human uroepithelial cells line  (ATCC)

Journal: Advanced Science

Article Title: m 5 C‐Modified tRF3b‐Cys GCA ‐23 Suppresses Bladder Cancer Malignancy by Repressing H3K18 Lactylation via Stabilizing RBM4

doi: 10.1002/advs.202522294

Figure Lengend Snippet: Identification of the novel m5C‐modified tsRNA m 5 C‐tRF3b‐CysGCA‐23 (mtRC) in transformed urothelial cells and bladder cancer (BC) cells. (A‐C) Heatmaps showing differentially expressed m 5 C‐tsRNAs identified by Arraystar Human m 5 C small RNA modification microarray in SV‐HUC‐1 vs. Cd‐SV‐HUC‐1 (A), SV‐HUC‐1 versus T24 (B), and Cd‐SV‐HUC‐1 vs. T24 (C). (D, E) Venn diagrams showing five commonly upregulated (D) and one commonly downregulated (E) m 5 C ‐tsRNAs across the three comparisons. (F) Venn diagram showing the intersection between tRNAs containing m 5 C modification (from tRNA bisulfite sequencing) and the six commonly regulated m 5 C ‐tsRNAs. (G) Flow chart of mtRC detection using m 5 C‐MeRIP‐3′/5′‐adaptor ligation RT‐PCR Method. (H) mtRC levels in SV‐HUC‐1, Cd‐SV‐HUC‐1, and T24 cells. Statistical significance was determined by one‐way ANOVA followed by Tukey's multiple comparisons test. (I) Representative Northern blot assay confirming mtRC expression in SV‐HUC‐1 and Cd‐SV‐HUC‐1 cells. (J) The abundance of mtRC was detected in BC tissues ( n = 12) compared to para normal tissues ( n = 12). Statistical significance was determined by two‐tailed paired t‐test. (K) The abundance of mtRC was detected in CdCl 2 ‐induced multi‐stage bladder tissues and normal tissues ( n = 5 rats per group). (L) mtRC expression was quantified in urine specimens from BC patients ( n = 12) and healthy donors ( n = 12). Statistical significance was determined by two‐tailed unpaired t‐test. (M) The abundance of mtRC was detected in multi‐stage urine specimens from previously established multi‐stage rat bladder cancer ( n = 5 rats per group). (K,M) Data are presented as box plots showing the median and quartiles. Statistical significance was determined by one‐way ANOVA with linear trend analysis to assess the time‐dependent effect (P trend < 0.0001). (N) Analysis of the correlation between mtRC levels and the degree of carcinogenesis. Data are presented as mean ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human uroepithelial cell line SV‐HUC‐1 (RRID:CVCL_3798) was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in the F‐12K medium with 10% FBS (Gibco).

Techniques: Modification, Transformation Assay, RNA modification, Microarray, Methylation Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: m 5 C‐Modified tRF3b‐Cys GCA ‐23 Suppresses Bladder Cancer Malignancy by Repressing H3K18 Lactylation via Stabilizing RBM4

doi: 10.1002/advs.202522294

Figure Lengend Snippet: The m 5 C‐tRF3b‐Cys GCA ‐23 suppresses BC growth. (A) mtRC levels were decreased in SV‐HUC‐1, Cd‐SV‐HUC‐1 and T24 cells after transfected inhibitor. Statistical significance was determined by two‐tailed unpaired t‐test. (B) Overexpression of tRC in SV‐HUC‐1, Cd‐SV‐HUC‐1 and T24 cells. Statistical significance was determined by two‐tailed unpaired t‐test. (C) Overexpression of mtRC in SV‐HUC‐1, Cd‐SV‐HUC‐1 and T24 cells. Statistical significance was determined by two‐tailed unpaired t‐test. (D–F) Cell proliferative capacity was significantly increased by small interfering RNA in SV‐HUC‐1, Cd‐SV‐HUC‐1 and T24 cells. Statistical significance was determined by repeated Measures ANOVA. (G–I) Cell proliferation assay indicated that tRC (without m 5 C modification) overexpression does not affect cell proliferation in SV‐HUC‐1, Cd‐SV‐HUC‐1, and T24 cells. Statistical significance was determined by repeated measures ANOVA. (J, K) Overexpression of mtRC significantly downregulated the ability of proliferation in Cd‐SV‐HUC‐1 and T24 cells. Statistical significance was determined by repeated measures ANOVA. (L–N) Both tumor weight (M) and tumor volume (N) were markedly decreased with mtRC‐agomir treatment compared to the NS antagomir group, whereas tRC overexpression did not influence tumor growth. (Each group n = 5). (O) tRC‐agomir treatment significantly increased tRC expression. (P)The mtRC‐agomir treatment considerably increased mtRC expression. Data are presented as mean ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human uroepithelial cell line SV‐HUC‐1 (RRID:CVCL_3798) was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in the F‐12K medium with 10% FBS (Gibco).

Techniques: Transfection, Two Tailed Test, Over Expression, Small Interfering RNA, Proliferation Assay, Modification, Expressing

Journal: Advanced Science

Article Title: m 5 C‐Modified tRF3b‐Cys GCA ‐23 Suppresses Bladder Cancer Malignancy by Repressing H3K18 Lactylation via Stabilizing RBM4

doi: 10.1002/advs.202522294

Figure Lengend Snippet: NSUN6 regulates the abundance of mtRC. (A) IGV of tRNA bisulfite sequencing data revealed that the m 5 C methylation site of mtRC is located at C72 (parental tRNA). (B) Flow chart of m 5 C immunoprecipitation combined with bisulfite conversion assay (m 5 C‐BS‐RNA) (C, D) The m 5 C site on mtRC was detected using the m 5 C IP combined with bisulfite conversion assay (BS‐RNA). (E) m 5 C‐specific methylated small RIP microarray analysis in NSUN6‐knockdown Cd‐SV‐HUC‐1 and control cells. (F,G) After the knockdown of NSUN6, the levels of tsRNAs modified by m 5 C, which is mediated by NSUN6, are significantly reduced. (H) The stable cells with knockout NSUN6 were constructed using the CRISPR/Cas9 gene editing technology. NSUN6‐knockout stable Cd‐SV‐HUC‐1 cell lines were established and overexpressed NSUN6 T24 cells. (I,J) Dot blot (G) and Northwestern blot (H) assays revealed that NSUN6 knockdown significantly reduced the m 5 C levels, whereas NSUN6 overexpression enhanced the m 5 C levels in T24 cells. (K,L) The levels of mtRC in NSUN6‐depleted (K) and overexpressed T24 cells (L) were detected using m 5 C IP‐3′/5′ adaptor ligation RT‐PCR. Statistical significance was determined by two‐tailed unpaired t‐test. (M) Construction of knockout NSUN6 and overexpressed NSUN6 stable J82 cells. (N) Northwestern blot assays revealed that NSUN6 knockdown significantly reduced the m 5 C levels, whereas NSUN6 overexpression enhanced the m 5 C levels in J82 cells. (O,P) The levels of mtRC in NSUN6‐depleted (O) and overexpressed J82 cells (P) were detected using m 5 C IP‐3′/5′ adaptor ligation RT‐PCR. Statistical significance was determined by two‐tailed unpaired t‐test. Data are presented as mean ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Human uroepithelial cell line SV‐HUC‐1 (RRID:CVCL_3798) was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in the F‐12K medium with 10% FBS (Gibco).

Techniques: Methylation Sequencing, Methylation, Immunoprecipitation, Microarray, Knockdown, Control, Modification, Knock-Out, Construct, CRISPR, Dot Blot, Over Expression, Ligation, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Advanced Science

Article Title: m 5 C‐Modified tRF3b‐Cys GCA ‐23 Suppresses Bladder Cancer Malignancy by Repressing H3K18 Lactylation via Stabilizing RBM4

doi: 10.1002/advs.202522294

Figure Lengend Snippet: mtRC binds to RBM4 and upregulates its expression. (A) A Venn analysis on the pull‐down proteins using RNA pull‐down assays. (B) RNA pull‐down assays revealed that RBM4 interacted with mtRC. (C) The interaction between RBM4 and mtRC was further validated by RIP assays. Statistical significance was determined by two‐tailed unpaired t‐test. (D) Profile of RBM4 protein. (E) RBM4 protein structure analysis. (F) Vectors carrying GFP‐tagged truncated and full‐length RBM4 were generated. Transfected proteins were verified by WB. (G) RIP assays demonstrated that mtRC interacts with domain II of RBM4. Statistical significance was determined by two‐tailed unpaired t‐test. (H–J) WB and immunofluorescence results demonstrated that mtRC overexpression upregulated the level of RBM4, whereas overexpression of tRC did not affect RBM4 protein levels. Statistical significance was determined by two‐tailed unpaired t‐test. (K) Nucleocytoplasmic separation assay indicated that most mtRC was distributed in the cytoplasm. (L, M) CHX chase results indicated that overexpression of mtRC in T24 and Cd‐SV‐HUC‐1 cells enhanced RBM4 protein stability. Statistical significance was determined by repeated measures ANOVA. (N–P) Deubiquitination assays indicated that mtRC repressed ubiquitin/proteasome‐dependent degradation of RBM4 protein. Data are presented as mean ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human uroepithelial cell line SV‐HUC‐1 (RRID:CVCL_3798) was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in the F‐12K medium with 10% FBS (Gibco).

Techniques: Expressing, Two Tailed Test, Generated, Transfection, Immunofluorescence, Over Expression, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: m 5 C‐Modified tRF3b‐Cys GCA ‐23 Suppresses Bladder Cancer Malignancy by Repressing H3K18 Lactylation via Stabilizing RBM4

doi: 10.1002/advs.202522294

Figure Lengend Snippet: RBM4 is expressed at a low level in BC and inhibits cell proliferation. (A,B) Immunofluorescence indicated that the expression level of RBM4 was gradually downregulated with the increasing extent of carcinogenesis. Data are presented as box plots showing the median and quartiles. Statistical significance was determined by one‐way ANOVA with linear trend analysis to assess the time‐dependent effect (P trend < 0.0001). (C) the RBM4 protein levels were examined in Cd‐SV‐HUC‐1 and BC cells using WB. (D) The stable cells with knockout RBM4 were constructed using the CRISPR/Cas9 gene editing technology. (E) Construction of knockout RBM4 stable cells and overexpressed RBM4 cells. (F,G) CCK‐8 assays revealed that RBM4 depletion strongly upregulated Cd‐SV‐HUC‐1 and 5637 cell growth. Statistical significance was determined by repeated measures ANOVA. (H) CCK‐8 assays revealed that RBM4 overexpression downregulated the proliferation ability of T24 cells. Statistical significance was determined by repeated measures ANOVA. (I,J) EdU assays also revealed that RBM4 knockdown enhances cell proliferation. Statistical significance was determined by two‐tailed unpaired t‐test. (K) EdU assays also revealed that RBM4 overexpression suppresses the proliferation ability of cells. Statistical significance was determined by two‐tailed unpaired t‐test. (L) BC patient‐derived organoid experimental results also indicated that overexpression of RBM4 significantly inhibits BC organoids growth. (M,N) CCK‐8 assays demonstrated that RBM4 overexpression suppressed cell proliferative capability in mtRC‐downregulated Cd‐SV‐HUC‐1 cells. Statistical significance was determined by repeated measures ANOVA. (O,P) A decrease of RBM4 significantly rescued the proliferation in mtRC‐overexpressing Cd‐SV‐HUC‐1 cells. Statistical significance was determined by repeated measures ANOVA. (Q–S) mtRC overexpression markedly inhibited tumor growth, whereas RBM4 silencing partially reversed this inhibitory effect, as reflected by increased tumor weight (R) and tumor volume (S) compared with the mtRC overexpression group (Each group n = 5). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human uroepithelial cell line SV‐HUC‐1 (RRID:CVCL_3798) was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in the F‐12K medium with 10% FBS (Gibco).

Techniques: Immunofluorescence, Expressing, Knock-Out, Construct, CRISPR, CCK-8 Assay, Over Expression, Knockdown, Two Tailed Test, Derivative Assay

Journal: Advanced Science

Article Title: m 5 C‐Modified tRF3b‐Cys GCA ‐23 Suppresses Bladder Cancer Malignancy by Repressing H3K18 Lactylation via Stabilizing RBM4

doi: 10.1002/advs.202522294

Figure Lengend Snippet: RBM4 suppresses glycolytic capacity in BC cells. (A) DEGs were detected by RNA‐seq analysis in Cd‐SV‐HUC‐1 cells with RBM4 knockdown compared to control cells using RNA‐seq. (B) DEGs were assessed for pathway enrichment in metabolism using the KEGG pathway analysis. (C) Pathway analysis of DEGs involved in the glycolytic pathway. (D) ECAR results revealed that RBM4 knockdown accelerated glycolysis in Cd‐SV‐HUC‐1 cells. Statistical significance was determined by two‐tailed unpaired t‐test. (E) ECAR assays indicated that overexpression of mtRC inhibited glycolysis in T24 cells. Statistical significance was determined by two‐tailed unpaired t‐test. (F) ECAR experiment indicated that overexpression of RBM4 rescued glycolysis in mtRC knockdown Cd‐SV‐HUC‐1 cells. Statistical significance was determined by two‐tailed unpaired t‐test. (G) Schematic illustration of RT‐qPCR primer design targeting two ALDOC isoforms (wild‐type and exon 7–skipped). (H) RBM4 knockdown did not alter the exon inclusion pattern of ALDOC. Statistical significance was determined by two‐tailed unpaired t‐test. (I) Schematic illustration of RT‐qPCR primer design targeting two STAT1 isoforms (wild‐type and exon 3–skipped). (J) RBM4 knockdown did not alter the exon inclusion pattern of STAT1. Statistical significance was determined by two‐tailed unpaired t‐test. (K) RT–qPCR showed that RBM4 knockdown upregulated STAT1 mRNA levels. Statistical significance was determined by two‐tailed unpaired t‐test. (L) RNA immunoprecipitation (RIP) assays showed that RBM4 bound to STAT1 mRNA, while STAT1 mRNA enrichment was substantially reduced upon RBM4 silencing. Statistical significance was determined by two‐tailed unpaired t‐test. (M) RBM4 knockdown increased STAT1 mRNA stability. Statistical significance was determined by repeated measures ANOVA. (N) RBM4 depletion resulted in elevated STAT1 protein expression. (O–U) STAT1 knockdown in RBM4‐silenced cells reduced the expression of multiple glycolytic genes. Statistical significance was determined by two‐tailed unpaired t‐test. Data are presented as mean ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human uroepithelial cell line SV‐HUC‐1 (RRID:CVCL_3798) was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in the F‐12K medium with 10% FBS (Gibco).

Techniques: RNA Sequencing, Knockdown, Control, Two Tailed Test, Over Expression, Quantitative RT-PCR, RNA Immunoprecipitation, Expressing

Journal: Advanced Science

Article Title: m 5 C‐Modified tRF3b‐Cys GCA ‐23 Suppresses Bladder Cancer Malignancy by Repressing H3K18 Lactylation via Stabilizing RBM4

doi: 10.1002/advs.202522294

Figure Lengend Snippet: Effects of histone lactylation inhibition by mtRC on BC cells proliferation. (A) the total lactate produced was measured in T24 cells with overexpressed mtRC. (B) Schematic of glycolysis, indicating the strategies used in this study to inhibit lactate production and histone lactylation. (C,D) Cells were treated with varying concentrations of the glycolysis inhibitors 2‐DG or oxamate for 24 h to assess their effects on pan‐Kla and histone lactylation. (E) the levels of pan‐Kla and histone lactylation upon LDH depletion were assessed using Western blotting. (F) mtRC reduced intracellular levels of pan‐Kla and H3K18la. (G) Lactate addition restored H3K18la in LDHA/LDHB‐knockdown SV‐HUC‐1 and T24 cells. (H) Lactate addition restored H3K18la in mtRC‐overexpressing cells. (I) The levels of pan‐Kla and histone lactylation were measured in SV‐HUC‐1 and BC cell lines. (J–O) CCK‐8 (J,K) and EdU (L–O) experiments showed that LDHA/LDHB knockdown strongly reduced the proliferation of Cd‐SV‐HUC‐1 and T24 cells, whereas sodium lactate partially restored growth. (J,K) Statistical significance was determined by repeated measures ANOVA. (N, O) Statistical significance was determined by two‐tailed unpaired t‐test. (P–S) CCK‐8 (P,Q) and EdU (R,S) assays showed that mtRC overexpression significantly suppressed cell proliferation, and this inhibitory effect was partially rescued by sodium lactate treatment. (P,Q) Statistical significance was determined by repeated measures ANOVA. (R,S) Statistical significance was determined by two‐tailed unpaired t‐test. Data are presented as mean ± SEM. ** P < 0.01, *** P < 0.001, *** P < 0.0001.

Article Snippet: Human uroepithelial cell line SV‐HUC‐1 (RRID:CVCL_3798) was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in the F‐12K medium with 10% FBS (Gibco).

Techniques: Inhibition, Produced, Western Blot, Knockdown, CCK-8 Assay, Two Tailed Test, Over Expression