliferation, migration, and invasion of NPC cells were detected by CCK-8, plate colony formation, and Transwell assays, and the effect of AXL knockdown on tumor growth in nude mice was detected by subcutaneous tumor formation assay. The sequence of AXL upstream 2.0 kb promoter region was obtained by UCSC online database. The PROMO online database was used to predict AXL transcription factors with 0% fault tolerance, and the JASPAR online database was used to predict the binding sites of ETS1 to AXL. Real-time PCR and Western blotting were used to detect the effect of ETS1 on AXL protein and mRNA expression. The AXL upstream 2.0 kb promoter region was divided into 8 fragments, each of which was 250 bp in length. Primers were designed for 8 fragments. The binding of ETS1 to AXL promoter region was detected by chromatin immuno-precipitation (ChIP) assay to determine the direct regulatory relationship between ETS1 and AXL. Rescue assay was used to determine whether ETS1 affected the proliferation, migration, and invasion of NPC cells through AXL. Bioinformatics analysis showed that AXL was highly expressed in NPC tissues (P<0.05), and AXL expression was positively correlated with OS, DFI, DSS, and PFI in HNSC patients. Western blotting and immunohistochemical results showed that AXL was highly expressed in NPC cell lines and tissues compared with the normal nasopharyngeal epithelial cell line and tissues. Real-time PCR and Western blotting results showed that knockdown and overexpression efficiency in the stable cell lines met the requirements of subsequent experiments. The results of CCK-8, plate colony formation, Transwell assays and subcutaneous tumor formation in nude mice showed that down-regulation of AXL significantly inhibited the proliferation, migration, invasion of NPC cells and tumor growth (all P<0.05), and the up-regulation of AXL significantly promoted the proliferation, migration, and invasion of NPC cells (all P<0.05).As predicted by PROMO and JASPAR online databases, ETS1 was a transcription factor of AXL and had multiple binding sites in the AXL promoter region. Real-time PCR and Western blotting results showed that knockdown or overexpression of ETS1 down-regulated or up-regulated AXL protein and mRNA expression levels. ChIP assay result showed that ETS1 bound to AXL promoter region and directly regulate AXL expression. Rescue assay showed that AXL rescued the effects of ETS1 on proliferation, migration and invasion of NPC cells (P<0.05). AXL is highly expressed in NPC cell lines and tissues, which can promote the malignant progression of NPC, and its expression is regulated by transcription factor ETS1. 目的: 鼻咽癌(nasopharyngeal carcinoma，NPC)是一种高侵袭性的上皮源性恶性肿瘤，具有独特的地理和种族分布特征，多发于中国南方和东南亚地区，其治疗主要依靠放射治疗和化学治疗。但NPC通常发现于晚期，且常出现局部复发和远处转移，患者预后较差。受体酪氨酸激酶AXL在多种肿瘤中表达上调，参与肿瘤增殖、迁移、侵袭等过程，与肿瘤不良预后相关。本研究旨在检测AXL在NPC细胞系及组织中的表达，探讨AXL在NPC中的生物学功能及表达调控的分子机制。方法: 利用基因表达综合(Gene Expression Omnibus，GEO)数据库中的GSE68799、GSE12452、GSE53819三个数据集分析AXL在正常鼻咽上皮组织及NPC组织中的表达水平，利用癌症基因组图谱(The Cancer Genome Atlas，TCGA)数据库分析AXL与头颈部鳞状细胞癌(head and neck squamous cell carcinoma，HNSC)患者预后的关系，包括总生存时间(overall survival，OS)、无病生存期(disease-free interval，DFI)、疾病特异性生存期(disease-specific survival，DSS)及无进展生存期(progression-free interval，PFI)4个预后指标；采用蛋白质印迹法检测AXL在正常鼻咽上皮细胞系及NPC细胞系中的蛋白质表达水平，免疫组织化学实验检测AXL在正常鼻咽上皮组织及NPC组织中的表达；利用慢病毒干扰载体包装病毒感染5-8F和Fadu细胞建立稳定敲低AXL的细胞系，利用慢病毒表达载体包装病毒感染C666-1和HK-1细胞建立稳定过表达AXL的细胞系，并通过real-time PCR及蛋白质印迹法检测稳转细胞系的干扰和过表达效率；利用CCK-8、平板克隆形成实验及Transwell实验检测敲低或过表达AXL对NPC细胞增殖、迁移及侵袭能力的影响，利用裸鼠皮下成瘤实验检测敲低AXL对裸鼠体内肿瘤生长的影响；利用UCSC在线数据库查询并获取AXL上游2.0 kb启动子区域的碱基序列，将序列输入PROMO在线数据库中以容错率0%为条件预测AXL的转录因子，并利用JASPAR在线数据库预测ETS1与AXL的结合位点；通过real-time PCR及蛋白质印迹法检测ETS1表达改变对AXL蛋白及mRNA表达水平的影响；将AXL上游2.0 kb启动子区域划分为8个片段，每个片段长度为250 bp，分别针对8个片段设计引物，利用染色质免疫沉淀(chromatin immuno-precipitation，ChIP)实验检测ETS1与AXL启动子区域的结合，明确ETS1对AXL的直接调控关系；利用功能回复实验检测ETS1是否通过AXL影响NPC细胞的增殖、迁移及侵袭能力。结果: 生物信息学分析发现AXL在NPC组织中高表达(P<0.05)，且AXL高表达与HNSC患者更短的OS、DFI、DSS及PFI呈正相关。蛋白质印迹及免疫组织化学实验结果显示：相对于正常鼻咽上皮细胞系及组织，AXL在NPC细胞系及组织中高表达。Real-time PCR及蛋白质印迹实验结果显示：稳转细胞系的干扰及过表达效率满足后续实验要求。CCK-8、平板克隆形成实验、Transwell实验及裸鼠皮下成瘤实验结果表明：AXL表达下调显著抑制NPC细胞的增殖、迁移、侵袭及肿瘤生长(均P<0.05)，AXL表达上调显著促进NPC细胞的增殖、迁移及侵袭(均P<0.05)。PROMO及JASPAR在线数据库预测结果显示：ETS1是AXL的转录因子，且与AXL启动子区域存在多个结合位点。Real-time PCR及蛋白质印迹法结果显示：敲低或过表达ETS1能够下调或上调AXL蛋白及mRNA的表达水平。ChIP实验结果显示：ETS1能与AXL的启动子区域结合，直接调控AXL的表达。功能回复实验结果显示：AXL能够回复ETS1对NPC细胞增殖、迁移及侵袭能力的影响(P<0.05)。结论: AXL在NPC细胞系及组织中高表达，能促进NPC的恶性进展，其表达受转录因子ETS1的调控。. Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignant tumor with unique geographical and ethnic distribution characteristics. NPC is mostly found in south China and Southeast Asia, and its treatment mainly depends on radiotherapy and chemotherapy. However, NPC is usually found in the late stage, and local recurrence and distant metastasis are common, leading to poor prognosis. The receptor tyrosine kinase AXL is up-regulated in various tumors and it is involved in tumor proliferation, migration, invasion, and other processes, which are associated with poor prognosis of tumors. This study aims to detect the expression of AXL in NPC cell lines and tissues, and to investigate its biological function of AXL and the underlying molecular mechanisms in regulation of NPC. The expression levels of AXL in normal nasopharyngeal epithelial tissues and NPC tissues were analyzed by GSE68799, GSE12452, and GSE53819 data sets based on Gene Expression Omnibus (GEO) database. The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between AXL and prognosis of head and neck squamous cell carcinoma (HNSC). The indicators of prognosis included overall survival (OS), disease-free interval (DFI), disease-specific survival (DSS), and progression-free interval (PFI). Western blotting assay was used to detect the AXL protein expression levels in normal nasopharyngeal epithelial cell line and NPC cell lines. Immunohistochemical method was used to detect AXL expression levels in normal nasopharyngeal epithelial tissues and NPC tissues. Cell lines with stable AXL knockdown were established by infecting 5-8F and Fadu cells with lentivirus interference vector, and cell lines with stable AXL overexpression were established by infecting C666-1 and HK-1 cells with lentivirus expression vector. Real-time PCR and Western blotting were used to detect the efficiency of knockdown and overexpression in stable cell lines. The effects of AXL knockdown or overexpression on proliferation, migration, and invasion of NPC cells were detected by CCK-8, plate colony formation, and Transwell assays, and the effect of AXL knockdown on tumor growth in nude mice was detected by subcutaneous tumor formation assay. The sequence of AXL upstream 2.0 kb promoter region was obtained by UCSC online database. The PROMO online database was used to predict AXL transcription factors with 0% fault tolerance, and the JASPAR online database was used to predict the binding sites of ETS1 to AXL. Real-time PCR and Western blotting were used to detect the effect of ETS1 on AXL protein and mRNA expression. The AXL upstream 2.0 kb promoter region was divided into 8 fragments, each of which was 250 bp in length. Primers were designed for 8 fragments. The binding of ETS1 to AXL promoter region was detected by chromatin immuno-precipitation (ChIP) assay to determine the direct regulatory relationship between ETS1 and AXL. Rescue assay was used to determine whether ETS1 affected the proliferation, migration, and invasion of NPC cells through AXL. Bioinformatics analysis showed that AXL was highly expressed in NPC tissues (P<0.05), and AXL expression was positively correlated with OS, DFI, DSS, and PFI in HNSC patients. Western blotting and immunohistochemical results showed that AXL was highly expressed in NPC cell lines and tissues compared with the normal nasopharyngeal epithelial cell line and tissues. Real-time PCR and Western blotting results showed that knockdown and overexpression efficiency in the stable cell lines met the requirements of subsequent experiments. The results of CCK-8, plate colony formation, Transwell assays and subcutaneous tumor formation in nude mice showed that down-regulation of AXL significantly inhibited the proliferation, migration, invasion of NPC cells and tumor growth (all P<0.05), and the up-regulation of AXL significantly promoted the proliferation, migration, and invasion of NPC cells (all P<0.05).As predicted by PROMO and JASPAR online databases, ETS1 was a transcription factor of AXL and had multiple binding sites in the AXL promoter region. Real-time PCR and Western blotting results showed that knockdown or overexpression of ETS1 down-regulated or up-regulated AXL protein and mRNA expression levels. ChIP assay result showed that ETS1 bound to AXL promoter region and directly regulate AXL expression. Rescue assay showed that AXL rescued the effects of ETS1 on proliferation, migration and invasion of NPC cells (P<0.05). AXL is highly expressed in NPC cell lines and tissues, which can promote the malignant progression of NPC, and its expression is regulated by transcription factor ETS1.
The doctoral degree regulations at German faculties partially show fundamental differences. Against this background in 2016 the Medical Faculty Association (MFT) published a position paper on teaching scientific competence in medical studies. This study project aims to analyze the quality of doctoral degree regulations in Germany 9 years later. In this study 39 current doctoral degree regulations (PromO) from German medical faculties with the right to award doctorates were analyzed along with the supervision agreements, if available. The standard of the statutes was assessed according to the scoring system of Sorg et al. The mean value of the total score of all doctoral regulations was 68.4 points (SD ± 8.4) and has increased compared to 2016 (57.5 points, SD ± 9.4, p < 0.001). Supervision agreements and cumulative doctorates have been strongly codified since 2016. Methodological training and plagiarism checks have become more mandatory compared to 2016 but are not widespread. The MFT requirement of at least 9 months of research activity was only addressed by 1 medical faculty. Not all doctorates have to be published. The key points of the MFT position paper have not been fully implemented; however, the quality of doctoral regulations in Germany has improved over the last 9 years. HINTERGRUND: Die Promotionsordnungen an deutschen Fakultäten weisen teils grundlegende Unterschiede auf. Vor diesem Hintergrund wurde ein Positionspapier des Medizinischen Fakultätentages (MFT) zur Vermittlung von Wissenschaftskompetenz im Medizinstudium im Jahr 2016 verfasst. Das vorliegende Studienprojekt soll nun 9 Jahre später die Qualität der Promotionsordnungen in Deutschland untersuchen. Es wurden die 39 aktuellen Promotionsordnungen (PromO) aller deutschen medizinischen Fakultäten mit Promotionsrecht herangezogen und analysiert. Zusätzlich wurden die Betreuungsvereinbarungen, falls vorhanden, aus den gleichen Quellen miteinbezogen. Der Anspruch der Satzungen wurde systematisch gemäß dem Scoringsystem nach Sorg et al. bewertet. Der Mittelwert der Gesamtpunktzahl aller Promotionsordnungen beträgt 68,4 Punkte (SD: ± 8,4) und hat sich im Vergleich zu 2016 (57,5 Punkte, SD: ± 9,4) signifikant erhöht (p < 0,001). Die Verpflichtung zum Abschluss einer Betreuungsvereinbarung und die Möglichkeit der kumulativen Promotion wurden seit 2016 stark kodifiziert. Die Einführung in Methodenkenntnisse und die Überprüfung auf Plagiate sind im Vergleich zu 2016 vermehrt vorgeschrieben, aber nicht flächendeckend verbreitet. Die vom MFT geforderten mindestens 9 Monate andauernde Forschungstätigkeit wurde nur von einer medizinischen Fakultät adressiert. Nicht alle Promotionen müssen publiziert werden. Die Kernpunkte des MFT-Positionspapiers wurden nicht vollständig umgesetzt. Die Qualität der Promotionsordnungen in Deutschland hat sich in den letzten 9 Jahren jedoch verbessert.
Approximately one in three people worldwide have been exposed to Toxoplasma gondii (T. gondii). Primary infection with T. gondii during pregnancy can cause severe complications. Our previous study demonstrated that deficiency of triggering receptor expressed on myeloid cells 2 (Trem2) exacerbates pregnancy-related complications in T. gondii-infected mice. However, understanding the mechanisms by which T. gondii modulates Trem2 expression in macrophages remains an unmet challenge. A mouse pregnancy model of T. gondii infection and an in vitro cellular stimulation model using soluble T. gondii antigens (sTgAg) were used to assess Trem2 expression. Recombinant plasmids containing the full-length Trem2 promoter were constructed to evaluate the effect of sTgAg on promoter activity, followed by the construction of truncated promoter plasmids to identify key regulatory regions. Transcription factors potentially binding to the Trem2 promoter were predicted using PROMO and JASPAR, with ATF3 identified as responsive to sTgAg stimulation via western blot analysis. The binding of ATF3 to the Trem2 promoter was validated by chromatin immunoprecipitation (ChIP) assays. Finally, ATF3 knockdown experiments were performed to determine its role in mediating the inhibitory effect of sTgAg on Trem2 expression. T. gondii significantly suppressed Trem2 expression in both mouse placentas and cellular models, with truncated promoter assays identifying key regulatory regions of the Trem2 promoter inhibited by sTgAg. ATF3 was identified as a transcription factor responsive to sTgAg stimulation, which bound to the Trem2 promoter. Importantly, knockdown of ATF3 restored Trem2 expression, demonstrating its critical role in mediating the inhibitory effect of sTgAg. We identified that sTgAg may target and inhibit Trem2 expression through the transcription factor ATF3, and inhibition of ATF3 activity may help maintain Trem2 expression in macrophages, providing a potential therapeutic approach to avert negative effects on pregnancy related to T. gondii infection.
HLA-G is a non-classical HLA class I molecule that promotes transplant tolerance. It engages the inhibitory receptor LILRB1 on immune effector cells, suppressing cytotoxic responses and inflammation, while promoting tolerogenic and regulatory immune phenotypes. Polymorphisms in the HLA-G 3' untranslated region (3'UTR) modulate HLA-G expression levels, and LILRB1 promoter variants influence receptor expression. The combined effect on kidney transplant (KTx) rejection has not been systematically studied. Living donor-recipient pairs undergoing KTx were genotyped for nine variants in the HLA-G 3'UTR region and two single nucleotide polymorphisms (SNPs) in the LILRB1 promoter (PROMO) regions. Haplotypes were arranged for both loci. Clinical endpoints were biopsy-proven T cell-mediated rejection (TCMR) within one year and antibody-mediated rejection (AMR) within five years post-transplant. Donor positivity for HLA-G 3'UTR-1 or UTR-2 or negative for UTR-3 haplotype were associated with a significantly higher risk of TCMR in both univariate or multivariate analyses. Recipients lacking the LILRB1-PROMO CG haplotype also had an increased TCMR risk. The combination of an HLA-G 3'UTR-2 positive donor with a LILRB1-PROMO CG haplotype negative recipient was found to be an independent predictor of TCMR. In contrast, HLA-G 3'UTR variants were not associated with AMR, while the presence of the recipient LILRB1-PROMO CG haplotype emerged as an independent AMR risk factor. Donor HLA-G 3'UTR and recipient LILRB1-PROMO haplotypes define a functional immunogenetic axis that differentially influence TCMR and AMR. These results support the clinical potential of HLA-G/LILRB1 genetic profiling to improve donor selection in living KTx and to guide the development of novel rejection therapies.
Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide. Adipocyte Plasma Membrane Associated Protein (APMAP) is a glycosylated type II transmembrane protein, and APMAP induces malignant metastasis of colorectal cancer. However, the role and mechanism of APMAP in NSCLC remain unknown. We aimed to study the APMAP regulation on NSCLC. APMAP mRNA levels in NSCLC tissues were tested by quantitative reverse transcription-PCR (qRT-PCR). APMAP protein levels in NSCLC tissues and cells were determined via Western blot. Also, the association between APMAP expressions and NSCLC clinicopathology was assessed using Chi-square test. After silencing APMAP (sh-APMAP) in NSCLC cells, APMAP's roles in NSCLC were revealed by Western blot, Cell Counting Kit-8 (CCK-8) assay, Transwell, Fe2+ level analysis, and immunofluorescence assays. Meanwhile, APMAP mechanism in NSCLC was verified through Human TFDB and PROMO databases, qRT-PCR, Western blot, dual-luciferase reporter assay, CCK-8 experiment, Transwell, analysis of Fe2+, reactive oxygen species levels, and Clinical Bioinformatics Home. Also, APMAP function in vivo was examined using a tumor xenograft model, immunohistochemistry assay, and Western blot. APMAP expressions were up-regulated in NSCLC tissues and cells. Moreover, patients with high APMAP expression exhibited a poorer overall survival rate. APMAP expression was closely related to TNM stage, distant metastasis, and tumor differentiation. Functionally, APMAP knockdown repressed NSCLC cell proliferation and invasion, and induced cell ferroptosis. Mechanistically, transcription factor YY1 induced APMAP expression. Meanwhile, APMAP expressions were decreased after interfering with YY1, while APMAP expressions were increased after overexpressing YY1. Also, co-transfection experiments demonstrated that YY1 overexpression enhanced the fluorescence intensity of APMAP-WT promoter. Importantly, YY1 overexpression promoted NSCLC cell proliferation and invasion, and repressed cell ferroptosis, yet these effects were reversed after APMAP knockdown. Moreover, knocking down APMAP reduced NSCLC proliferation in vivo, mainly through reduced tumor volume, reduced KI-67 and GPX4 expressions, and decreased p-pI3K, p-AKT, and p-mTOR protein levels. APMAP regulated by YY1 transcription enhanced NSCLC proliferation, invasion, and repressed cell ferroptosis via activating PI3K/AKT/mTOR.