Kupffer cells abrogate homing and repopulation of allogeneic hepatic progenitors in injured liver site.

BACKGROUND: Allogeneic hepatocyte transplantation is an emerging approach to treat acute liver defects. However, durable engraftment of the transplanted cells remains a daunting task, as they are actively cleared by the recipient's immune system. Therefore, a detailed understanding of the innate or adaptive immune cells-derived responses against allogeneic transplanted hepatic cells is the key to rationalize cell-based therapies. METHODS: Here, we induced an acute inflammatory regenerative niche (3-96 h) on the surface of the liver by the application of cryo-injury (CI) to systematically evaluate the innate immune response against transplanted allogeneic hepatic progenitors in a sustained micro-inflammatory environment. RESULTS: The resulting data highlighted that the injured site was significantly repopulated by alternating numbers of innate immune cells, including neutrophils, monocytes and Kupffer cells (KCs), from 3 to 96 h. The transplanted allo-HPs, engrafted 6 h post-injury, were collectively eliminated by the innate immune response within 24 h of transplantation. Selective depletion of the KCs demonstrated a delayed recruitment of monocytes from day 2 to day 6. In addition, the intrasplenic engraftment of the hepatic progenitors 54 h post-transplantation was dismantled by KCs, while a time-dependent better survival and translocation of the transplanted cells into the injured site could be observed in samples devoid of KCs. CONCLUSION: Overall, this study provides evidence that KCs ablation enables a better survival and integration of allo-HPs in a sustained liver inflammatory environment, having implications for rationalizing the cell-based therapeutic interventions against liver defects.

Pan-Cancer Analysis Reveals CENPI as a Potential Biomarker and Therapeutic Target in Adrenocortical Carcinoma.

Background: Centromere protein I (CENPI) has been shown to affect the tumorigenesis of breast and colorectal cancers. However, its biological role and prognostic value in other kinds of cancer, especially adrenocortical carcinoma (ACC), remained to be further investigated. Methods: Various bioinformatics tools were adopted for exploring the significance of differential expression of CENPI in several malignant tumors from databases such as Depmap portal, GTEx, and TCGA. ACC was selected for further analyzed, and information such as clinicopathological features, the prognostic outcome of diverse subgroups, differentially expressed genes (DEGs), co-expression genes, as well as levels of tumor-infiltrating immune cells (TIIC), was extracted from multiple databases. To verify the possibility of CENPI as a therapeutic target in ACC, drug sensitivity assay and si-RNA mediate knockdown of CENPI were carried out. Results: The pan-cancer analyses showed that the CENPI mRNA expression levels differed significantly among most cancer types. Additionally, a high precision in cancer prediction and close relation with cancer survival indicated that CENPI could be a potential candidate biomarker to diagnose and predict cancer prognosis. In ACC, CENPI was closely related to multiple clinical characteristics, such as pathological stage and primary therapy outcome. High CENPI levels predicted poor overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) of ACC patients, particularly for different clinical subgroups. Moreover, the expression of CENPI showed positive relationship to Th2 cells but negatively related to most of the TIICs. Furthermore, drug sensitivity assay showed that vorinostat inhibit CENPI expression and ACC cell growth. Additionally, si-RNA mediated knockdown of CENPI inhibited ACC cell growth and invasion and showed synergistic anti-proliferation effect with AURKB inhibitor barasertib. Conclusion: Pan-cancer analysis demonstrated that CENPI is a potential diagnostic and prognostic biomarker in various cancers as well as an anti-ACC therapeutic target.

A prognostic model based on prognosis-related ferroptosis genes for patients with acute myeloid leukemia.

Background: Acute myeloid leukemia (AML) is a heterogeneous disorder with an unpredictable prognosis. Ferroptosis, the iron-dependent cell death program, could serve as an alternative for overcoming drug resistance. However, its effect on AML remains largely unclear. Methods: We collected RNA sequencing data and relevant clinical information of AML patients from The Cancer Genome Atlas to construct a prognosis prediction model. Risk score was calculated with eight prognosis-related ferroptosis genes (PRFGs) discovered through univariate analysis and Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression. A nomogram was constructed by incorporating LASSO risk score, age, and cytogenetic risk based on univariate/multivariate Cox regression. Results: Of the 33 AML PRFGs identified from the TCGA-derived dataset, 8 genes were used to construct a gene signature to predict AML prognosis. Principal component analysis and heatmap showed significant differences between the low and high risk score groups. Next, LASSO risk score, age, and cytogenetic risk were incorporated into the nomogram to predict the overall survival (OS) of AML patients. According to survival analysis, patients with a low risk score had markedly increased OS as compared to those with a high risk score. Based on the results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, the differences between the two risk groups showed a close relationship with immune-related pathways and membrane transportation. The analysis of tumor-infiltrating immune cells and immune checkpoints revealed that the immunosuppressive tumor microenvironment possibly facilitated different prognostic outcomes between the two groups. Gene expression analyses showed that the mRNA expression levels of PARP1 and PARP3 (PARPs) were closely related to the different clinical subgroups and the analyzed OS in AML patients. Finally, the PARP inhibitor talazoparib and the ferroptosis inducer erastin exerted a synergistic anti-proliferative effect on AML cells. Conclusion: We constructed a nomogram by incorporating PRFGs, and the constructed nomogram showed a good performance in AML patient stratification and prognosis prediction. The combination of PARP inhibitors with ferroptosis inducers could be a novel treatment strategy for treating AML patients.

MiRNA-142-3P and FUS can be Sponged by Long Noncoding RNA DUBR to Promote Cell Proliferation in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) represents a frequently occurring adulthood acute leukemia (AL). Great progresses have been achieved in the treatment of AML, but its pathogenic mechanism remains unclear. This study reported the biological functions of lncRNA DUBR in AML pathogenic mechanism. As a result, lncRNA DUBR showed high expression level within AML, resulting in poor prognosis, especially in M4 AML. In vitro studies elucidated that knockdown of DUBR with small interfering RNA (siRNA) resulted in the suppression of survival and colony formation ability, as well as induction of apoptosis, in AML cells. RNA pull-down assay and computational revealed that DUBR could sponge with miRNA-142-3P and interact with FUS protein. MiRNA-142-3P have a negative correlation with DUBR and overexpression of miRNA-142-3P inhibited cell growth in AML. Meanwhile, DUBR promoted the expression of FUS protein, targeting inhibition of FUS significantly promoted cell apoptosis in AML cell lines. In conclusion, these results revealed new mechanism of lncRNA DUBR in AML malignant behavior, and suggested that the manipulation of DUBR expression could serve as a potential strategy in AML therapy.

Celebrex Adjuvant Therapy on Coronavirus Disease 2019: An Experimental Study.

Background: The pandemic of coronavirus disease 2019 (COVID-19) resulted in grave morbidity and mortality worldwide. There is currently no effective drug to cure COVID-19. Based on analyses of available data, we deduced that excessive prostaglandin E2 (PGE2) produced by cyclooxygenase-2 was a key pathological event of COVID-19. Methods: A prospective clinical study was conducted in one hospital for COVID-19 treatment with Celebrex to suppress the excessive PGE2 production. A total of 44 COVID-19 cases were enrolled, 37 cases in the experimental group received Celebrex as adjuvant (full dose: 0.2 g, bid; half dose: 0.2 g, qd) for 7-14 days, and the dosage and duration was adjusted for individuals, while seven cases in the control group received the standard therapy. The clinical outcomes were evaluated by measuring the urine PGE2 levels, lab tests, CT scans, vital signs, and other clinical data. The urine PGE2 levels were measured by mass spectrometry. The study was registered and can be accessed at http://www.chictr.org.cn/showproj.aspx?proj=50474. Results: The concentrations of PGE2 in urine samples of COVID-19 patients were significantly higher than those of PGE2 in urine samples of healthy individuals (mean value: 170 ng/ml vs 18.8 ng/ml, p < 0.01) and positively correlated with the progression of COVID-19. Among those 37 experimental cases, there were 10 cases with age over 60 years (27%, 10/37) and 13 cases (35%, 13/37) with preexisting conditions including cancer, atherosclerosis, and diabetes. Twenty-five cases had full dose, 11 cases with half dose of Celebrex, and one case with ibuprofen. The remission rates in midterm were 100%, 82%, and 57% of the full dose, half dose, and control group, respectively, and the discharged rate was 100% at the endpoint with Celebrex treatment. Celebrex significantly reduced the PGE2 levels and promoted recovery of ordinary and severe COVID-19. Furthermore, more complications, severity, and death rate were widely observed and reported in the COVID-19 group of elders and with comorbidities; however, this phenomenon did not appear in this particular Celebrex adjunctive treatment study. Conclusion: This clinical study indicates that Celebrex adjuvant treatment promotes the recovery of all types of COVID-19 and further reduces the mortality rate of elderly and those with comorbidities.

Synergistic modulation of signaling pathways to expand and maintain the bipotency of human hepatoblasts.

BACKGROUND: The limited proliferative ability of hepatocytes is a major limitation to meet their demand for cell-based therapy, bio-artificial liver device, and drug tests. One strategy is to amplify cells at the hepatoblast (HB) stage. However, expansion of HBs with their bipotency preserved is challenging. Most HB expansion methods hardly maintain the bipotency and also lack functional confirmation. METHODS: On the basis of analyzing and manipulating related signaling pathways during HB (derived from human induced pluripotent stem cells, iPSCs) differentiation and proliferation, we established a specific chemically defined cocktails to synergistically regulate the related signaling pathways that optimize the balance of HB proliferation ability and stemness maintenance, to expand the HBs and investigate their capacity for injured liver repopulation in immune-deficient mice. RESULTS: We found that the proliferative ability progressively declines during HB differentiation process. Small molecule activation of Wnt or inhibition of TGF-ß pathways promoted HB proliferation but diminished their bipotency, whereas activation of hedgehog (HH) signaling stimulated proliferation and sustained HB phenotypes. A cocktail synergistically regulating the BMP/WNT/TGF-ß/HH pathways created a fine balance for expansion and maintenance of the bipotency of HBs. After purification, colony formation, and expansion for 20 passages, HBs retained their RNA profile integrity, normal karyotype, and ability to differentiate into mature hepatocytes and cholangiocytes. Moreover, upon transplantation into liver injured mice, the expanded HBs could engraft and differentiate into mature human hepatocytes and repopulate liver tissue with restoring hepatocyte mass. CONCLUSION: Our data contribute to the understanding of some signaling pathways for human HB proliferation in vitro. Simultaneous BMP/HGF induction, activation of Wnt and HH, and inhibition of TGF-ß pathways created a reliable method for long-term stable large-scale expansion of HBs to obtain mature hepatocytes that may have substantial clinical applications.

NEAT1/hsa-mir-98-5p/MAPK6 axis is involved in non-small-cell lung cancer development.

Long noncoding RNAs (lncRNAs) or microRNAs belong to the two most important noncoding RNAs and they are involved in a lot of cancers, including non-small-cell lung cancer (NSCLC). Therefore, currently, we focused on the biological and clinical significance of lncRNA nuclear enriched abundant transcript 1 (NEAT1) and hsa-mir-98-5p in NSCLC. It was observed that NEAT1 was upregulated while hsa-mir-98-5p was downregulated respectively in NSCLC cell lines compared to human normal lung epithelial BES-2B cells. Inhibition of NEAT1 can suppress the progression of NSCLC cells and hsa-mir-98-5p can reverse this phenomenon. Bioinformatics search was used to elucidate the correlation between NEAT1 and hsa-mir-98-5p. Additionally, a novel messenger RNA target of hsa-mir-98-5p, mitogen-activated protein kinase 6 (MAPK6), was predicted. Overexpression and knockdown studies were conducted to verify whether NEAT1 exhibits its biological functions through regulating hsa-mir-98-5p and MAPK6 in vitro. NEAT1 was able to increase MAPK6 expression and hsa-mir-98-5p mimics can inhibit MAPK6 via downregulating NEAT1 levels. We speculated that NEAT1 may act as a competing endogenous lncRNA to upregulate MAPK6 by attaching hsa-mir-98-5p in lung cancers. Taken these together, NEAT1/hsa-mir-98-5p/MAPK6 is involved in the development and progress in NSCLC. NEAT1 could be recommended as a prognostic biomarker and therapeutic indicator in NSCLC diagnosis and treatment.

DNA methylation biomarkers for the occurrence of lung adenocarcinoma from TCGA data mining.

The development of lung cancer is a combination of multifactor, multistage, and multiple genetic alterations processes. DNA methylation is an important factor. Currently, the study on the genome-scale epigenetic modification for studying the pathogenesis of lung cancer is still lacking. Here, we aimed to identify the epigenetic modifications of lung cancer, thus to provide scientific basis for the personalized medicine, and research of classification screening for lung adenocarcinoma patients. The DNA methylation data, and the corresponding clinical information of lung adenocarcinoma samples were extracted from the Cancer Genome Atlas (TCGA) database. We explored the association of DNA methylation and gene transcription expression of lung adenocarcinoma by identifying the differentially expressed genes, DNA methylated locis, functional gene clusters, and the relevant genes associated with the survival. We identified 17 differentially expressed genes which had differentially methylated locis, 4 functional gene clusters regulated by methylation, and 522 genes, which were relevant to the survival time of patients. Our study suggested that methylation controlled the gene expression in a variety of ways, which had high/low expression and hyper-/hypo-methylation. Genes of different methylation status showed the different survival curve. The genes and methylated locis identified in this study could be potential biomarkers and therapeutic targets for lung adenocarcinoma.

Generation of WAe001-A-15, a human embryonic stem cell line with miR-122 doxycycline-inducible expression.

MiR-122 is the most abundant miRNA in the human liver accounting for 52% of the entire hepatic miRNome. Previous studies have demonstrated that miR-122 is a valuable therapeutic target for liver diseases, including viral hepatitis, fibrosis, steatosis, and hepatocarcinoma. Here, we constructed a miR-122 doxycycline-inducible expression human embryonic stem cell line WAe001-A-15 using the piggyBac transposon system. The cell line retained its pluripotency, in vitro differentiation potential, normal morphology, and karyotype.

Generation of a SMO homozygous knockout human embryonic stem cell line WAe001-A-16 by CRISPR/Cas9 editing.

The human SMO protein encoded by the smoothened (SMO) gene acts as a positive mediator for Hedgehog signaling. This pathway regulates many cellular activities, developmental morphogenesis, and tumorigenesis. Using CRISPR/Cas9 to edit human embryonic stem cell line WA01 (H1), we established a SMO mutant cell line (WAe001-A-16). This cell line has a 40bp homozygous deletion in exon 2 of SMO leading to a shift in the open reading frame and early termination at amino acid position 287. WAe001-A-16 maintains a normal karyotype, parental cell morphology, pluripotency markers, and the capacity to differentiate into all three germline layers.

Pathologic lymph node ratio is a predictor of esophageal carcinoma patient survival: a literature-based pooled analysis.

The positive lymph node ratio (LNR) has been suggested as a predictor of survival in patients with esophageal carcinoma (EC). However, existed evidences did not completely agree with each other. We sought to examine whether LNR was associated with overall survival (OS). Electronic database was searched for eligible literatures. The primary outcome was the relationship between LNR and OS, which was presented as hazard ratio (HR) with 95% confidence intervals (CIs). All statistical analyses were performed using STATA 11.0 software. A total of 18 relevant studies which involved 7,664 cases were included. Patients with an LNR of 0.3 or greater had an increased risk of death compared to those with an LNR of less than 0.3(HR = 2.33; 95% CI 2.03-2.68; P<0.01). Similarly, patients with an LNR greater than 0.5 was also associated with a decreased OS(HR = 1.95; 95% CI 1.52-2.50; P<0.01). No publication bias was found. This meta-analysis confirmed that LNR was a significant predictor of survival in patients with EC and should be considered in prognostication.

Generation of two MEN1 knockout lines from a human embryonic stem cell line.

The MEN1 gene is cytogenetically located at 11q13.1 and encodes the nuclear protein menin, which is involved in cell proliferation, apoptosis, differentiation, and metabolism. Here, we generated two MEN1 knockout human embryonic stem cell lines, WAe001-A-4 and WAe001-A-5, by targeting exon-2 and exon-9 of MEN1 using the CRISPR/Cas9 technique. These cell lines maintained their pluripotency, in vitro differentiation potential, normal morphology, and karyotype. These human MEN1-mutated cell lines not only enlarge the pool of lab resources but also provide ideal models to dissect the detailed physio-pathological roles of the menin protein.

Modeling Congenital Hyperinsulinism with ABCC8-Deficient Human Embryonic Stem Cells Generated by CRISPR/Cas9.

Congenital hyperinsulinism (CHI) is a rare genetic disorder characterized by excess insulin secretion, which results in hypoglycemia. Mutation of sulfonylurea receptor 1 (SUR1), encoded by the ABCC8 gene, is the main cause of CHI. Here, we captured the phenotype of excess insulin secretion through pancreatic differentiation of ABCC8-deficient stem cells generated by the CRISPR/Cas9 system. ABCC8-deficient insulin-producing cells secreted higher insulin than their wild-type counterparts, and the excess insulin secretion was rescued by nifedipine, octreotide and nicorandil. Further, we tested the role of SUR1 in response to different potassium levels and found that dysfunction of SUR1 decreased the insulin secretion rate in low and high potassium environments. Hence, pancreatic differentiation of ABCC8-deficient cells recapitulated the CHI disease phenotype in vitro, which represents an attractive model to further elucidate the function of SUR1 and to develop and screen for novel therapeutic drugs.

Generation of non-integrated induced pluripotent stem cells from a 59-year-old female with multiple endocrine neoplasia type 1 syndrome.

Urine resource cells were collected from a 59-year-old female patient with multiple endocrine neoplasia type 1 syndrome (MEN1) for generating iPS cells with episomal plasmids carrying Oct4, Sox2, Klf4 and miR-302-367. The patient sustained a heterozygous G>T transition mutation on the exon 9 of Men1 gene that was confirmed by sequencing analysis on the obtained iPSC lines. Karyotyping indicated the chromosomes with normal appearances and numbers. Their pluripotency was demonstrated by gene expression, as well as their abilities for differentiating into three germ layers. This cell line provides an ideal model for studying MEN1.

Generation of non-integrated induced pluripotent stem cells from a 23-year-old male with multiple endocrine neoplasia type 1 syndrome.

Urine resource cells were collected from a 23-year-old male with multiple endocrine neoplasia type 1 syndrome (MEN1) for generating iPS cells with episomal plasmids. Two stable iPSC lines with free of episomal plasmid were established. The patient has a heterozygous G>T mutation on the exon 9 of Men1 gene that was confirmed by sequencing analysis on all resulted cell lines. Karyotyping indicated the chromosomes with normal appearances and numbers. Their pluripotency was demonstrated by gene expression and their abilities for differentiating into three germ layers. These iPSC lines provide valuable in vitro resources for pathological study on MEN1 syndrome.

SGEF is overexpressed in prostate cancer and contributes to prostate cancer progression.

The purpose of this study was to investigate the potential roles of the SH3-containing guanine nucleotide exchange factor (SGEF) in human prostate cancer. Experimental data showed that SGEF was overexpressed in human prostate cancer cells and specimens. The reduction of SGEF expression through an SGEF-targeting siRNA in androgen-independent C4-2 and C4-2B cells suppressed both anchorage-dependent and anchorage-independent growth. In addition, the androgen receptor (AR) antagonist bicalutamide further strengthened this inhibitory effect due to the suppression of the elevated AR transactivation after knockdown of SGEF. Collectively, our results provide the first demonstration that SGEF is a novel promoter of human prostate cancer progression and development.