OBJECTIVES: Bladder cancer is a common malignancy with high incidence and poor prognosis. N⁶-methyladenosine (m⁶A) modification is widely involved in diverse physiological processes, among which the m⁶A recognition protein YTH N⁶-methyladenosine RNA binding protein F2 (YTHDF2) plays a crucial role in bladder cancer progression. This study aims to elucidate the molecular mechanism by which O-linked N-acetylglucosamine (O-GlcNAc) modification of YTHDF2 regulates its downstream target, period circadian regulator 1 (PER1), thereby promoting bladder cancer cell proliferation. METHODS: Expression of YTHDF2 in bladder cancer was predicted using The Cancer Genome Atlas (TCGA). Twenty paired bladder cancer and adjacent normal tissues were collected at the clinical level. Normal bladder epithelial cells (SV-HUC-1) and bladder cancer cell lines (T24, 5637, EJ-1, SW780, BIU-87) were examined by quantitative real-time PCR (RT-qPCR), Western blotting, and immunohistochemistry for expression of YTHDF2, PER1, and proliferation-related proteins [proliferating cell nuclear antigen (PCNA), minichromosome maintenance complex component 2 (MCM2), Cyclin D1]. YTHDF2 was silenced in 5637 and SW780 cells, and cell proliferation was assessed by Cell Counting Kit-8 (CCK-8), colony formation, and EdU assays. Bioinformatics was used to predict glycosylation sites of YTHDF2, and immunoprecipitation (IP) was performed to detect O-GlcNAc modification levels of YTHDF2 in tissues and cells. Bladder cancer cells were treated with DMSO, OSMI-1 (O-GlcNAc inhibitor), or Thiamet G (O-GlcNAc activator), followed by cycloheximide (CHX), to assess YTHDF2 ubiquitination by IP. YTHDF2 knockdown and Thiamet G treatment were further used to evaluate PER1 mRNA stability, PER1 m⁶A modification, and cell proliferation. TCGA was used to predict PER1 expression in tissues; SRAMP predicted potential PER1 m⁶A sites. Methylated RNA immunoprecipitation (MeRIP) assays measured PER1 m⁶A modification. Finally, the effects of knocking down YTHDF2 and PER1 on 5637 and SW780 cell proliferation were assessed. RESULTS: YTHDF2 expression was significantly upregulated in bladder cancer tissues compared with adjacent tissues (mRNA: 2.5-fold; protein: 2-fold), which O-GlcNAc modification levels increased 3.5-fold (P<0.001). YTHDF2 was upregulated in bladder cancer cell lines, and its knockdown suppressed cell viability (P<0.001), downregulated PCNA, MCM2, and CyclinD1 (all P<0.05), reduced colony numbers 3-fold (P<0.01), and inhibited proliferation. YTHDF2 exhibited elevated O-GlcNAc modification in cancer cells. OSMI-1 reduced YTHDF2 protein stability (P<0.01) and enhanced ubiquitination, while Thiamet G exerted opposite effects (P<0.001). Thiamet G reversed the proliferation-suppressive effects of YTHDF2 knockdown, promoting cell proliferation (P<0.01) and upregulating PCNA, MCM2, and CyclinD1 (all P<0.05). Mechanistically, YTHDF2 targeted PER1 via m⁶A recognition, promoting PER1 mRNA degradation. Rescue experiments showed that PER1 knockdown reversed the inhibitory effect of YTHDF2 knockdown on cell proliferation, upregulated PCNA, MCM2, and Cyclin D1 (all P<0.05), and promoted bladder cancer cell proliferation (P<0.001). CONCLUSIONS O-GlcNAc modification YTHDF2 promotes bladder cancer development by downregulating the tumor suppressor gene PER1 through m⁶A-mediated post-transcriptional regulation.
Humans ; Urinary Bladder Neoplasms/metabolism* ; RNA-Binding Proteins/genetics* ; Cell Proliferation ; Cell Line, Tumor ; Disease Progression ; Acetylglucosamine/metabolism* ; Adenosine/metabolism* ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor

Gliomas are the most common central nervous system tumours; they are highly aggressive and have a poor prognosis. RGS16 belongs to the regulator of G-protein signalling (RGS) protein family, which plays an important role in promoting various cancers, such as breast cancer, pancreatic cancer, and colorectal cancer. Moreover, previous studies confirmed that let-7c-5p, a well-known microRNA, can act as a tumour suppressor to regulate the progression of various tumours by inhibiting the expression of its target genes. However, whether RGS16 can promote the progression of glioma and whether it is regulated by miR let-7c-5p are still unknown. Here, we confirmed that RGS16 is upregulated in glioma tissues and that high expression of RGS16 is associated with poor survival. Ectopic deletion of RGS16 significantly suppressed glioma cell proliferation and migration both in vitro and in vivo. Moreover, RGS16 was validated as a direct target gene of miR let-7c-5p. The overexpression of miR let-7c-5p obviously downregulated the expression of RGS16, and knocking down miR let-7c-5p had the opposite effect. Thus, we suggest that the suppression of RGS16 by miR let-7c-5p can promote glioma progression and may serve as a potential prognostic biomarker and therapeutic target in glioma.
Humans ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; MicroRNAs/metabolism* ; Glioma/genetics* ; Genes, Tumor Suppressor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor

Genes, p53 ; Genetic Predisposition to Disease ; Germ-Line Mutation ; Humans ; Li-Fraumeni Syndrome/genetics* ; Tumor Suppressor Protein p53/genetics*

BACKGROUND: The pathogenesis of osteosarcoma (OS) is still unclear, and it is still necessary to find new targets and drugs for anti-OS. This study aimed to investigate the role and mechanism of the anti-OS effects of miR-296-5p. METHODS: We measured the expression of miR-296-5p in human OS cell lines and tissues. The effect of miR-296-5p and its target gene staphylococcal nuclease and tudor domain containing 1 on proliferation, migration, and invasion of human OS lines was examined. The Student's t test was used for statistical analysis. RESULTS: We found that microRNA (miR)-296-5p was significantly downregulated in OS cell lines and tissues (control vs. OS, 1.802 ± 0.313 vs. 0.618 ± 0.235, t = 6.402, P < 0.01). Overexpression of miR-296-5p suppressed proliferation, migration, and invasion of OA cells. SND1 was identified as a target of miR-296-5p by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of SND1 abrogated the effects induced by miR-296-5p upregulation (miRNA-296-5p vs. miRNA-296-5p + SND1, 0.294 ± 0.159 vs. 2.300 ± 0.277, t = 12.68, P = 0.003). CONCLUSION Our study indicates that miR-296-5p may function as a tumor suppressor by targeting SND1 in OS.
Bone Neoplasms/genetics* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Endonucleases/genetics* ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; MicroRNAs/genetics* ; Osteosarcoma/genetics*

OBJECTIVE: To explore whether tumor suppressor gene Foxo1 and PTEN play a critical role in the tumorigenesis of mouse natural killer-cell lymphoma. METHODS: NKp46-iCre mice were crossed with mice carrying floxed Foxo1 alleles (Foxo1) as well as floxed PTEN alleles (PTEN) to generate mice in which Foxo1 and PTEN in NK cells were knock-out, referred as Foxo1PTEN. The growth and development of the mice and tumor formation were observed. The flow cytometry was used to detect the percentages of NK cells in main lymphatic organs. B16F10 metanoma model of tumor metastasis was utilized to investigate NK cell-mediated tumor surveillance in vivo after NK cells special deletion of Foxol and PTEN. RESULTS: The mouse model with NK cell-special Foxo1 and PTEN double knockout was established. Compared with control group (Foxo1PTEN mice), Foxo1PTEN mice were born alive and appeared to be healthy over a period of 46 weeks. No spontaneous tumor formation was observed at this stage. There were no significant differences in NK cell percentages of gated lymphocytes from various organs including blood, bone marrow, peripheral lymph node and spleen between Foxo1PTEN mice and Foxo1PTEN mice [PB: 4.76%±0.46% vs 4.17%±0.64% (P＞0.05, n=8); BM: 1.13%±0.23% vs 1.31%±0.10% (P＞0.05, n=8) ; LN: 0.50%±0.10% vs 0.85%±0.20% (P＞0.05, n=8); SP: 4.41%±0.65% vs 3.50%±0.24% (P＞0.05, n=8)]. B16F10 melanoma metastasis model of tumor was established, No differences in median survival time were observed in the 2 types of mice (P＞0.05, n=13). CONCLUSION The simultaneous deletion of the Foxo1 and PTEN genes may not plays significant role in the tumorigenesis of mouse natural killer-cell lymphoma and NK cell-mediated tumor surveillance in vivo.
Animals ; Cell Transformation, Neoplastic ; Forkhead Box Protein O1 ; Genes, Tumor Suppressor ; Killer Cells, Natural ; Lymphoma ; Mice ; Mice, Knockout

Carbonic Anhydrase IV ; genetics ; Chloride Channels ; genetics ; Colorectal Neoplasms ; genetics ; Databases, Genetic ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; Inhibin-beta Subunits ; genetics

BACKGROUND: BRCA1 mutated breast cancer cells exhibit the elevated cell proliferation and the higher metastatic potential. G protein-coupled receptor 30 (GPR30) has been shown to regulate growth of hormonally responsive cancers, such as ovarian and breast cancers, and high expression of GPR30 is found in estrogen receptor (ER)-negative breast cancer cells. ER-negative breast cancer patients often have a mutation in the tumor suppressor gene, BRCA1. This study explored antiproliferative effects of genistein, a chemopreventive isoflavone present in legumes, and underlying molecular mechanisms in triple negative breast cancer cells with or without functionally active BRCA1.METHODS: Expression of BRCA1, GPR30 and Nrf2 was measured by Western blot analysis. Reactive oxygen species (ROS) accumulation was monitored by using the fluorescence-generating probe, 2’,7’-dichlorofluorescein diacetate. The effects of genistein on breast cancer cell viability and proliferation were assessed by the MTT, migration and clonogenic assays.RESULTS: The expression of GPR30 was dramatically elevated at both transcriptional and translational levels in BRCA1 mutated breast cancer cells compared to cells with wild-type BRCA1. Notably, there was diminished Akt phosporylation in GPR30 silenced cells. Treatment of BRCA1 silenced breast cancer cells with genistein resulted in the down-regulation of GPR30 expression and the inhibition of Akt phosphorylation as well as the reduced cell viability, migration and colony formation. Genistein caused cell cycle arrest at the G₂/M phase in BRCA1-mutant cells through down-regulation of cyclin B1 expression. Furthermore, BRCA1-mutant breast cancer cells exhibited higher levels of intracellular ROS than those in the wild-type cells. Genistein treatment lowered the ROS levels through up-regulation of Nrf2 expression.CONCLUSIONS: Lack of functional BRCA1 activates GPR30 signaling, thereby stimulating Akt phosphorylation and cell proliferation. Genistein induces G2/M phase arrest by down-regulating cyclin B1 expression, which is attributable to its suppression of GPR30 activation and Akt phosphorylation in BRCA1 impaired breast cancer cells.
Blotting, Western ; Breast Neoplasms ; Breast ; Cell Cycle Checkpoints ; Cell Proliferation ; Cell Survival ; Cyclin B1 ; Down-Regulation ; Estrogens ; Fabaceae ; Genes, Tumor Suppressor ; Genistein ; Humans ; Phosphorylation ; Reactive Oxygen Species ; Triple Negative Breast Neoplasms ; Up-Regulation

OBJECTIVE: To investigate the effect of the long chain non-coding RNA H19 (lncRNA H19) on the invasion and migration of oral cancer cells and its related molecular mechanism. METHODS: The expression levels of lncRNA H19, miR-107, and cyclin-dependent kinase 6 (CDK6) in the immortalized oral epithelial cell line HIOEC and the oral cancer cell line CAL27 were detected by real-time quantitative polymerase chain reaction. CAL27 cells were transfected with siRNA H19, miR-107 mimics, pcDNA H19, or anti-miR-107, and the effects of H19 and miR-107 on the invasion and migration of cells were examined via Transwell assay. The TargetScan database predicted the targeting of H19, miR-107, and CDK6. Double luciferase reporter gene assay was performed to detect interactions among H19, miR-107, and CDK6. Western blot analysis was conducted to examine the effects of H19 and miR-107 on the protein level of the target gene CDK6. RESULTS: Compared with that in HIOEC cells, the expression of H19 was significantly increased in CAL27 cells (P<0.05). After transfection with siRNA H19, the expression of H19 decreased, and the invasion and migration ability of CAL27 cells were inhibited (P<0.05). H19 could bind specifically to the 3'-UTR of miR-107 to modulate the expression of miR-107. Compared with that in HIOEC cells, the expression of miR-107 significantly decreased in CAL27 cells (P<0.05). The expression of miR-107 increased after transfection with siRNA H19, and anti-mir-107 co-transfection could promote the invasion and migration ability of siRNA H19 in CAL27 cells (P<0.05). Compared with that in HIOEC cells, CDK6 expression significantly increased in CAL27 cells (P<0.05), and the expression level of the gene was coregulated by H19 and miR-107 (P<0.05). CONCLUSIONS lncRNA H19 plays an important role in the development of oral cancer. It can regulate the invasion and migration of oral cancer cells by targeting the miR-107/CDK6 signaling axis.
Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; MicroRNAs ; Mouth Neoplasms ; RNA, Long Noncoding

A variety of clonal cytogenetic abnormalities have been reported in aggressive natural killer (NK)-cell lymphoma and leukemia. Recent chromosomal microarray studies have shown both gain and loss of 1q and loss of 7p as recurrent abnormalities in aggressive NK-cell leukemia. Here, we report a case of aggressive NK-cell leukemia with complex chromosomal gains and losses, as confirmed by chromosomal microarray analysis. The patient showed an aggressive clinical course, which was complicated by hemophagocytic lymphohistiocytosis. Conventional cytogenetic analysis revealed trisomy 3 and 1q gain only. However, chromosomal microarray analysis detected an additional gain of 1q21.1–q24.2 and a loss of 1q24.2–q31.3. These abnormal lesions might play a role in the pathogenesis of aggressive NK-cell leukemia by inactivating tumor suppressor genes or by activating oncogenes. These results suggest that chromosomal microarray analysis may be used to provide further genetic information for patients with hematological malignancies, including aggressive NK-cell leukemia.
Chromosome Aberrations ; Cytogenetic Analysis ; Genes, Tumor Suppressor ; Hematologic Neoplasms ; Humans ; Leukemia ; Lymphohistiocytosis, Hemophagocytic ; Lymphoma ; Microarray Analysis ; Oncogenes ; Trisomy

Objective: To explore the clinical implications and prognostic value of TP53 gene mutation and deletion in patients with myelodysplastic syndromes (MDS) . Methods: 112-gene targeted sequencing and interphase fluorescence in situ hybridization (FISH) were used to detect TP53 mutation and deletion in 584 patients with newly diagnosed primary MDS who were admitted from October 2009 to December 2017. The association of TP53 mutation and deletion with several clinical features and their prognostic significance were analyzed. Results: Alterations in TP53 were found in 42 (7.2%) cases. Of these, 31 (5.3%) cases showed TP53 mutation only, 8 (1.4%) cases in TP53 deletion only, 3 (0.5%) cases harboring both mutation and deletion. A total of 37 mutations were detected in 34 patients, most of them (94.6%) were located in the DNA binding domain (exon5-8) , the remaining 2 were located in exon 10 and splice site respectively. Patients with TP53 alterations harbored significantly more mutations than whom without alterations (z=-2.418, P=0.016) . The median age of patients with TP53 alterations was higher than their counterparts[60 (21-78) years old vs 52 (14-83) years old, z=-2.188, P=0.029]. TP53 alterations correlated with complex karyotype and International prognostic scoring system intermediate-2/high significantly (P<0.001) . Median overall survival of patients with TP53 alterations was shorter than the others[13 (95%CI 7.57-18.43) months vs not reached, χ(2)=12.342, P<0.001], while the significance was lost during complex karyotype adjusted analysis in multivariable model. Conclusion: TP53 mutation was more common than deletion in MDS patients. The majority of mutations were located in the DNA binding domain. TP53 alterations were strongly associated with complex karyotype and always coexisted with other gene mutations. TP53 alteration was no longer an independent prognostic factor when complex karyotype were occurred in MDS.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Genes, p53 ; Humans ; In Situ Hybridization, Fluorescence ; Middle Aged ; Mutation ; Myelodysplastic Syndromes/genetics* ; Prognosis ; Tumor Suppressor Protein p53 ; Young Adult