SiRNA combination and application thereof

Abstract

The invention discloses a siRNA combination and application thereof, and relates to the technical field of biology, and the siRNA combination comprises one or more of a first siRNA, a second siRNA, a third siRNA, a fourth siRNA, a fifth siRNA, a sixth siRNA, a seventh siRNA, an eighth siRNA, a ninth siRNA and a tenth siRNA. According to the invention, 10 kinds of siRNAs are designed to silence and knock down HNF4alpha, so that the multiplication capacity of prostatic cancer stem cells is remarkably inhibited; in addition, knock-down of HNF4alpha significantly inhibits the tumor formation ability of prostatic cancer stem cells in mice, and inhibits the malignant degree of prostatic cancer tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a siRNA combination and application thereof. Background Art

[0002] Prostate cancer stem cells (PCSCs) are considered to be the main cause of castration-resistant prostate cancer (CRPC) progression. Among all CRPC progression mechanisms, abnormal activation of the androgen receptor (AR) signaling pathway still plays a dominant role in tumor progression, and blocking the androgen signaling pathway is still an important therapeutic approach.

[0003] Although treatments targeting AR have been approved in recent years, such as abiraterone and the second-generation antiandrogen drug Enzalutamide, which inhibit the synthesis of androgens by inhibiting 17-α hydroxylase (CYP-17) in the adrenal glands, testicles, and tumors, or directly target AR to block the activation of the AR signaling pathway, long-term effects are still limited due to drug resistance caused by a variety of complex reasons such as AR overexpression, mutation, and activation of AR signaling bypass.

[0004] Therefore, while blocking the AR signaling pathway, combining new chemotherapy and biological immunotherapy has become a new option for clinical treatment. However, chemotherapy drugs such as docetaxel and cabazitaxel are not ideal due to severe drug toxicity and late drug tolerance. Summary of the invention

[0005] The main purpose of the present invention is to propose an siRNA combination and its application, aiming to provide a new CRPC treatment method.

[0006] To achieve the above object, the present invention provides an siRNA combination, wherein the siRNA combination includes one or more of a first siRNA, a second siRNA, a third siRNA, a fourth siRNA, a fifth siRNA, a sixth siRNA, a seventh siRNA, an eighth siRNA, a ninth siRNA, and a tenth siRNA;

[0007] The sense strand sequence of the first siRNA is shown in SEQ ID NO.1, and the antisense strand sequence of the first siRNA is shown in SEQ ID NO.2;

[0008] The sense strand sequence of the second siRNA is shown in SEQ ID NO.3, and the antisense strand sequence of the second siRNA is shown in SEQ ID NO.4;

[0009] The sense strand sequence of the third siRNA is shown in SEQ ID NO.5, and the antisense strand sequence of the third siRNA is shown in SEQ ID NO.6;

[0010] The sense strand sequence of the fourth siRNA is shown in SEQ ID NO.7, and the antisense strand sequence of the fourth siRNA is shown in SEQ ID NO.8;

[0011] The sense strand sequence of the fifth siRNA is shown in SEQ ID NO.9, and the antisense strand sequence of the fifth siRNA is shown in SEQ ID NO.10;

[0012] The sense strand sequence of the sixth siRNA is shown in SEQ ID NO.11, and the antisense strand sequence of the sixth siRNA is shown in SEQ ID NO.12;

[0013] The sense strand sequence of the seventh siRNA is shown in SEQ ID NO.13, and the antisense strand sequence of the seventh siRNA is shown in SEQ ID NO.14;

[0014] The sense strand sequence of the eighth siRNA is shown in SEQ ID NO.15, and the antisense strand sequence of the eighth siRNA is shown in SEQ ID NO.16;

[0015] The sense strand sequence of the ninth siRNA is shown in SEQ ID NO.17, and the antisense strand sequence of the ninth siRNA is shown in SEQ ID NO.18;

[0016] The sense strand sequence of the tenth siRNA is shown as SEQ ID NO.19, and the antisense strand sequence of the tenth siRNA is shown as SEQ ID NO.20.

[0017] The present invention also provides a use of the aforementioned siRNA combination in inhibiting HNF4α gene expression.

[0018] The present invention also provides a use of the aforementioned siRNA combination in preparing a prostate cancer stem cell growth inhibitor.

[0019] The present invention also provides a use of the aforementioned siRNA combination in preparing a drug for treating prostate cancer.

[0020] The present invention also provides an HNF4α antagonist, comprising the aforementioned siRNA combination.

[0021] The present invention also provides a prostate cancer stem cell growth inhibitor, comprising the aforementioned siRNA combination.

[0022] The present invention also provides a drug for treating prostate cancer, comprising the aforementioned siRNA combination.

[0023] In one embodiment, the medicament further comprises a pharmaceutically acceptable carrier.

[0024] In one embodiment, the type of the drug includes oral, intravenous, subcutaneous or intramuscular injections.

[0025] In the technical solution of the present invention, 10 siRNAs were designed to silence and knock down HNF4α, thereby significantly inhibiting the proliferation ability of prostate cancer stem cells; in addition, knocking down HNF4α significantly inhibited the tumor-forming ability of prostate cancer stem cells in mice and inhibited the malignancy of prostate cancer tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0027] Figure 1 (A) is a graph showing the result of qRT-PCR detection of HNF4α mRNA expression level of VCaP cells under charcoal adsorption fetal bovine serum culture conditions in Example 1 provided by the present invention; Figure 1 (B) is a graph showing the result of qRT-PCR detection of HNF4α mRNA expression level in VCaP-CRPC model in Example 1 provided by the present invention; Figure 1 (C) is a graph showing the result of qRT-PCR detection of HNF4α mRNA expression levels in PCSCs derived from clinical pathological tissue samples in Example 1 provided by the present invention; Figure 1 Middle (D) is a microscope image of tumor multicellular spheres formed by LNCaP and VCaP cells under adherent and 3D suspension culture conditions in Example 1 provided by the present invention; Figure 1 Middle (E) is a result diagram of detecting the expression level of HNF4α mRNA in tumor multicellular spheres formed by prostate cancer LNCaP and VCaP cells by qRT-PCR in Example 1 provided by the present invention; Figure 1 Middle (F) is a graph showing the results of Western blot detection of the expression level of HNF4α protein in tumor multicellular spheres formed by LNCaP and VCaP cells in Example 1 provided by the present invention;

[0028] Figure 2(A) is the separation of HNF4α from prostate cancer cells by flow cytometry in Example 1 provided by the present invention + / hi (HNF4α high expression) and HNF4α - / lo Schematic diagram of (HNF4α low expression) cell population; Figure 2 (B) is a diagram showing the difference in tumorigenicity of VCaP cells with different HNF4α expression levels in mice in Example 1 provided by the present invention; Figure 2 Middle (C) is a microscope image of the multicellular sphere formation ability of LNCaP and VCaP cells with different HNF4α expression levels in Example 1 provided by the present invention; Figure 2 Middle (D) is a statistical result diagram showing the difference in multicellular sphere formation ability of LNCaP and VCaP cells with different HNF4α expression levels in Example 1 provided by the present invention; Figure 2 Middle (E) is a graph showing the expression levels of PCSC stem cell markers in prostate cancer LNCaP cells with high expression of HNF4α in Example 1 provided by the present invention; Figure 2 Middle (F) is a graph showing the expression levels of PCSC stem cell markers in prostate cancer VCaP cells with high expression of HNF4α in Example 1 provided by the present invention;

[0029] Figure 3 The target sites of the 10 HNF4α-siRNAs in HNF4α mRNA provided in Example 1 of the present invention;

[0030] Figure 4 The target positions of the 10 HNF4α-siRNAs in the HNF4α protein provided in Example 1 of the present invention;

[0031] Figure 5 The knockdown efficiency results of the 10 siRNAs on the HNF4α gene in prostate cancer cells provided in Example 2 of the present invention;

[0032] Figure 6 These are the results of the effects of the 10 siRNAs in Example 3 provided by the present invention on the proliferation of prostate cancer LNCaP cells.

[0033] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0034] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0035] Prostate cancer stem cells (PCSCs) are considered to be the main cause of castration-resistant prostate cancer (CRPC) progression. Among all CRPC progression mechanisms, abnormal activation of the androgen receptor (AR) signaling pathway still plays a dominant role in tumor progression, and blocking the androgen signaling pathway is still an important therapeutic approach.

[0036] Although treatments targeting AR have been approved in recent years, such as abiraterone and the second-generation antiandrogen drug Enzalutamide, which inhibit the synthesis of androgens by inhibiting 17-α hydroxylase (CYP-17) in the adrenal glands, testicles, and tumors, or directly target AR to block the activation of the AR signaling pathway, long-term effects are still limited due to drug resistance caused by a variety of complex reasons such as AR overexpression, mutation, and activation of AR signaling bypass.

[0037] Therefore, while blocking the AR signaling pathway, combining new chemotherapy and biological immunotherapy has become a new option for clinical treatment. However, chemotherapy drugs such as docetaxel and cabazitaxel are not ideal due to severe drug toxicity and late drug tolerance.

[0038] In view of this, the present invention provides a siRNA combination, the siRNA combination comprising one or more of a first siRNA, a second siRNA, a third siRNA, a fourth siRNA, a fifth siRNA, a sixth siRNA, a seventh siRNA, an eighth siRNA, a ninth siRNA, and a tenth siRNA;

[0039] The sense strand sequence of the first siRNA is shown in SEQ ID NO.1, and the antisense strand sequence of the first siRNA is shown in SEQ ID NO.2;

[0040] The sense strand sequence of the second siRNA is shown in SEQ ID NO.3, and the antisense strand sequence of the second siRNA is shown in SEQ ID NO.4;

[0041] The sense strand sequence of the third siRNA is shown in SEQ ID NO.5, and the antisense strand sequence of the third siRNA is shown in SEQ ID NO.6;

[0042] The sense strand sequence of the fourth siRNA is shown in SEQ ID NO.7, and the antisense strand sequence of the fourth siRNA is shown in SEQ ID NO.8;

[0043] The sense strand sequence of the fifth siRNA is shown in SEQ ID NO.9, and the antisense strand sequence of the fifth siRNA is shown in SEQ ID NO.10;

[0044] The sense strand sequence of the sixth siRNA is shown in SEQ ID NO.11, and the antisense strand sequence of the sixth siRNA is shown in SEQ ID NO.12;

[0045] The sense strand sequence of the seventh siRNA is shown in SEQ ID NO.13, and the antisense strand sequence of the seventh siRNA is shown in SEQ ID NO.14;

[0046] The sense strand sequence of the eighth siRNA is shown in SEQ ID NO.15, and the antisense strand sequence of the eighth siRNA is shown in SEQ ID NO.16;

[0047] The sense strand sequence of the ninth siRNA is shown in SEQ ID NO.17, and the antisense strand sequence of the ninth siRNA is shown in SEQ ID NO.18;

[0048] The sense strand sequence of the tenth siRNA is shown as SEQ ID NO.19, and the antisense strand sequence of the tenth siRNA is shown as SEQ ID NO.20.

[0049] In the technical solution of the present invention, 10 siRNAs (as shown in Table 1) were designed to silence and knock down HNF4α, thereby significantly inhibiting the proliferation ability of prostate cancer stem cells; in addition, knocking down HNF4α significantly inhibited the tumor-forming ability of prostate cancer stem cells in mice and inhibited the malignancy of prostate cancer tumors.

[0050] It should be noted that HNF4α (Hepatocyte Nuclear Factor 4Alpha) is an orphan nuclear receptor with no ligand or a ligand that has not yet been found. It binds to its action element in the form of a dimer, is located upstream of the transcription factor "regulatory network", and is widely involved in the regulation of human metabolism and the expression and regulation of genes related to cell differentiation. HNF4α protein has the characteristics of a fat-soluble hormone receptor, contains a DNA binding domain (DBD) and a ligand domain (LBD), and can directly enter the cell nucleus to regulate the transcriptional expression of target genes, thereby regulating various physiological activities of the body.

[0051] It should be noted that, in the present invention, siRNA, i.e., small interfering RNA (small interfering RNA), can guide the degradation of target mRNA (HNF4α mRNA) during RNA interference (RNAi), thereby specifically inhibiting or shutting down the expression of HNF4α gene.

[0052] In addition, each siRNA comprises a sense strand and an antisense strand, and the base pairs thereof are 15 to 30, preferably, the base pairs are 21 or 25. The antisense strand comprises a complementary region to the mRNA encoding HNF4α.

[0053] In the scheme of the present invention, the siRNA combination can be any one of the above 10 siRNAs, or two or more of the above 10 siRNAs, all of which are within the protection scope of the present invention.

[0054] Table 1 Sequences of ten siRNAs

[0055]

[0056]

[0057] It should be noted that the sources of the siRNA include shRNA, siRNA, miRNA, dsRNA, stRNA, small molecule compounds, small nucleic acid drugs, nucleic acid aptamers, or antibodies or antigen-binding fragments thereof. In addition, siRNA can also be improved by modification to increase its stability, improve the uptake rate of siRNA by cells, and effectively inhibit the expression of the target gene.

[0058] In some embodiments of the present invention, the sense strand and / or antisense strand of the siRNA comprises at least one nucleotide modification. The nucleotide modification comprises at least one of a phosphate backbone modification, a ribose modification, and a base modification. Specifically, the nucleotide modification comprises at least one of a 5' terminal cholesterol modification, a 5' terminal phosphorylation modification, a 2' methoxy modification of deoxyribose, a 5-methylcytosine modification of the base, and a 5' terminal fluorescent group modification. The modification can be performed by a variety of techniques, such as chemical synthesis.

[0059] In addition, the relevant properties of siRNA can be further improved by connecting it to a suitable ligand to form a ligand-RNA conjugate to reduce the clearance rate in the circulation, enhance targeted accumulation and cellular uptake, and thus regulate its pharmacokinetics and pharmacodynamics. The ligand includes small molecules, lipids, peptides, antibodies, proteins, carbohydrates and non-coding RNA.

[0060] The present invention also provides a use of the aforementioned siRNA combination in inhibiting the expression of HNF4α gene. The siRNA combination of the present invention can specifically inhibit or shut down the expression of HNF4α gene by interfering with the mRNA of HNF4α.

[0061] The present invention also provides an application of the aforementioned siRNA combination in preparing a prostate cancer stem cell growth inhibitor. The present invention finds that the HNF4α gene is specifically highly expressed in prostate cancer stem cells. Since the siRNA combination can inhibit the expression of the HNF4α gene, the siRNA combination can inhibit the growth of prostate cancer stem cells.

[0062] The present invention also provides a use of the aforementioned siRNA combination in the preparation of a drug for treating prostate cancer. Since PCSCs are the main cause of castration-resistant prostate cancer progression, the siRNA combination can inhibit the growth of prostate cancer stem cells, and thus the siRNA combination can be used to inhibit or even treat prostate cancer.

[0063] The present invention also provides an HNF4α antagonist, including the aforementioned siRNA combination. The siRNA combination affects the expression of HNF4α protein in cells by interfering with the mRNA expression of HNF4α, that is, when the siRNA successfully pairs with the mRNA of HNF4α and guides its degradation, the mRNA level of HNF4α decreases, thereby reducing the synthesis of HNF4α protein. Therefore, the siRNA combination can be used to inhibit the expression of HNF4α protein.

[0064] The present invention also provides a prostate cancer stem cell growth inhibitor, comprising the aforementioned siRNA combination. The siRNA combination inhibits the proliferation of prostate cancer stem cells by interfering with the mRNA expression of HNF4α, so the siRNA combination can be used to prepare a prostate cancer stem cell growth inhibitor.

[0065] The present invention also provides a drug for treating prostate cancer, comprising the aforementioned siRNA combination. The siRNA combination inhibits the proliferation of prostate cancer stem cells by interfering with the mRNA expression of HNF4α, thereby inhibiting the development of prostate cancer. Therefore, the siRNA combination can be used to treat or inhibit prostate cancer.

[0066] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include N-acetylglucosamine (GalNAc), lipid-based delivery systems (such as micelles, liposomes and lipid nanoparticles), polymer-based RNA delivery systems or nanoparticle-based RNA delivery systems, etc.

[0067] In some embodiments of the present invention, the type of the drug includes oral, intravenous, subcutaneous or intramuscular injections. Each route of administration has its indications and contraindications, and the choice of which method depends on factors such as the characteristics of the drug itself, the goal of treatment, and the health status of the patient.

[0068] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0069] Example 1 Design of HNF4α-siRNA

[0070] 1. Expression of HNF4α in prostate cancer stem cells

[0071] (1) Prostate cancer VCaP cells were cultured in fetal bovine serum (Charcoal-stripped FBS, Catalog No. 12676-029, purchased from GIBCO) for 48 h, and the expression level of HNF4α mRNA in the cultured VCaP cells was detected. The detection results are as follows: Figure 1 As shown in (A).

[0072] (2) Severe Combined Immunodeficient mice (SCID mice) were used to construct a CRPC mouse model to obtain the VCaP-CRPC model. The specific construction method was as follows: prostate cancer VCaP cells (2×10 6The cells / 50 μL were mixed with matrix gel at a ratio of 1:1 and injected subcutaneously into the axilla of 6- to 8-week-old male SCID mice. The tumor volume was observed and measured once a week. The tumor volume was about 0.8 cm 3 The host mice were anesthetized and castrated and kept normally for about 14 weeks. The recurrent tumor xenografts grew to about 1.2 cm 3 The mice were humanely killed by overdose of anesthesia and the tumors were removed. Tumor specimens were collected before castration and 8 weeks after castration (tumor recurrence after castration) to detect the expression level of HNF4α mRNA in tumor tissues. The test results are shown in Figure 1 As shown in (B).

[0073] (3) Prostate cancer tissues were obtained from surgical resection and CD44 + / CD133 + Positive PCSC cells (i.e., CD44 + / CD133 + positive prostate cancer stem cells), and CD44 - / CD133 - Negative prostate cancer cells were used as the control group (Control), and the difference in HNF4α mRNA expression levels of the two cells was detected. The test results are as follows Figure 1 As shown in (C).

[0074] (4) Two prostate cancer cells, LNCaP and VCaP, were subjected to adherent culture and 3D suspension culture. The conditions for adherent culture were as follows: RMPI1640 containing 10% fetal bovine serum and DMEM culture medium (purchased from Gibco) were used to culture at 37°C, 5% carbon dioxide, and a relative humidity of 90%-95%. The method for 3D suspension culture was as follows: prostate cancer cells (LNCaP cells and VCaP cells) were digested into single cells by trypsin, washed three times with phosphate buffered saline (PBS), and resuspended in DMEM / F12 culture medium (DMEM / F12 culture medium, specific components: 10 ng / mL EGF, 10 ng / ml bFGF, 2% B-27, 5 μg / mL insulin, 1% knockout replacement serum, 1% penicillin and streptomycin) at 1×10 5 The cells were inoculated into a 100 mm ultra-low adsorption culture dish at a concentration of 100 cells / mL and cultured at 37°C for 5 days. The culture medium was replaced every 2-3 days for a total of 2 weeks. Cell spheres with a diameter greater than 80 μm (such as Figure 1D) was used for subsequent experimental studies. Among them, prostate cancer cell lines VCaP (Cat. No. CRL-2876) and LNCaP cells (Cat. No. CRL-1740) were purchased from the American Type Culture Collection (ATCC), and multicellular spheres were obtained by 3D culture. The appearance of cells obtained by the two culture methods was observed, and the results are as follows Figure 1 As shown in (D); the expression levels of HNF4α mRNA and protein in LNCaP and VCaP cells after 3D suspension culture were detected, and the results were shown in Figure 1 As shown in (E) and (F).

[0075] Among them, the expression level of HNF4α mRNA was detected by Real-time PCR method: total RNA of cell samples was extracted by TRIzol method, treated with deoxyribonuclease I (DNaseI), and then incubated with a mixture of PrimeScript RTase and oligo-dT (PrimeScript RT Master Mix purchased from TaKaRa, Japan) at 37°C for 15 min to complete reverse transcription cDNA synthesis, and then heat-inactivated reverse transcriptase (RTase) at 85°C for 5 s. 25 ng cDNA, 100 nmol / L primer pair and fluorescent quantitative reagent mixture (SYBR Premix Ex Taq, ROX Plus purchased from TaKaRa, Japan) were mixed and PCR reaction was carried out at 95°C for 20 s in a real-time PCR system, followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 30 s.

[0076] The expression level of HNF4α protein was detected by Western blot method: after prostate cancer multicellular spheres were obtained by 3D suspension culture, the total proteins of multicellular spheres and adherent cells were lysed and extracted using cold lysis buffer (20mM PIPES, 0.1% SDS, 1mM EDTA, 1mM EGTA, 10mM monothioglycerol, 1mM PMSF, 5mM leupeptin, 0.25M sucrose). The extracted total proteins were separated by SDS-PAGE and transferred to PVDF membrane, incubated with primary antibody [recombinant Anti-HNF-4-alpha antibody (purchased from Abcam, catalog number ab92378)] and secondary antibody [HRP anti-rabbit IgG antibody (purchased from Abcam, catalog number ab288151)], and then detected by chemiluminescence detection method.

[0077] Result analysis:

[0078] Depend on Figure 1As shown in (A), in VCaP cells cultured in fetal bovine serum treated with activated carbon, the expression level of HNF4α mRNA gradually increased with the extension of treatment time.

[0079] Depend on Figure 1 As can be seen in (B), blue represents before castration, and purple represents after castration. In the CRPC mouse model, the HNF4α mRNA expression level in the tumor-bearing tissue of mice after castration was significantly higher than that before castration.

[0080] Depend on Figure 1 In (C), we can see that in CD44 + / CD133 + In positive prostate cancer stem cells, the expression level of HNF4α mRNA was significantly higher than that in the Control group.

[0081] Depend on Figure 1 As shown in (D), prostate cancer LNCaP cells and VCaP multicellular spheroids were successfully obtained through the 3D suspension culture method.

[0082] Depend on Figure 1 As shown in (E), the expression level of HNF4α mRNA in prostate cancer LNCaP multicellular spheroids and VCaP multicellular spheroids was significantly higher than that in the cell groups growing on the wall.

[0083] Depend on Figure 1 As can be seen from (F), the expression level of HNF4α protein in prostate cancer LNCaP cells and VCaP multicellular spheroids was significantly higher than that in adherent cell groups.

[0084] In summary, by analyzing the endogenous mRNA expression level and endogenous protein expression level of HNF4α in prostate cancer stem cells, it was found that the expression of HNF4α in prostate cancer stem cells was significantly higher than that in the adherent cells of the control group at both the mRNA and protein levels ( Figure 1 ).

[0085] 2. Correlation analysis between HNF4α expression level and PCSC stemness function

[0086] HNF4α high expression (HNF4α + / hi ) and low expression of HNF4α (HNF4α - / lo ) cell population, the sorting diagram is as follows Figure 2 As shown in (A).

[0087] The HNF4α isolated above + / hi and HNF4α - / loThe cell population was used for mouse subcutaneous tumor formation experiment. The specific experimental method was as follows: HNF4α high expression (HNF4α + / hi ) and low expression of HNF4α (HNF4α - / lo ) VCaP cells, the cell density reached about 80-90%. The cells were digested with trypsin and resuspended to a final concentration of 2×10 6 The cell suspension and matrix gel were diluted at a ratio of 1:1 at 4°C to prepare a final concentration of 1×10 6 The solution was injected into the left armpit of nude mice subcutaneously with a volume of 100 μL. The injected nude mice were returned to the cage for continued feeding, and the tumor growth and tumor volume were observed weekly. According to the experimental design, the tumor volume did not exceed 1000 mm. 3 The nude mice were euthanized, the tumors were removed, and the images were taken to compare the HNF4α + / hi and HNF4α - / lo The difference in tumorigenicity of VCaP cells in mice, as shown in the photo Figure 2 As shown in (B).

[0088] The HNF4α isolated above + / hi and HNF4α - / lo The cell population was cultured in 3D suspension. The specific culture method was the same as in step 1. After culture, prostate cancer multicellular spheres were obtained. Microscope images of two multicellular spheres with different HNF4α expression levels obtained by culture are shown in Figure 2. Figure 2 As shown in (C).

[0089] HNF4α + / hi and HNF4α - / lo The number of multicellular spheres formed by LNCaP and VCaP cells was counted and the analysis results were as follows: Figure 2 As shown in (D).

[0090] PCSC stem cell markers (including CD133, CK14, CD24, CD44, β-catenin, SOX2, Oct3 / 4, Nanog, CD49, etc.) were detected by real-time PCR (the method is the same as in step 1) in LNCaP and VCaP cells with high expression of HNF4α. The results of stem cell marker detection in LNCaP cells with high expression of HNF4α are as follows: Figure 2 As shown in (E), the results of stemness marker detection of VCaP cells with high expression of HNF4α are as follows Figure 2 As shown in (F).

[0091] Result analysis:

[0092] Depend on Figure 2 As shown in (B), HNF4α was found + / hi The VCaP cells have a stronger tumor-forming ability in mice and the tumors they form are larger.

[0093] Figure 2 (B), (C) and (D) represent HNF4α + / hi and HNF4α - / lo The differences in the tumor-forming ability of cell populations in mice and in their multicellular formation ability in vitro showed that high levels of HNF4α expression promoted the stemness and tumorigenicity of prostate cancer stem cells. Conversely, cells with low levels of HNF4a expression were less malignant.

[0094] Depend on Figure 2 As shown in (E) and (F), qRT-PCR detection of HNF4α + / hi The expression levels of PCSC stem cell markers in LNCaP and VCaP cells were significantly higher than those in HNF4α - / lo Cell population.

[0095] 3. Design of HNF4α-siRNA

[0096] HNF4α protein has a total of 598 amino acids and a molecular weight of 66.6KD. The ligand binding domain (LBD) is usually occupied by several bulky hydrophobic residue side chains in the ligand binding region. Therefore, HNF4α lacks the "classical" binding site of the coactivator. Its mRNA nucleotide sequence is shown in SEQ ID NO.21, and its amino acid sequence is shown in SEQ ID NO.22. Figure 3 As shown, in this example, 10 (#1, #2, #3, #4, #5, #6, #7, #8, #9, #10) HNF4α-siRNAs were designed to target HNF4α mRNA. The specific targeting positions in the HNF4α protein are shown in Figure 4 .

[0097] SEQ ID NO.21 is as follows:

[0098]

[0099] SEQ ID NO.22 is as follows:

[0100] MRLSKTLVDMDMADYSAALDPAYTTLEFENVQVLTMGNDTSPSEGTNLNAPNSLGVSALCAICGDRATGKHYGASSCDGCKGFFRRSVRKNHMYSCRFSRQCVVDKDKRNQCRYCRLK KCFRAGMKKEAVQNERDRISTRRSSYEDSSLPSINALLQAEVLSRQITSPVSGINGDIRAKKIASIADVCESMKEQLLVLVEWAKYIPAFCELPLDDQVALLRAHAGEHLLLGATKRSM VFKDVLLLGNDYIVPRHCPELAEMSRVSIRILDELVLPFQELQIDDNEYAYLKAIIFFDPDAKGLSDPGKIKRLRSQVQVSLEDYINDRQYDSRGRFGELLLLLPTLQSITWQMIEQIQ FIKLFGMAKIDNLLQEMLLGGSPSDAPHAHHPLHPHLMQEHMGTNVIVANTMPTHLSNGQMCEWPRPRGQAATPETPQPSPPGGSGSEPYKLLPGAVATIVKPLSAIPQPTITKQEVI.

[0101] Example 2 Effect of HNF4α-siRNA on HNF4α expression in prostate cancer cells

[0102] The prostate cancer cell line LNCaP was purchased from the American Model Culture Collection and cultured at 37°C in Gibco RPMI 1640 Medium containing 10% fetal bovine serum (FBS).

[0103] 1. The steps for transfecting HNF4α siRNA in the prostate cancer cell line LNCaP are as follows:

[0104] (1) Briefly centrifuge the centrifuge tube containing the synthesized siRNA powder (2000 rpm) to ensure that the siRNA is at the bottom of the centrifuge tube.

[0105] (2) Use appropriate amount of DEPC-DW (or RNase-free water) to dissolve the 10 siRNAs designed in Example 1 respectively to a concentration of 100 nmol / mL. Use a pipette to pipette up and down 3 to 5 times (or briefly vortex). Briefly centrifuge at low speed to allow the liquid to accumulate at the bottom of the centrifuge tube.

[0106] (3) One day before transfection, 3.0×10 5 For LNCaP cells, place 2.5 mL of growth medium without antibiotics in each well to ensure a 50-60% confluency rate during transfection.

[0107] (4) Remove the growth medium in the 6-well plate and add 1.5 mL of fresh serum-free growth medium. The original growth medium in step (3) and step (4) is RMPI1640, purchased from Gibco.

[0108] (5) For each transfected well, prepare the siRNA and Lipofectamine RNAiMAX mixture as follows:

[0109] siRNA dilution: in 250 μL Add 100 pmol siRNA to serum-free medium and mix gently;

[0110] Lipofectamine RNAiMAX dilution: Lipofectamine RNAiMAX should be mixed before use, then diluted in 250 μL I. Add 5 μL of Lipofectamine RNAiMAX to serum-free medium for dilution and incubate at room temperature for 5 minutes;

[0111] Mix the diluted siRNA and Lipofectamine RNAiMAX and incubate at room temperature for 20 minutes.

[0112] (6) Add the siRNA and Lipofectamine RNAiMAX mixture from (5) to a 6-well plate containing LNCaP cells, with a final volume of 2 mL per well, and gently shake to mix.

[0113] (7) The cells were cultured in an incubator at 37° C. and 5% carbon dioxide for 5 to 6 hours, the culture medium containing serum was replaced and the cells were cultured for 24 to 48 hours to obtain HNF4α gene knockout cells.

[0114] 2. Detection of knockdown efficiency of HNF4α gene by siRNA using Real-time PCR

[0115] (1) The total RNA of the HNF4α knockout cell sample obtained in step 1 was extracted using the TRIzol method. After treatment with deoxyribonuclease I (DNaseI), the reverse transcription cDNA synthesis was completed by incubation with a mixture of PrimeScript RTase and oligo-dT (PrimeScript RT MasterMix, TaKaRa) at 37°C for 15 min, and then the reverse transcriptase (RTase) was heat inactivated at 85°C for 5 s.

[0116] (2) 25 ng of cDNA, 100 nmol / L primer pair and fluorescent quantitative reagent mixture (SYBR Premix ExTaq, ROX Plus, TaKaRa) were mixed and PCR reaction was carried out at 95°C for 20 s in a real-time PCR system (StepOne, Applied Biosystems), followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 30 s.

[0117] The test results are shown in Figure 5 ,From the experimental results, we can see that all 10 siRNAs have obvious knockdown efficiency on HNF4α.

[0118] Example 3 Effect of HNF4α-siRNA on Proliferation of Prostate Cancer LNCaP Cells

[0119] Using the transfection method in Example 2, the LNCaP cell line was transfected with Control siRNA (Cat. No. siBDM2500, purchased from RiboBio Corporation) and 10 HNF4α-siRNAs, and an MTT cell viability experiment was performed. After 3 days of cell culture, the proliferation of LNCaP cells was obtained by measuring the absorbance value at OD 570nm. The experimental results are shown in Figure 6 .

[0120] Depend on Figure 6 It can be seen that compared with the Control siRNA control group, the LNCaP cell viability values ​​in the 10 siRNA treatment groups were significantly lower, indicating that the cell proliferation of LNCaP cells in the 10 siRNA treatment groups was significantly inhibited.

[0121] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A siRNA combination, characterized in that: The siRNA combination includes one or more of a first siRNA, a second siRNA, a third siRNA, a fourth siRNA, a fifth siRNA, a sixth siRNA, a seventh siRNA, an eighth siRNA, a ninth siRNA, and a tenth siRNA; The sense strand sequence of the first siRNA is shown in SEQ ID NO.1, and the antisense strand sequence of the first siRNA is shown in SEQ ID NO.2; The sense strand sequence of the second siRNA is shown in SEQ ID NO.3, and the antisense strand sequence of the second siRNA is shown in SEQ ID NO.4; The sense strand sequence of the third siRNA is shown in SEQ ID NO.5, and the antisense strand sequence of the third siRNA is shown in SEQ ID NO.6; The sense strand sequence of the fourth siRNA is shown in SEQ ID NO.7, and the antisense strand sequence of the fourth siRNA is shown in SEQ ID NO.8; The sense strand sequence of the fifth siRNA is shown in SEQ ID NO.9, and the antisense strand sequence of the fifth siRNA is shown in SEQ ID NO.10; The sense strand sequence of the sixth siRNA is shown in SEQ ID NO.11, and the antisense strand sequence of the sixth siRNA is shown in SEQ ID NO.12; The sense strand sequence of the seventh siRNA is shown in SEQ ID NO.13, and the antisense strand sequence of the seventh siRNA is shown in SEQ ID NO.14; The sense strand sequence of the eighth siRNA is shown in SEQ ID NO.15, and the antisense strand sequence of the eighth siRNA is shown in SEQ ID NO.16; The sense strand sequence of the ninth siRNA is shown in SEQ ID NO.17, and the antisense strand sequence of the ninth siRNA is shown in SEQ ID NO.18; The sense strand sequence of the tenth siRNA is shown as SEQ ID NO.19, and the antisense strand sequence of the tenth siRNA is shown as SEQ ID NO.

20.

2. Use of the siRNA combination as claimed in claim 1 in inhibiting HNF4α gene expression.

3. Use of the siRNA combination as claimed in claim 1 in the preparation of a prostate cancer stem cell growth inhibitor.

4. Use of the siRNA combination according to claim 1 in the preparation of a drug for treating prostate cancer.

5. A HNF4α antagonist, characterized in that Comprising the siRNA combination as claimed in claim 1.

6. A prostate cancer stem cell growth inhibitor, characterized in that Comprising the siRNA combination as claimed in claim 1.

7. A drug for treating prostate cancer, characterized in that: Comprising the siRNA combination as claimed in claim 1.

8. The drug for treating prostate cancer according to claim 7, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

9. The drug for treating prostate cancer according to claim 7, characterized in that: The types of the drug include oral, intravenous, subcutaneous or intramuscular.

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