LncRNA BCLET variant confers bladder cancer susceptibility through alternative splicing of MSANTD2 exon 1

Abstract

Background: Alternative splicing (AS)-related single nucleotide polymorphisms (SNPs) are associated with risk of cancers, but the potential mechanism has not been fully elucidated.

Methods: Two-stage case-control studies comprising 1630 cases and 2504 controls were conducted to investigate the association between the AS-SNPs and bladder cancer susceptibility. A series of assays were used to evaluate the functional effect of AS-SNPs on bladder cancer risk.

Results: We observed that SNP rs558814 A>G located in lncRNA BCLET (Bladder Cancer Low-Expressed Transcript, ENSG00000245498) can decrease the risk of bladder cancer (odds ratio [OR] = 0.84, 95% confidence interval [CI] = 0.76-0.92, p = 3.26 × 10^-4 ). Additionally, the G allele of rs558814 had transcriptional regulatory effects and facilitated the expression of BCLET transcripts, including BCLET-long and BCLET-short. We also found decreased BCLET expression in bladder cancer tissues and cells, and BCLET transcript upregulation substantially inhibited tumor growth of both bladder cancer cells and xenograft models. Mechanistically, BCLET recognized and regulated AS of MSANTD2 to participate in bladder carcinogenesis, preferentially promoting the production of MSANTD2-004.

Conclusions: SNP rs558814 was associated with the expression of BCLET, which mainly increased the expression of MSANTD2-004 through AS of MSANTD2.

Keywords: alternative splicing; bladder cancer; genetic variations; lncRNA BCLET; molecular mechanism.

FIGURE 1

Alternative splicing‐single nucleotide polymorphism (AS‐SNP) selection and genetic variants associated with bladder cancer risk. (A) Flowchart of the AS‐SNP screen. (B) Circle Manhattan map of 9 AS‐SNPs. The ‐log10 p‐values of AS‐SNPs in the discovery stage according to their chromosomal positions. The red dotted circle indicates p < 0.05. (C) Functional annotation scores of nine candidate AS‐SNPs from RegulomeDB, HaploRefg, CancerSpliceQTL and SNPinfo. (D) SNP rs558814 at 11q24.2 is located in the intron of ENSG00000245498 (RP1F1‐677M14.7).

FIGURE 2

Single nucleotide polymorphism rs558814 regulated BCLET transcripts through transcriptional regulation. The expression of BCLET in The Cancer Genome Atlas (TCGA) data (A), 51 paired bladder cancer and adjacent normal tissues (B), and the bladder epithelial cell SV‐HUC‐1 and bladder cancer cells EJ, J82, and T24 (C). (D) The prediction of the protein coding ability of BCLET‐long and BCLET‐short by the CPC online tool. (E) Schematic diagram of the transcripts, expression and primers of BCLET. (F) The association between rs558814 genotypes and the expression of BCLET in TCGA bladder cancer database. (G‐I) The association between rs558814 genotypes and the expression of total BCLET (G), BCLET‐long (H) and BCLET‐short (I) in 24 bladder adjacent tissues. (J) The effect of rs558814 on the transcriptional activity of BCLET by luciferase reporter assays in bladder cancer cells.

FIGURE 3

BCLET transcripts significantly inhibited tumor proliferation in vitro and in vivo. The effect of BCLET transcripts overexpression on cell viability was detected by CCK‐8 (A) and colony formation assays (B) in T24 cell lines. Tumor burdens after inoculation with LV‐BCLET‐long/LV‐BCLET‐short and LV‐NC were examined in nude mice (C) and their xenografts (D). (E) The tumor volumes were measured. (F) The tumor weights in the nude mice were determined after 4 weeks. (G) Representative images of HE and immunohistochemical staining for Ki67 and MSANTD2 in mouse tumors (scale bars = 50 μm).

FIGURE 4

BCLET increased the expression level of the MSANTD2‐004 isoform through alternative splicing of MSANTD2 exon 1. (A) The location diagram of BCLET and MSANTD2 transcripts. (B) Diagram and expression of the MSANTD2 isoform in bladder cancer tissue. (C) Schematic diagram of primer design. (D) Calculation of the splicing ratio for MSANTD2‐004. The ratio of MSANTD2‐004 expression to the total expression of MSANTD2 was used to evaluate the splicing ratio of the MSANTD2‐004 transcript, and the values were converted by log2. (E) The coexpression analysis of BCLET and MSANTD2‐004 in bladder cancer tissues (left) and bladder adjacent tissues (right). (F) Correlation analysis between BCLET expression level and the MSANTD2‐004 splicing ratio in bladder cancer tissues (left) and bladder adjacent tissues (right). The values were converted by log2. (G) The expression level of MSANTD2‐004 in the bladder cancer cells transfected with the BCLET overexpression vector (left) and lncRNA Smart Silencer (right). (H) The splicing ratio of MSANTD2‐004 in the bladder cancer cells transfected with the BCLET overexpression vector (left) and lncRNA Smart Silencer (right). (I) Schematic diagram of the RNA immunoprecipitation (RIP) experiment. (J) RIP experiments were used to investigate the relationship between BCLET and MSANTD2 in bladder cancer cells.

FIGURE 5

MSANTD2‐004 regulated the bladder cancer cells malignant phenotype. (A) CCK8 assay. (B) Cell clone formation. (C) Cell migration. (D) Cell invasion. (E) Cell apoptosis. (F) Graphical representation of the regulation and function of BCLET rs558814 in bladder cancer. Single nucleotide polymorphism (SNP) rs558814 A>G in BCLET was significantly associated with a reduced risk of bladder cancer. Furthermore, BCLET mediated cell proliferation, Transwell activity, and tumorigenesis to participate in bladder oncogenesis. BCLET ultimately bound MSANTD2 and increased the expression of MSANTD2‐004 by modulating alternative splicing (AS) of MSANTD2 exon 1.