An antisense amido-bridged nucleic acid gapmer oligonucleotide targeting SRRM4 alters REST splicing and exhibits anti-tumor effects in small cell lung cancer and prostate cancer cells

Abstract

Background: Antisense oligonucleotide (ASO) medicine for clinical applications has been becoming a reality. We previously developed a gapmer ASO targeting Ser/Arg repetitive matrix 4 (SRRM4) that is abnormally expressed in small cell lung cancer (SCLC). However the detailed mechanism of ASO through repressing SRRM4 has not been completely elucidated. Further, effectiveness of SRRM4 ASO to prostate cancer (PCa) cells expressing SRRM4 similar to SCLC remains to be elucidated. RE1-silencing transcription factor (REST) is a tumor suppressor, and its splicing isoform (sREST) is abnormally expressed by SRRM4 and causes carcinogenesis with neuroendocrine phenotype in SCLC. The present study aimed to understand the contribution of REST splicing by SRRM4 ASO administration.

Methods: SRRM4 expression and REST splicing were analyzed by RT-qPCR and conventional RT-PCR after treating SRRM4 ASO, and cell viability was analyzed in vitro. Exogenous reconstitution of Flag-tagged REST plasmid in SCLC cells and the splice-switching oligonucleotide (SSO) specific for REST was analyzed for cell viability. Furthermore, we expanded the application of SRRM4 ASO in PCa cells abnormally expressing SRRM4 mRNA in vitro.

Results: SRRM4 ASO successfully downregulated SRRM4 expression, followed by repressed cell viability of SCLC and PCa cells in a dose-dependent manner. Administration of SRRM4 ASO then modified the alternative splicing of REST, resulting reduced cell viability. REST SSO specifically modified REST splicing increased REST expression, resulting in reduced cell viability.

Conclusions: Our data demonstrate that a gapmer ASO targeting SRRM4 (SRRM4 ASO) reduces cell viability through splicing changes of REST, followed by affecting REST-controlled genes in recalcitrant tumors SCLC and PCa cells.

Keywords: Antisense oligonucleotide; Gapmer; Prostate cancer; REST/NRSF; SCLC; SRRM4; Small cell lung cancer.

Fig. 1

SRRM4 ASO affects the cell viability and promote REST splicing change. a SRRM4 mRNA expression analysis in SCLC cells transfected with SRRM4 ASO. SCLC cells (H146 and H209) were transfected with various amounts of ASO (AmNA_7174) and negative control oligonucleotide (AmNA_26). Cells (0.5 × 10⁶) were transfected with SRRM4 ASO and collected 24 h after transfection. RT-qPCR was performed using specific primers, and the 2^−ΔΔct values of SRRM4 mRNA expression were normalized to endogenous actin in non-treated cells (NT) as 1.0. The assay was performed 3 times to confirm reproducibility. Data are presented as the mean ± SEM of three independent experiments (n = 3). ***P < 0.001. b REST splicing was analyzed by RT-PCR. Cells were collected after 72 h following transfection and total mRNA was prepared. RT-PCR was performed using specific primers for either REST or β-actin. PCR products were analyzed by electrophoresis using polyacrylamide gels followed by staining. One of three independent experiments is shown. c After 72 h of transfection, cell viability was measured by CellTiter-Glo 3D assay. The assay was performed 3 times to confirm reproducibility. Data are presented as the mean ± SEM of three independent cell cultures (n = 3). Statistic differences were analyzed using one-way analysis of variance (ANOVA) followed by the Student’s t-test ***P < 0.001

Fig. 2

Reconstitution of REST by the exogenous vector in SCLC cells reduced cell viability. a SCLC cells (H146) were transfected with each Flag-tagged REST or sREST expression plasmid (5 or 15 μg/1.0 × 10⁶ cells) by electroporation. Then, 24 h post-transfection, cells were collected and cell lysates were prepared. All samples were analyzed by SDS-PAGE followed by immunoblot analysis using anti-Flag antibody. n.s.; non-specific band b SCLC cells (H146) were transfected with each plasmid (5.0 μg /1.0 × 10⁶ cells) by electroporation. After 72 h of transfection, cell viability was measured by CCK-8 assay. The assay was performed 3 times to confirm reproducibility. Data are present as the mean ± S.D. in triplicate. **P < 0.01

Fig. 3

REST splicing analysis and cell viability in SCLC cells transfected with REST SSO. a Scheme of REST splicing change by REST SSO. b SCLC cells (H146) were transfected with SSO (AmNA_[+ 21/ + 40]) or negative control oligonucleotide. Cells were collected after 48 h of transfection and total RNA was prepared. RT-PCR was performed using specific primers for REST and actin and PCR products were analyzed using polyacrylamide gels; c Cell viability was measured by CellTiter-Glo 3D assay every 24 h until 72 h of culturing. The assay was performed 3 times to confirm reproducibility. Data are presented as the mean ± SEM of three independent experiments (n = 3). ***P < 0.001

Fig. 4

SRRM4 ASO decreased cell viability in SRRM4-expressing prostate cancer (PCa) cells. a PCa cells expressing SRRM4 (22Rv1 and VCaP) were transfected with various amounts of ASO (AmNA_7174) and negative control oligonucleotide (AmNA_26). Cells were collected after 24 h following transfection and RT-qPCR was performed using specific primers and 2^−ΔΔct values of SRRM4 mRNA expression were normalized with endogenous actin in non-treated cells (NT) as 1.0. The assay was performed multiple times with different amount of AmNA_7174 to confirm reproducibility. Data are presented as the mean ± SEM of three independent experiments (n = 3). ***P < 0.001. (t test); b After 72 h of transfection, cell viability was measured by CellTiter-Glo 3D assay. The assay was performed multiple times with different amount of AmNA_7174 to confirm reproducibility. Data are presented as the mean ± SEM of three independent experiments (n = 3). ***P < 0.001; c Cells were collected after 48 h following transfection. RT-PCR was performed using specific primers for REST and β-actin. PCR products were analyzed by performing electrophoresis using polyacrylamide gels. One of three independent experiments is shown