SiRNA for inhibiting PRPF8 expression and application thereof

Abstract

The invention discloses siRNA for inhibiting expression of PRPF8 and application of the siRNA, and relates to the field of breast cancer. The siRNA comprises siRNA PRPF8-1 and siRNA PRPF8-2; the nucleotide sequence of the siRNA PRPF8-1 is as shown in SEQ ID No. 1 (sequence identifier number 1); the nucleotide sequence of the siRNA PRPF8-2 is as shown in SEQ ID No. 2 (sequence identifier number 2). The siRNA is applied to preparation of drugs for improving the drug resistance of the triple negative breast cancer. The PRPF8 expression inhibitor can enhance the tamoxifen sensitivity of triple negative breast cancer cells, IC50 is remarkably reduced, and the PRPF8 expression inhibitor can be used for clinical treatment of triple negative breast cancer.

Description

Technical Field

[0001] The invention belongs to the field of breast cancer, and specifically relates to an application of a PRPF8 expression inhibitor in tamoxifen resistance of triple-negative breast cancer. Background Art

[0002] The clinical treatment of triple-negative breast cancer (TNBC) is difficult. The expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in its cells is negative, making endocrine therapy and HER2 targeted therapy commonly used in the clinical treatment of breast cancer almost ineffective. In addition, TNBC cells are relatively highly heterogeneous and easily develop drug resistance, which seriously affects the efficacy of radiotherapy and chemotherapy. Clinically, less than 30% of TNBC patients have a good response to chemotherapy, and most other patients can only relieve symptoms or control tumor growth through chemotherapy.

[0003] In response to the various difficulties in TNBC treatment, solutions such as early screening technology, multi-drug / multi-therapy combination, and new targeted therapy drug development are being actively researched and promoted. Among them, finding new effective therapeutic targets has always been the focus and difficulty of TNBC treatment. Although several immunotherapy drugs have entered the clinic, the therapeutic effect is very limited. Therefore, exploring new angles to develop effective TNBC targeted therapeutic drugs or treatment methods is our inevitable way to conquer TNBC. It is worth noting that there have been no reports on the study of TNBC therapeutic drugs from the perspective of alternative splicing.

[0004] Publication No. CN107106704A, invention patent "Methods and compositions for treating cancer" discloses that an inhibitory nucleic acid domain (an inhibitory nucleic acid domain containing siRNA) can inhibit (e.g., can target) the expression of gene products upregulated in cancer cells and / or the expression of genes required for cell growth and / or survival. The inhibitory nucleic acid inhibits the expression of genes selected from the group consisting of: Plk1, MCL1, EphA2, PsmA2, MSI1, BMI1, XBP1, PRPF8, PFPF38A, RBM22, USP39, RAN, NUP205 and NDC80. And cancers including triple-negative breast cancer are given.

[0005] This patent is for siRNA designed for the EpCAM gene, which is not highly specific to the PRPF8 gene. The impact of PRPF8 knockout on triple-negative breast cancer and its impact on tamoxifen resistance are unclear. Summary of the invention

[0006] The purpose of the present invention is to provide a siRNA for inhibiting the expression of PRPF8 and an application thereof.

[0007] The present invention provides an siRNA for inhibiting the expression of PRPF8, wherein the siRNA is a double-stranded RNA molecule consisting of a sense strand and an antisense strand, including siRNA PRPF8-1 and siRNA PRPF8-2;

[0008] The nucleotide sequence of the sense strand of the siRNA PRPF8-1 is shown in SEQ ID No.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID No.2; the nucleotide sequence of the sense strand of the siRNA PRPF8-2 is shown in SEQ ID No.3; and the nucleotide sequence of the antisense strand is shown in SEQ ID No.4.

[0009] The nucleotide sequence of the positive strand of the siRNA PRPF8-1 is GTACCATACTCCCAATGTTdTdT.

[0010] The antisense strand nucleotide sequence of the siRNA PRPF8-1 is AACATTGGGAGTATGGTACdTdT.

[0011] The positive strand nucleotide sequence of the siRNAPRPF8-2 is GCATGCGCCTCATGAAACAdTdT.

[0012] The antisense strand nucleotide sequence of the siRNAPRPF8-2 is TGTTTCATGAGGCGCATGCdTdT.

[0013] The invention discloses an application of siRNA for inhibiting PRPF8 expression, wherein the siRNA is applied in the preparation of a drug for improving drug resistance of triple-negative breast cancer.

[0014] Furthermore, the drug resistance of triple-negative breast cancer refers to resistance to tamoxifen.

[0015] Furthermore, the improvement of drug resistance of triple-negative breast cancer is achieved by knocking out PRPF8.

[0016] A pharmaceutical composition of the present invention comprises the siRNA for inhibiting PRPF8 expression according to claim 1.

[0017] The present invention has the following beneficial effects:

[0018] The PRPF8 expression inhibitor of the present invention can enhance the tamoxifen sensitivity of triple-negative breast cancer cells, and the IC50 is significantly reduced, and can be used for the clinical treatment of triple-negative breast cancer. The present invention enhances the tamoxifen sensitivity of HCC 1937 cells by knocking down PRPF8. The IC50 of the control group NC is 12.44 μM, while the IC50 of the PRPF8 knockdown group 8-1 is 7.40 μM, and the IC50 of 8-2 is 7.42 μM, which is significantly lower than that of the NC group (p<0.0001). BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the effect of PRPF8 knockdown on Tamoxifen resistance of HCC 1937 cells; (a). Survival rate of HCC 1937 cells after PRPF8 knockdown for 48 hours and Tamoxifen treatment for 72 hours; (b). Tamoxifen IC50 of HCC 1937 cells after PRPF8 knockdown. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0021] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention, but are not intended to limit the present invention.

[0022] Example 1

[0023] Cell culture: HCC 1937 cell line was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cell culture was performed in an air incubator at 37°C using L15 medium supplemented with 10% calf serum.

[0024] siRNA transfection:

[0025] After PRPF8 knockdown for 48 h, the cells were counted and plated in a 96-well plate at 1×10 4 Each group of cells was cultured at 37°C in a cell culture incubator with 4 replicate wells. Six Tamoxifen concentrations of 0μM, 5μM, 10μM, 15μM, 20μM, and 25μM were prepared as needed. After the cells adhered overnight, Tamoxifen culture solutions of various concentrations were added. After 72 hours, the absorbance of each well at OD450nm was detected by using CCK8 reagent on a microplate reader.

[0026] The results show:

[0027] After PRPF8 was knocked down for 48 hours, CCK8 reagent was used to detect the changes in Tamoxifen resistance of HCC 1937 cells. The experimental results showed that after PRPF8 was knocked down, the IC50 of the control group NC was 12.44μM, while the IC50 of the knockdown groups 8-1 and 8-2 was 7.40μM and 7.42μM, respectively. The sensitivity of HCC1937 cells to Tamoxifen was significantly increased (see Appendix Figure 1 ). The drug used in the patent "Methods and compositions for treating cancer" is paclitaxel, and the experiment does not test the effect of siRNA transfection on the drug resistance of breast cancer cells. "Methods and compositions for treating cancer" tests the inhibitory ability of its designed siRNA on breast cancer cells, which has nothing to do with the change of drug resistance of breast cancer cells. There is a significant difference from this embodiment.

Claims

1. A siRNA for inhibiting PRPF8 expression, characterized in that The siRNA is a double-stranded RNA molecule consisting of a sense strand and an antisense strand, including siRNA PRPF8-1 and siRNA PRPF8-2; The nucleotide sequence of the sense strand of the siRNA PRPF8-1 is shown in SEQ ID No.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID No.2; the nucleotide sequence of the sense strand of the siRNA PRPF8-2 is shown in SEQ ID No.3; and the nucleotide sequence of the antisense strand is shown in SEQ ID No.

4.

2. The use of a siRNA for inhibiting PRPF8 expression as claimed in claim 1, characterized in that The siRNA is used in the preparation of improving the drug resistance of triple-negative breast cancer.

3. The use according to claim 2, characterized in that The drug resistance of triple-negative breast cancer refers to resistance to tamoxifen.

4. The use according to claim 2 or 3, characterized in that The improvement of drug resistance of triple-negative breast cancer is achieved by knocking out PRPF8.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the siRNA for inhibiting PRPF8 expression according to claim 1.

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