BACKGROUND: Angiogenesis is described as a complex process in which new microvessels sprout from endothelial cells of existing vasculature. This study aimed to determine whether long non-coding RNA (lncRNA) H19 induced the angiogenesis of gastric cancer (GC) and its possible mechanism. METHODS: Gene expression level was determined by quantitative real-time polymerase chain reaction and western blotting. Cell counting kit-8, transwell, 5-Ethynyl-2'-deoxyuridine (EdU), colony formation assay, and human umbilical vein endothelial cells (HUVECs) angiogenesis assay as well as Matrigel plug assay were conducted to study the proliferation, migration, and angiogenesis of GC in vitro and in vivo . The binding protein of H19 was found by RNA pull-down and RNA Immunoprecipitation (RIP). High-throughput sequencing was performed and next Gene Ontology (GO) as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted to analyze the genes that are under H19 regulation. Methylated RIP (me-RIP) assay was used to investigate the sites and abundance among target mRNA. The transcription factor acted as upstream of H19 was determined through chromatin immunoprecipitation (ChIP) and luciferase assay. RESULTS: In this study, we found that hypoxia-induced factor (HIF)-1α could bind to the promoter region of H19, leading to H19 overexpression. High expression of H19 was correlated with angiogenesis in GC, and H19 knocking down could inhibit cell proliferation, migration and angiogenesis. Mechanistically, the oncogenic role of H19 was achieved by binding with the N 6 -methyladenosine (m 6 A) reader YTH domain-containing family protein 1 (YTHDF1), which could recognize the m 6 A site on the 3'-untransated regions (3'-UTR) of scavenger receptor class B member 1 (SCARB1) mRNA, resulting in over-translation of SCARB1 and thus promoting the proliferation, migration, and angiogenesis of GC cells. CONCLUSION HIF-1α induced overexpression of H19 via binding with the promoter of H19, and H19 promoted GC cells proliferation, migration and angiogenesis through YTHDF1/SCARB1, which might be a beneficial target for antiangiogenic therapy for GC.
Humans ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Endothelial Cells/metabolism* ; Gene Expression Regulation ; Gene Expression Regulation, Neoplastic/genetics* ; Hypoxia ; MicroRNAs/genetics* ; RNA ; RNA, Long Noncoding/metabolism* ; RNA-Binding Proteins/metabolism* ; Scavenger Receptors, Class B/metabolism* ; Stomach Neoplasms/genetics*

BACKGROUND: Long non-coding RNA colon cancer-associated transcript 1 (CCAT1) is involved in transforming multiple cancers into malignant cancer types. Previous studies underlining the mechanisms of the functions of CCAT1 primarily focused on its decoy for miRNAs (micro RNAs). However, the regulatory mechanism of CCAT1-protein interaction associated with tumor metastasis is still largely unknown. The present study aimed to identify proteome-wide CCAT1 partners and explored the CCAT1-protein interaction mediated tumor metastasis. METHODS: CCAT1-proteins complexes were purified and identified using RNA antisense purification coupled with the mass spectrometry (RAP-MS) method. The database for annotation, visualization, and integrated discovery and database for eukaryotic RNA binding proteins (EuRBPDB) websites were used to bioinformatic analyzing CCAT1 binding proteins. RNA pull-down and RNA immunoprecipitation were used to validate CCAT1-Vimentin interaction. Transwell assay was used to evaluate the migration and invasion abilities of HeLa cells. RESULTS: RAP-MS method worked well by culturing cells with nucleoside analog 4-thiouridine, and cross-linking was performed using 365 nm wavelength ultraviolet. There were 631 proteins identified, out of which about 60% were RNA binding proteins recorded by the EuRBPDB database. Vimentin was one of the CCAT1 binding proteins and participated in the tumor metastasis pathway. Knocked down vimetin ( VIM ) and rescued the downregulation by overexpressing CCAT1 demonstrated that CCAT1 could enhance tumor migration and invasion abilities by stabilizing Vimentin protein. CONCLUSION CCAT1 may bind with and stabilize Vimentin protein, thus enhancing cancer cell migration and invasion abilities.
Humans ; HeLa Cells ; RNA, Long Noncoding/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Vimentin/metabolism* ; MicroRNAs/metabolism* ; Colonic Neoplasms/genetics* ; RNA-Binding Proteins/metabolism* ; Gene Expression Regulation, Neoplastic/genetics* ; Cell Movement/genetics*

BACKGROUND: Previous studies have examined the bulk transcriptome of peripheral blood immune cells in acquired immunodeficiency syndrome patients experiencing immunological non-responsiveness. This study aimed to investigate the characteristics of specific immune cell subtypes in acquired immunodeficiency syndrome patients who exhibit immunological non-responsiveness. METHODS: A single-cell transcriptome sequencing of peripheral blood mononuclear cells obtained from both immunological responders (IRs) (CD4 + T-cell count >500) and immunological non-responders (INRs) (CD4 + T-cell count <300) was conducted. The transcriptomic profiles were used to identify distinct cell subpopulations, marker genes, and differentially expressed genes aiming to uncover potential genetic factors associated with immunological non-responsiveness. RESULTS: Among the cellular subpopulations analyzed, the ratios of monocytes, CD16 + monocytes, and exhausted B cells demonstrated the most substantial differences between INRs and IRs, with fold changes of 39.79, 11.08, and 2.71, respectively. In contrast, the CD4 + T cell ratio was significantly decreased (0.39-fold change) in INRs compared with that in IRs. Similarly, the ratios of natural killer cells and terminal effector CD8 + T cells were also lower (0.37-fold and 0.27-fold, respectively) in the INRs group. In addition to several well-characterized immune cell-specific markers, we identified a set of 181 marker genes that were enriched in biological pathways associated with human immunodeficiency virus (HIV) replication. Notably, ISG15 , IFITM3 , PLSCR1 , HLA-DQB1 , CCL3L1 , and DDX5 , which have been demonstrated to influence HIV replication through their interaction with viral proteins, emerged as significant monocyte marker genes. Furthermore, the differentially expressed genes in natural killer cells were also enriched in biological pathways associated with HIV replication. CONCLUSIONS We generated an atlas of immune cell transcriptomes in HIV-infected IRs and INRs. Host genes associated with HIV replication were identified as markers of, and were found to be differentially expressed in, different types of immune cells.
Humans ; Acquired Immunodeficiency Syndrome ; Transcriptome/genetics* ; HIV ; HIV Infections/genetics* ; Leukocytes, Mononuclear/metabolism* ; CD4-Positive T-Lymphocytes/metabolism* ; Virus Replication ; Membrane Proteins/metabolism* ; RNA-Binding Proteins/metabolism*

The genomic instability may lead to an initiation of cancer in many organisms. Homologous recombination repair (HRR) is vital in maintaining cellular genomic stability. RAD51 associated protein 1 (RAD51AP1), which plays a crucial role in HRR and primarily participates in forming D-loop, was reported as an essential protein for maintaining cellular genomic stability. However, recent studies showed that RAD51AP1 was significantly overexpressed in various cancer types and correlated with poor prognosis. These results suggested that RAD51AP1 may play a significant pro-cancer effect in multiple cancers. The underlying mechanism is still unclear. Cancer stemness-maintaining effects of RAD51AP1 might be considered as the most reliable mechanism. Meanwhile, RAD51AP1 also promoted resistance to radiation therapy and chemotherapy in many cancers. Thus, researches focused on RAD51AP1, and its regulatory molecules may provide new targets for overcoming cancer progression and treatment resistance. Here, we reviewed the latest research on RAD51AP1 in cancers and summarized its differential expression and prognostic implications. In this review, we also outlined the potential mechanisms of its pro-cancer and drug resistance-promoting effects to provide several potential directions for further research.
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Humans ; DNA-Binding Proteins/metabolism* ; RNA-Binding Proteins/metabolism* ; Lung Neoplasms ; DNA Repair ; Genomic Instability ; Rad51 Recombinase/metabolism*

BACKGROUND: There have been many significant advances in the diagnosis and treatment of non-small cell lung cancer (NSCLC). However, the mechanism underlying the progression of NSCLC is still not clear. Plant homodomain finger-like domain-containing protein 5A (PHF5A) plays an important role in processes of chromatin remodeling, morphological development of tissues and organs and maintenance of stem cell pluripotency. This study aims to investigate the role of PHF5A in the proliferation and migration of NSCLC. METHODS: A549 and PC-9 PHF5A overexpression cell lines were constructed. PHF5A expression was decreased in H292 and H1299 cells by using siRNA. Flow cytometry was used to detect the cell cycle. MTT assay and clone formation assay were used to examine the proliferative ability of NSCLC, while migration assay and wound healing assay were performed to evaluate the ability of migration. Western blot analysis was used to measure the expressions of PI3K, p-AKT and the associated downstream factors. RESULTS: Up-regulation of PHF5A in A549 and PC-9 cells increased the proliferation rate, while down-regulation of PHF5A in H292 and H1299 cells inhibited the proliferation rate at 24 h, 48 h and 72 h (P<0.05). The metastatic ability was elevated in the PHF5A-overexpresion groups, while reduced in the PHF5A-down-regulation group (P<0.05). In addition, reduced expression of PHF5A induced cell cycle arrest at G1/S phase (P<0.05). Furthermore, decreased expression of PHF5A reduced the expression levels of PI3K, phosphorylation of AKT, c-Myc (P<0.05) and elevated the expression of p21 (P<0.05). CONCLUSIONS These results demonstrated that PHF5A may play an important role in progression of NSCLC by regulating the PI3K/AKT signaling pathway.
Humans ; Carcinoma, Non-Small-Cell Lung/pathology* ; Lung Neoplasms/pathology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cell Movement/genetics* ; Gene Expression Regulation, Neoplastic ; Trans-Activators/genetics* ; RNA-Binding Proteins/metabolism*

OBJECTIVES: Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor of head and neck. Screening of target genes for malignant tumor therapy is one of the focuses of cancer research, with proto-oncogene and tumor suppressor gene as the breakthrough. It has become an urgent need to find the target gene related to the treatment and prognosis of LSCC.This study aims to explore the role of Lin28B and C-myc in LSCC by detecting the expressions of these two proteins and analyze the correlation between the expression of Lin28B and C-myc and clinicopathological features and prognosis of LSCC. METHODS: We detected the expression of Lin28B and C-myc proteins in 102 specimens of LSCC and 90 specimens of adjacent tissues by immunochemistry, and analyzed the correlation between Lin28B and C-myc protein expressions in LSCC as well as the correlation between the expressions of the two proteins and the clinicopathological features of LSCC. At the same time, the Kaplan-Meier method was used to analyze the relation between Lin28B and C-myc protein levels with the postoperative survival rate of LSCC patients. RESULTS: The protein levels of Lin28B and C-myc in the LSCC tissnes were significantly higher than those in the adjacent tissues (both P<0.05),and there was a positive correlation between the expression of Lin28B and C-myc in LSCC (r＝0.476, P<0.05). The expression of Lin28B protein was closely related to age, lymph node metastasis, clinical stage, tumor size, and pathological differentiation of LSCC patients (all P<0.05). while the expression of C-myc protein was closely related to lymph node metastasis, clinical stage, tumor size, and pathological differentiation of LSCC patients (all P<0.05). A relevant survival analysis showed that in patients with higher level of Lin28B (P＝0.001) or C-myc protein (P<0.001), the postoperative survival rate was relatively low. CONCLUSIONS Lin28B and C-myc proteins are highly expressed in LSCC with a positive correlation. Furthermore, they are closely related to lymph node metastasis, clinical stage, tumor size, pathological differentiation and prognosis, suggesting that both Lin28B and C-myc might be involved in the occurrence and development of LSCC.
Humans ; Squamous Cell Carcinoma of Head and Neck ; Proto-Oncogene Proteins c-myc/metabolism* ; Laryngeal Neoplasms/diagnosis* ; Carcinoma, Squamous Cell/genetics* ; Lymphatic Metastasis ; Prognosis ; Head and Neck Neoplasms ; Biomarkers, Tumor/metabolism* ; RNA-Binding Proteins/genetics*

RBM46 is a germ cell-specific RNA-binding protein required for gametogenesis, but the targets and molecular functions of RBM46 remain unknown. Here, we demonstrate that RBM46 binds at specific motifs in the 3'UTRs of mRNAs encoding multiple meiotic cohesin subunits and show that RBM46 is required for normal synaptonemal complex formation during meiosis initiation. Using a recently reported, high-resolution technique known as LACE-seq and working with low-input cells, we profiled the targets of RBM46 at single-nucleotide resolution in leptotene and zygotene stage gametes. We found that RBM46 preferentially binds target mRNAs containing GCCUAU/GUUCGA motifs in their 3'UTRs regions. In Rbm46 knockout mice, the RBM46-target cohesin subunits displayed unaltered mRNA levels but had reduced translation, resulting in the failed assembly of axial elements, synapsis disruption, and meiotic arrest. Our study thus provides mechanistic insights into the molecular functions of RBM46 in gametogenesis and illustrates the power of LACE-seq for investigations of RNA-binding protein functions when working with low-abundance input materials.
Animals ; Mice ; 3' Untranslated Regions/genetics* ; Cell Cycle Proteins/metabolism* ; Gametogenesis/genetics* ; Meiosis/genetics* ; Nuclear Proteins/genetics* ; RNA-Binding Proteins/genetics*

OBJECTIVE: To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells. METHODS: Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated. RESULTS: Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing. CONCLUSION RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.
Humans ; rab GTP-Binding Proteins/metabolism* ; Triple Negative Breast Neoplasms ; Cell Line, Tumor ; rab27 GTP-Binding Proteins/metabolism* ; RNA, Small Interfering/genetics* ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic

OBJECTIVE: Bo investigate the regulatory relationship between NKD1 and YWHAE and the mechanism of NKD1 for promoting tumor cell proliferation. METHODS: HCT116 cells transfected with pcDNA3.0-NKD1 plasmid, SW620 cells transfected with NKD1 siRNA, HCT116 cells with stable NKD1 overexpression (HCT116-NKD1 cells), SW620 cells with nkd1knockout (SW620-nkd1^-/- cells), and SW620-nkd1^-/- cells transfected with pcDNA3.0-YWHAE plasmid were examined for changes in mRNA and protein expression levels of YWHAE using qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NKD1 to the promoter region of YWHAE gene. The regulatory effect of NKD1 on YWHAE gene promoter activity was analyzed by dual-luciferase reporter gene assay, and the interaction between NKD1 and YWHAE was analyzed with immunofluorescence assay. The regulatory effect of NKD1 on glucose uptake was examined in the tumor cells. RESULTS: In HCT116 cells, overexpression of NKD1 significantly enhanced the expression of YWHAE at both the mRNA and protein levels, while NKD1 knockout decreased its expression in SW620 cells (P < 0.001). ChIP assay showed that NKD1 protein was capable of binding to the YWHAE promoter sequence; dual luciferase reporter gene assay showed that NKD1 overexpression (or knockdown) in the colon cancer cells significantly enhanced (or reduced) the transcriptional activity of YWHAE promoter (P < 0.05). Immunofluorescence assay demonstrated the binding of NKD1 and YWHAE proteins in colon cancer cells. NKD1 knockout significantly reduced glucose uptake in colon cancer cells (P < 0.01), while YWHAE overexpression restored the glucose uptake in NKD1-knockout cells (P < 0.05). CONCLUSION NKD1 protein activates the transcriptional activity of YWHAE gene to promote glucose uptake in colon cancer cells.
Humans ; Colonic Neoplasms ; HCT116 Cells ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; RNA, Messenger ; Glucose ; Calcium-Binding Proteins/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism* ; 14-3-3 Proteins/metabolism*

OBJECTIVE: To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells. METHODS: Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level. RESULTS: Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 0.05). CONCLUSIONS TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.
Animals ; Male ; Mice ; GTP-Binding Proteins/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; RNA, Messenger ; Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism* ; Signal Transduction ; Spermatocytes ; Tubulin/genetics*