siRNA knockdown of CREPT inhibits tumor cell proliferation

Abstract

The application discloses siRNA for inhibiting expression of a CREPT gene, wherein a positive sense chain nucleotide sequence of the siRNA is shown in any one of SEQ ID No. 1-3. The siRNA can inhibit the CREPT. Some siRNAs can inhibit growth of mouse colon cancer cells and growth of NIH3T3 fibrosarcoma cells. The liposome delivery drug of the si-CREPT through a delivery system can treat mouse liver cancer, and significantly reduce tumor load of the mouse liver cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and in particular relates to siRNA for knocking down CREPT, a pharmaceutical composition containing the siRNA, and application of inhibiting tumor cell proliferation and treating tumors. BACKGROUND

[0002] CREPT is a target for screening anti-tumor drugs in the molecular network of cell signaling and cell cycle regulation. It mainly targets Wnt, STAT3 signaling pathway and cell cycle regulatory protein CDK / cydin D, etc. It is also found that CREPT protein plays an important role in tumor cell exosome secretion. CREPT is highly expressed in tumor tissues, and forms a ring with the CCND1 genome to recruit RNA polymerase II to circulate and promote cell cycle and tumor proliferation. CREPT can promote the transcription of Wnt target genes. CREPT acts as a co-activator of the b-catenin / TCF4 transcription complex, enhances the Wnt signaling pathway, and thus promotes cell proliferation and cell infiltration. CREPT plays a role in tumor occurrence through multiple pathways.

[0003] Therefore, there is an urgent need to inhibit tumor cell proliferation and treat tumors by knocking down CREPT. However, there are few successful reports in the prior art.

[0004] In addition, the published patent document WO02 / 44321A2 describes in the specification that the nucleoside placed at the center of the siRNA opposite to the target RNA cleavage site is an important specificity determinant and even a single nucleoside change reduces RNAi to an undetectable level (see the first paragraph on page 50 of the specification). This also increases the difficulty of using siRNA to inhibit the expression and function of CREPT. SUMMARY

[0005] To solve the problems of the prior art, the purpose of the present application is to provide siRNA for inhibiting the CREPT gene, wherein the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID No. 1-3.

[0006] The nucleotide sequence of the sense strand of the siRNA for inhibiting the CREPT gene of the present application can have a homology of ≥75% with SEQ ID No. 1-3.

[0007] Homology refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Sequence analysis is a method for finally determining the degree of homology, and DNA-DNA hybridization or DNA-RNA hybridization is also a useful estimation approach.

[0008] For example, the sense strand nucleotide sequence of the siRNA of the present application inhibiting the CREPT gene can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 90%, 100% homology with SEQ ID No. 1.

[0009] or the sense strand nucleotide sequence of the siRNA of the present application inhibiting the CREPT gene can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 90%, 100% homology with SEQ ID No. 2.

[0010] or the sense strand nucleotide sequence of the siRNA of the present application inhibiting the CREPT gene can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 90%, 100% homology with SEQ ID No. 3.

[0011] As certain embodiments of the present application, the siRNA further comprises a 3' overhang beyond the double-stranded complementary structure, the end of the 3' overhang being a dangling nucleoside.

[0012] In the present application, by adding a dangling nucleoside at the 3' of the siRNA, the stability of the siRNA is increased, its half-life in vivo can be prolonged, and a higher RNAi inhibitory activity is maintained, thereby exerting a better therapeutic effect.

[0013] Bases, also known as nucleobases or nitrogenous bases, are nitrogen-containing compounds that form nucleosides, which are components of nucleotides. Bases can form base pairs with each other and stack on each other, and they are important components of long-chain helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

[0014] The dangling nucleoside of the 3' overhang of the siRNA is in a dangling state because it does not form a base pair with the antisense strand.

[0015] As certain embodiments of the present application, the end of the 3' overhang is two dangling nucleosides, and the dangling nucleosides are deoxynucleosides.

[0016] As certain embodiments of the present application, the dangling nucleosides are dTdT.

[0017] As certain embodiments of the present application, the sense strand nucleotide sequence of the siRNA is as shown in any one of SEQ ID Nos. 4 to 6.

[0018] The sense strand nucleotide sequence of the siRNA of the present application for inhibiting the CREPT gene can have ≥ 75% homology with SEQ ID Nos. 4 to 6.

[0019] For example, the sense strand nucleotide sequence of the siRNA of the present application for inhibiting the CREPT gene can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 90%, 100% homology with SEQ ID No. 4.

[0020] Or the sense strand nucleotide sequence of the siRNA of the present application for inhibiting the CREPT gene can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 90%, 100% homology with SEQ ID No. 5.

[0021] Or the sense strand nucleotide sequence of the siRNA of the present application for inhibiting the CREPT gene can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 90%, 100% homology with SEQ ID No. 6.

[0022] As certain embodiments of the present application, the siRNA includes modified nucleotides; the modifications include phosphodiester bond modification, nucleotide base modification, siRNA end modification, and / or amino acid modification.

[0023] Phosphodiester bond modification refers to the modification of the structure of the phosphodiester bond of siRNA by chemical means, which can improve the stability of siRNA in cells and the ability to resist nuclease degradation. Common modifications include 5'-thiophosphate group, 2'-oxymethyl (2'-O-Me), 2'-fluorine (2'-F) and phosphodiester choline (PC).

[0024] Nucleotide base modification is to modify the nucleotide base of siRNA, which can improve its pairing efficiency and specificity with mRNA. Common modifications include 2'-oxymethyl, 2'-amino and N-methyl diisothiouronium.

[0025] siRNA end modification is to introduce a chemical modification group at the 3' end or 5' end of siRNA, which can change the structure and hydrophilicity of siRNA, thereby improving the stability of siRNA in cells. Common modifications include hydroxyl (OH), phosphodiester (PO4) and choline (Chol).

[0026] Amino acid modification is to introduce amino acid modification in siRNA, which can improve the specificity of siRNA in cells. Common modifications include N-acetyl cysteine (Ac-Cys) and N-acetyl lysine (Ac-Lys)

[0027] The present application also provides a pharmaceutical composition for inhibiting CREPT gene and treating tumors, which comprises an effective amount of the siRNA and a delivery system.

[0028] As certain embodiments of the present application, the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID No. 4-6.

[0029] As certain embodiments of the present application, the delivery system is selected from a lipid-based delivery system, siRNA-ligand conjugate, nucleotide-derived nanoparticle, polymer-based siRNA delivery system, metal nanoparticle, mesoporous silica nanoparticle, or exosome.

[0030] As certain embodiments of the present application, the lipid-based delivery system is neutral lipid nanoparticle (LNP).

[0031] As certain embodiments of the present application, the neutral lipid nanoparticle (LNP) is composed of cholesterol, distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phospho-choline, DSPC), poly(ethylene glycol) lipids (PEG-lipid) and ionizable lipids.

[0032] Distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phospho-choline) is a cylindrical liposome, which is used for the synthesis of liposome and is a lipid component in the lipid nanoparticle (LNP) system.

[0033] Polyethylene glycol lipid provides polyethylene glycolation in lipid nanoparticles and other nanocarriers, which can be widely used to improve their stability and plasma half-life.

[0034] Ionizable lipids are lipids that are designed to exhibit net neutrality at physiological pH when formulated into lipid nanoparticles (LNP) as a component of the LNP formulation.

[0035] The siRNA of the present application can be delivered in various ways, such as vector-mediated delivery, targeted delivery, or direct injection of siRNA into tumor tissue. Vector-mediated delivery is to package siRNA into a complex using a carrier such as a nanoparticle to improve its cell membrane penetration ability and resistance to nuclease degradation. The carrier is mainly divided into viral vectors and non-viral vectors. Viral vectors have low efficiency and can cause immune response when entering the human body, so they are less used now. Among the non-viral vectors, nanoparticles and conjugate coupling are commonly used. Targeted delivery is to guide siRNA into tumor cells by using antibodies, proteins, or low-molecular-weight compounds against tumor cell surface molecules, etc., to improve the targeting of siRNA. Common targeting substances include RGD peptide, anti-HER2 antibody, etc.

[0036] As certain embodiments of the present application, the pharmaceutical composition further comprises a second drug and a pharmaceutically acceptable carrier; the second drug is a drug for treating tumors.

[0037] The present application also provides a pharmaceutical combination product comprising an effective amount of the siRNA and a second drug for simultaneous or sequential administration.

[0038] As certain embodiments of the present application, the pharmaceutical combination product further comprises an siRNA delivery system.

[0039] As certain embodiments of the present application, the pharmaceutical combination product further comprises a pharmaceutically acceptable carrier.

[0040] The present application also provides the use of the siRNA or the pharmaceutical composition described above in the preparation of a drug for inhibiting the CREPT gene and treating tumors.

[0041] As certain embodiments of the present application, the tumor is selected from colon cancer, fibroblastoma, or liver cancer.

[0042] As certain embodiments of the present application, the subject of the use is a mammal.

[0043] As certain embodiments of the present application, the subject of the use is a mouse or a human.

[0044] As described above, the siRNA of the present application knocks down CREPT to inhibit tumor cell proliferation, which has the following beneficial effects:

[0045] The three si-RNA of the present application can effectively inhibit CREPT in mouse cells.

[0046] The si-crept-2 of the present application can inhibit the growth of mouse MC38 colon cancer cells, and si-crept-2 and si-crept-3 can inhibit the growth of NIH3T3 fibroblast cells.

[0047] The liposome-delivered si-CREPT drug can treat mouse liver cancer and significantly reduce the tumor burden of mouse liver cancer. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The efficiency of CREPT-si-RNA knockdown and its effect on cell proliferation in four mouse cells are shown. Figure 1 A is the efficiency of CREPT knockdown in mouse melanoma cells and its effect on cell proliferation, Figure 1 B is the efficiency of CREPT knockdown in mouse colon cancer cells and its effect on cell proliferation, Figure 1 C is the efficiency of CREPT knockdown in mouse fibroblasts and its effect on cell proliferation. D. The efficiency of CREPT knockdown in mouse lung cancer cells and its effect on cell proliferation;

[0049] Figure 2 The effect of LNP-encapsulated siRNA targeting CREPT on the proliferation of orthotopic liver cancer in vivo is shown. Figure 2 A is a schematic diagram of the drug treatment strategy of LNP-encapsulated siRNA in the orthotopic model of liver cancer, Figure 2 B is the effect of siRNA targeting CREPT on Hepa-1-6 tumor formation, Figure 2 C shows the fluorescence detection of the effect of siRNA targeting CREPT on tumor growth. DETAILED DESCRIPTION

[0050] In order to make the technical means, creative features, purposes and effects realized by the present application easy to understand, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0051] [Experimental materials]

[0052] B16 mouse melanoma cells, Ponsay Life Science Co., Ltd., Catalog No. CL-0319

[0053] MC38 mouse colon cancer cells, Fenghui Biotech, Cat No. CL0203

[0054] NIH3T3 mouse fibroblast cells, Pnuo Life Science, Cat No. CL-0006

[0055] LLC mouse lung cancer cells, Pnuo Life Science, Cat No. CL-0140

[0056] Hepa-1-6 liver cancer cells, Fenghui Biotech, Cat No. CL-0451

[0057] C57BL / 6 mice, purchased from Jan Suoyao Kang

[0058] Iodophor, Hisun Group, Cat No. 6935941200155

[0059] Meloxicam, Merck, Cat No. 71125-38-7

[0060] Neutral lipid nanoparticle (LNP), prepared according to the method described in the literature: Alung-selective delivery of mRNA encoding broadly neutralizing antibody against SARS-CoV-2 infection. Wanbo Tai, Kai Yang, Yunfeng Chen, Mingyao Tian, Ye Xiang, Guocan Yu, Gong Cheng, Nat Commun. 2023; 14: 8042. Page 4, right column, last paragraph to page 5, right column, first paragraph.

[0061] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0062] Example 1

[0063] Three si-RNAs were designed according to the CDS sequence of mouse CREPT, and the nucleotide sequences are shown in Table 1 as SEQ ID No. 1-3. The overhanging nucleotides of 3' end of double-stranded complementary structure of the above-mentioned three si-RNAs were added with dTdT suspension nucleotides, respectively, to obtain si-Crept-1#, si-Crept-2# and si-Crept-3#, and the nucleotide sequences are shown in Table 1 as SEQ ID No. 4-6. Among them, si-Crept-1#, si-Crept-2# have homologous fragments in the human genome. In addition, a blank control si-mus-NC was designed, and the nucleotide sequence is shown as SEQ ID No. 7.

[0064] Table 1 siRNA sequences for inhibiting CREPT

[0065] si-RNA 1 GGCAGAUGAAGGCUGUAAAnn* SEQ ID No. 1 si-RNA 2 GCGGCGAGUUCAUACAGCAnn* SEQ ID No. 2 si-RNA 3 CUCGGAUGCUGGUGGAAUAnn* SEQ ID No. 3 si-mouse Crept-1 GGCAGAUGAAGGCUGUAAATT SEQ ID No. 4 si-mouse Crept-2 GCGGCGAGUUCAUACAGCATT SEQ ID No. 5 si-mouse Crept-3 CUCGGAUGCUGGUGGAAUATT SEQ ID No. 6 si-mus-NC UUCUCCGAACGUGUCACGUTT SEQ ID No. 7

[0066] n is any deoxynucleoside

[0067] Example 2

[0068] To verify the knockdown efficiency of the three si-RNAs and the effect of knocking down CREPT on the growth rate of the cells in mice, we selected four different mouse cells, including B16 mouse melanoma cells, MC38 mouse colon cancer cells, NIH3T3 mouse fibroblast cells, and LLC mouse lung cancer cells for verification. We plated the four cells in four 6-cm dishes respectively one day before transfection, and the next day, we transfected each cell with control si-NC, si-Crept-1#, si-Crept-2#, and si-Crept-3# respectively. We collected the cells at 24 h, 48 h, 72 h, and 96 h after transfection, lysed the cells, and verified the knockdown efficiency by western blot.

[0069] The results showed that at 24 h and 48 h after transfection, the expression of CREPT in the four cells was significantly reduced compared with the control group si-NC, indicating that the three si-RNAs can effectively inhibit CREPT in mouse cells. However, since the knockdown of si-RNA is a transient effect, the results showed that the expression of CREPT increased with the extension of time to 72-96 h.

[0070] To verify the effect of inhibiting the expression of CREPT in mouse cells on the proliferation efficiency of mouse cells, we transfected the three si-RNAs in the four cells respectively, digested the cells at 24 h after transfection and plated them in 96-well plates (1000 cells / well), added CCK-8 reagent at 6 h after plating, measured the OD450 value, measured the CCK-8 of each group of cells for 5 days, and drew the cell proliferation curve for comparison of the effect of knocking down CREPT on cell proliferation in mouse cells. The results showed that in B16 cells and LLC cells, knocking down CREPT with the three si-RNAs did not significantly affect cell growth. In mouse MC38 cells, si-crept-2 could inhibit cell growth, and in NIH3T3 cells, si-crept-2 and si-crept-3 could inhibit cell growth efficiency.

[0071] Example 3

[0072] To detect the effect of siRNA targeting CREPT on the proliferation of orthotopic liver cancer cells, we injected liver cancer cells Hepa-1-6 into the liver of mice to construct an orthotopic liver cancer model, and the experimental process is as follows:

[0073] First, the mouse was anesthetized in the anesthetic chamber, the hair on the abdomen of the mouse was removed with an electric shaver, and the abdomen was disinfected with an alcohol cotton ball once. Then the mouse was anesthetized again with a respiratory mask, and the mouse was kept anesthetized throughout the operation. During the operation, the anesthetic state of the mouse was confirmed by touching the mouse to observe whether it had a reaction. Using disinfected scissors, a 1 cm long incision was made in the middle of the mouse's abdomen, and the liver lobe was identified after the skin and peritoneum were cut. The insulin needle with Hepa-1-6 cells was inserted along the liver lobe, and 60 microliters of Hepa-1-6 cells (about 1.6X 10 6 ) were slowly injected. After injection, the injection site was pressed with a cotton ball for about 1 minute, then the needle was slowly rotated out, and then a 5-0 suture needle with a thread was used to suture the peritoneum twice. After the skin was sutured twice, iodine was used to disinfect the suture site, and meloxicam was injected subcutaneously to relieve pain in the mouse. Then the mouse was transferred to an electric blanket for about 10 minutes, during which the heart rate and eye color of the mouse were observed. After the mouse woke up, it was sent back to the original cage for feeding. Three days later, the mouse was injected with meloxicam to further reduce the pain caused by the operation.

[0074] The next day, the mice with orthotopic tumors were divided into two groups according to Figure 2 A shows the subsequent drug treatment experiment. 100 microliters of si-NC or si-CREPT drug (si-mouseCrept-2#) wrapped by neutral lipid nanoparticle LNP (dose about 100 μg) were injected once through the tail vein, and the injection was repeated every 4 days for a total of 7 times (including 1 injection on the second day), and the mice were dissected after 34 days for observation and photography. Figure 2 A shows the drug treatment strategy of LNP-wrapped siRNA on the orthotopic liver cancer model. Figure 2 B shows the effect of siRNA inhibitor of CREPT on the formation of Hepa-1-6 tumors in the liver. The results show that compared with the si-NC control group (i.e. si-NC sequence in Table 1), the siRNA inhibitor of CREPT significantly reduced the tumor burden of liver cancer in mice Figure 2 B-C).

[0075] In summary, the three si-RNA of the application can effectively inhibit the expression of CREPT in mouse cells. si-mousecrept-2# can inhibit the growth of mouse MC38 cells, and si-mousecrept-2# and si-mousecrept-3# can inhibit the growth of NIH3T3 fibrosarcoma cells. Liposome delivery of si-CREPT (si-mousecrept-2#) can treat mouse liver cancer and significantly reduce the tumor burden of mouse liver cancer.

[0076] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with reference to the preferred embodiments above, the present application is not intended to be limited thereto. Any person skilled in the art, without departing from the technical scope of the present application, can make some changes or modifications to the above disclosed methods and technical contents to obtain equivalent embodiments with equivalent changes. Any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical scope of the present application, shall still fall within the scope of the technical solution of the present application.

Claims

1. siRNA for inhibiting a CREPT gene, characterized in that, The sense strand nucleotide sequence of the siRNA is shown as SEQ ID No.

2.

2. The siRNA of claim 1, wherein The siRNA comprises 3' overhang beyond the double-stranded complementary structure.

3. The siRNA of claim 1, wherein The end of the 3' overhang is two dangling nucleosides, which are deoxynucleosides.

4. The siRNA of claim 1, wherein The dangling nucleosides are dTdT.

5. The siRNA of claim 1, wherein The sense strand nucleotide sequence of the siRNA is shown as SEQ ID No.

5.

6. The siRNA of claim 1, wherein The siRNA comprises modified nucleotides. The modification comprises at least one of phosphodiester bond modification, nucleotide base modification, siRNA end modification, and amino acid modification.

7. A pharmaceutical composition for inhibiting a CREPT gene and treating a tumor, comprising the compound of claim 1 or 2. The pharmaceutical composition comprises an effective amount of the siRNA according to any one of claims 1-6 and a delivery system.

8. The pharmaceutical composition of claim 4, wherein, The sense strand nucleotide sequence of the siRNA is shown as SEQ ID No.

5.

9. The pharmaceutical composition of claim 7, wherein, The delivery system is selected from the group consisting of a lipid-based delivery system, a siRNA-ligand conjugate, a nucleotide-derived nanoparticle, a polymer-based siRNA delivery system, a metal nanoparticle, a mesoporous silica nanoparticle, and an exosome.

10. The pharmaceutical composition of claim 9, wherein, The lipid-based delivery system is a neutral lipid nanoparticle.

11. The pharmaceutical composition of claim 9, wherein, The neutral lipid nanoparticle is composed of cholesterol, distearoylphosphatidylcholine, polyethylene glycol lipid, and ionizable lipid.

12. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition further comprises a second drug and a pharmaceutically acceptable carrier; the second drug is a drug for treating tumors.

13. Use of the siRNA according to any one of claims 1 to 6 or the pharmaceutical composition according to any one of claims 7 to 12, characterized in that, The use is for preparing a drug for treating colon cancer and liver cancer.

14. Use according to claim 13, characterized in that, The subject of the use is a mammal.

15. Use according to claim 14, characterized in that, The subject of the use is a mouse or a human.

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