Method for inhibiting tumor cell proliferation by knocking down CREPT through siRNA

Abstract

The invention discloses siRNA (small interfering Ribonucleic Acid) for inhibiting the expression of a CREPT gene. The nucleotide sequence of a positive-sense strand of the siRNA is shown as any one of SEQ ID No. 1 to SEQ ID No. 3. The siRNA can be used for inhibiting CREPT (Cathode Reactive Part of siRNA can inhibit the growth of mouse colon cancer cells and the growth of NIH3T3 fibroblast cells. And the drug can be delivered through the liposome of the delivery system si-CREPT, so that the mouse liver cancer can be treated, and the tumor load of the mouse liver cancer can be remarkably reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to siRNA for knocking down CREPT, a pharmaceutical composition containing the siRNA, and applications in inhibiting tumor cell proliferation and treating tumors. Background Art

[0002] CREPT is a target for anti-tumor drug screening within the molecular network of cell signaling and cell cycle regulation. It primarily targets the Wnt and STAT3 signaling pathways and the cell cycle regulatory proteins CDK / cydin D. CREPT has also been found to play a crucial role in exosome secretion by tumor cells. CREPT is highly expressed in tumor tissues and loops the CCND1 genome, recruiting RNA polymerase II to promote the cell cycle and tumor proliferation. CREPT can promote the transcription of Wnt target genes. CREPT acts as a co-activator of the β-catenin / TCF4 transcriptional complex, enhancing the Wnt signaling pathway and promoting cell proliferation and invasion. CREPT plays a role in tumorigenesis through multiple pathways.

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

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

[0005] To address the deficiencies of the prior art, the present invention aims to provide siRNA for inhibiting the CREPT gene, wherein the positive chain nucleotide sequence of the siRNA is shown in any one of SEQ ID Nos. 1 to 3.

[0006] The positive chain nucleotide sequence of the siRNA for inhibiting the CREPT gene of the present invention may 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 the amino acid sequences of two protein molecules. Sequence analysis is the ultimate method for determining the degree of homology, while DNA-DNA hybridization or DNA-RNA hybridization are also useful estimation methods.

[0008] For example, the positive chain nucleotide sequence of the siRNA that inhibits the CREPT gene of the present invention 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%, or 100% homology with SEQ ID No. 1.

[0009] Or the positive chain nucleotide sequence of the siRNA that inhibits the CREPT gene of the present invention 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%, or 100% homology with SEQ ID No. 2.

[0010] Or the positive chain nucleotide sequence of the siRNA that inhibits the CREPT gene of the present invention 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%, or 100% homology with SEQ ID No. 3.

[0011] As certain embodiments of the present invention, the siRNA further comprises a 3' overhang extending beyond the double-stranded complementary structure, and the end of the 3' overhang is a hanging nucleoside.

[0012] In the present invention, by adding a dangling nucleoside at the 3' end of siRNA, the stability of siRNA is increased, its half-life in the body can be prolonged, and a high RNAi inhibitory activity can be 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 and stack with each other. They are important components of long helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

[0014] The dangling nucleosides at the 3' overhang of the siRNA do not form base pairs with the antisense strand and thus remain dangling.

[0015] As certain embodiments of the present invention, the 3' overhang ends with two dangling nucleosides, and the dangling nucleosides are deoxynucleosides.

[0016] In certain embodiments of the present invention, the pendant nucleoside is dTdT.

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

[0018] The positive chain nucleotide sequence of the siRNA for inhibiting the CREPT gene of the present invention may have a homology of ≥75% with SEQ ID No. 4-6.

[0019] For example, the positive chain nucleotide sequence of the siRNA that inhibits the CREPT gene of the present invention 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%, or 100% homology with SEQ ID No. 4.

[0020] Or the positive chain nucleotide sequence of the siRNA that inhibits the CREPT gene of the present invention 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%, or 100% homology with SEQ ID No. 5.

[0021] Or the positive chain nucleotide sequence of the siRNA that inhibits the CREPT gene of the present invention 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%, or 100% homology with SEQ ID No.6.

[0022] In certain embodiments of the present invention, the siRNA comprises modified nucleotides; the modifications include phosphodiester bond modifications, nucleotide base modifications, siRNA end modifications, and / or amino acid modifications.

[0023] Phosphodiester modification refers to chemically altering the structure of the siRNA phosphodiester bond, which can improve the stability of siRNA in cells and its resistance to nuclease degradation. Common modifications include 5'-phosphorothioate, 2'-oxymethyl (2'-O-Me), 2'-fluoro (2'-F), and phosphodiester choline (PC).

[0024] Nucleotide base modifications are modifications to the siRNA's nucleotide bases, which can improve its pairing efficiency and specificity with mRNA. Common modifications include 2'-oxymethyl, 2'-amino, and N-methyldiisosulfonium urea.

[0025] siRNA end modification involves the introduction of chemical modification groups into the 3' or 5' end of the siRNA, which can alter the structure and hydrophilicity of the siRNA, thereby improving its intracellular stability. Common modifications include hydroxyl (OH), phosphodiester (PO4), and choline (Chol).

[0026] Amino acid modification is the introduction of amino acid modifications into siRNA to improve the specificity of siRNA in cells. Common modifications include N-acetylcysteine ​​(Ac-Cys) and N-acetyllysine (Ac-Lys).

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

[0028] As certain embodiments of the present invention, the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID Nos. 4 to 6.

[0029] As certain embodiments of the present invention, the delivery system is selected from lipid-based delivery systems, siRNA-ligand conjugates, nucleotide-derivatized nanoparticles, polymer-based siRNA delivery systems, metal nanoparticles, mesoporous silica nanoparticles, or exosomes.

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

[0031] As certain embodiments of the present invention, the neutral lipid nanoparticles (LNPs) are composed of cholesterol, distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phospho-choline (DSPC)), polyethylene glycol lipids (poly(ethylene glycol) lipids (PEG-lipid)) and ionizable lipids.

[0032] Distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphatidylcholine) is a cylindrical liposome used in the synthesis of liposomes and is the lipid component in lipid nanoparticle (LNP) systems.

[0033] PEGylated lipids are widely used to provide PEGylation for lipid nanoparticles and other nanocarriers to improve their stability and plasma half-life.

[0034] Ionizable lipids are components of lipid nanoparticle (LNP) formulations. When formulated into LNPs, these lipids are designed to exhibit net neutrality at physiological pH.

[0035] The siRNA of the present invention can be delivered in a variety of ways, such as vector-mediated delivery, targeted delivery or direct injection of siRNA into tumor tissue. Vector-mediated delivery is the use of carriers such as nanoparticles to package siRNA into a complex to improve its cell membrane penetration ability and resistance to nuclease degradation. Vectors are mainly divided into viral vectors and non-viral vectors. Viral vectors are less efficient and will cause an immune response after entering the human body, and are now less used. Among non-viral vectors, nanoparticles and conjugate coupling are more commonly used. Targeted delivery is the use of antibodies, proteins or low molecular weight compounds against tumor cell surface molecules to guide siRNA into tumor cells, thereby improving the targeting of siRNA. Commonly used targeting substances include RGD peptides, anti-HER2 antibodies, etc.

[0036] As certain embodiments of the present invention, 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 invention also provides a pharmaceutical combination product, which comprises an effective amount of the siRNA and a second drug for simultaneous or sequential administration.

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

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

[0040] The present invention also provides the use of the siRNA or the pharmaceutical composition, wherein the use is in preparing drugs for inhibiting the CREPT gene and treating tumors.

[0041] According to certain embodiments of the present invention, the tumor is selected from colon cancer, fibroblastoma, or liver cancer.

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

[0043] As certain embodiments of the present invention, the subjects of application are mice and humans.

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

[0045] The three si-RNAs 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 both si-crept-2 and si-crept-3 can inhibit the growth of NIH3T3 fibroblast cells.

[0047] Liposome-delivered drugs of si-CREPT can treat liver cancer in mice and significantly reduce the tumor burden of liver cancer in mice. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0049] Figure 2 Shown is an in vivo validation of the effect of LNP-encapsulated siRNA targeting CREPT on the proliferation of in situ hepatocellular carcinoma. Figure 2 A is a schematic diagram of the drug delivery strategy of LNP-encapsulated siRNA in an in situ liver cancer model. Figure 2 B is to detect the effect of siRNA targeting CREPT on Hepa-1-6 tumor formation, Figure 2 C shows the fluorescence detection of the effect of siRNA treatment targeting CREPT on tumor growth. DETAILED DESCRIPTION

[0050] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0051] [Experimental Materials]

[0052] B16 mouse melanoma cells, Purnocell Life Science Co., Ltd., catalog number CL-0319

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

[0054] NIH3T3 mouse fibroblasts, Purnocell Life Science Co., Ltd., catalog number CL-0006

[0055] LLC mouse lung cancer cells, Purnosai Life Science Co., Ltd., catalog number CL-0140

[0056] Hepatocellular carcinoma cell line Hepa-1-6, Fenghui Biotechnology, catalog number CL-0451

[0057] Mice C57BL / 6 mice were purchased from Jicui Yaokang

[0058] Iodine tincture, Haishi Hainuo Group, catalog number 6935941200155

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

[0060] Neutral lipid nanoparticles (LNPs). For preparation methods, see the reference: 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. See page 4, right column, last paragraph, to page 5, right column, first paragraph.

[0061] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0062] Example 1

[0063] Three si-RNAs were designed based on the CDS sequence of mouse CREPT. Their nucleotide sequences are shown in SEQ ID Nos. 1 to 3 in Table 1. dTdT pendant nucleosides were added to the 3' overhangs of these three si-RNAs beyond the double-stranded complementary structure to generate si-Crept-1#, si-Crept-2#, and si-Crept-3#, respectively. Their nucleotide sequences are shown in SEQ ID Nos. 4 to 6 in Table 1. si-Crept-1# and si-Crept-2# have homologous fragments in the human genome. A blank control, si-mus-NC, was also designed, and its nucleotide sequence is shown in SEQ ID No. 7.

[0064] Table 1 siRNA sequences that inhibit CREPT

[0065] si-RNA1 GGCAGAUGAAGGCUGUAAAnn* SEQ ID No.1 si-RNA2 GCGGCGAGUUCAUACAGCAnn* SEQ ID No. 2 si-RNA3 CUCGGAUGCUGGUGGAAUAnn* SEQ ID No. 3 si-mouseCrept-1# GGCAGAUGAAGGCUGUAAATT SEQ ID No.4 si-mouseCrept-2# GCGGCGAGUUCAUACAGCATT SEQ ID No.5 si-mouseCrept-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 CREPT knockdown on cell growth in mouse cells, we selected four different mouse cell lines: B16 mouse melanoma cells, MC38 mouse colon cancer cells, NIH3T3 mouse fibroblasts, and LLC mouse lung cancer cells. The day before transfection, the four cell lines were plated in four 6 cm culture dishes. The next day, each cell line was transfected with the control si-NC, si-Crept-1#, si-Crept-2#, and si-Crept-3#. Cells were harvested 24, 48, 72, and 96 hours after transfection, lysed, and knockdown efficiency verified by western blot.

[0069] The results showed that at 24 and 48 hours after transfection, CREPT expression in the four cell types was significantly reduced compared to the control si-NC group, indicating that all three si-RNAs can effectively inhibit CREPT in mouse cells. However, since si-RNA knockdown is a transient effect, the results showed that CREPT expression began to rise again over time, reaching 72-96 hours.

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

[0071] Example 3

[0072] To test the effect of siRNA targeting CREPT on the proliferation of in situ liver cancer cells, we injected Hepa-1-6 liver cancer cells into the liver of mice to establish an in situ liver cancer model. The experimental process is as follows:

[0073] First, the mouse was anesthetized in an anesthesia chamber. The mouse's abdominal hair was shaved with an electric shaver and the abdomen was disinfected with an alcohol cotton ball. The mouse was then anesthetized using a breathing mask. The mouse was kept in anesthesia throughout the operation. The anesthesia status of the mouse was confirmed by touching the mouse to see if it reacted. Sterilized scissors were used to cut the skin and peritoneum in the middle of the mouse's abdomen, with a length of about 1 cm. After the liver lobe was visible, the injection site was determined. An insulin needle with Hepa-1-6 cells was inserted into the liver lobe parallel to the liver lobe and 60 μl (the number of Hepa-1-6 cells was approximately 1.6 x 10 6 ), after the injection, use a cotton ball to press the injection site for about 1 minute, then slowly rotate the needle out, and then use a 5-0 suture needle with thread to suture the peritoneum twice. After suturing the skin twice, use iodine to disinfect the suture site, and subcutaneously inject meloxicam to relieve the pain of the mice. Then transfer the mice to an electric blanket for about 10 minutes, during which time observe the heart rate and whether the eyes of the mice are white. After waking up, they can be returned to the original cage for breeding. Three days later, the mice are injected with meloxicam again to further reduce the pain caused by the surgery to the mice.

[0074] Starting from the next day, the mice with orthotopic tumors were divided into two groups. Figure 2 A shows the subsequent drug treatment experiment. A single injection of 100 μL of neutral lipid nanoparticle (LNP)-encapsulated si-NC or si-CREPT (si-mouseCrept-2#) (dose approximately 100 μg) was administered via the tail vein. Starting on day 10, injections were continued every four days for a total of seven injections (including one injection on the second day). After 34 days, the mice were dissected and photographed. Figure 2 A shows a schematic diagram of the drug delivery strategy of LNP-encapsulated siRNA in an in situ liver cancer model. Figure 2 B shows the effect of CREPT's siRNA inhibitor on Hepa-1-6 tumor formation in the liver. The results showed that compared with the tumor volume of the si-NC control group (i.e., the si-NC sequence in Table 1), the CREPT siRNA inhibitor significantly reduced the tumor burden of mouse liver cancer ( Figure 2 BC).

[0075] In summary, all three si-RNAs in this application effectively inhibit CREPT in mouse cells. si-mousecrept-2# inhibited the growth of mouse MC38 cells, while both si-mousecrept-2# and si-mousecrept-3# inhibited the growth of NIH3T3 fibroblast cells. Liposome-delivered si-CREPT (si-mouseCrept-2#) was shown to treat liver cancer in mice, significantly reducing tumor burden.

[0076] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An siRNA for inhibiting the CREPT gene, characterized in that: The nucleotide sequence of the positive strand of the siRNA is shown in any one of SEQ ID Nos. 1 to 3.

2. The siRNA according to claim 1, wherein The siRNA includes a 3' overhang extending beyond the double-stranded complementary structure; Preferably, the 3' overhang ends with two dangling nucleosides, and the dangling nucleosides are deoxynucleosides; Preferably, the pendant nucleoside is dTdT; Preferably, the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID No. 4 to 6.

3. The siRNA according to claim 1, wherein The siRNA comprises modified nucleotides; Modifications include phosphodiester bond modifications, nucleotide base modifications, siRNA end modifications, and / or amino acid modifications.

4. A pharmaceutical composition for inhibiting CREPT gene and treating tumors, characterized in that: The pharmaceutical composition comprises an effective amount of the siRNA according to any one of claims 1 to 3 and a delivery system.

5. The pharmaceutical composition according to claim 4, characterized in that The nucleotide sequence of the positive strand of the siRNA is shown in any one of SEQ ID No. 4 to 6.

6. The pharmaceutical composition according to claim 4, characterized in that The delivery system is selected from lipid-based delivery systems, siRNA-ligand conjugates, nucleotide-derivatized nanoparticles, polymer-based siRNA delivery systems, metal nanoparticles, mesoporous silica nanoparticles, or exosomes; Preferably, the lipid-based delivery system is a neutral lipid nanoparticle (LNP); Preferably, the neutral lipid nanoparticles (LNP) are composed of cholesterol, distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phospho-choline (DSPC)), polyethylene glycol lipids (poly(ethylene glycol) lipids (PEG-lipid)) and ionizable lipids.

7. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition further comprises a second drug and a pharmaceutically acceptable carrier; the second drug is a drug for treating tumors.

8. Use of the siRNA according to any one of claims 1 to 3, or the pharmaceutical composition according to any one of claims 4 to 7, characterized in that: The application is application in preparing drugs for inhibiting CREPT gene and treating tumors.

9. The use according to claim 8, characterized in that The tumor is selected from colon cancer, fibroblastoma, or liver cancer.

10. The use according to claims 8 to 9, characterized in that: The object of the application is a mammal; Preferably, the subjects of application are mice and humans.

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