Application of siRNA targeting RNF24 in preparation of medicine for treating liver cancer

Abstract

The application relates to the technical field of gene targeting regulation, and provides application of siRNA targeting RNF24 in preparation of a liver cancer treatment drug. The sense strand and the antisense strand of the siRNA targeting RNF24 have nucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 10, the siRNA can treat liver cancer by inhibiting the expression of an RNF24 gene and reducing the synthesis of a protein coded by the RNF24 gene, and can reduce the proliferation and migration abilities of liver cancer cells. The application solves the problems that existing liver cancer molecular targeted treatment drugs are limited, sequence silencing efficiency and specificity are difficult to be considered in siRNA targeted treatment, stability is insufficient, and delivery efficiency is low.

Description

Technical Field

[0001] This invention belongs to the field of gene targeting and regulation technology, and in particular relates to the application of siRNA targeting RNF24 in the preparation of drugs for treating liver cancer. Background Technology

[0002] Liver cancer is a malignant tumor originating in the liver. Because early-stage liver cancer often lacks typical clinical symptoms, by the time symptoms and signs appear, the disease has usually progressed to the middle or late stages, placing a heavy burden on families and society and posing a serious challenge to modern medicine and society. Drugs for molecularly targeted therapy of liver cancer are very limited; therefore, in-depth research into the molecular mechanisms of liver cancer to find potential targets for drug development is of great significance.

[0003] Small interfering RNA (siRNA) is a double-stranded RNA molecule that achieves post-transcriptional gene silencing by binding complementary to target mRNA through the RNA interference pathway and mediating its degradation. It is an important tool for gene function research and targeted therapy. However, current siRNA design technologies still face challenges: not only must the silencing efficiency and specificity of the sequence design be considered, but the stability issues of siRNA itself being easily degraded by nucleases, the low efficiency of active cellular uptake, and the need to avoid triggering the body's innate immune activation and causing side effects, must also be addressed. Therefore, exploring the pathogenesis of liver cancer and optimizing the sequence design and chemical modification of siRNA to target the pathogenic targets of liver cancer is urgently needed. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides the application of siRNA targeting RNF24 in the preparation of drugs for treating liver cancer, with the aim of solving the problems mentioned in the background art.

[0005] This invention provides the application of siRNA targeting RNF24 in the preparation of drugs for treating liver cancer, wherein the siRNA is selected from siRNA1, siRNA2, siRNA3, siRNA4 or siRNA5; The siRNA1 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.2; The siRNA2 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.4; The siRNA3 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.5 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.6; The siRNA4 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.7 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.8; The siRNA5 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.9 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.10.

[0006] Furthermore, the drug treats liver cancer by inhibiting the expression of the RNF24 gene and reducing the synthesis of the protein encoded by the RNF24 gene.

[0007] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0008] Furthermore, the pharmaceutically acceptable carriers include lipid carriers, viral carriers, or polymer carriers.

[0009] Furthermore, the drug is formulated into a clinically acceptable dosage form.

[0010] Furthermore, the clinically acceptable dosage forms include injections, aerosols, or patches.

[0011] Furthermore, the drug reduces the proliferation and migration ability of liver cancer cells.

[0012] The present invention has the following technical effects: (1) The sense and antisense strands of the siRNA targeting RNF24 have nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.10. By inhibiting the expression of the RNF24 gene and reducing the synthesis of the protein encoded by the RNF24 gene, it can treat liver cancer and reduce the proliferation and migration ability of liver cancer cells.

[0013] (2) To solve the problems of limited existing molecular targeted therapy drugs for liver cancer, difficulty in achieving both sequence silencing efficiency and specificity, insufficient stability and low delivery efficiency in siRNA targeted therapy. Attached Figure Description

[0014] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1 This is a graph showing the detection results of RT-PCR and Western blot in Example 1 of the present invention, wherein: Figure 1 Figure A shows the analysis results of RNF24 mRNA expression levels in each group of liver cancer cells. ** indicates p<0.01, and *** indicates p<0.001. Figure 1 Figure B in the graph shows the analysis results of the protein expression level of RNF24 in each group of liver cancer cells.

[0015] Figure 2 This is a graph showing the detection results of the CCK8 detection method in Embodiment 2 of the present invention. *** indicates p < 0.001, where: Figure 2 In the figure, A represents the cell viability statistics of liver cancer cells RNF24 after treatment with siRNA1; Figure 2 In the figure, B represents the cell viability statistics of liver cancer cells RNF24 after treatment with siRNA2. Figure 2 In the figure, C represents the cell viability statistics of liver cancer cells RNF24 after treatment with siRNA3; Figure 2 In the figure, D represents the cell viability statistics of liver cancer cells RNF24 after treatment with siRNA4; Figure 2 E in the figure represents the cell viability statistics of liver cancer cells after treatment with RNF24 siRNA5.

[0016] Figure 3 This is a diagram showing the results of a clone formation experiment in Example 3 of the present invention, wherein: Figure 3 In the diagram, A represents a microscopic image of the clonogenic assay of liver cancer cells in each group. Figure 3 B in the figure is a quantitative statistical graph of the clonogenic assay of liver cancer cells in each group, and *** indicates p<0.001.

[0017] Figure 4 This is a diagram showing the Transwell experimental results of Embodiment 4 of the present invention, wherein: Figure 4 In the image, A represents a microscopic image of each group of liver cancer cells obtained through the Transwell experiment. Figure 4 B in the figure is a quantitative statistical graph of liver cancer cells in each group using the Transwell assay. *** indicates p<0.001.

[0018] Figure 5 This is a scratch test result diagram of Embodiment 5 of the present invention, wherein: Figure 5 In the image, A represents the microscopic images of the scratch test on liver cancer cells in each group. Figure 5 B in the figure is a graph showing the quantitative statistics (healing rate) of the scratch test for liver cancer cells in each group. ** indicates p<0.01, and *** indicates p<0.001. Detailed Implementation

[0019] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0021] This invention provides the application of siRNA targeting RNF24 in the preparation of drugs for treating liver cancer, wherein the siRNA is selected from siRNA1, siRNA2, siRNA3, siRNA4 or siRNA5; siRNA1 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.2; siRNA2 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.4; siRNA3 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.5 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.6; siRNA4 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.7 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.8; siRNA5 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.9 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.10.

[0022] In some embodiments, the drug treats liver cancer by inhibiting the expression of the RNF24 gene and reducing the synthesis of the protein encoded by the RNF24 gene.

[0023] In some embodiments, the medicament may also include a pharmaceutically acceptable carrier.

[0024] In some embodiments, pharmaceutically acceptable carriers include lipid carriers, viral carriers, or polymer carriers.

[0025] In some embodiments, the drug is formulated into a clinically acceptable dosage form.

[0026] In some embodiments, clinically acceptable dosage forms include injections, aerosols, or patches.

[0027] In some embodiments, the drug reduces the proliferation and migration ability of liver cancer cells.

[0028] Experimental materials: (1) Cell culture: The liver cancer cells were MHCC97-H cell line. The MHCC97-H cell line was cultured in high glucose DMEM medium containing 1% penicillin / streptomycin and 10% FBS, and then in a saturated humidity incubator at 37°C and 5% CO2.

[0029] (2) Design and screening of siRNA targeting RNF24 in liver cancer cells, nucleotide sequence: RNF24 siRNA1, positive strand: GGUACUGCUAUAUUUGUCUTT (SEQ ID NO.1), negative strand: AGACAAAAUAUAGCAGUACCTT (SEQ ID NO.2). RNF24 siRNA2, positive strand: CACAGAAAGUGCCUUAUUATT (SEQ ID NO.3), negative strand: UAUAAGGCACUUUCUGUGTT (SEQ ID NO.4); RNF24 siRNA3, positive strand: GGGCAGAGAACAUUGUAUATT (SEQ ID NO.5), negative strand: UUACAAUGUUCUCUGCCCTT (SEQ ID NO.6). RNF24 siRNA4, positive strand: GCACAUGCCAGUUCUACATT (SEQ ID NO.7), negative strand: UGUAGAACUGGCAUGUUGCTT (SEQ ID NO.8). RNF24 siRNA5, positive strand: CUGCCUCUCAACAUAUAUATT (SEQ ID NO.9), negative strand: UAUAUAUGUUGAGAGGCAGTT (SEQ ID NO.10).

[0030] Example 1: Hepatocellular carcinoma cells were divided into four groups: control group (using a universal Scramble sequence); siRNF24-1 group (using RNF24 siRNA1 to knock down RNF24); siRNF24-2 group (using RNF24 siRNA2 to knock down RNF24); siRNF24-3 group (using RNF24 siRNA3 to knock down RNF24); siRNF24-4 group (using RNF24 siRNA4 to knock down RNF24); and siRNF24-5 group (using RNF24 siRNA5 to knock down RNF24). The mRNA expression level of RNF24 in each group of hepatocellular carcinoma cells was analyzed by RT-PCR (reverse transcription polymerase chain reaction); the protein expression level of RNF24 was analyzed by Western blotting.

[0031] The results of RT-PCR and Western blot detection are as follows: Figure 1As shown, the results indicate that RNF24 siRNAs (RNF24siRNA1, RNF24 siRNA2, RNF24 siRNA3, RNF24 siRNA4, and RNF24 siRNA5) significantly knocked down RNF24 mRNA. Figure 1 A in the protein (A) and protein (B) Figure 1 The expression level of B in the text.

[0032] Example 2: Hepatocellular carcinoma cells were divided into four groups: control group (using a universal Scramble sequence); siRNF24-1 group (using RNF24 siRNA1 to knock down RNF24); siRNF24-2 group (using RNF24 siRNA2 to knock down RNF24); siRNF24-3 group (using RNF24 siRNA3 to knock down RNF24); siRNF24-4 group (using RNF24 siRNA4 to knock down RNF24); and siRNF24-5 group (using RNF24 siRNA5 to knock down RNF24). To analyze the effect of RNF24 siRNA on the short-term proliferation ability of hepatocellular carcinoma cells, each group of hepatocellular carcinoma cells was treated with RNF24 siRNA for 48 hours, and the viability of hepatocellular carcinoma cells was detected by CCK8 assay.

[0033] The results of the CCK8 test method are as follows: Figure 2 As shown, the results indicated that, compared with the control group, RNF24 siRNA (RNF24siRNA1, RNF24 siRNA2, RNF24 siRNA3, RNF24 siRNA4 and RNF24 siRNA5) significantly reduced the short-term proliferation ability of liver cancer cells.

[0034] Example 3: Hepatocellular carcinoma cells were divided into four groups: control group (using a universal Scramble sequence); siRNF24-1 group (using RNF24 siRNA1 to knock down RNF24); siRNF24-2 group (using RNF24 siRNA2 to knock down RNF24); siRNF24-3 group (using RNF24 siRNA3 to knock down RNF24); siRNF24-4 group (using RNF24 siRNA4 to knock down RNF24); and siRNF24-5 group (using RNF24 siRNA5 to knock down RNF24). To analyze the effect of RNF24 siRNA on the long-term proliferation ability of hepatocellular carcinoma cells, a colony formation assay was performed in each group. Specifically, after transfecting hepatocellular carcinoma cells with RNF24 siRNA for 48 hours, 1000 cells per well were evenly seeded in 6-well plates and cultured in a 37°C incubator containing 5% CO2 for 14 days. After fixation and staining, the hepatocellular carcinoma cells were photographed and statistically analyzed.

[0035] Cloning experiment results as follows Figure 3 As shown, compared with the control group, RNF24 siRNA (RNF24 siRNA1, RNF24 siRNA2, RNF24 siRNA3, RNF24 siRNA4 and RNF24 siRNA5) significantly reduced the long-term proliferation ability of liver cancer cells.

[0036] Example 4: Hepatocellular carcinoma cells were divided into four groups: control group (using a universal Scramble sequence); siRNF24-1 group (using RNF24 siRNA1 to knock down RNF24); siRNF24-2 group (using RNF24 siRNA2 to knock down RNF24); siRNF24-3 group (using RNF24 siRNA3 to knock down RNF24); siRNF24-4 group (using RNF24 siRNA4 to knock down RNF24); and siRNF24-5 group (using RNF24 siRNA5 to knock down RNF24). To analyze the effect of RNF24 siRNA on the migration ability of hepatocellular carcinoma cells, Transwell assays were performed on each group.

[0037] Transwell experimental results are as follows: Figure 4 As shown, the results indicated that, compared with the control group, RNF24 siRNA (RNF24siRNA1, RNF24 siRNA2, RNF24 siRNA3, RNF24 siRNA4 and RNF24 siRNA5) significantly reduced the migration ability of liver cancer cells.

[0038] Example 5: Hepatocellular carcinoma cells were divided into four groups: control group (using a universal Scramble sequence); siRNF24-1 group (using RNF24 siRNA1 to knock down RNF24); siRNF24-2 group (using RNF24 siRNA2 to knock down RNF24); siRNF24-3 group (using RNF24 siRNA3 to knock down RNF24); siRNF24-4 group (using RNF24 siRNA4 to knock down RNF24); and siRNF24-5 group (using RNF24 siRNA5 to knock down RNF24). To analyze the effect of RNF24 siRNA on the migration ability of hepatocellular carcinoma cells, scratch assays were performed on each group of hepatocellular carcinoma cells. Specifically, after RNF24 siRNA transfection into hepatocellular carcinoma cells for 48 hours, scratch assays were performed, and images were taken and the migration area was counted at 0 hours and 48 hours.

[0039] The results of the scratch test are as follows Figure 5 As shown, the results indicated that, compared with the control group, RNF24 siRNA (RNF24 siRNA1, RNF24 siRNA2, RNF24 siRNA3, RNF24 siRNA4 and RNF24 siRNA5) significantly reduced the migration ability of liver cancer cells.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of siRNA targeting RNF24 in the preparation of drugs for treating liver cancer, characterized by: The siRNA is selected from siRNA1, siRNA2, siRNA3, siRNA4 or siRNA5; The siRNA1 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.2; The siRNA2 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.4; The siRNA3 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.5 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.6; The siRNA4 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.7 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.8; The siRNA5 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO.9 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.

10.

2. The application as described in claim 1, characterized in that: The drug treats liver cancer by inhibiting the expression of the RNF24 gene and reducing the synthesis of the protein encoded by the RNF24 gene.

3. The application as described in claim 2, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

4. The application as described in claim 3, characterized in that: Pharmaceutically acceptable carriers include lipid carriers, viral carriers, or polymer carriers.

5. The application as described in claim 2, characterized in that: The drug is formulated into a clinically acceptable dosage form.

6. The application as described in claim 5, characterized in that: Clinically acceptable dosage forms include injections, aerosols, or patches.

7. The application as described in claim 2, characterized in that: The drug reduces the proliferation and migration ability of liver cancer cells.

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