DLEU2 facilitates bladder cancer progression through miR-103a-2-5p/SOS1 axis

Microarray raw data analyses

Employing the raw data sourced from the NCBI GEO database, the expression patterns of lncRNAs, miRNAs, and mRNAs were examined in both normal bladder tissues and BC tissues. Differentially expressed lncRNAs, miRNAs, and mRNAs were detected according to the standards of P < 0.05, and |fold change| ≥ 2. Heat maps, volcano plots, and Venn diagrams were constructed to visualize the results of differential expression analyses. Interactions between lncRNA and miRNA, as well as between miRNAs with mRNAs were predicted using databases such as lncbase, targetscan, miRDB, and BiBiServ.

Cell culture with transfection

Human BC cells (5637, T24, SW780, J82, and 253J) along with the normal bladder epithelial cell line (SV-HUC-1) were procured from the Shanghai Academy of Life Science (Shanghai, China). Cells were cultured in RPMI 1640 medium (Gibco) fortified with 10% FBS (Gibco) and 1% (penicillin + streptomycin) (Sigma) at 37 °C with 5% CO₂.

Small interfering RNAs (SiRNAs) targeting DLEU2 (si-DLEU2-1, si-DLEU2-2, si-DLEU2-3) and SOS1 (si-SOS1) negative control siRNA (si-NC), DLEU2 overexpression (ov-DLEU2), SOS1 overexpression (ov-SOS1), and their corresponding negative control vectors (pcDNA3.1 plasmid) (ov-NC), miR-103a-2-5p mimic, miR-103a-2-5p inhibitor, and miR mimic control (mimic NC), along with inhibitor NC, were procured from GenePharma (Shanghai, China). Transfections were carried out using Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer’s protocol. Transfection efficiency was confirmed via reverse transcription quantitative polymerase chain reaction (RT-qPCR). All experiments were performed in triplicate. Detailed information on cell lines is available at https://www.cellosaurus.org/. The siRNA sequences are listed in Table 1.

RT-qPCR

Total cellular RNA was isolated from cells through the utilization of the TRIzol reagent (Invitrogen) with DNase treatment. RNA purity (OD260/OD280 > 1.8) was assessed using a BioPhotometer Plus spectrophotometer (Eppendorf, Hamburg, Germany), and RNA integrity was appraised by subjecting it 1% denatured agarose gel electrophoresis. Reverse transcription was carried out using the MicroRNA RT (Applied Biosystems) for miR-103a-2-5p and the RT Reagent kit (TaKaRa) for lncRNAs and mRNAs. Quantitative PCR (qPCR) was executed with the ABI PCR system (Applied Biosystems). Fold changes in the transcripts were computed using the 2^−ΔΔCT method. U6 served as the internal reference for miRNA (miR-103a-2-5p; NR_029519.1), and GAPDH for mRNA (DLEU2; NR_152566.1 and SOS1; NM_001382394). Primer sequences are detailed in Table 2 and were validated using BLAST.

EdU assays

BC cells were transfected for 48 h and co-cultured with EdU reagent (RiboBio) for 2 h. Cells were fixed using 4% formaldehyde, permeabilized, and stained with Apollo 643 reagent. To visualize the nuclei, they were counterstained with DAPI (Sigma), and images of these cells were captured employing a fluorescence microscope (Olympus).

Cell proliferation assays

Cell proliferation assay was assessed by means of the Cell Counting Kit-8 (CCK-8) method. Transfected BC cells were seeded in plates and incubated for 0 and 24 h. At each of these time points, 10 µL of CCK-8 solution was added into each well. Subsequently, the plates were further incubated for an additional 2 h at 37 °C. Optical density (OD) was measured at 450 nm using a microplate reader (Bio-Rad).

Wound healing assays

After transfection, cells were seeded into 24-well plates and cultured for 24 h to form a confluent monolayer. A sterile pipette tip was used to create a wound in the cell layer. After washing, the cells were cultured in complete media for 48 h and wound closure was monitored and photographed. Measurements were repeated at least three times for statistical analysis.

Transwell assay

Cell migration was assessed using a Transwell migration chamber (Sigma). Cells were seeded in serum-free media within the upper chamber, while the lower chamber filled with RPMI 1640 medium was supplemented with 20% fetal bovine serum (FBS). After 24 h, cells on the upper membrane surface were removed and cells on the lower surface were then fixed with 4% polyformaldehyde (PFA), stained with 0.1% crystal violet (Beyotime), and counted in five random fields under a microscope.

Luciferase reporter assay

Luciferase reporter assays were performed using the psi-CHECK2 vector (Promega). Constructs containing wild-type (WT) or mutant (MUT) sequences of DLEU2 and SOS1 3′-UTRs were synthesized by GenePharma. The constructs were co-transfected with miRNA mimic or miRNA inhibitors into 239T cells. After 48 h, luciferase activity was measured using the Dual-Luciferase Assay kit (Promega). Renilla luciferase activity was normalized against firefly luciferase activity.

Western blotting

Total protein extraction was achieved by using RIPA lysis buffer containingprotease inhibitors (Beyotime). Proteins (30 µg per sample) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes supplied by Millipore. Membranes were first blocked and then incubated overnight at 4 °C with primary antibodies: anti-SOS1 antibody (1:1,000, ab140621, Abcam) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1,500, ab181602, Abcam). After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,500, ab205718, Abcam) for 2 h at 25 °C. Protein bands were made visible with the help of an enhanced chemiluminescence detection kit (Beyotime) and imaged with the ChemiDoc™ XRS systems (Bio-Rad).

Statistical analysis

The data underwent analysis by means of IBM SPSS 22.0 and are presented as means ± standard deviation (SD). To determine statistical significance, the t-test was utilized when making comparisons between two groups. For comparisons among multiple groups, one-way analysis of varience (ANOVA) followed by Tukey’s post-hoc test was employed. A P value of less than 0.05 was regarded as indicating statistical significance.

DLEU2 was markedly elevated in BC

To identify abnormally expressed lncRNAs in BC, sequencing data from BC tissues (n = 4) along with adjacent normal tissues (n = 4) were evaluated using the GEO (GSE138118) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138118). The top 10 differentially expressed lncRNAs were selected (Figs. 1A and 1B). Among them, DLEU2 showed the highest upregulation (22.48 fold change) and CECR9 the most downregulated (4.55 fold change) in J82 BC cells compared to SV-HUC-1 normal bladder epithelial cells, as confirmed by RT-qPCR (Fig. 1C). Considering these observations, DLEU2 was singled out for more in-depth investigation.

DLEU2 overexpression could facilitate BC cell proliferation, migration

DLEU2 expression was evaluated across BC cell lines to explore its functional role in BC. DLEU2 expression was highest in J82 cells and lowest in T24 cells (Fig. 2A). J82 and T24 cells were thus chosen for subsequent experiments. DLEU2 overexpression was achieved in T24 cells by (Fig. 2B) transfecting an ov-DLEU2 vector, while DLEU2 knockdown in J82 cells was achieved using si-DLEU2. Si-DLEU2-1, demonstrating the most effective knockdown, was used for all further analyses. Functional assays, including proliferation (Edu), transwell, and scratch assays revealed that DLEU2 overexpression significantly enhanced T24 cell proliferation along with migration, while DLEU2 knockdown in J82 cells suppressed these functions (Figs. 2C–2F).

DLEU2 sponged miR-103a-2-5p

Given the predominant cytoplasmic localization of DLEU2 in J82 cells (Fig. 3A), its potential interaction with miRNAs was investigated. The lncbase and GEO databases identified 9 miRNAs downregulated in BC (Figs. 3B and 3C). qRT-PCR analysis confirmed that miR-103a-2-5p was the miRNA showing the greatest degree of downregulation within BC cells and was inversely correlated with DLEU2 expression (Figs. 3D and 3E). BiBiServ analysis and dual-luciferase assays validated a direct interaction between DLEU2 and miR-103a-2-5p (Fig. 3F). Additionally, RNA binding protein immunoprecipitation (RIP) assays demonstrated significant enrichment of miR-103a-2-5p with DLEU2, confirming their physical interaction (Fig. 3G). These outcomes implied that DLEU2 functions in the capacity of a sponge for miR-103a-2-5p, which is downregulated in BC cells.

DLEU2 regulates the proliferation and migration of BC by regulating miR-103a-2-5p

Overexpression of miR-103a-2-5p in J82 cells suppressed proliferation and migration compared to control cells (Figs. 4A–4E). Co-transfection of miR-103a-2-5p mimics and ov-DLEU2 reversed the inhibitory effects of miR-103a-2-5p overexpression on these functions, indicating that DLEU2 counteracts the effects of miR-103a-2-5p.

Conversely, miR-103a-2-5p knockdown in T24 cells enhanced proliferation and migration, effects that were mitigated by simultaneous DLEU2 knockdown (Fig. 5). These findings demonstrate that DLEU2 regulates BC cell behavior by modulating miR-103a-2-5p activity.

SOS1 was a target of miR-103a-2-5p

To identify downstream targets of miR-103a-2-5p, TargetScan software and miRDB were used to predict potential mRNA targets. Integration with GEO data revealed 208 upregulated mRNAs, including 10 associated with the mitogen-activated protein kinases (MAPK) pathway, a key pathway altered in BC (Figs. 6A and 6B). Among these, SOS1 exhibited the most significant downregulation upon miR-103a-2-5p overexpression (Fig. 6C). BiBiServ analysis and dual luciferase assays confirmed that miR-103a-2-5p directly binds the SOS1-3′UTR (Fig. 6D). Western blot analysis showed that SOS1 protein levels were highest in J82 cells, while lowest in T24 cells (Fig. 6E); DLEU2 overexpression upregulated SOS1 protein levels in T24 cells, while DLEU2 knockdown reduced SOS1 protein levels in J82 cells (Fig. 6F). miR-103a-2-5p overexpression downregulated SOS1 protein expression, which was reversed by DLEU2 overexpression in J82 cells (Fig. 6G). Conversely, miR-103a-2-5p knockdown upregulated SOS1 protein expression, counteracted by DLEU2 knockdown (Fig. 6G). These results establish SOS1 as a direct target of miR-103a-2-5p and suggest that DLEU2 modulates SOS1 expression through miR-103a-2-5p.

miR-103a-2-5p regulates the proliferation and migration of BC by target SOS1

In J82 cells, SOS1 silencing significantly reduced SOS1 protein expression, as verified by western blot. However, co-treatment with SOS1 knockdown and a miR-103a-2-5p inhibitor restored SOS1 protein levels (Fig. 7A). Functional assay, including cell proliferation, Edu incorporation, transwell migration, and scratch assays demonstrated that SOS1 silencing reduced J82 cell proliferation and migration. Conversely, SOS1 knockdown combined with miR-103a-2-5p inhibition reversed these effects (Figs. 7B–7E). These findings indicate that SOS1 downregulation suppresses J82 cell proliferation and migration, while miR-103a-2-5p inhibition counteracts this suppression.

In T24 cells, SOS1 overexpression increased SOS1 protein expression, whereas co-expression of SOS1 overexpression and miR-103a-2-5p decreased SOS1 protein levels (Fig. 8A). Functional assays revealed that SOS1 overexpression enhanced T24 cells proliferation and migration. However, co-expression of miR-103a-2-5p reversed these effects by suppressing SOS1 expression (Figs. 8B–8E). These outcomes suggest that miR-103a-2-5p acts as a negative regulator of SOS1, thereby modulating BC cell proliferation and migration.