Dysregulated RNA splicing is a well-recognized characteristic of colorectal cancer (CRC); however, its intricacies remain obscure, partly due to challenges in profiling full-length transcript variants at the single-cell level. Here, we employ high-depth long-read scRNA-seq to define the full-length transcriptome of colorectal epithelial cells in 12 CRC patients, revealing extensive isoform diversities and splicing alterations. Cancer cells exhibited increased transcript complexity, with widespread 3'-UTR shortening and reduced intron retention. Distinct splicing regulation patterns were observed between intrinsic-consensus molecular subtypes (iCMS), with iCMS3 displaying even higher splicing factor activities and more pronounced 3'-UTR shortening. Furthermore, we revealed substantial shifts in isoform usage that result in alterations of protein sequences from the same gene with distinct carcinogenic effects during tumorigenesis of CRC. Allele-specific expression analysis revealed dominant mutant allele expression in key oncogenes and tumor suppressors. Moreover, mutated PPIG was linked to widespread splicing dysregulation, and functional validation experiments confirmed its critical role in modulating RNA splicing and tumor-associated processes. Our findings highlight the transcriptomic plasticity in CRC and suggest novel candidate targets for splicing-based therapeutic strategies.
Humans ; Colorectal Neoplasms/metabolism* ; RNA Isoforms/metabolism* ; Single-Cell Analysis ; RNA Splicing ; Gene Expression Regulation, Neoplastic ; RNA, Neoplasm/metabolism* ; Transcriptome

BACKGROUND: Trichorhinophalangeal syndrome (TRPS) patients tend to have alopecia that appears to be androgenetic, and this genetic model might give clues to the pathogenesis of hair loss or hair morphogenesis. OBJECTIVE: This study was conducted to identify additional genetic evidence of TRPS and hair morphogenesis from a TRPS patient. METHODS: From one TRPS type I patient, we extracted RNA and profiled whole transcriptome in non-balding and balding scalp areas using high-throughput RNA sequencing. RESULTS: We found a total of 26,320 genes, which comprised 14,892 known genes with new isoforms and 4,883 novel genes from the non-balding and balding areas. Among these, a total of 1,242 genes showed different expression in the two scalp areas (p<0.05 and log2 fold-change >0). Several genes related to the skin and hair, alopecia, and the TRPS1 gene were validated by qRT-PCR. Twelve of 15 genes (KRT6C, KRTAP3-1, MKI67, GPRC5D, TYRP1, DSC1, PMEL, WIF1, SOX21, TINAG, PTGDS, and TRPS1) were down-regulated (10 genes: p<0.01; SOX21 and PTGDS: p>0.05), and the three other genes (HBA2, GAL, and DES) were up-regulated (p<0.01) in the balding scalp. Many genes related to keratin and hair development were down-regulated in the balding scalp of the TRPS type I patient. In particular, the TRPS1 gene might be related to androgen metabolism and hair morphogenesis. CONCLUSION: Our result could suggest a novel perspective and evidence to support further study of TRPS and hair morphogenesis.
Alopecia ; Gene Expression Profiling* ; Hair ; High-Throughput Nucleotide Sequencing ; Humans ; Metabolism ; Models, Genetic ; Morphogenesis ; Protein Isoforms ; RNA ; Scalp* ; Skin ; Transcriptome*

The methylcytosine dioxygenases TET proteins (TET1, TET2, and TET3) play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA (valproic acid)-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neuronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demonstrated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma.
Animals ; Catalytic Domain ; Cell Differentiation ; drug effects ; physiology ; Cell Line, Tumor ; DNA-Binding Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; GTPase-Activating Proteins ; genetics ; metabolism ; Immunohistochemistry ; Mice ; Microscopy, Fluorescence ; Neuroblastoma ; metabolism ; pathology ; Protein Isoforms ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; metabolism ; Valproic Acid ; pharmacology

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; drug effects ; Female ; Guanine Nucleotide Exchange Factors ; antagonists & inhibitors ; genetics ; metabolism ; Humans ; Indoles ; pharmacology ; MCF-7 Cells ; Maleimides ; pharmacology ; Protein Isoforms ; genetics ; metabolism ; Protein Kinase C ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Receptor, ErbB-2 ; genetics ; metabolism ; Tetradecanoylphorbol Acetate ; toxicity

OBJECTIVETo investigate the expression of osteopontin (OPN) splice variant mRNA, including the three isoforms OPN-A, OPN-B, and OPN-C, to explore its correlation with clinicopathologic features in gastric cancer, and to elucidate their role in tumor invasion and distant metastasis of gastric cancer.

METHODSThe expression of OPN-A, OPN-B and OPN-C mRNA were detected by real-time reverse transcriptase-polymerase chain reaction in 66 gastric cancer tissues. The relationship between the expression of OPN-A, OPN-B and OPN-C mRNA and clinicopathologic features of gastric cancer was analyzed.

RESULTSThe expression of OPN-C mRNA in the gastric cancer tissue was 3.21-fold higher than that in peritumoral mucosal tissue, showing a significant difference between them (P < 0.001). OPN-C mRNA expression was correlated with the depth of tumor invasion, tumor diameter, lymph node meatastasis, distant meatastasis and had no correlation with differentiation grades. The low expression of OPN-C mRNA was correlated with long survival benefit (P = 0.03). The expression of OPN-A and OPN-B mRNA had no significant relationship with clinicopathologic features of gastric cancer.

CONCLUSIONSOne of the isoform of osteopontin (OPN) OPN-C mRNA is overexpressed in gastric cancer. The overexpression of OPN-C mRNA may reflect the progression and is associated with the prognosis of gastric cancer. OPN-C mRNA may have value as a prognostic biomarker in gastric cancer. However, the expression of OPN-A and OPN-B are not correlated with the progression and metastasis of gastric cancer.

Disease Progression ; Gastric Mucosa ; metabolism ; Humans ; Lymph Nodes ; Lymphatic Metastasis ; Neoplasm Invasiveness ; Neoplasm Proteins ; genetics ; Osteopontin ; genetics ; Prognosis ; Protein Isoforms ; genetics ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Stomach Neoplasms ; genetics ; mortality ; pathology

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.
Acanthamoeba castellanii/cytology/*genetics/physiology ; Amebiasis/*parasitology ; Amino Acid Sequence ; Autophagy ; Cell Membrane/metabolism ; DNA, Protozoan/chemistry/genetics ; Gene Dosage ; Gene Silencing ; Genes, Reporter ; Humans ; Molecular Sequence Data ; Phagosomes/metabolism ; Protein Isoforms ; Protozoan Proteins/*genetics/metabolism ; RNA, Messenger/genetics ; RNA, Protozoan/genetics ; RNA, Small Interfering/chemical synthesis/genetics ; Recombinant Fusion Proteins ; Sequence Alignment

Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; Apoptosis ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Neoplasms ; pathology ; Phosphorylation ; Protein Isoforms ; RNA-Binding Proteins ; chemistry ; metabolism ; Transcription Factors ; antagonists & inhibitors

Cellular senescence is an irreversible cell cycle arrest triggered by the activation of oncogenes or mitogenic signaling as well as the enforced expression of tumor suppressors such as p53, p16(INK4A) and promyelocytic leukemia protein (PML) in normal cells. E2F-binding protein 1 (E2FBP1), a transcription regulator for E2F, induces PML reduction and suppresses the formation of PML-nuclear bodies, whereas the down-regulation of E2FBP1 provokes the PML-dependent premature senescence in human normal fibroblasts. Here we report that the depletion of E2FBP1 induces the accumulation of PML through the Ras-dependent activation of MAP kinase signaling. The cellular levels of p16(INK4A) and p53 are elevated during premature senescence induced by depletion of E2FBP1, and the depletion of p16(INK4A), but not p53 rescued senescent cells from growth arrest. Therefore, the premature senescence induced by E2FBP1 depletion is achieved through the p16(INK4A)-Rb pathway. Similar to human normal fibroblasts, the growth inhibition induced by E2FBP1 depletion is also observed in human tumor cells with intact p16(INK4A) and Rb. These results suggest that E2FBP1 functions as a critical antagonist to the p16(INK4A)-Rb tumor suppressor machinery by regulating PML stability.
Cell Line, Tumor ; Cells, Cultured ; Cellular Senescence ; genetics ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; antagonists & inhibitors ; genetics ; physiology ; DNA-Binding Proteins ; deficiency ; genetics ; physiology ; Down-Regulation ; Fibroblasts ; Gene Expression Regulation ; Humans ; Intranuclear Inclusion Bodies ; metabolism ; MAP Kinase Signaling System ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Promyelocytic Leukemia Protein ; Protein Isoforms ; Protein Stability ; RNA Interference ; Retinoblastoma Protein ; antagonists & inhibitors ; genetics ; physiology ; Transcription Factors ; deficiency ; genetics ; metabolism ; physiology ; Transfection ; Tumor Suppressor Protein p53 ; physiology ; Tumor Suppressor Proteins ; genetics ; metabolism ; physiology ; Ubiquitination ; ras Proteins ; metabolism

OBJECTIVE: To study the mRNA expression of muscle phenotype and collagen of soft palate and pathology in obstructive sleep apnea hypopnea syndrome (OSAHS). METHOD: We used the Real-time PCR to test the mRNA expression of soft palate muscle myosin heavy chain (MyHC) phenotype and collagen in 12 OSAHS patients and 8 control patients. We also distinguished the muscle isoforms I , II with ATPase staining, then counted the numbers of isoforms muscle fiber. RESULT: The mRNA expression of OSAHS group was more than control group in II A MyHC phenotype (P<0.01). The number of OSAHS group muscle fibre I isoform was less than control group with pH4. 3 ATPase staining (P<0.05). CONCLUSION Compare to control group, the enhancement happened in the mRNA expression of II A MyHC phenotype which can increase the velocity and power but de crease the enduring quality of muscle in OSAHS, and the reduce be in the I MyHC isoform of muscle fiber that can cause muscle velocity become slower and persistency become longer in OSAHS patients.
Case-Control Studies ; Female ; Humans ; Male ; Middle Aged ; Muscle Fibers, Skeletal ; metabolism ; pathology ; Myosin Heavy Chains ; metabolism ; Palate, Soft ; metabolism ; pathology ; Phenotype ; Protein Isoforms ; metabolism ; RNA, Messenger ; genetics ; Sleep Apnea, Obstructive ; metabolism ; pathology

Chronic and heavy alcohol consumption is one of the causes of heart diseases. However, the effects of ethanol on insulin sensitivity in myocardium has been unclear. To investigate the effects of ethanol on the expression of AMP-activated protein kinase (AMPK), myocyte enhancer factor 2 (MEF2) and glucose transporter 4 (GLUT4), all of which are involved in the regulation of insulin sensitivity, in the myocardium, we performed three parts of experiments in vivo and in vitro. I: Rats were injected with 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR, 0.8 mg.kg(-1)) for 2 h. II: Rats received different dose (0.5, 2.5 or 5 g.kg(-1).d(-1)) of ethanol for 22-week. III: Primary neonatal rat cardiomyocytes were isolated and treated with or without 100 mM ethanol or 1 mM AICAR for 4 h. The cardiac protein and mRNA expression of AMPKalpha subunits, MEF2 and GLUT4 were observed by western-blotting and RT-PCR, respectively. Serum TNFalpha levels were assessed by ELISA. The results showed chronic ethanol exposure induced insulin resistance. Ethanol decreased the mRNA levels of AMPKalpha1 and alpha2, the protein levels of total- and phospho-AMPKalpha in cardiomyocytes. Similarly, ethanol showed inhibitory effects on both the mRNA and protein levels of MEF2A and 2D, and GLUT4 in a dose-response-like fashion. Correlation analysis implied an association between phospho-AMPKalpha and MEF2A or MEF2D, and between the levels of MEF2 protein and GLUT4 transcription. In addition, ethanol elevated serum TNFalpha level. Taken together, chronic ethanol exposure decreases the expression of AMPKalpha and MEF2, and is associated with GLUT4 decline in rat myocardium.
AMP-Activated Protein Kinases/genetics/*metabolism ; Aminoimidazole Carboxamide/analogs & derivatives/pharmacology ; Animals ; Enzyme Activation/drug effects ; Ethanol/*administration & dosage/*pharmacology ; Feeding Behavior/*drug effects ; Gene Expression Regulation/drug effects ; Glucose Transporter Type 4/genetics/*metabolism ; Insulin/pharmacology ; Insulin Resistance ; Male ; Myocardium/*enzymology ; Myogenic Regulatory Factors/antagonists & inhibitors/genetics/*metabolism ; Protein Isoforms/antagonists & inhibitors/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Wistar ; Ribonucleotides/pharmacology ; Time Factors ; Tumor Necrosis Factor-alpha/blood