SiRNA targeting SPHK1 super enhancer and application of siRNA in treatment of triple negative breast cancer

Abstract

The invention discloses siRNA of a targeted SPHK1 super enhancer and application of the siRNA in treatment of triple negative breast cancer. The invention reveals that abnormal expression of sphingosine kinase SPHK1 is driven by a specific super enhancer for the first time, SPHK1-SE-targeted double-stranded small interfering RNA (siRNA-SE) is designed by positioning an SE core regulation region on the upstream of an SPHK1 gene, a non-coding regulation element is specifically silenced, and the expression level of SPHK1 mRNA and protein is remarkably reduced. Further research finds that siRNA-SE can effectively reverse up-regulation of SPHK1 expression in TNBC cells induced by Niraparib, the killing effect of Niraparib on tumor cells is enhanced, the synergistic anti-tumor effect is achieved, the treatment sensitivity of Niraparib in the TNBC cells is remarkably improved, and a treatment means based on SE precise intervention is provided for TNBC.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor molecular biology and targeted therapy, and specifically relates to an siRNA targeting the SPHK1 super enhancer and its application in treating triple-negative breast cancer. Background Art

[0002] Triple-negative breast cancer (TNBC) relies heavily on chemotherapy for clinical treatment due to its high expression of estrogen receptor (ER), progesterone receptor (PR), and HER2. However, its aggressive nature, high metastatic potential, and acquired drug resistance contribute to a dismal prognosis. Recent studies have revealed that super enhancers (SEs) contribute to the malignant progression of TNBC by driving the aberrant expression of cell identity genes. SEs are populated by high-density transcription factor / cofactor complexes, whose activity is dynamically regulated by epigenetic marks such as H3K27ac, and are closely associated with phenotypes such as tumor metabolic reprogramming and apoptosis resistance. However, the specific mechanisms by which SEs regulate a key oncogene (SPHK1) in TNBC remain largely undefined, hindering the development of SE-targeted therapeutic strategies.

[0003] Sphingosine kinase (SPHK1), a key enzyme in lipid metabolism, catalyzes the conversion of sphingosine to sphingosine-1-phosphate (SP1-phosphate), regulating chemotherapy resistance in TNBC by balancing pro-survival signals (SP1-phosphate) with pro-apoptotic signals (SP1-phosphate). Although SPHK1 is abnormally overexpressed in TNBC, the location of its upstream SE, its transcription factor recruitment mechanism, and its pharmacological regulatory mechanisms remain largely unknown. While existing targeted interventions, such as the PARP inhibitor niraparib, can kill BRCA-ness tumors through a "synthetic lethality" effect, their efficacy is limited in most TNBCs and can exacerbate drug resistance by upregulating SPHK1 expression through the SE mechanism. New synergistic therapeutic strategies are urgently needed. Summary of the Invention

[0004] The purpose of the present invention is to provide an siRNA targeting the SPHK1 super enhancer and its application in treating triple-negative breast cancer.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention first provides an siRNA targeting the SE core regulatory region of the SPHK1 gene, the sequence of the siRNA is as follows: Sense strand: 5′-GAUGUUGCCCAUGGAGCAATT-3′; Antisense strand: 5′-UUGCUCCAUGGGCAACAUCTT-3′; The nucleotide sequence of the SPHK1 gene is shown in SEQ ID No. 1; The SE core regulatory region is divided into SE1 and SE2. The nucleotide sequence of SE1 is shown in SEQ ID No. 2, and the nucleotide sequence of SE2 is shown in SEQ ID No. 3.

[0006] The present invention also provides the use of the above siRNA in inhibiting the expression of SPHK1 gene.

[0007] The present invention also provides the use of the above siRNA in enhancing the killing effect of Niraparib on TNBC cells.

[0008] Furthermore, the siRNA can reverse the upregulation of SPHK1 gene expression level induced by Niraparib.

[0009] The present invention also provides the use of the above siRNA in preparing a drug for treating triple-negative breast cancer.

[0010] The present invention also provides a method for enhancing the killing effect of Niraparib on tumor cells, which comprises co-administering the above-mentioned siRNA and Niraparib.

[0011] Furthermore, the tumor cells are triple-negative breast cancer cells.

[0012] The beneficial effects of the present invention are: This study reveals for the first time that the sphingosine kinase SPHK1 in triple-negative breast cancer is regulated by a specific super-enhancer, SPHK1-SE. It also innovatively develops a synergistic therapeutic approach combining siRNA-SE targeting non-coding regulatory elements with the PARP inhibitor (Niraparib). Using ChIP-seq and H3K27ac epigenomic analysis, the study pinpointed the core regions of SPHK1-SE: SE1 (chr17: 74366302-7436926) and SE2 (chr17: 7436926-7438101). The study also discloses siRNA sequences designed to target SPHK1-SE. RT-qPCR and Western blot analysis confirm that SPHK1 is regulated by the super-enhancer in TNBC cells. Western blot analysis confirms that the introduction of siRNA-SE into TNBC cells reverses niraparib-induced SPHK1 upregulation, confirming that siRNA targeting SPHK1-SE enhances the cytotoxicity of niraparib against TNBC cells. This invention breaks through the traditional gene coding region targeting strategy and pioneers the "SE silencing + PARP inhibition" synergistic therapy, proving that siRNA targeting SPHK1-SE combined with Niraparib is a new and effective treatment strategy for triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Venn diagram of 331 TNBC-specific SE target genes and upregulated differentially expressed genes in the transcriptome data of MDA-MB-231 cells treated with Niraparib for 48 h.

[0014] Figure 2 : GraphPad Prism software was used to draw the H3K27ac signal diagram of 331 TNBC-specific SE target genes (marked Figure 1 7 genes at the intersection).

[0015] Figure 3 : IGV displays the H3K27ac signal peaks of 7 gene-SE in 3 TNBC cell lines.

[0016] Figure 4 : RT-qPCR verified the SPHK1 mRNA level in TNBC cells treated with 4 μM JQ1 for 24 hours, and verified that the BRD4 inhibitor (JQ1) could reverse the upregulation of SPHK1 expression caused by Niraparib.

[0017] Figure 5 : RT-qPCR verified the SPHK1 mRNA level in TNBC cells treated with 4 μM OTX-015 for 24 hours, and verified that the BRD4 inhibitor (OTX-015) could reverse the upregulation of SPHK1 expression caused by Niraparib.

[0018] Figure 6 : Western Blot verified that BRD4 inhibitors (JQ1 and OTX-015) could reverse the upregulation of SPHK1 protein expression caused by Niraparib.

[0019] Figure 7 : The genomic coordinates of SE1 (chr17: 74366302-7436926) and SE2 (chr17: 7436926-7438101) were determined using the NCBI database.

[0020] Figure 8 : Western Blot analysis confirmed that the introduction of siRNA-SE into TNBC cells led to downregulation of SPHK1 protein.

[0021] Figure 9 :Western blot analysis was used to determine that the introduction of siRNA-SE into TNBC cells could reverse Niraparib-induced SPHK1 upregulation.

[0022] Figure 10: siRNA targeting SPHK1-SE enhanced the cytotoxic effect of Niraparib on TNBC cells. DETAILED DESCRIPTION

[0023] To illustrate the technical content, characterization and performance analysis methods of the present invention in detail, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0024] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used can be purchased commercially.

[0025] The main cells and experimental materials involved in this invention are: (1) Cell lines: TNBC cell lines, including MDA-MB-231 and MDA-MB-468, were purchased from the cell bank of Shanghai Institute of Biochemistry and Cell Biology (SIBCB, Shanghai, China).

[0026] (2) Compounds: Niraparib (Pubchem CID: 24958200, GlpBio, GC17802), JQ1 (Pubchem CID: 46907787, GlpBio, GC13822), OTX-015 (Pubchem CID: 9936746, GlpBio, GC17973).

[0027] (3) Antibodies: Anti-SPHK1 (1:1000, proteintech, Lot No: 10670-1-AP); Anti-GAPDH (1:1000, proteintech, Lot No:60004-1-IG); Anti-mouse IgG, HRP-linkedAntibody (1:5000, CST, Cat# 7076, Lot No: 36) and Anti-rabbit IgG, HRP-linkedAntibody (1:5000, CST, Cat# 7074, LotNo: 31).

[0028] Example 1 1. Cell line culture: MDA-MB-231 (ER⁻ / PR⁻ / HER2⁻, BRCA1 wild-type) and MDA-MB-468 (ER⁻ / PR⁻ / HER2⁻, BRCA1 wild-type) cells were cultured in RPMI-1640 medium (Sigma-Aldrich, R8758) supplemented with 10% fetal bovine serum (FBS; Gibco, 10099141) and 1% penicillin-streptomycin (HyClone TM , SV30010). All cell lines were authenticated by short tandem repeat (STR) profiling within six months before the experiment and were incubated at 37°C in 5% CO2.

[0029] 2. Transcriptomic Sequencing Analysis: Use Beyoool from collected cells TM Total RNA was extracted using a 400 μg / ml reagent (Beyotime, R0011), and poly(A) mRNA was enriched using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, E7490). TM The purified mRNA was reverse transcribed into cDNA using IV Reverse Transcriptase (Invitrogen, 18090010) and oligo(dT)20 primer. To construct sequencing libraries, NEBNext® Ultra TM cDNA was fragmented using the DNALibrary Prep Kit (NEB, E7645) and ligated to an Illumina TruSeq ® Adapters (Illumina, 20015965) were added with paired-end index barcodes by 8 rounds of PCR amplification. TM Libraries were quantified using a fluorescence-based assay (Thermo Fisher) and paired-end sequenced on an Illumina NovaSeq 6000 platform (150 bp, approximately 40 million reads per sample). Raw sequencing data were quality assessed using FastQC and aligned to the human reference genome (GRCh38) using STAR (v2.7.9a). Transcript quantification was performed using featureCounts (v2.0.3). Differential gene expression analysis was performed using DESeq2, using stringent thresholds. Functional annotation of differentially expressed genes was performed using the DAVID bioinformatics resource (v2023q1), supplemented by KEGG pathway enrichment analysis.

[0030] 3. Multidimensional Omics Analysis: ChIP-seq data for H3K27ac / H3K4me1 / H3K4me3 and DNase I-seq data from different cell lines (MDA-MB-231, MDA-MB-468, and HCC1937) were retrieved from the GEO database to analyze the regulation of SPHK1 by super-enhancers in TNBC. Peaks were visualized using IGV software. The GEO database accession numbers for the H3K27ac-ChIP-seq data from the different cell lines are GSM2500251, GSM4874881, and GSM2258902, respectively. The DNase I-seq data accession number is GSM2242136. The super-enhancer landscape was defined using the ROSE algorithm.

[0031] TNBC has corresponding specific SEs, with a total of 331 TNBC-specific SE target gene pairs. The intersection of the 331 TNBC-specific SE-regulated target genes and the transcriptome differentially expressed genes in MDA-MB-231 cells treated with PARP1 inhibitors was taken, and the corresponding Venn diagram was drawn ( Figure 1 ), there are 7 genes at the intersection, namely SPHK1 (sequence shown in SEQ ID No. 1), LAMA1 (ID: 284217), SEL1L3 (ID: 23231), ADAMTS9 (ID: 56999), NXPH4 (ID: 11247), TFCP2L1 (ID: 29842), LAMP3 (ID: 27074). Then, GraphPad Prism software was used to draw the H3K27ac signal graph of 331 SE target genes. The horizontal axis represents the log2 (FoldChange) value of each gene, and the vertical axis represents the H3K27ac signal value of Gene-SE. The 7 genes at the intersection are marked with red dots, and it was found that SPHK1-SE has the strongest H3K27ac signal value ( Figure 2 ). H3K27ac-Chip-seq data of different cell lines (MDA-MB-231, MDA-MB-468, HCC1937) were retrieved by searching the GEO database. The data numbers are GSM2500251, GSM4874881, and GSM2258902. The peaks of H3K27ac of the seven genes (SPHK1, LAMA1, SEL1L3, ADAMTS9, NXPH4, TFCP2L1, and LAMP3) selected by IGV software analysis were obtained. Figure 3 ), SPHK1-SE was found to be the most active.

[0032] Example 2 RT-qPCR Verification of SPHK1 Regulation by Super Enhancers in TNBC Cells JQ1 and OTX-015, classic BET bromodomain inhibitors, competitively bind to the BD1 / BD2 domains of BRD4, blocking its interaction with acetylated histones (such as H3K27ac). This inhibits SE-mediated transcriptional complex assembly and downregulates the expression of SE-driven oncogenes (such as MYC and BCL2). They are commonly used to study super-enhancer activity. Cells (MDA-MB-231 and MDA-MB-468) were divided into four groups: control (DMSO-treated), niraparib, JQ1 / OTX-015 (testing JQ1 and OTX-015, respectively), and niraparib + JQ1 / OTX-015 (testing niraparib + JQ1 and niraparib + OTX-015, respectively). After 24 hours of cell culture, cells were treated with 25 μM niraparib for 24 hours in the niraparib group, 4 μM JQ1 in the JQ1 group, and 4 μM OTX-015 in the OTX-015 group. The niraparib + JQ1 group was treated with 25 μM niraparib and 4 μM JQ1, and the niraparib + OTX-015 group was treated with 25 μM niraparib and 4 μM OTX-015 for 24 hours. Cell samples from each group were collected, centrifuged at 12,000 rpm for 5 minutes, and washed three times with PBS. Total RNA was extracted from the cell samples using the HighPure RNA Purification Kit (heruibio, HRQ0151, Fujian, China) and treated with DNase I on the column to remove genomic DNA contamination. RNA integrity was verified by agarose gel electrophoresis (28S / 18S rRNA ratio >1.8), and RNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Subsequently, 1 μg of RNA was reverse transcribed into cDNA using HiScript ® II Q RT SuperMix (Vazyme, R223-01). cDNA was mixed with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) and loaded into RT 2 ProfilerPCR Array plates (Qiagen, PAHS-343ZA, Hilden, Germany). ®Amplification was performed on a 96-well system (Roche, 04729692001, Mannheim, Germany) using standardized conditions: initial denaturation at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and melting curve analysis (95°C for 15 seconds, 60°C for 60 seconds, and 95°C for 15 seconds). Target validation was performed using custom primers (Sangon Biotech, Shanghai, China; see Table 1) using the same SYBR reagents and cycling parameters. Data were analyzed using actin as an internal control and 2 −ΔΔCt Method for analysis.

[0033] Table 1 Primers used for real-time quantitative PCR experiments RT-qPCR experiments confirmed the decrease in SPHK1 gene mRNA levels, e.g. Figures 4 and 5 As shown in Figure 2, BRD4 inhibitor (JQ1 / OTX-015) was able to reverse Niraparib-induced upregulation of SPHK1 expression. Figure 6 The protein levels shown also confirmed that BRD4 inhibitors could reverse Niraparib-induced upregulation of SPHK1 protein expression. Taken together, these results provide strong evidence that SPHK1 expression in TNBC cells is regulated by super enhancers.

[0034] Western Blot analysis further confirmed that SPHK1 is regulated by super enhancers in TNBC cells: The cell samples from each group were collected, centrifuged at 12000 rpm for 5 min, and washed three times with PBS. Total protein extracts were prepared using RIPA lysis buffer (Beyotime Biotechnology, P0013B) supplemented with protease and phosphatase inhibitors (Beyotime Biotechnology, P1049). TMProtein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology, P0012S). Equal amounts of protein (20–30 µg) were separated by SDS-PAGE and transferred to a methanol-activated polyvinylidene fluoride (PVDF) membrane (Beyotime Biotechnology, P0965). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline (TBST) containing 0.1% Tween-20 for one hour at room temperature and then incubated overnight at 4°C with primary antibodies diluted in primary antibody dilution buffer (Beyotime Biotechnology, P0023A). After three 5-minute washes with TBST, the membrane was incubated with secondary antibodies for one hour at room temperature. After three additional TBST washes, protein bands were visualized using BeyoECL Plus substrate (Beyotime Biotechnology, P0018M), and densitometric quantification was performed using ImageJ software.

[0035] Example 3 Design of siRNA-SE targeting the SPHK1 super enhancer sequence and transfection The super enhancer (SE) peak corresponding to SPHK1 is located in the region 74366302-74368101 on chromosome 17. Based on the intensity of the peak, we divided them into SE1 and SE2. The genomic coordinates of SE1 (chr17:74366302-7436926) and SE2 (chr17: 7436926-7438101) were then confirmed using the NCBI database ( Figure 7To functionally validate the role of SE, siRNA-SE (si-SE) targeting the SPHK1-SE sequence was designed and chemically synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. (Shanghai, China). The sequences are shown in Table 2. One day before transfection, cells were trypsinized, and the cell concentration was adjusted and counted. Subsequently, 400,000 cells were seeded per well in a 6-well plate and incubated overnight at 37°C, 5% CO2. The cells (MDA-MB-231 and MDA-MB-468) were divided into the control group (DMSO-treated), the si-SE group, the niraparib group, and the si-SE + niraparib group. The niraparib group treated cells with 25 μM niraparib for 24 hours, the si-SE group was transfected with 75 pmol of siRNA, and the si-SE + niraparib group treated cells with 25 μM niraparib for 24 hours and then transfected with 75 pmol of siRNA. On the day of transfection, when the cell confluence reached approximately 60%, transfection was started. Using the siRNA-mate plus transfection kit, 75 pmol of siRNA was transfected into TNBC cells for 10 hours. Cells were collected 10 hours after transfection for subsequent experimental analysis. Western blot analysis showed ( Figures 8 and 9 ), introducing siRNA-SE into TNBC cells can reverse Niraparib-induced SPHK1 upregulation. These results indicate that the designed siRNA-SE is effective.

[0036] Table 2 siRNA sequences Example 4 Cell viability assay To investigate the effect of siRNA-mediated interference of SPHK1-SE on Niraparib IC 50 To investigate the effects of niraparib on the growth of MDA-MB-468 cells, MDA-MB-468 cells were seeded at a density of 5000 cells / well in 96-well plates and allowed to adhere overnight. The cells were divided into control (DMSO) + niraparib and si-SE + niraparib groups. After stable attachment, the experimental compounds were added to the culture medium at varying concentrations. After incubation at 37°C, 5% CO₂ for 72 hours, 20 μL of MTT reagent (5 mg / mL, phosphate buffered saline) was added to each well. The cells were incubated for a further 4 hours to promote mitochondria-dependent reduction of MTT to insoluble purple formazan crystals. The culture medium was then carefully aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan precipitate. Absorbance was measured at 570 nm using a microplate spectrophotometer. Data are presented as the mean ± SD of three independent experiments.

[0037] MTT results showed that ( Figure 10 siRNA knockdown of SE significantly enhanced the cytotoxic effect of Niraparib on TNBC cells. This suggests that the combination of siRNA targeting SPHK1-SE and Niraparib is a novel and effective therapeutic strategy.

[0038] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An siRNA targeting the SE core regulatory region of the SPHK1 gene, characterized by: The sequence of the siRNA is as follows: Sense strand: 5′-GAUGUUGCCCAUGGAGCAATT-3′; Antisense strand: 5′-UUGCUCCAUGGGCAACAUCTT-3′; The nucleotide sequence of the SPHK1 gene is shown in SEQ ID No. 1; The SE core regulatory region is divided into SE1 and SE2. The nucleotide sequence of SE1 is shown in SEQ ID No. 2, and the nucleotide sequence of SE2 is shown in SEQ ID No.

3.

2. Use of the siRNA according to claim 1 in inhibiting SPHK1 gene expression.

3. Use of the siRNA according to claim 1 in enhancing the killing effect of Niraparib on TNBC cells.

4. The use according to claim 3, characterized in that: The siRNA can reverse the upregulation of SPHK1 gene expression level induced by Niraparib.

5. Use of the siRNA according to claim 1 in the preparation of a drug for treating triple-negative breast cancer.

6. A method for enhancing the killing effect of Niraparib on tumor cells, characterized in that: The siRNA according to claim 1 is co-administered with Niraparib.

7. The method according to claim 6, characterized in that: The tumor cells are triple-negative breast cancer cells.

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