BACKGROUND: Ischemic acute kidney injury (AKI) is a common syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathogenesis of ischemic AKI remains incompletely understood, and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking. Here, this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing (scRNA-seq) analysis in kidneys. METHODS: In this study, scRNA-seq technology was applied to kidneys from two ischemic AKI patients, and three human public scRNA-seq datasets were collected as controls. Differentially expressed genes (DEGs) and cell clusters of kidneys were determined. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, as well as the ligand-receptor interaction between cells, were performed. We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion (I/R) injury induced AKI mice through immunohistochemistry staining. RESULTS: 15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control. The injured proximal tubules (PT) displayed a proapoptotic and proinflammatory phenotype. PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47 , RASSF4 , EBAG9 , IER3 , SASH1 , SEPTIN7 , and NUB1 , which have not been reported in ischemic AKI previously. Several hub genes were validated in kidneys from human AKI and renal I/R injury mice, respectively. Furthermore, PT highly expressed DEGs enriched in endoplasmic reticulum stress, autophagy, and retinoic acid-inducible gene I (RIG-I) signaling. DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain (NOD)-like receptor signaling, estrogen signaling, interleukin (IL)-12 signaling, and IL-17 signaling. Overexpressed genes in kidney-resident immune cells including macrophages, natural killer T (NKT) cells, monocytes, and dendritic cells were associated with leukocyte activation, chemotaxis, cell adhesion, and complement activation. In addition, the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI. CONCLUSION Together, this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients, altered signaling pathways, and potential cell-cell crosstalk in the development of AKI. These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.
Humans ; Mice ; Animals ; Transcriptome/genetics* ; Ligands ; Kidney/metabolism* ; Acute Kidney Injury/metabolism* ; Ischemia/metabolism* ; Reperfusion Injury/metabolism* ; Sequence Analysis, RNA ; Adaptor Proteins, Signal Transducing/metabolism* ; Tumor Suppressor Proteins/metabolism*

Royal jelly (RJ) from honeybee has been widely used as a health promotion supplement. The major royal jelly proteins (MRJPs) have been identified as the functional component of RJ. However, the question of whether MRJPs have anti-senescence activity for human cells remains. Human embryonic lung fibroblast (HFL-I) cells were cultured in media containing no MRJPs (A), MRJPs at 0.1 mg/ml (B), 0.2 mg/ml (C), or 0.3 mg/ml (D), or bovine serum albumin (BSA) at 0.2 mg/ml (E). The mean population doubling levels of cells in media B, C, D, and E were increased by 12.4%, 31.2%, 24.0%, and 10.4%, respectively, compared with that in medium A. The cells in medium C also exhibited the highest relative proliferation activity, the lowest senescence, and the longest telomeres. Moreover, MRJPs up-regulated the expression of superoxide dismutase-1 (SOD1) and down-regulated the expression of mammalian target of rapamycin (MTOR), catenin beta like-1 (CTNNB1), and tumor protein p53 (TP53). Raman spectra analysis showed that there were two unique bands related to DNA synthesis materials, amide carbonyl group vibrations and aromatic hydrogens. These results suggest that MRJPs possess anti-senescence activity for the HFL-I cell line, and provide new knowledge illustrating the molecular mechanism of MRJPs as anti-senescence factors.
Animals ; Bees ; Cattle ; Cell Line ; Cell Proliferation ; Cellular Senescence/drug effects* ; Culture Media ; Dose-Response Relationship, Drug ; Fatty Acids/chemistry* ; Fibroblasts/drug effects* ; Humans ; Insect Proteins/chemistry* ; Lung/drug effects* ; Serum Albumin/metabolism* ; Spectrum Analysis, Raman ; Superoxide Dismutase-1/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Tumor Suppressor Protein p53/metabolism* ; beta Catenin/metabolism*

In recent years, large numbers of non-coding RNAs (ncRNAs) have been identified in C. elegans but their functions are still not well studied. In C. elegans, CEP-1 is the sole homolog of the p53 family of genes. In order to obtain transcription profiles of ncRNAs regulated by CEP-1 under normal and UV stressed conditions, we applied the 'not-so-random' hexamers priming strategy to RNA sequencing in C. elegans, This NSR-seq strategy efficiently depleted rRNA transcripts from the samples and showed high technical replicability. We identified more than 1,000 ncRNAs whose apparent expression was repressed by CEP-1, while around 200 were activated. Around 40% of the CEP-1 activated ncRNAs promoters contain a putative CEP-1-binding site. CEP-1 regulated ncRNAs were frequently clustered and concentrated on the X chromosome. These results indicate that numerous ncRNAs are involved in CEP-1 transcriptional network and that these are especially enriched on the X chromosome in C. elegans.
Animals ; Binding Sites ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; High-Throughput Nucleotide Sequencing ; Promoter Regions, Genetic ; RNA, Untranslated ; metabolism ; Sequence Analysis, RNA ; Transcriptome ; radiation effects ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Ultraviolet Rays ; X Chromosome

OBJECTIVETo analyze immunophenotypes and gene mutations of colorectal precancerous lesions and adenocarcinoma, and to compare the difference of carcinogenetic mechanisms between the two precancerous lesions.

METHODSFifty-three cases of colorectal serrated lesions including 30 hyperplastic polyps, 20 sessile serrated adenomas (SSA) and 3 mixed polyps were collected from January 2006 to June 2012.Forty-five cases of traditional adenomas and 50 cases of colorectal adenocarcinomas were also recruited. Thirty hyperplastic polyps, 20 cases of SSA, 3 mixed polyps and 45 traditional adenomas were investigated by immunohistochemistry for the expression of DNA mismatch repair (MMR) proteins (MLH1, MSH2 and MSH6) and DNA methyltransferase MGMT. Mutations of KRAS, BRAF and PIK3CA genes in 10 cases of SSAs, 10 traditional adenomas, 1 mixed polyps and 50 colorectal adenocarcinomas were analyzed by PCR followed by direct Sanger sequencing.

RESULTS(1) Only 3 cases of hyperplastic polyps lost MLH1 expression, and none of SSAs or traditional adenomas showed loss of MLH1. The negative expression rates of MSH2, MSH6 and MGMT in hyperplastic polyps and SSA were significantly higher than those of traditional adenomas. (2) KRAS mutation was found in 5/10 cases of SSAs, 5/10 traditional adenomas and 1/1 mixed polyps. (3) Colorectal adenocarcinomas harbored the mutations of KRAS (48%, 24/50), BRAF (6%, 3/50) and PIK3CA (4%, 2/50).

CONCLUSIONSImmunophenotypic and gene mutation profiles are different between colorectal serrated lesion and traditional adenoma. Alterations of MMR and MGMT expression play important roles in the pathogenesis of "serrated neoplasm". KRAS mutation is a significant genetic change in the early phase of colorectal carcinogenesis.

Adaptor Proteins, Signal Transducing ; metabolism ; Adenocarcinoma ; genetics ; metabolism ; Adenoma ; genetics ; metabolism ; Aged ; Class I Phosphatidylinositol 3-Kinases ; Colonic Polyps ; genetics ; metabolism ; Colorectal Neoplasms ; genetics ; metabolism ; DNA Mismatch Repair ; DNA Modification Methylases ; metabolism ; DNA Repair Enzymes ; metabolism ; DNA, Neoplasm ; metabolism ; DNA-Binding Proteins ; metabolism ; Female ; Humans ; Hyperplasia ; Immunophenotyping ; Male ; Middle Aged ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; metabolism ; Nuclear Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; genetics ; Point Mutation ; Precancerous Conditions ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins B-raf ; genetics ; Proto-Oncogene Proteins p21(ras) ; Sequence Analysis, DNA ; Tumor Suppressor Proteins ; metabolism ; ras Proteins ; genetics

OBJECTIVETo investigate the expression and promoter methylation status of p73 gene in ovarian epithelial tumors and their clinicopathological correlations.

METHODSTissue microarrays (TMA) consisting of 68 ovarian cancers, 37 ovarian borderline tumors and 21 ovarian benign tumors were constructed. p73 expression was detected by immunohistochemistry (EnVision method). Fresh-frozen tissue samples from 13 cases of ovarian carcinomas and 5 cases of borderline tumors were evaluated for the presence of p73 promoter methylation using bisulfite sequencing.

RESULTSOverall, 92.6% (63/68) ovarian carcinomas expressed p73, with a mean value of 32% (percentage of p73 positive cells in the tumor). The mean value of p73 expression rate (40%) in serous carcinoma (26/26) was higher than those of other cancer types (P = 0.006). The mean value of p73 expression rate (40%) in type II ovarian carcinoma was significantly higher than that in type I ovarian carcinoma (24%, P = 0.010). The expression of p73 was not associated with FIGO stage and histological grade (both P > 0.05). The mean values of p73 expression in ovarian borderline tumor (30/37) and benign tumor (12/21) were 16% and 15%, respectively. Of the two groups, the mean value of p73 expression rate in serous type was higher than that in mucous type (P = 0.003, P = 0.026). Ovarian carcinomas had a higher level of p73 expression than borderline tumors and benign tumors (both P < 0.05), while that between ovarian borderline tumors and benign tumors had no statistical difference (P > 0.05). Among serous tumors (49/53), the mean value of p73 expression in the carcinoma group (26/26) was significantly higher than those in the borderline tumor group (12/14) and benign tumor group (11/13; P = 0.024 and P = 0.002, respectively), while that between borderline tumor group and benign tumor group had no statistical difference (P = 0.428). Among mucous tumors (15/27), the mean value of p73 expression in carcinoma group (6/7) was higher than that in benign tumor group (1/8; P = 0.032). No statistical difference of p73 expression was seen between the carcinoma group and ovarian borderline tumor group (8/12) and between the borderline tumor group and benign tumor group (P = 0.234, P = 0.201, respectively). p73 promotor methylation was found in 8 of 13 cases of carcinomas but at different methylation levels with a mean value of 8.0%. Two of 5 ovarian borderline tumors showed detectable p73 promotor methylation with a mean value of 9.0%. Compared with the borderline tumors, ovarian carcinomas showed a similar p73 methylation level (P > 0.05). The p73 methylation level in ovarian carcinomas was not associated with histological type, pathogenetic type, histological grade and FIGO stage (all P > 0.05).

CONCLUSIONSMost of ovarian epithelial tumors express p73 protein with mean values higher in ovarian carcinomas than those in the borderline and benign tumors. Ovarian serous carcinomas have the highest expression level of p73. A simple linear correlation does not exist between the promoter methylation and protein expression of p73.

Adult ; Aged ; Cystadenocarcinoma, Mucinous ; metabolism ; pathology ; Cystadenocarcinoma, Serous ; metabolism ; pathology ; Cystadenofibroma ; metabolism ; pathology ; Cystadenoma, Mucinous ; metabolism ; pathology ; Cystadenoma, Serous ; metabolism ; pathology ; DNA Methylation ; DNA-Binding Proteins ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Middle Aged ; Neoplasm Grading ; Neoplasm Staging ; Neoplasms, Glandular and Epithelial ; metabolism ; pathology ; Nuclear Proteins ; metabolism ; Oligonucleotide Array Sequence Analysis ; Ovarian Neoplasms ; metabolism ; pathology ; Promoter Regions, Genetic ; Tumor Protein p73 ; Tumor Suppressor Proteins ; metabolism ; Young Adult

Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Basigin ; analysis ; Biomarkers, Tumor ; analysis ; genetics ; Carcinoma, Transitional Cell ; drug therapy ; genetics ; metabolism ; Gene Expression Profiling ; Humans ; In Situ Hybridization, Fluorescence ; Inhibitor of Apoptosis Proteins ; analysis ; Mutation ; Neoplasm Recurrence, Local ; metabolism ; Nuclear Proteins ; analysis ; Receptor, Fibroblast Growth Factor, Type 3 ; analysis ; genetics ; Tumor Suppressor Protein p53 ; analysis ; genetics ; Urinary Bladder Neoplasms ; drug therapy ; genetics ; metabolism

Acid Anhydride Hydrolases ; genetics ; metabolism ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Biomarkers, Tumor ; analysis ; Carcinoma, Squamous Cell ; diagnosis ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; DNA Methylation ; drug effects ; Genes, p16 ; Humans ; Laryngeal Neoplasms ; diagnosis ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism

We studied the expression of BRCA1, ERCC1, and RRM1 which play an important role in DNA repair systems in breast cancer. Immunohistochemical staining for EGFR, BRCA1, ERCC1, and RRM1 were performed by using a tissue microarray made from 230 breast cancer patients. Patients were classified into luminal A, luminal B, HER-2, and triple negative breast cancer (TNBC) types according to ER, PR, and HER-2 expression. The expression of ERCC1, RRM1, and BRCA1 were correlated (P < 0.05). The expression level of ERCC1 was the lowest in TNBC type (P = 0.031), ERCC1 negativity was more prominent in TNBC and luminal B groups than luminal A and HER-2 groups (P = 0.013). Cases with EGFR overexpression showed high expression of RRM1 and BRCA1 (P = 0.046, and 0.004, respectively). In conclusion, the expression of ERCC1 is particularly lower in TNBCs than other types of breast cancers.
Adult ; BRCA1 Protein/*genetics/metabolism ; Breast Neoplasms/*genetics/metabolism/pathology ; DNA Repair ; DNA-Binding Proteins/*genetics/metabolism ; Disease-Free Survival ; Endonucleases/*genetics/metabolism ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; Middle Aged ; Phenotype ; Prognosis ; Protein Array Analysis ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Tumor Markers, Biological/*genetics/metabolism ; Tumor Suppressor Proteins/*genetics/metabolism

In carcinoma ex pleomorphic adenoma (CXPA), pleomorphic adenoma (PA) and diverse carcinoma components showing luminal (ductal) or non-luminal (myoepithelial) differentiation coexist. To elucidate the clinicopathological implications of cellular differentiation in CXPA and the potential role of p53, vascular endothelial growth factor (VEGF), c-erbB-2, c-kit, and glucose transporter 1 (Glut-1) in carcinogenesis, we analyzed 11 CXPAs with luminal differentiation (CXPAs-LD) and 6 CXPAs with non-luminal differentiation (CXPAs-NLD) and compared protein expressions in residual PAs and carcinomas by immunohistochemistry. Among the CXPAs-LD, 5 were invasive and 8 were histologically high-grade tumors. The 5-year survival rate was 72.7%. P53, c-erbB-2, VEGF, and Glut-1 were more immunoreactive in carcinoma components than in PAs (P = 0.008, 0.004, 0.002, and 0.024, respectively); c-erbB-2 overexpression was associated with high histological grade (P = 0.024). Carcinoma components frequently lacked c-kit expression (P = 0.009). CXPAs-NLD were all low-grade and invasive with a larger mean tumor size (5.2 cm) than CXPAs-LD (3.3 cm) (P = 0.040). The patients remained disease-free without significant immunohistochemical expression. The immunoprofiles and clinical course of CXPA differed according to cellular differentiation. Therefore, it is important to report the histological subtype and to assess potential biomarkers in diagnostic and therapeutic trials.
Adenoma, Pleomorphic/*immunology/metabolism/*pathology ; Adult ; Aged ; Carcinoma/*immunology/metabolism/*pathology ; Cell Differentiation ; Female ; Glucose Transport Proteins, Facilitative/metabolism ; Humans ; Male ; Middle Aged ; Proto-Oncogene Proteins c-kit/metabolism ; Receptor, erbB-2/metabolism ; Salivary Gland Neoplasms/*immunology/metabolism/*pathology ; Tumor Markers, Biological/*analysis ; Tumor Suppressor Protein p53/metabolism ; Vascular Endothelial Growth Factor A/metabolism

OBJECTIVETo develop a method for the detection of RRM1, ERCC1 and BRCA1 gene expression by SYBR real-time fluorescent quantitative PCR in non-small cell lung cancer tissues and peripheral blood.

METHODSThe plasmid standard of RRM1, ERCC1, BRCA1 and β-actin genes was constructed. SYBR real-time PCR was performed, and the standard curve was established. The expressions of RRM1, ERCC1 and BRCA1 mRNA in non-small cell lung cancer tissues and peripheral blood were detected.

RESULTThe standard curve presented linearity. The liquate curves of standard gene were all single apex, indicating that a good specificity was obtained.

CONCLUSIONThe developed SYBR real-time fluorescent quantitative PCR has advantage of convenient operation, low cost, good specificity and high veracity.

Actins ; genetics ; BRCA1 Protein ; blood ; genetics ; Carcinoma, Non-Small-Cell Lung ; blood ; metabolism ; DNA-Binding Proteins ; blood ; genetics ; Endonucleases ; blood ; genetics ; Female ; Humans ; Lung Neoplasms ; blood ; metabolism ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; RNA, Messenger ; analysis ; blood ; Tumor Suppressor Proteins ; blood ; genetics