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12.09.2019 | Original Article

Downregulation of SRSF3 by antisense oligonucleotides sensitizes oral squamous cell carcinoma and breast cancer cells to paclitaxel treatment

Zeitschrift:
Cancer Chemotherapy and Pharmacology
Autoren:
Yanan Sun, Lingyan Yan, Jihua Guo, Jun Shao, Rong Jia
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1007/​s00280-019-03945-9) contains supplementary material, which is available to authorized users.
Yanan Sun and Lingyan Yan contributed equally to this work.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Abstract

Purpose

Paclitaxel (PTX) is widely used in the chemotherapy of many cancers, including breast cancer and oral squamous cell carcinoma (OSCC). However, many patients respond poorly to PTX treatment. The SRSF3 oncogene and several splicing factors play important roles in OSCC tumorigenesis. This study aimed to understand the function of splicing factors in PTX treatment and improve the therapeutic effects of PTX treatment.

Methods

Splicing factors regulated by PTX treatment were screened in CAL 27 cell by reverse transcription polymerase chain reaction. The function of SRSF3 in PTX treatment was analyzed by gain-of-function or loss-of-function assay in OSCC cell lines CAL 27 and SCC-9 and breast cancer cell line MCF-7. Alternative splicing of SRSF3 exon 4 in cancer tissues or cells was analyzed by RT-PCR and online program TSVdb. SRSF3-specific antisense oligonucleotide (ASO) SR-3 was used to downregulate SRSF3 expression and enhance the effect of PTX treatment.

Results

PTX treatment decreased SRSF3 expression, and SRSF3 overexpression rescued the growth inhibition caused by PTX in both OSCC and breast cancer cells. Moreover, we found that PTX treatment could repress SRSF3 exon 4 (containing an in-frame stop codon) exclusion and then decrease the SRSF3 protein expression. Increased exclusion of SRSF3 exon 4 is correlated with poor survival in OSCC and breast cancer patients. SR-3 downregulated SRSF3 protein expression and significantly increased the sensitivity of cancer cells to PTX treatment.

Conclusions

SRSF3 downregulation by ASO sensitizes cancer cells to PTX treatment.

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Zusatzmaterial
Figure S1 The expression of hnRNP proteins in CAL 27 cells with PTX treatment. PTX treatment CAL 27 cells were treated with 0 nM, 7.8 nM, or 15.6 nM PTX for 48 h. The mRNA expression levels of hnRNP proteins were analyzed by RT-PCR (PPTX 2311 kb)
280_2019_3945_MOESM1_ESM.pptx
Figure S2 PTX regulated the alternative splicing of SRSF3 exon 4. Long or short isoform of SRSF3 were detected respectively using two pairs of isoform-specific primers. (A) OSCC cell line CAL27 and breast cancer cell line MCF-7 were treated with various concentration of PTX or (B) 20 nM SR-3 or non-sense antisense oligonucleotide for 48 h. The mRNA expression levels of long and short isoform of SRSF3 were analyzed separately by RT-PCR using isoform-specific primers. Data are mean ± SE, n = 3. (PPTX 2106 kb)
280_2019_3945_MOESM2_ESM.pptx
Figure S3 PTX and SR-3 treatment significantly downregulated SRSF3 expression and altered the splicing profile of SRSF3 in OSCC cell line SCC-9. (A) OSCC cell line SCC-9 was treated with 0 nM, 31.25 nM, or 62.5 nM PTX for 48 h. The mRNA expression levels of SRSF3 were analyzed by RT-PCR and qRT-PCR. (B) The expression of SRSF3 protein was analyzed by Western blot. (C-D) RT-PCR or qRT-PCR was used to analyze the alternative splicing of exon 4 in SCC-9 cells treated with PTX at the indicated concentration by using a pair of primers (C) or two pairs of isoform-specific primers (D). (E–G) SCC-9 were treated with 20 nM SR-3 or NS antisense oligonucleotides.) RT-PCR or qRT-PCR was used to analyze the alternative splicing of exon 4 by using a pair of primers (E) or two pairs of isoform-specific primers (F). The expression of full-length SRSF3 protein was analyzed by Western blot (G). Data are mean ± SE, n = 3 (PPTX 299 kb)
280_2019_3945_MOESM3_ESM.pptx
Figure S4 Anti-SRSF3 antisense oligonucleotide (SR-3) downregulated CDC25B and PLK1. CAL 27 or MCF-7 cells were treated with 20 nM SR-3 or non-specific antisense oligonucleotide (NS). (A) The mRNA levels of CDC25B and PLK1 were analyzed by RT-PCR. Data are the mean ± SD. (B) The histograms summarized the relative mRNA levels of CDC25B and PLK1. Data are mean ± SD, n = 3 (PPTX 88 kb)
280_2019_3945_MOESM4_ESM.pptx
Figure S5 SR-3 sensitizes SCC-9 to PTX treatment. Cells were divided into four groups: PTX + SR-3, PTX + NS, DMSO + SR-3, and DMSO + NS. DMSO and NS served as controls. (A) SCC-9 were transfected with 20 nM SR-3 or NS on Day 0. Twenty-four hours after transfection, cells were treated with 31.25 nM PTX or DMSO. Cell numbers were counted on Day 3. (B) SRSF3 expression in each group was detected through western blots. β-actin served as a loading control. Relative expression levels of SRSF3 protein were normalized to β-actin. (C-D) Cell apoptosis were analyzed by flow cytometry on Day3. (C) Annexin V-FITC/PI were applied to detect the proportion of apoptotic cells. Analysis of the apoptotic rate of cells in all groups are shown in (D). Data are mean ± SE, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (PPTX 135 kb)
280_2019_3945_MOESM5_ESM.pptx
Figure S6 The expression of truncated SRSF3 protein increased upon PTX and SR-3 ASO treatment. Truncated SRSF3 protein was analyzed by an antibody recognizing both full-length and truncated SRSF3 protein (clone 7B4) in CAL 27 (A) or MCF-7 (B). GAPDH served as a loading control. Relative expression levels of truncated SRSF3 protein were normalized to GAPDH (PPTX 73 kb)
280_2019_3945_MOESM6_ESM.pptx
Supplementary material 7 (DOCX 14 kb)
280_2019_3945_MOESM7_ESM.docx
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