Journal: International Journal of Biological Sciences

Article Title: Loss of UFL1 confers enzalutamide resistance of prostate tumors by regulating METTL16-mediated m6A modification of EEF1A1 mRNA

doi: 10.7150/ijbs.124214

Figure Lengend Snippet: UFL1 is downregulated in ENZR CRPC. (A) Analysis of UFL1 level in human PCa samples from the TCGA-PRAD dataset. (B) Representative IHC staining images for UFL1 in PCa tissues. (C) Statistical analysis of UFL1 protein levels by IHC. (D) WB examination demonstrating UFL1 protein levels in fresh PCa tumors and paired normal tissues. (E-H) Correlation of UFL1 mRNA level with Gleason score (E), clinical stage (F), lymph node metastasis (G), and distant metastasis (H). (I-J) UFL1 levels in PCa cell lines. LNCaP-Parental, LNCaP-ENZR, C4-2B-Parental, and C4-2B-ENZR cells were procured and examined by qRT-PCR (I) and WB (J). (K) Representative IHC images of UFL1 staining in tumor sections from xenograft mice. (L) UFL1 expression in the public GSE44927 dataset. (M-N) Representative IHC images demonstrating UFL1 level in CSPC and CRPC tissues. (O) Examination of UFL1 expression in human PCa samples from GEO datasets. (P-Q) In the published PCa cohort ( GSE116918 ) and TCGA-PRAD dataset, reduced UFL1 expression was correlated with worse overall survival. Scale bar, 50 µm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human PCa cell lines (LNCaP and C4-2B) along with HEK293T cells were procured from the American Type Culture Collection (ATCC) and cultured per the supplier's protocols.

Techniques: Immunohistochemistry, Quantitative RT-PCR, Staining, Expressing

Journal: International Journal of Biological Sciences

Article Title: Loss of UFL1 confers enzalutamide resistance of prostate tumors by regulating METTL16-mediated m6A modification of EEF1A1 mRNA

doi: 10.7150/ijbs.124214

Figure Lengend Snippet: METTL16 facilitates PCa cell growth, survival, and ENZR. (A) Violin plots illustrating CERES scores for all METTL family members across 11 PCa cell lines. (B) Analysis of METTL16 expression in human PCa samples from GEO datasets. (C) Representative IHC staining images for METTL16 and a statistical graph of METTL16 protein levels. (D) METTL16 expression in PCa cell lines. LNCaP-Parental, LNCaP-ENZR, C4-2B-Parental, and C4-2B-ENZR cells were collected, and total lysates were analyzed by WB (D). (E-J) Cell proliferation assessment utilized CCK-8 assay and colony formation assay in LNCaP-ENZR and C4-2B-ENZR cells following transfection with siNC or METTL16 siRNA. (K-L) Cell viability determination occurred in the designated cell lines during ENZ treatment. LNCaP-ENZR and C4-2B-ENZR cells underwent transfection as specified and received graded ENZ doses over 3 days. Cell viability quantification employed CCK-8 assays. (M-N) Recovery effects of wild-type METTL16 and catalytically inactive METTL16 mutants on METTL16 knockdown-mediated cell proliferation inhibition in LNCaP-ENZR and C4-2B-ENZR cells. (O-P) Cell viability determination in specified cell lines under ENZ treatment. Scale bar, 50 µm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human PCa cell lines (LNCaP and C4-2B) along with HEK293T cells were procured from the American Type Culture Collection (ATCC) and cultured per the supplier's protocols.

Techniques: Expressing, Immunohistochemistry, CCK-8 Assay, Colony Assay, Transfection, Knockdown, Inhibition

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: The rs11067228 locus is a functional enhancer in PCa cells. (A) H3K27ac ChIP-seq data of the rs11067228 locus in C4-2B, LNCaP and 22Rv1 PCa lines and in the normal prostate epithelial cell line RWPE1. (B, C, D) Assessment of luciferase activity in 22Rv1, C4-2B and LNCaP cells transduced with a reporter harboring either the non-risk (G) rs11067228-associated enhancer region or the risk (A) allele. The pGL3 promoter vector lacking the enhancer served as control. Firefly luciferase signals were normalized to Renilla signals. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05. (E) Transwell assays of WT 22Rv1 cells and three enhancer KO lines. Cells migrating to the lower chambers were stained with 0.1% crystal violet (left). Scale bar =100 μm. Quantification of corresponding migrated cells is at right. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001. (F) Analysis of colony formation in soft agar of WT 22Rv1 cells and three enhancer KO lines (left). Scale bar =100 μm. Quantification is at right. Data represent means ± S.E.M. of three independent experiments. **P < 0.01.

Article Snippet: The human PCa cell lines 22Rv1, LNCaP and C4-2B were obtained from the American Type Culture Collection (ATCC).

Techniques: Functional Assay, ChIP-sequencing, Luciferase, Activity Assay, Transduction, Plasmid Preparation, Control, Staining

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: Deletion of rs11067228-related enhancer in PCa cells promotes widespread transcriptomic changes and decreases in malignant phenotypes. (A) Heatmap of differentially-expressed genes in 3 different WT and enhancer KO 22Rv1 lines based on RNA-seq (|log2fold-change| >1.3; p-adjusted value <0.05). (B) KEGG pathway analysis showing biological processes associated with downregulated genes in KO relative to WT cells (Top 10). (C) Gene Set Enrichment Analysis (GSEA) of genes differentially expressed in enhancer KO versus WT 22Rv1 cells (|NES| >1 and NOM p-val <0.05). (Upper) Genes associated with drug metabolism cytochrome p450. (Lower) Genes associated with drug metabolism other enzymes. (D, E) Analysis of LDH release in enzalutamide-treated 22Rv1 WT cells and three similarly-treated enhancer KO lines, as an indicator of cytotoxicity. Release was assayed 24 (D) and 48 (E) hours after treatment. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001. (F) MTS assay in enzalutamide-treated 22Rv1 WT cells and three similarly-treated enhancer KO lines, as an indicator of cell viability. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001. (G) Real-time qPCR validation of transcript levels of indicated NE-related genes ( SYP , CHGA and CHGB ) in WT 22Rv1 and three enhancer KO lines. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001. (H) Western blot showing levels of NE-related proteins expression in 22Rv1 WT and three enhancer KO lines.

Article Snippet: The human PCa cell lines 22Rv1, LNCaP and C4-2B were obtained from the American Type Culture Collection (ATCC).

Techniques: RNA Sequencing, MTS Assay, Biomarker Discovery, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: The rs11067228 risk enhancer is a hub for intra- and inter-chromosomal interactions. (A) Circos plot showing genome-wide interactions indicated by curves extending from the enhancer bait locus. For each curve, cis-interactions are depicted in orange and trans-interactions are depicted in blue. Interactions reproducible in two biological replicates are shown. (B) Comparison of fold-changes in gene expression based on RNA-seq and corresponding 4C-Seq signal counts. Boxed genes indicate overlapping down-regulated genes in enhancer KO relative to WT lines with numbers of genome-wide interactions, as indicated by 4C-Seq (log2fold-change < -1.3; p-adjusted value <0.05). (C) Real-time qPCR validation of transcript levels of potential target genes of the risk enhancer based on analysis in enhancer KO cells. Shown are means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (D-G) 3C-PCR and sequencing confirmation of interaction of the rs11067228-associated enhancer with loci harboring NFASC (D), UGT2B15 (E), TBX3 (F) and SRRM4 (G) genes. Chromatograms confirm respective enhancer and target gene sequences flanking a HindIII linker sequence. M: marker. G-DNA: Genomic DNA, serving as PCR template. 3C: PCR products amplified from the DNA fragments in the 3C library. Forward and reverse PCR primers were designed based on sequences at both enhancer and target gene regions, respectively. (H-O) RT-qPCR and western blot analysis validation of mRNA levels of 4 target genes in WT 22Rv1 cells, 3 enhancer KO lines and enhancer-KO cells subjected to CRISPRa to overexpress indicated target genes individually. Data represents means ± S.E.M. of three independent experiments. *** P < 0.001.

Article Snippet: The human PCa cell lines 22Rv1, LNCaP and C4-2B were obtained from the American Type Culture Collection (ATCC).

Techniques: Genome Wide, Comparison, Gene Expression, RNA Sequencing, Biomarker Discovery, Sequencing, Marker, Amplification, Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: UGT2B15 and SRRM4 are essential for the development and maintenance of neuroendocrine differentiation of PCa cells. (A, B) Results of soft agar colony formation assays of WT 22Rv1 cells and three lines each of enhancer KO lines, plus enhancer KO cells subjected to CRISPRa to re-express either UGT2B15 (A) or SRRM4 (B) (left). Scale bar =100 μm. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001. (C, D) Transwell assays of cells described in (A, B) (left). Scale bar =100 μm. Cells migrated to lower chambers were stained with 0.1% crystal violet. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001. (E-H) LDH assays in enzalutamide-treated cells corresponding to those described in (A, B). LDH release was assayed after 24 (E, G) and 48 (F, H) hours of treatment. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (I) MTS assays in enzalutamide-treated cells corresponding to those described in (A, B). Data represent means ± S.E.M. of three independent experiments. ***P < 0.001. (J) Western blot showing NE-related protein ( SYP , CHGA and CHGB ) expression in wild-type 22Rv1 cells, as well as three lines each of enhancer-deleted lines plus enhancer-deleted cells re-expressing (by CRISPRa) SRRM4 . (K) Real-time qPCR validation of transcript levels of NE-related genes in cells corresponding to those described in J. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001.

Article Snippet: The human PCa cell lines 22Rv1, LNCaP and C4-2B were obtained from the American Type Culture Collection (ATCC).

Techniques: Staining, Western Blot, Expressing, Biomarker Discovery

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: SRRM4 knockdown blocks PCa cell neuroendocrine differentiation. (A-G, R) Validation of target gene knockdown in PCa cells. Expression of indicated target genes, as measured by real-time qPCR and western blot analysis. sgRNAs targeting gene promoter regions were used in CRISPR-interference (CRISPRi) assays. Data represents means ± S.E.M. of three independent experiments. *** P < 0.001. (H, I) Soft agar colony formation assays in WT control 22Rv1 cells and in cells made deficient in indicated target genes using CRISPRi (left). Scale bar =100 μm. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001. (J, K) Transwell assays of cells described in (H, I) (left). Scale bar =100 μm. Cells that had migrated to lower chambers were stained with 0.1% crystal violet. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001. (L-O) LDH assays of enzlutamide-treated cells described in (H, I). Assays were performed after 24 (L, N) and 48 (M, O) hours of treatment. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (P) MTS assays of enzlutamide-treated cells described in (H, I). Data represent means ± S.E.M. of three independent experiments. ***P < 0.001. (Q) Real-time qPCR validation of transcript levels of indicated NE-related genes ( SYP , CHGA and CHGB ) in cells made deficient in SRRM4 using CRISPRi. Data represent means ± SEM of three independent experiments. ***P < 0.001. (R) Western blot showing NE-related protein expression in cells made deficient in SRRM4 using CRISPRi.

Article Snippet: The human PCa cell lines 22Rv1, LNCaP and C4-2B were obtained from the American Type Culture Collection (ATCC).

Techniques: Knockdown, Biomarker Discovery, Expressing, Western Blot, CRISPR, Control, Staining

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: SNP rs11067228 is a functional SNP in 22Rv1 cells. (A) Transwell assays in 22Rv1 cells homozygous for the non-risk (G/G, WT) versus risk (A/A, MUT) alleles of rs11067228. Scale bar =100 μm. Cells that had migrated to lower chambers were stained with 0.1% crystal violet. Quantification is at right. Data represent means ± SEM of three independent experiments. **P < 0.01. (B) Soft agar colony formation analysis of WT (G/G) versus MUT (A/A) 22Rv1 cells. Scale bar =100 μm. Quantification is at right. Data represent means ± SEM of three independent experiments. **P < 0.01. (C, D) LDH assays comparing WT (G/G) and MUT (A/A) cells treated with enzalutamide. LDH release was assayed at 24 (C) and 48 (D) hours after treatment. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (E) MTS assays comparing WT (G/G) and MUT (A/A) cells treated with enzalutamide. Data represent means ± S.E.M. of three independent experiments. **P < 0.01. (F) Real-time qPCR validation of transcript levels of indicated NE-related genes in WT (G/G) versus MUT (A/A) cells. Data represent means ± SEM of three independent experiments. ***P < 0.001, **P < 0.01. (G) Heatmap of differentially-expressed genes in two samples of 22Rv1 cells harboring the non-risk (G) allele (WT) and two groups of MUT cells harboring the risk (A) allele, based on RNA-seq (|log2fold change| >1 and p-adjusted value <0.05). (H) KEGG pathway showing biological processes of genes upregulated in MUT relative to WT cells (Top 10). (I) Overlap of genes down-regulated in enhancer KO versus WT lines (p-adjusted value <0.05) (green), with the number of genes up-regulated in lines harboring MUT (A/A) versus WT (G/G) cells (log2fold-change >1; p-adjusted value <0.05) (blue). (J, K) Real-time qPCR and western blot analysis validation of transcript levels of indicated target genes of the risk enhancer in MUT and WT cells. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (L) Real-time qPCR validation of transcript levels of EMT-related genes in WT (G/G), enhancer KO, and MUT (A/A) cells. Data represent means ± SEM of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05. (M-O) Photograph showing 22Rv1 xenografts in nude mice with WT (G/G), enhancer KO, enhancer KO cells subjected to CRISPRa to re-express SRRM4 and MUT (A/A). Tumor volume was measured once a week at indicated time points. Tumor weight was measured when sacrificed (n = 6 per group). Data represent means ± SEM of three independent experiments. ***P < 0.001, **P < 0.01, n.s., not significant. (P) Representative immunohistochemistry staining of indicated 22Rv1 xenografts in (M). Scale bar =100 μm.

Article Snippet: The human PCa cell lines 22Rv1, LNCaP and C4-2B were obtained from the American Type Culture Collection (ATCC).

Techniques: Functional Assay, Staining, Biomarker Discovery, RNA Sequencing, Western Blot, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: The transcription factor SOX4 preferentially bind to the rs11067228 risk allele to modulate target gene expression. (A) Schematic showing DNA-protein pull-down assays and mass spectrometry (MS) analysis to identify TFs binding to the rs11067228 risk allele. (B) Overlap in the number of TFs significantly enriched at the risk (A) relative to non-risk (G) allele (orange) with numbers of predicted TF motifs analyzed in the JASPAR database (blue). (C) The sequence of a potential SOX4 binding motif differs in the risk (A) versus non-risk (G) alleles of rs11067228. (Upper) Predicted preferential SOX4 binding site, based on the JASPAR database. (Lower) Shown are actual sequences of non-risk and risk alleles. (D) Quantification of ChIP-qPCR analysis showing SOX4 enrichment at the rs11067228 locus in indicated WT (G/G) versus MUT (A/A) cells. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, n.s., not significant. (E) Quantification of ChIP-qPCR analysis showing H3K27ac enrichment at the rs11067228 locus in indicated WT (G/G) versus MUT (A/A) cells. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, n.s., not significant. (F) Transwell assays of MUT 22Rv1 cells and two lines each of SOX4 knockdown lines, plus SOX4 knockdown cells subjected to CRISPRa to re-express SRRM4 . Cells migrated to lower chambers were stained with 0.1% crystal violet. Scale bar =100 μm. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, n.s., not significant. (G) Results of soft agar colony formation assays of cells described in (F) (left). Scale bar =100 μm. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (H, I) LDH assays in enzalutamide-treated cells corresponding to those described in (F). LDH release was assayed after 24 (H) and 48 (I) hours of treatment. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05. (J) Real-time qPCR validation of transcript levels of NE-related genes ( SYP , CHGA and CHGB ) in cells described in (F). Data represent means ± S.E.M. of three independent experiments. ***P < 0.001.

Article Snippet: The human PCa cell lines 22Rv1, LNCaP and C4-2B were obtained from the American Type Culture Collection (ATCC).

Techniques: Targeted Gene Expression, Mass Spectrometry, Binding Assay, Sequencing, ChIP-qPCR, Knockdown, Staining, Biomarker Discovery