Testing multiplexed anti-ASFV CRISPR-Cas9 in reducing African swine fever virus.

African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies. IMPORTANCE: ASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.

Long-term effects of chronic exposure to electronic cigarette aerosol on the cardiovascular and pulmonary system in mice: A comparative study to cigarette smoke.

Electronic cigarettes (e-cigarettes) have rapidly gained popularity as alternatives to traditional combustible cigarettes. However, their long-term health impact remains uncertain. This study aimed to investigate the effects of chronic exposure to e-cigarette aerosol (ECA) in mice compared to conventional cigarette smoke (CS) exposure. The mice were exposed to air (control), low, medium, or high doses of ECA, or a reference CS dose orally and nasally for eight months. Various cardiovascular and pulmonary assessments have been conducted to determine the biological and prosthetic effects. Histopathological analysis was used to determine structural changes in the heart and lungs. Biological markers associated with fibrosis, inflammation, and oxidative stress were investigated. Cardiac proteomic analysis was applied to reveal the shared and unique protein expression changes in ECA and CS groups, which related to processes such as immune activation, lipid metabolism, and intracellular transport. Overall, chronic exposure to ECA led to adverse cardiovascular and pulmonary effects in mice, although they were less pronounced than those of CS exposure. This study provides evidence that e-cigarettes may be less harmful than combustible cigarettes for the long-term health of the cardiovascular and respiratory systems in mice. However, further human studies are needed to clarify the long-term health risks associated with e-cigarette use.

Role of steroid receptor-associated and regulated protein in tumor progression and progesterone receptor signaling in endometrial cancer.

BACKGROUND: Steroid receptor-associated and regulated protein (SRARP) suppresses tumor progression and modulates steroid receptor signaling by interacting with estrogen receptors and androgen receptors in breast cancer. In endometrial cancer (EC), progesterone receptor (PR) signaling is crucial for responsiveness to progestin therapy. The aim of this study was to investigate the role of SRARP in tumor progression and PR signaling in EC. METHODS: Ribonucleic acid sequencing data from the Cancer Genome Atlas, Clinical Proteomic Tumor Analysis Consortium, and Gene Expression Omnibus were used to analyze the clinical significance of SRARP and its correlation with PR expression in EC. The correlation between SRARP and PR expression was validated in EC samples obtained from Peking University People's Hospital. SRARP function was investigated by lentivirus-mediated overexpression in Ishikawa and HEC-50B cells. Cell Counting Kit-8 assays, cell cycle analyses, wound healing assays, and Transwell assays were used to evaluate cell proliferation, migration, and invasion. Western blotting and quantitative real-time polymerase chain reaction were used to evaluate gene expression. The effects of SRARP on the regulation of PR signaling were determined by co-immunoprecipitation, PR response element (PRE) luciferase reporter assay, and PR downstream gene detection. RESULTS: Higher SRARP expression was significantly associated with better overall survival and disease-free survival and less aggressive EC types. SRARP overexpression suppressed growth, migration, and invasion in EC cells, increased E-cadherin expression, and decreased N-cadherin and Wnt family member 7A ( WNT7A ) expression. SRARP expression was positively correlated with PR expression in EC tissues. In SRARP -overexpressing cells, PR isoform B (PRB) was upregulated and SRARP bound to PRB. Significant increases in PRE-based luciferase activity and expression levels of PR target genes were observed in response to medroxyprogesterone acetate. CONCLUSIONS: This study illustrates that SRARP exerts a tumor-suppressive effect by inhibiting the epithelial-mesenchymal transition via Wnt signaling in EC. In addition, SRARP positively modulates PR expression and interacts with PR to regulate PR downstream target genes.

A mechanism study of DUSP1 in inhibiting malignant progression of endometrial carcinoma by regulating ERK/AP-1 axis and dephosphorylation of EPHA2.

Background: Endometrial carcinoma is one of the most common female malignancies worldwide. Based on our preliminary investigation, DUSP1 was identified as a potential biomarker for endometrial carcinoma prognosis, but its function and mechanism remained unclear. Methods: In this study, genes highly correlated with DUSP1 in endometrial cancer were found through correlation analysis, and the promoter sequence of DUSP1 was analyzed by PROMO program. Next-generation phosphorylation mass spectrometry was used to explore new downstream target proteins and pathways of DUSP1 in endometrial carcinoma. The mRNA and protein expression levels were detected by real-time quantitative PCR, immunohistochemistry and Western blotting. The cell survival and proliferation were analyzed by CCK8 assay, cell apoptosis was analyzed by Annexin-V-APC and PI dual staining assay, and the cell invasion was analyzed by Transwell method. Results: (1) There was a high correlation between the expression of DUSP1 and the genes involved in AP-1 complex and its co-expression network. (2) Promoter sequence analysis predicted that the members of AP-1 complex might be the upstream transcriptional regulators of DUSP1. (3) Transfection experiments proved DUSP1 can inhibit tumor growth and invasion, and promote apoptosis by regulating ERK pathway. (4) The results of phosphorylation mass spectrometry showed that overexpression of DUSP1 mainly dephosphorylated EPHA2 in endometrial carcinoma, and co-immunoprecipitation verified the protein interaction between DUSP1 and EPHA2. (5) Overexpression or knockdown of EPHA2 significantly changed the phosphorylation level of EPHA2. (6) The expression of EPHA2 protein was high in patients with more aggressive endometrial cancer. (7) Using EPHA2 inhibitor could significantly slow down the growth rate of tumor cells. Conclusion: (1) There exists a mutual regulation relationship between DUSP1 and AP-1 co-expression network in endometrial carcinoma. (2) It is reported for the first time that DUSP1 phosphatase acts on the ser899 site of EphA2 in endometrial carcinoma. (3) DUSP1 can inhibit tumor growth and invasion, and promote apoptosis by regulating MAPK pathway through directly dephosphorylating ERK, or by dephosphorylating EPHA2.

Pharmacokinetics and tissue distribution of bleomycin-induced idiopathic pulmonary fibrosis rats treated with cryptotanshinone.

Introduction: Cryptotanshinone(CTS), a compound derived from the root of Salvia miltiorrhiza, has been linked to various of diseases, particularly pulmonary fibrosis. In the current study, we investigated the benefit of CTS on Sprague-Dawley (SD) rats induced by bleomycin (BLM) and established high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods to compare pharmacokinetics and tissue distribution in subsequent normal and modulated SD rats. Methods: The therapeutic effect of CTS on BLM-induced SD rats was evaluated using histopathology, lung function and hydroxyproline content measurement, revealing that CTS significantly improved SD rats induced by BLM. Additionally, a simple, rapid, sensitive and specific HPLC-MS/MS method was developed to determine the pharmacokinetics of various components in rat plasma. Results: Pharmacokinetic studies indicated that CTS was slowly absorbed by oral administration and had low bioavailability and a slow clearance rate. The elimination of pulmonary fibrosis in 28-day rats was slowed down, and the area under the curve was increased compared to the control group. Long-term oral administration of CTS did not accumulate in vivo, but the clearance was slowed down, and the steady-state blood concentration was increased. The tissue distribution study revealed that CTS exposure in the lungs and liver. Discussion: The lung CTS exposure was significantly higher in the model group than in the control group, suggesting that the pathological changes of pulmonary fibrosis were conducive to the lung exposure of CTS and served as the target organ of CTS.

Low-dose AAV-CRISPR-mediated liver-specific knock-in restored hemostasis in neonatal hemophilia B mice with subtle antibody response.

AAV-delivered CRISPR/Cas9 (AAV-CRISPR) has shown promising potentials in preclinical models to efficiently insert therapeutic gene sequences in somatic tissues. However, the AAV input doses required were prohibitively high and posed serious risk of toxicity. Here, we performed AAV-CRISPR mediated homology-independent knock-in at a new target site in mAlb 3'UTR and demonstrated that single dose of AAVs enabled long-term integration and expression of hF9 transgene in both adult and neonatal hemophilia B mice (mF9 -/-), yielding high levels of circulating human Factor IX (hFIX) and stable hemostasis restoration during entire 48-week observation period. Furthermore, we achieved hemostasis correction with a significantly lower AAV dose (2 × 109 vg/neonate and 1 × 1010 vg/adult mouse) through liver-specific gene knock-in using hyperactive hF9R338L variant. The plasma antibodies against Cas9 and AAV in the neonatal mice receiving low-dose AAV-CRISPR were negligible, which lent support to the development of AAV-CRISPR mediated somatic knock-in for treating inherited diseases.

Trypsin inhibitor LH011 inhibited DSS-induced mice colitis via alleviating inflammation and oxidative stress.

Background: Ulcerative colitis (UC) is one type of inflammatory bowel disease, characterized by inflammation with infiltration and activation of macrophages in colonic tissue. LH011 is a trypsin inhibitor with potential anti-inflammatory effect. Purpose: Here, we aim to assay the effects of LH011 on UC and further investigate the potential mechanisms in vitro and in vivo. Methods: Dextran sulfate sodium (DSS, 3.5%, w/v) was used to induce UC, and lipopolysaccharide (LPS) was used to induce inflammation in RAW 264.7 cells. LH011 was administrated to mice in vivo or to RAW 264.7 cells in vitro at different concentrations. The cytokines (IL-1ß, IL-6, and TNF-α) and the changes of NF-κB and Nrf2 pathways were detected. Results: The results showed that LH011 improved DSS-induced mice colitis, including loss of weight, disease activity index (DAI), and colonic pathological damage. In addition, LH011 inhibited the expressions of IL-1ß, IL-6, and TNF-α and strengthened the anti-oxidative capacity. Mechanically, LH011 downregulated the nuclear localization of NF-κB p65 and upregulated the protein expression of Nrf2. Conclusion: These results demonstrated that LH011 alleviated inflammation and oxidative stress during UC by inhibiting TLR4/NF-κB and activating Nrf2/Keap1/HO-1 signaling pathways.

Evolving AAV-delivered therapeutics towards ultimate cures.

Gene therapy has entered a new era after decades-long efforts, where the recombinant adeno-associated virus (AAV) has stood out as the most potent vector for in vivo gene transfer and demonstrated excellent efficacy and safety profiles in numerous preclinical and clinical studies. Since the first AAV-derived therapeutics Glybera was approved by the European Medicines Agency (EMA) in 2012, there is an increasing number of AAV-based gene augmentation therapies that have been developed and tested for treating incurable genetic diseases. In the subsequent years, the United States Food and Drug Administration (FDA) approved two additional AAV gene therapy products, Luxturna and Zolgensma, to be launched into the market. Recent breakthroughs in genome editing tools and the combined use with AAV vectors have introduced new therapeutic modalities using somatic gene editing strategies. The promising outcomes from preclinical studies have prompted the continuous evolution of AAV-delivered therapeutics and broadened the scope of treatment options for untreatable diseases. Here, we describe the clinical updates of AAV gene therapies and the latest development using AAV to deliver the CRISPR components as gene editing therapeutics. We also discuss the major challenges and safety concerns associated with AAV delivery and CRISPR therapeutics, and highlight the recent achievement and toxicity issues reported from clinical applications.

MicroRNA-92a -mediated endothelial to mesenchymal transition controls vein graft neointimal lesion formation.

PURPOSE: Long-term failure of vein grafts due to neointimal hyperplasia remains an important problem in coronary artery bypass graft surgery. Endothelial to mesenchymal transition (EndMT) contributes to vein graft vascular remodeling. However, there is little study on microRNA-mediated EndMT contributions to neointimal formation in vein graft. We hypothesized that microRNA-92a (miR-92a) might play an important role in determining EndMT contributions to neointimal formation. METHODS: miR-92a and EndMT-related proteins detected by qRT-PCR and Western blot in vitro and in vivo. Adeno-associated virus 6 (AAV6) delivery gene therapy was used to inhibit neointimal formation in vivo. The intimal hyperplasia of vein grafts was measured by HE staining, the expression of EndMT-related protein in vein grafts was measured by immunofluorescence. Immunohistochemistry and luciferase assay were used to detect potential targets of miR-92a. RESULTS: The expression of miR-92a was found to be upregulated in neointimal hyperplasic lesions after vein grafting. Using cultured human umbilical vein endothelial cells (HUVECs), we show that TGF-ß1 treatment of HUVECs significantly increased miR-92a expression and induced EndMT, characterized by suppression of endothelial-specific markers (CD31 and VE-cadherin) and an increase in mesenchymal-specific markers (a-SMA and vimentin), while inhibition of miR-92a expression blunted EndMT in cultured HUVECs. Furthermore, AAV6 mediated miR-92a suppression gene therapy effectively resulted in decreased EndMT and less neointimal formation in vein grafts in vivo. We further identified that integrin alpha 5 (ITGA5) is a potential target gene involved in the development of neointima formation in these vein grafts. CONCLUSION: This data suggests that neointimal formation does not solely rely on vascular smooth muscle cell phenotypic switching but is also related to EndMT, and miR-92a-mediated EndMT is an important mechanism underlying neointimal formation in vein grafts.

Calcium and TRPV4 promote metastasis by regulating cytoskeleton through the RhoA/ROCK1 pathway in endometrial cancer.

Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable cation channel that has been associated with several types of cancer. However, its biological significance, as well as its related mechanism in endometrial cancer (EC) still remains elusive. In this study, we examined the function of calcium in EC, with a specific focus on TRPV4 and its downstream pathway. We reported here on the findings that a high level of serum ionized calcium was significantly correlated with advanced EC progression, and among all the calcium channels, TRPV4 played an essential role, with high levels of TRPV4 expression associated with cancer progression both in vitro and in vivo. Proteomic and bioinformatics analysis revealed that TRPV4 was involved in cytoskeleton regulation and Rho protein pathway, which regulated EC cell migration. Mechanistic investigation demonstrated that TRPV4 and calcium influx acted on the cytoskeleton via the RhoA/ROCK1 pathway, ending with LIMK/cofilin activation, which had an impact on F-actin and paxillin (PXN) levels. Overall, our findings indicated that ionized serum calcium level was significantly associated with poor outcomes and calcium channel TRPV4 should be targeted to improve therapeutic and preventive strategies in EC.

Gelsolin Promotes Cancer Progression by Regulating Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma and Correlates with a Poor Prognosis.

Gelsolin (GSN), a cytoskeletal protein, is frequently overexpressed in different cancers and promotes cell motility. The biological function of GSN in hepatocellular carcinoma (HCC) and its mechanism remain unclear. The expression of GSN was assessed in a cohort of 188 HCC patients. The effects of GSN on the migration and invasion of tumour cells were examined. Then, the role of GSN in tumour growth in vivo was determined by using a cancer metastasis assay. The possible mechanism by which GSN promotes HCC progression was explored. As a result, GSN was overexpressed in HCC tissues. High GSN expression was significantly correlated with late Edmondson grade, encapsulation, and multiple tumours. Patients with high GSN expression had worse overall survival (OS) and disease-free survival (DFS) than those with low GSN expression. GSN expression was identified as an independent risk factor in both OS (hazard risk (HR) = 1.620, 95% confidence interval (CI) = 1.105-2.373, P < 0.001) and DFS (HR = 1.744, 95% CI = 1.205-2.523, P=0.003). Moreover, GSN knockdown significantly inhibited the migration and invasion of HCC tumour cells, while GSN overexpression attenuated these effects by regulating epithelial-mesenchymal transition (EMT) In conclusion, GSN promotes cancer progression and is associated with a poor prognosis in HCC patients. GSN promotes HCC progression by regulating EMT.

Deregulation of cell adhesion molecules is associated with progression and poor outcomes in endometrial cancer: Analysis of The Cancer Genome Atlas data.

Cell adhesion molecules (CAMs) determine the behavior of cancer cells during metastasis. Although some CAMs are dysregulated in certain types of cancer and are associated with cancer progression, to the best of our knowledge, a comprehensive study of CAMs has not been undertaken, particularly in endometrial cancer (EC). In the present study the expression of 225 CAMs in EC patients with various clinicopathological phenotypes were evaluated by statistical analysis using publicly available data from The Cancer Genome Atlas database. The Kaplan-Meier method, and univariate and multivariate Cox proportional hazards regression models were used for survival analyses. Among the differentially expressed CAMs that were associated with aggressive clinicopathological phenotypes, 10 CAM genes were independent prognostic factors compared with other clinicopathological prognostic factors, including stage, grade, age, lymph node status, peritoneal cytology and histological subtype. A total of six genes (L1 cell adhesion molecule, mucin 15, cell surface associated, cell adhesion associated, oncogene regulated, immunoglobulin superfamily member 9B, protocadherin 9 and protocadherin ß1) were selected for integrative analysis. The six-gene signature was demonstrated to be an independent prognostic factor and could effectively stratify patients with different risks. Patients with more high-expression CAMs had a higher risk of poor overall survival (OS) rate. The mortality risk for patients with elevation of >4 CAMs was 11 times of that in those without elevation of these 6 CAMs. Similar results were obtained when relapse-free survival (RFS) time was used during the analysis. Prognostic reliability of the six-gene model was validated using data of an independent cohort from the International Cancer Genome Consortium. In conclusion, a combination of CAM alterations contributed to progression and aggressiveness of EC. The six-gene signature was effective for predicting worse OS and RFS in patients with EC and could be complementary to the present clinical prognostic criteria.

Responses of AOA and AOB activity and DNA/cDNA community structure to allylthiourea exposure in the water level fluctuation zone soil.

Ammonia oxidation is mainly performed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Allylthiourea (ATU) has been found to specifically inhibit ammonia oxidation. However, the effect of ATU on AOA and AOB transcription has been infrequently studied. In the present study, we examined the responses of AOA and AOB activity and DNA/cDNA community structure to ATU exposure. The ammonia oxidation activity in the 100-mg/L ATU group was 4.3% of that in the control group after 7 days. When exposed to ATU, the gene abundance of AOA was favored compared with that of AOB, and there were no statistically significant differences in the abundance of AOB amoA in DNA and cDNA between the two groups. Compared with the control group, the gene abundance of AOA significantly increased by 5.23 times, while the transcription of AOA significantly decreased by 0.70 times. Moreover, the transcriptional ratio of AOA in the ATU group was only 0.05 times as high as that in the control group. ATU selectively affected AOB and completely inhibited Nitrosomonas europaea and Bacterium amoA.22.HaldeII.kultur at the genetic level. Under ATU exposure, all AOA clusters were transcribed, but three AOB clusters were not transcribed. Our results indicated that the ammonia oxidation potential of the soil of water level fluctuation areas, based on ATU inhibition, was associated mainly with AOA amoA gene abundance and AOB community shifts in DNA and cDNA.

The abundance and community structure of active ammonia-oxidizing archaea and ammonia-oxidizing bacteria shape their activities and contributions in coastal wetlands.

Aerobic ammonia oxidation, an important part of the global nitrogen cycle, is thought to be jointly driven by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in coastal wetlands. However, the activities and contributions of AOA and AOB in coastal wetlands have remained largely unknown. Here, we investigated the oxidation capability of AOA and AOB in four types of typical coastal wetlands (paddy, estuary, shallow and reed wetland) in the Bohai region in China using DNA-based stable-isotope probing (DNA-SIP), quantitative PCR and high-throughput sequencing techniques. We found that the community structure of AOB varied substantially, and the AOA structure was more stable across different coastal wetlands. The rate of AOA was 0.12, 0.84, 0.45 and 0.93 µg N g-1 soil d-1 in paddy, estuary, shallow and reed wetlands, and the rate of AOB was 5.61, 10.72, 0.74 and 1.16 µg N g-1 soil d-1, respectively. We found that the contribution of AOA gradually increased from paddy to estuary to shallow wetland and finally to reed wetland, with values of 2.03%, 7.25%, 37.53% and 44.51%, respectively. Our results provide new insight into the mechanisms of the differences in activities and the contributions of AOA and AOB in different coastal wetlands, and our findings may contribute to further understanding of the global nitrogen cycle.

The preventive effect of Chinese herbal preparation Xuebijing against hyperactive inflammation after hepato-pancreato-biliary surgery.

BACKGROUND: Hepato-pancreato-biliary (HPB) surgery is a primary treatment for benign and malignant diseases of the liver, biliary tract, and pancreas. Hyperactive inflammation has been indicated as a critical risk factor of post-operation death after HPB surgery. Xuebijing is an anti-inflammatory intravenous herbal preparation made from traditional Chinese medicines. Emerging evidence has implicated a protective role of Xuebijing against hyperactive inflammation. METHODS: A retrospective cohort study was conducted. We analyzed a total of 638 cases of HPB surgery, including hepatectomy, Whipple's surgery, and surgeries for cholelithiasis, which were divided into a Xuebijing treatment group and a conventional treatment group according to whether they were treated with Xuebijing injection or not. Clinical data related to liver function and inflammation were compared between the two groups after operation, including liver function index, white blood cell (WBC) count, neutrophil percentage (NE%), C-reactive protein (CRP), serum interleukin-6 (IL-6), body temperature, mortality, incidence of adverse reaction, length of postoperative hospital stay, and hospitalization cost. RESULTS: Xuebijing injection was found to decrease the levels of inflammatory markers in the blood significantly, including WBC, NE%, CRP, IL-6, and reduce the incidence of postoperative fever without prolonging in-hospital length or increasing cost compared to the conventional treatment group. Moreover, our data demonstrated that Xuebijing injection did not impact liver function after hepatectomy. CONCLUSIONS: These results suggest that Xuebijing injection alleviates hyperactive inflammation caused by HPB surgery, and support the application of Xuebijing injection as a safe therapeutic approach against hyperactive inflammation in patients with HPB surgery.

Expression and clinical significance of KLK5-8 in endometrial cancer.

Kallikrein-related peptidase (KLK) family is one of the major serine proteases in tumor microenvironment, which plays a crucial role in cancer invasion and metastasis. A number of KLK family members have been found to be upregulated or downregulated in some cancers, and some KLKs may be potential biomarkers for cancers. However, little is known about the role of KLKs in endometrial carcinoma (EC). In this study, we analyzed the mRNA sequencing data of EC from The Cancer Genome Atlas (TCGA) public database and found that the higher expression of KLK family members 5-8 (KLK5-8) was associated with an aggressive clinicopathologic phenotype and worse prognosis in EC patients. High expression of KLK5-8 was also confirmed in our patients with advanced stage and high-grade EC, as well as in a highly invasive cell line. Our study also demonstrated the differences between the subcellular localization of KLK5-8 and the co-expression of different splicing variants of KLK5-8 in EC cells, suggesting that various isoforms of KLK5-8 may work synergistically to regulate invasion and migration. We found that the elevation of more KLKs in a patient's sample indicated a higher risk of worse survival. Combination of KLK5-8 was shown to be an independent prognostic factor for overall survival by multivariate Cox regression (hazard ratio: 2.215, 95% confidence interval: 1.045-4.694, P=0.038), and may be a promising biomarker.

Predicting prognosis of endometrioid endometrial adenocarcinoma on the basis of gene expression and clinical features using Random Forest.

Traditional clinical features are not sufficient to accurately judge the prognosis of endometrioid endometrial adenocarcinoma (EEA). Molecular biological characteristics and traditional clinical features are particularly important in the prognosis of EEA. The aim of the present study was to establish a predictive model that considers genes and clinical features for the prognosis of EEA. The clinical and RNA sequencing expression data of EEA were derived from samples from The Cancer Genome Atlas (TCGA) and Peking University People's Hospital (PKUPH; Beijing, China). Samples from TCGA were used as the training set, and samples from the PKUPH were used as the testing set. Variable selection using Random Forests (VSURF) was used to select the genes and clinical features on the basis of TCGA samples. The RF classification method was used to establish the prediction model. Kaplan-Meier curves were tested with the log-rank test. The results from this study demonstrated that on the basis of TCGA samples, 11 genes and the grade were selected as the input features. In the training set, the out-of-bag (OOB) error of RF model-1, which was established using the '11 genes', was 0.15; the OOB error of RF model-2, which was established using the 'grade', was 0.39; and the OOB error of RF model-3, established using the '11 genes and grade', was 0.15. In the testing set, the classification accuracy of RF model-1, model-2 and model-3 was 71.43, 66.67 and 80.95%, respectively. In conclusion, to the best of our knowledge, the VSURF was used to select features relevant to EEA prognosis, and an EEA predictive model combining genes and traditional features was established for the first time in the present study. The prediction accuracy of the RF model on the basis of the 11 genes and grade was markedly higher than that of the RF models established by either the 11 genes or grade alone.

Intra-tumor heterogeneity for endometrial cancer and its clinical significance.

BACKGROUND: Management of tumors has become more complex owing to tumor heterogeneity. Fewer studies have been performed on intra-tumor heterogeneity of endometrial cancer (EC) until now. Therefore, it is of great clinical value to explore the intra-tumor heterogeneity of EC based on clinical features and gene expression profiles. METHODS: A total of 1688 patients with EC were screened and 114 patients were finally selected, including specimens from 84 patients with primary EC without relapse (PE) and the paired metastases (P-M) specimens, as well as specimens from 30 patients with primary EC with relapse (RPE) and the paired relapsed EC (P-RE) specimens. Microarray and RNA-seq were used to detect gene expression of EC samples. Clinicopathological characteristics and molecular data were compared between PE and P-M groups and between RPE and P-RE groups to explore the intra-tumor heterogeneity of EC. RESULTS: The clinical intra-tumor spatial heterogeneity of pathological type, grade, ER status, and PR status between PE and P-M were 17.9%, 13.1%, 28.6%, and 28.6%, respectively. The clinical intra-tumor spatiotemporal heterogeneity of pathological type, grade, ER status, and PR status between RPE and P-RE were 16.7%, 33.3%, 25.0%, and 37.5%, respectively. Cluster analysis sorts EC samples based on progression type of lesion and their pathological type. There were differentially expressed genes between PE and P-M and between RPE and P-RE, of which gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis were mainly enriched in cell proliferation, the p53 signaling pathway, etc. CONCLUSIONS:: Clinical and molecular data showed that there was spatiotemporal heterogeneity in intra-tumor of EC, which may add to the complexity of diagnosis and therapeutics for EC. Considering the intra-tumor heterogeneity, sequential chemotherapy and precision medicine may be a more suitable treatment plan for EC.

Homology-independent multiallelic disruption via CRISPR/Cas9-based knock-in yields distinct functional outcomes in human cells.

BACKGROUND: Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes. RESULTS: Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1-/- cells and attenuated cytokine-induced cell death in FAT10-/- clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones. CONCLUSION: Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.

New Turns for High Efficiency Knock-In of Large DNA in Human Pluripotent Stem Cells.

The groundbreaking CRISPR technology is revolutionizing biomedical research with its superior simplicity, high efficiency, and robust accuracy. Recent technological advances by a coupling CRISPR system with various DNA repair mechanisms have further opened up new opportunities to overcome existing challenges in knocking-in foreign DNA in human pluripotent stem cells, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). In this review, we summarized the very recent development of CRISPR-based knock-in strategies and discussed the results obtained as well as potential applications in human ESC and iPSC.