LNCaP (cat. no. TCHu73), VCaP (cat. no. TCHu220) and PC-3 (cat. no. TCHu158) human prostate cancer cell lines were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. DU145 (cat. no. HTB-81) human prostate cancer cells were obtained from American Type Culture Collection. C4-2 (cat. no. CL-0046) human prostate cancer cells were obtained from Procell Life Science & Technology Co., Ltd. LNCaP, VCaP, C4-2 and DU145 cells were maintained in DMEM (HyClone; Cytiva) supplemented with 10% FBS (Cellmax) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). PC-3 cells were cultured with DMEM/F12 (HyClone; Cytiva) with 10% FBS (Cellmax) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). All cells were kept in a humidified incubator supplied with 5% CO₂ at 37°C.

VIM-AS1 overexpression vector (VIM-AS1 OE; cat. no. PG1-21050014; plasmid backbone, pcDNA3.1), IGF2BP2 overexpression vector (IGF2BP2 OE; cat. PB1-20100010; plasmid backbone, pcDNA3.1) and HMGCS1 overexpression vector (HMGCS1 OE; cat. no. PB1-20100011; plasmid backbone, pcDNA3.1), and pcDNA3.1 empty vector (Vector; cat. no. CL-0046) were purchased from Genecreate. VIM-AS1 short hairpin RNA (shRNA/sh) (sh-VIM-AS1; target sequence, 5′-GCT CCC TTT GGA TGA CAT AGA-3′; plasmid backbone, GV344) and normal scramble short hairpin RNA (sh-NC; control sequence, 5′-TTC TCC GAA CGT GTC ACG T-3′; plasmid backbone, GV344) were purchased from Shanghai GeneChem Co., Ltd. IGF2BP2 small interfering RNA (si-IGF2BP2), HMGCS1 small interfering RNA (si-HMGCS1) and non-targeting control small interfering RNA (siRNA/si) (si-NC; sequence) were purchased from Guangzhou RiboBio Co., Ltd and the sequences of siRNAs are listed in Table SI. The vector and siRNA transfections were performed as described previously (20). Briefly, ~1×10⁶ prostate cancer cells, which were seeded in six-well plates, were transfected with 0.5 µg vector or siRNAs using 2.5 µl Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) at 37°C for 48 h according to the manufacturer's protocol. Cells were collected for reverse transcription-quantitative PCR (RT-qPCR), western blotting, Cell Counting Kit-8 (CCK-8) assays, 5-ethynyl-2′-deoxyuridine (EdU) assays, colony formation assays, chemosensitivity assays, TUNEL assays and flow cytometry analysis 48 h after siRNA transfection. shRNA infections were performed as described previously (21). sh-VIM-AS1 (lentiviral plasmid, GV344; GeneChem, Inc.) and sh-NC were packaged in 293T cells (cat. no. CRL-3216; American Type Culture Collection) using a 2nd generation system with the ratio of lentiviral construct, packaging plasmid (Helper 1.0; GeneChem, Inc.) and envelope plasmid (Helper 2.0; GeneChem, Inc.) (20 µg:15 µg:10 µg). The culture medium was centrifugated at 1,000 × g for 5 min at 4°C after 293T cells were cultured for 48 h at 37°C. The supernatant containing viral particles was collected. After C4-2 cells were infected with sh-VIM-AS1 and sh-NC (multiplicity of infection, 50) at 37°C for 48 h, the efficiency was initially validated by assessing VIM-AS1 expression using RT-qPCR. The cells were then selected using puromycin at a concentration of 2 µg/ml for 2 weeks to obtain C4-2 cells with stable knockdown of VIM-AS1. Subsequently, the cells were maintained in complete medium with puromycin at a concentration of 0.5 µg/ml and collected for RT-qPCR, western blotting, CCK-8 assays, EdU assays, colony formation assays, chemosensitivity assays, TUNEL assays and flow cytometry analysis 48 h after shRNA infection.

A Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corp.) was used to isolate cytoplasmic and nuclear RNA of C4-2 cells according to the manufacturer's protocol. Total RNA from prostate cancer cells and resected tumor tissues was isolated using TRIzol^® solution (Invitrogen; Thermo Fisher Scientific, Inc.). For VIM-AS1, HMGCS1 mRNA and IGF2BP2 mRNA expression analysis, the aforementioned RNA extracts were reverse transcribed using a RevertAid First Strand cDNA Synthesis Kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol, followed by amplification and quantification using a 2× SYBR Green PCR MasterMix Kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol. CFX96 real time PCR detection system (Bio-Rad Laboratories, Inc.) was used for quantitative detection with the following thermocycling conditions: Initial denaturation at 95°C for 2 min, followed by 25 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min. The relative expression levels were determined using the 2^−ΔΔCq method (22). GAPDH was used as the internal control. The primers used in the present study were purchased from Sangon Biotech Co., Ltd. and are shown in Table SI.

Total protein from prostate cancer cells was extracted using RIPA lysis buffer (cat. no. G2002; Wuhan Servicebio Technology Co., Ltd.) supplemented with protease inhibitors (Beijing Solarbio Science & Technology Co., Ltd.). After determining the concertation of each proteins using a BCA protein assay kit (cat. no. G2026; Wuhan Servicebio Technology Co., Ltd.), a total of 30 µg protein per lane was loaded on a 10% SDS-gel, resolved using SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 5% nonfat dry milk at room temperature for 2 h, and then incubated with anti-HMGCS1 (cat. no. ab155787; 1:500; Abcam), anti-IGF2BP2 (cat. no. ab117809; 1:500; Abcam) or anti-GAPDH (cat. no. ab9485; 1:500; Abcam) primary antibodies overnight at 4°C, and subsequently incubated with an HRP-labeled goat-anti-rabbit (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) secondary antibody for 2 h at room temperature. The bands were visualized using Immobilon™ Western Chemilum HRP Substrate (cat. no. WBKLS0100, MilliporeSigma) and analyzed using Quantity One software version 4.6.6 (Bio-Rad Laboratories, Inc.).

A total of 3×10³ cells/well were plated into a 96-well plate. After adherence, cells were treated with different concentrations (0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 10, 20, 30, 40, 50 µM) of enzalutamide at 37°C for 72 h. Subsequently, 10 µl/well CCK-8 solution (cat. no. K1018; Dojindo Molecular Technologies, Inc.) was added to the culture medium. The cells were incubated with CCK-8 reagent at 37°C for 1 h and the optical density values at 450 nm were measured using a microplate reader. The IC₅₀ value was calculated as previously described (23).

A total of 500 C4-2 cells infected with sh-NC and sh-VIM-AS1 and LNCaP cells transfected with Vector and VIM-AS1 OE per well were plated into a 6-well plate. After 10 days of incubation at 37°C, the colonies were washed with PBS, fixed with 90% methanol at room temperature for 10 min and stained with crystal violet for 15 min at room temperature. Subsequently, the colonies (>50 cells) were imaged using a digital camera (version D3200; Nikon Corporation) at ×4 magnification and quantified using Image J software (version 1.5; National Institutes of Health).

TUNEL assays were performed using an In Situ Cell Death Detection Kit (cat. no. 11684817910; Roche Diagnostics GmbH) according to the manufacturer's instructions. Briefly, 1×10⁵ cells/well were seeded in 96-well plates and cultured with complete medium for 48 h at 37°C. Subsequently, cells were stained with 50 µl terminal deoxynucleotidyl transferase for 1 h in the dark at 37°C and 450 µl fluorescein-labeled deoxyuridine triphosphate solution for 1 h in the dark at 37°C after cell fixation by 4% paraformaldehyde for 30 min at room temperature and permeabilization by 0.5% TRITON X-100 for 90 sec at 4°C. Next, cells were stained with TUNEL reaction mixture and DAPI-containing mounting media (0.5 mg/ml; cat. no. S2110; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h in the dark at 37°C. Three visual fields were randomly selected for observation using a laser scanning confocal microscope (FV1000; Olympus Corporation) and corresponding software (FV10-ASW Viewer; version 4.2; Olympus Corporation) at ×200 magnification.

EdU assays were conducted using a Cell-Light™ EdU DNA Cell Proliferation Kit (cat. no. C10310-1; Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. Briefly, 1×10⁵ cells/well were seeded in 96-well plates and cultured with complete medium for 48 h at 37°C. Cells were stained with 100 µl 50 µM EdU solution for 2 h in the dark at room temperature. The cells were stained with Apollo^®567 (Guangzhou RiboBio Co., Ltd.) for 30 min at 4°C and DAPI for 10 min at 4°C after cell fixation by 4% paraformaldehyde for 30 min at room temperature and permeabilization by 0.5% TRITON X-100 for 90 sec at 4°C. Three visual fields were randomly selected for observation using a laser scanning confocal microscope (FV1000; Olympus Corporation) and corresponding software (FV10-ASW Viewer; version 4.2; Olympus Corporation) at ×200 magnification.

Cell apoptosis and cell cycle analyses were performed using an Annexin V Alexa fluor 488/PI Cell Apoptosis Kit and DNA Content Quantitation Kit (cat. no. CA1040; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. Briefly, 1×10⁶ cells/well were seeded in 6-well plates and cultured for 48 h at 37°C. For cell apoptosis, 1×10⁶ cells were harvested and stained with annexin-V and PI for 30 min at 4°C. For cell cycle analysis, 1×10⁶ cells were harvested and stained with PI for 30 min at 4°C after fixing with 70% ethanol for 30 min at 4°C. Early cell apoptosis (lower right quadrant-prophase apoptosis; Beijing Solarbio Science & Technology Co., Ltd.) and cell cycle (Beijing Solarbio Science & Technology Co., Ltd.) distribution analyses were performed using a flow cytometer (CytoFLEX LX; Beckman Coulter, Inc.). All data were analyzed with Flowjo version 10.0.6 (Becton, Dickinson and Company).

Total RNA was isolated from transfected LNCaP cells. Subsequently, 1.3 µg total RNA was sent for RNA sequencing (RNA-seq; Genecreate Co., Ltd.). RNA libraries for RNA-seq were prepared using a NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (cat. no. NEB+e7770; New England BioLabs, Inc.) according to the manufacturer's protocols (New England BioLabs, Inc.). The loading concentration of the constructed library was detected using the Agilent High Sensitivity DNA kit (cat. no. 5067-4626; Agilent Technologies, Inc.) and an Agilent 2100 bioanalyzer (Agilent Technologies, Inc), and was 6 pM for RNA-seq. Libraries which consisted of cDNA fragments of 200 bp in length were sequenced on the Illumina NovaSeq 6000 (150 base pairs; paired ends; Illumina, Inc.) using a MiniSeq High Output Reagent Kit (cat. no. FC-420-1002; Illumina, Inc.). Sequence reads were trimmed for adaptor sequence/low-quality sequence using fastp (version 0.12.0; https://github.com/OpenGene/fastp) (24). Trimmed sequence reads were mapped to hg38 using Hisat2 software (version 2.2.1; http://daehwankimlab.github.io/hisat2/) (25). Reads per kilobase of exon per megabase of library size were calculated using featureCounts (version 1.5.3; http://bioinf.wehi.edu.au/featureCounts/) (26). Differentially expressed genes were identified according to |log2(fold change)|>1 and P<0.05 using the R program (version 4.03; http://www.r-project.org). Next, Gene Ontology (GO; http://geneontology.org/) (27,28) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.kegg.jp/) (29) analysis, and Gene Set Enrichment Analysis (GSEA; http://www.gsea-msigdb.org/gsea/index.jsp) were used to further analyze the differentially expressed genes. The significance cult-off level was P<0.05.

Cells were lysed in NP40 lysis buffer (cat. no. N8030; Beijing Solarbio Science & Technology Co., Ltd.), and 1 mg cell extracts were incubated with a biotin-labeled VIM-AS1 (Genecreate Co., Ltd.; sequence, 5′-UCC CUG AGA UGA UGA AGA GGA CCA GUG CCC AUU CCA GGA-3′) or a normal scrambled control (NC; Genecreate Co., Ltd.; sequence, 5′-UAU CAC GUA GCC GUU GCA UUU GCC GUA GCC CUG UGG GCC-3′) probe at 4°C for 6 h. Subsequently, cell extracts, biotin-labeled VIM-AS1 or NC, and 40 µl streptavidin agarose beads were mixed and incubated on a rotator overnight at 4°C. After washing with PBS, precipitates were pulled down by centrifugation at 3,000 × g for 5 min at 4°C. The potential binding proteins in the retrieved precipitates were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) detection (Genecreate Co., Ltd.). HPLC-MS analysis was performed on a Orbitrap Exploris 480-mass spectrometer (Thermo Fisher Scientific, Inc.) that was coupled to a Nanospray Flex™ (Thermo Fisher Scientific, Inc.) for 60 min. The mass spectrometer was operated in positive ion mode to monitor the m/z transitions for all peptides. Target peptides (two for each protein) were measured in multiple reaction monitoring (MRM). Peptide ions between 350 and 1,200 m/z were scanned in the Orbitrap detector (Thermo Fisher Scientific, Inc.) every 3 sec with a resolution of 1.2×10⁵ (maximum fill time 50 msec; automatic gain control target 4×10⁵). A scheduled MRM acquisition method was constructed using manually optimized decluttering potentials, collision energies, collision cell entrance and exit potentials.

A total of 1×10⁶ cells were seeded in 6-well plates and cultured overnight. The following day, 5 µg/ml actinomycin D (cat. no. HY-17559; MedChemExpress) was added to cells to inhibit gene transcription for 2, 4, 6 or 8 h at 4°C. At the indicated times, the mRNA levels of HMGCS1 were determined using RT-qPCR as aforementioned.

RIP assays were performed using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (cat. no. 17-704; MilliporeSigma) according to the manufacturer's instructions. Briefly, a total of 1×10⁷ cells were collected, centrifuged at 4°C for 5 min at 1,000 × g, washed in pre-cooled PBS, and then lysed using complete RIP Lysis buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.). A total of 5 µg anti-IGF2BP2 (cat. no. ab117809; 1:500; Abcam) or lgG (cat. no. ab172730; 1:500; Abcam) was added to 50 µl protein A/G magnetic bead suspension for 30 min at room temperature. Subsequently, 100 µl of the aforementioned lysates were incubated with the beads-antibody complex overnight at 4°C. After washing with RIP wash buffer (part of the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit) three times, Protease K buffer (cat. no. P1120; Beijing Solarbio Science & Technology Co., Ltd.) was added to the immunoprecipitated product, followed by incubation at 55°C for 30 min. Following centrifugation at 4°C for 5 min at 1,000 × g, the immunoprecipitated RNA was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and analyzed using RT-qPCR as aforementioned.

FISH assays were performed using a RiboTM lncRNA FISH probe Mix Kit (cat. no. c10910; Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. Oligonucleotide modified Cy-3-labeled probes for VIM-AS1 (5′-TAG GAC TTC CTA GTA CTT CTG A-3), GAPDH and U6 were designed and synthesized by Genecreate. C4-2 cells were seeded on 20-mm confocal dishes (Corning, Inc). After overnight incubation, C4-2 cells were fixed with 4% paraformaldehyde for 20 min at 4°C and permeabilized using Triton X-100 for 90 sec at 4°C. Next, 250 µl prehybridization solution with 1% blocking solution (Guangzhou RiboBio Co., Ltd.) was added to C4-2 cells and cells were incubated at 42°C for 1 h. Subsequently, C4-2 cells were incubated with 100 µl hybridization buffer (Guangzhou RiboBio Co., Ltd.) supplemented with 1% blocking solution and 2.5 µl 20 µM 22-nucleotide CY-3-labeled-VIM-AS1, CY-3-labeled-GAPDH or CY-3-labeled-U6 FISH probe at 37°C overnight in a dark moist chamber. The following day, cells were washed three times in 2X sodium citrate buffer for 5 min at 42°C and stained with DAPI at 4°C for 10 min. Images were acquired using a laser scanning confocal microscope (FV1000; Olympus Corporation) and corresponding software (FV10-ASW Viewer; version 4.2; Olympus Corporation) at a magnification of ×400.

All animal experiments were performed in accordance with the relevant national ethical regulations and were approved by the Animal Care and Use Committee of the Air Force Medical University (approval no: 20220967; Xi'an, China). All mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in an animal room at a controlled temperature of 22°C and 40% humidity, with a 12 h light/dark cycle, and ad libitum access to food and water. Female BALB/c nude mice (6 weeks old; n=12; weight, ~20 g) were used to establish the C4-2 xenograft models. 100 µl C4-2 cells (5×10⁶/100 µl PBS) infected with sh-VIM-AS1 or sh-NC were injected subcutaneously into the left upper limb of the nude mice. For the following 5 weeks, the health and behavior of the mice were monitored twice a week, and tumor growth was monitored using a vernier caliper twice a week and an in vivo imaging system once a week. When the humane endpoints were reached or the tumor volume of mice in the sh-VIM-AS1 or sh-NC groups was closed 1,000 mm³, the mice would be euthanized. According to the requirements of the Animal Center of Air Force Medical University (Xi'an, China), humane endpoints were reached when the xenograft tumor diameter was >20 mm, or signs of unrelieved pain or distress without recovery were observed. At the end of 35 days of observation, all 12 mice had not reached human endpoints but the largest tumor volume was 892.4 mm³. Therefore, all 12 mice were sacrificed by acute exsanguination following isoflurane inhalation (5% for induction and 2% for maintenance of 5 min), cardiac arrest was then used to verify death based on a lack of a heartbeat. The tumors were excised and assessed using immunohistochemistry and RT-qPCR, and the tumor volumes and weights were measured.

Resected tumor tissues were fixed with 4% paraformaldehyde at 4°C for 24 h. The tissues were embedded in paraffin and sectioned (4 µm). After conventional dewaxing and rehydration with descending alcohol series at room temperature, the sections were incubated with 3% H₂O₂ for 15 min. The sections were incubated with 5% normal goat serum (SL038; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 1 h. Subsequently, Ki-67 antibody (cat. no. GB121141; 1:1,000; Wuhan Servicebio Technology Co., Ltd.) or HMGCS1 antibody (cat. no. ab155787; 1:500; Abcam) were added for incubation at 4°C overnight after antigen retrieval with citric acid buffer (pH 6.0) for 1 min 40 sec at 100°C in a pressure cooker. Subsequently, the sections were incubated with HRP-conjugated secondary antibody (cat. no. G1214; 1:200; Wuhan Servicebio Technology Co., Ltd.) at room temperature for 1 h. The sections were rinsed three times using TBS with 0.1% Tween-20 for 5 min and visualized using DAB solution (cat. no. G1212; 1:1,000; Wuhan Servicebio Technology Co., Ltd.) for 10 min at room temperature. Images were acquired using a light microscope at a magnification of ×200.

RNA-seq expression profiles and corresponding clinical information for VIM-AS1 in prostate cancer were downloaded from The Cancer Genome Atlas (TCGA; https://portal.gdc.cancer.gov/projects/TCGA-PRAD; project ID, TCGA-PRAD, mRNA sequencing data) and the Gene Expression Omnibus (GEO) database (dataset no. 32269, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32269) (30) and analyzed using the R program (version 4.03; http://www.r-project.org). The m6A modification status was predicted using Whistle (http://180.208.58.19/whistle/index.html). The gene (HMGCS1) was entered in the search box. m6A predicted modification sites were listed. P<0.05 was considered to indicate a statistically significant difference.

GraphPad Prism version 8.02 (GraphPad Software; Dotmatics) and SPSS version 22.0 (IBM Corp.) were used to perform statistical analysis. Data are presented as the mean ± SD or as scatter dot plots. All experiments were repeated three times. Unpaired Student's t-test was used to compare the difference between two groups, and one-way ANOVA followed by post hoc tests (least significant difference test for three groups and Bonferroni test for more than three groups) was used for comparisons among multiple groups. A Kruskal-Wallis test followed by a Bonferroni test was used to compare the difference of VIM-AS1 expression in patients with prostate cancer with different T stage and normal controls. Kaplan-Meier survival curves and the log-rank test were used to compare the differences in disease-free survival of patients with prostate cancer with different VIM-AS1 expression in TCGA dataset (project ID, TCGA-PRAD; mRNA data; cutoff-high, 75%; cutoff-low, 25%). The following parameters were selected: Disease-free survival, split patients by lower quartile, follow-up threshold: 14 years. Log rank P-values <0.05 for the Kaplan-Meier plots of patients with prostate cancer with different VIM-AS1 expression were considered to indicate a statistically significant difference. P-values were determined using a two-sided test. P<0.05 was considered to indicate a statistically significant difference.

A previous study indicated that VIM-AS1 was upregulated in C4-2 cells compared with LNCaP cells (14). To further assess the differential expression profiles of VIM-AS1 during the progression of prostate cancer, VIM-AS1 expression was analyzed using TCGA and GEO (GSE32269). VIM-AS1 expression was consistently increased in advanced prostate cancer based on the data from TCGA (P<0.001; Fig. 1A). In addition, VIM-AS1 was upregulated in the tissues of patients with CRPC compared with those of patients in the HSPC group based on the GSE32269 dataset from GEO (P<0.001; Fig. 1B). Furthermore, Kaplan-Meier survival data indicated that patients with prostate cancer with high VIM-AS1 expression exhibited worse disease-free survival compared with patients with low VIM-AS1 expression (P=0.003; Fig. 1C). Increased VIM-AS1 expression was also observed in androgen-independent prostate cancer cell lines (DU145, PC-3 and C4-2) compared with LNCaP and VCaP cell lines, which are androgen-dependent prostate cancer cell lines (Fig. 1D). These data indicated that VIM-AS1 expression was upregulated in advanced prostate cancer.

To examine the effects of VIM-AS1, VIM-AS1 was stably overexpressed in LNCaP cells and VIM-AS1 expression was knocked down in C4-2 cells. RT-qPCR demonstrated that transfection with the VIM-AS1 overexpression vector increased VIM-AS1 expression in LNCaP cells (P<0.05; Fig. 1E), whereas sh-VIM-AS1 transfection stably knocked down VIM-AS1 expression in C4-2 cells (P<0.05; Fig. 1F). Furthermore, it was demonstrated that the IC₅₀ of enzalutamide was increased after overexpression of VIM-AS1 in LNCaP cells (P<0.05; Figs. 2A and S1A). However, the IC₅₀ of enzalutamide was decreased after the knockdown of VIM-AS1 in C4-2 cells (P<0.05; Figs. 3A and S1A). Colony formation and EdU assays indicated that the proliferation of VIM-AS1-overexpressing LNCaP cells was significantly increased compared with that of the control group (P<0.05; Figs. 2B and C, and S1B and C), and sh-VIM-AS1-infected C4-2 cells exhibited significantly reduced proliferation compared with the sh-NC-infected cells (P<0.05; Figs. 3B and C, and S1B and C). In addition, TUNEL and flow cytometry analyses demonstrated that overexpression of VIM-AS1 significantly reduced apoptosis, increased the percentage of cells in the S stage, and decreased the percentage of cells in the G1 stage in LNCaP cells (P<0.05; Figs. 2D-F and S1D-F), whereas cell apoptosis and the percentage of cells in the G1 stage were markedly enhanced and the percentage of cells in the S stage was decreased following VIM-AS1 knockdown in C4-2 cells (P<0.05; Figs. 3D-F and S1D-F). Collectively, these data suggested that aberrant upregulation of VIM-AS1 served an important role in promoting proliferation and driving enzalutamide resistance in prostate cancer in vitro.

A nude mouse model was used to validate the effects of knockdown of VIM-AS1 on the proliferation of prostate cancer cells in vivo. C4-2 cells with stable knockdown of VIM-AS1 were injected into nude mice via subcutaneous injection. Small animal in vivo imaging and growth curves indicated that the mice injected with the VIM-AS1 knockdown C4-2 cells exhibited slower tumor growth than the mice in the sh-NC group (Fig. 4A and B). Similarly, the sizes and weights of the dissected tumors from nude mice indicated that knockdown of VIM-AS1 suppressed tumor growth of prostate cancer cells in vivo (Fig. 4C and D). VIM-AS1 expression was lower in the resected tissues from the sh-VIM-AS1 group compared with the sh-NC group (Fig. S2A). Ki-67 and TUNEL assays indicated that the subcutaneous tumors in the VIM-AS1 knockdown group had fewer Ki-67-positive cells (P<0.05; Fig. 4E) and more TUNEL-positive cells (P<0.05; Fig. 4F) compared with those in the sh-NC group. Overall, these results indicated that knockdown of VIM-AS1 expression in C4-2 cells markedly suppressed tumor growth of prostate cancer in vivo.

Next, it was attempted to determine the mechanism through which VIM-AS1 regulated the proliferation and enzalutamide sensitivity of prostate cancer cells. First, RNA-seq was used to identify the target genes in VIM-AS1-overexpressing LNCaP cells and vector-transfected cells. A total of 67 genes were found to be statistically upregulated by |log2(fold change)|>1 and 187 genes were found to be statistically downregulated by |log2(fold change)|>1 in VIM-AS1-overexpressing LNCaP cells (Fig. 5A). Notably, HMGCS1 was one of the most significantly upregulated genes in VIM-AS1-overexpressing cells (Fig. 5B). KEGG analysis showed that Steroid biosynthesis, Fat digestion and absorption and HIF-1 signaling pathway etc. were among the top 20 most enriched pathways (Fig. 5C). GO analysis demonstrated the 'steroid biosynthetic process', 'unfolded protein binding' and 'double-stranded RNA binding' were among the top 20 most enriched functions (Fig. 5D). Furthermore, GSEA demonstrated that the gene signatures of 'Steroid biosynthesis' were enriched in VIM-AS1-overexpressing cells (Fig. 5E). Notably, a previous study has reported that HMGCS1 was actively involved in steroid biosynthesis (31). As indicated by IHC analyses (Fig. S2B), RT-qPCR (Figs. 5F and S2C), and western blotting (Fig. 5G), the mRNA and protein levels of HMGCS1 were upregulated in LNCaP cells transfected with the VIM-AS1 overexpression vector in vitro (Fig. 5F and G), but downregulated in C4-2 cells transfected with sh-VIM-AS1 compared with the vector-transfected cells in vitro (Fig. 5F and G) and in vivo (Fig. S2B and C) (all P<0.05). Next, it was assessed whether VIM-AS1 regulated HMGCS1 expression by stabilizing HMGCS1 mRNA. To test this hypothesis, prostate cancer cells were treated with actinomycin D to measure the degradation of HMGCS1 mRNA. VIM-AS1 overexpression enhanced HMGCS1 mRNA stability, and VIM-AS1 knockdown significantly reduced HMGCS1 mRNA stability (at 4, 6 and 8 h; P<0.05; Fig. 5H). These results indicated that HMGCS1 may be involved in the functions of VIM-AS1 in prostate cancer cells.

To investigate the regulatory mechanisms of VIM-AS1 in prostate cancer, the subcellular localization of VIM-AS1 was further explored. RT-qPCR analysis of the nuclear and cytoplasmic fractions (Fig. 6A) and FISH analysis (Fig. 6B) showed that VIM-AS1 was present in both the cytoplasm and the nucleus of C4-2 cells. RNA pull-down assays were performed to identify VIM-AS1-interacting proteins. Several bands were identified as potential binding proteins that could be pulled down by biotinylated VIM-AS1 transcripts (Fig. 6C). By searching the functions of 13 potential VIM-AS1 binding proteins in published studies, IGF2BP2, a major differential band precipitated in LNCaP lysates of RNA pulldown (Fig. 6D) and mass spectrometry (Fig. S3), was confirmed as the only steroid biosynthesis-related protein with a molecular mass of 25-116 kDa (32). RNA pull-down assays and western blotting further confirmed that VIM-AS1 interacted with IGF2BP2 in LNCaP and C4-2 cells (Fig. 6E). The enrichment of VIM-AS1 in the precipitates of the IGF2BP2 antibody but not IgG antibody were further confirmed by RIP assays (Fig. 6F). VIM-AS1 knockdown or overexpression exhibited no influence on IGF2BP2 mRNA and protein levels (Fig. 6G and H). In summary, these results indicated that VIM-AS1 directly bound to IGF2BP2 protein but did not regulate its expression in prostate cancer cells.

As IGF2BP2 is a known N⁶-methyladenosine (m6A) reader and serves a pivotal role in downstream mRNA stabilization (33), it was next assessed whether IGF2BP2 was involved in the regulation of HMGCS1 expression. First, the HMGCS1 mRNA m6A modification status was predicted using Whistle (34). The results demonstrated that the 3′-untranslated region (3′-UTR) of HMGCS1 mRNA contained three m6A modification sites (Fig. S4). RT-qPCR and western blotting revealed that knockdown of IGF2BP2 or transfection with si-HMGCS1 inhibited VIM-AS1 overexpression vector transfection-induced HMGCS1 upregulation (Figs. 7A and B, and S5A and B). Overexpression of IGF2BP2 or HMGCS1 reversed sh-VIM-AS1-induced HMGCS1 downregulation (Figs. 7C and D, and S5C and D). mRNA stability assays demonstrated that overexpression of VIM-AS1 enhanced HMGCS1 mRNA stability, and enhancement of HMGCS1 mRNA stability was decreased following IGF2BP2 knockdown (Fig. 7E). Furthermore, knockdown of VIM-AS1 decreased HMGCS1 mRNA stability, and this was reversed by overexpression of IGF2BP2 (Fig. 7F). Collectively, these data suggested that the VIM-AS1/IGF2BP2 axis may exert roles in HMGCS1 post-transcriptional mRNA stabilization.

To further elucidate the functional interplay between HMGCS1 and VIM-AS1 in the regulation of proliferation and enzalutamide sensitivity of prostate cancer, HMGCS1 expression was knocked down in VIM-AS1-overexpressing LNCaP cells and HMGCS1 was overexpressed in the VIM-AS1 knockdown C4-2 cells. Functional experiments revealed that knockdown of HMGCS1 expression abrogated the increase in proliferation (Figs. 8B and C, and S5F and G) and cells at S stage (Figs. 8F and S5J), and decrease in enzalutamide sensitivity (Figs. 8A and S5E), apoptosis levels (Figs. 8E and S5I) and cells at G1 stage (Figs. 8F and S5J) induced by VIM-AS1 overexpression in LNCaP cells. Overexpression of HMGCS1 ameliorated the increase in enzalutamide sensitivity (Figs. 8A and S5E), apoptosis level (Figs. 8E and S5I) and cells at G1 stage (Figs. 8F and S5J), and reduced the increase in proliferation (Figs. 8B and C, and S5F and G) and cells at S stage (Figs. 8F and S5J) induced by VIM-AS1 knockdown in C4-2 cells. These results indicated that HMGCS1 mediated the VIM-AS1-induced increase in proliferation and enzalutamide resistance in prostate cancer cells.