SiRNA molecule of targeted human ANKRD10 gene and application of siRNA molecule

Abstract

The invention discloses a siRNA molecule of a targeted human ANKRD10 gene, the siRNA molecule is composed of a positive-sense strand and an antisense strand, and the specific sequence of the siRNA molecule is as follows: the positive-sense strand is 5 '-GGAGUUUGCUGUUGUAACANn-3', and the antisense strand is 5 '-UGUUACAACAUCCNn-3'; wherein N in the positive-sense strand and N in the antisense strand are the same or different and are respectively and independently cytosine C, uracil U, guanine G, adenine A, deoxycytosine dC, deoxyguanine dG, deoxyadenine dA or deoxythymine dT; n represents the number of N, and n = 0, 1 or 2. The invention further discloses application of the siRNA molecule of the targeted human ANKRD10 gene, and the siRNA molecule is applied to liver cancer treatment by efficiently inhibiting the expression level of ANKRD10.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an siRNA molecule targeting human ANKRD10 gene, and further relates to application of the siRNA molecule in preparation of a liver cancer treatment drug. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, and its morbidity and mortality rates are among the highest in the world, seriously threatening human health. Because of its insidious onset, rapid progression and resistance to traditional chemotherapy and radiotherapy, most patients have lost the opportunity for surgical resection or liver transplantation by the time of diagnosis, resulting in poor overall prognosis. In recent years, the application of molecular targeted drugs has brought new hope for HCC treatment. Drugs such as sorafenib and lenvatinib, represented by multi-target tyrosine kinase inhibitors (TKIs), can significantly delay tumor progression and improve the survival outcome of some patients by blocking angiogenesis signaling pathways and inhibiting tumor proliferation-related pathways. In addition, oral administration of targeted drugs and relatively controllable side effects provide a more feasible treatment for patients with advanced HCC. At present, the number of molecular targeted drugs for liver cancer treatment is very limited, and the key reason is the lack of effective molecular targets, and it is urgent to find new specific molecular targets.

[0003] ANKRD10 (ankyrin repeat domain-containing protein 10) is a protein containing ankyrin repeat domain. The human ANKRD10 gene is located in the 13q34 region of the chromosome, and there are two splice variants, encoding 420 and 220 amino acids, respectively. Ankyrin repeat domains are usually involved in protein-protein interactions and play an important role in various biological processes such as signal transduction, transcriptional regulation, and cell cycle regulation. Although the current research on ANKRD10 is relatively limited, studies have suggested that it may have a key function in cell proliferation, differentiation, and stress response. In recent years, with the advancement of high-throughput sequencing technology and multi-omics research of tumors, ANKRD10 has been found to be closely related to the occurrence and development of various malignant tumors. For example, in glioblastoma, ANKRD10 was identified as a methylation-driven gene, and its methylation status and expression pattern were associated with poor overall survival, suggesting that ANKRD10 may promote tumor progression and serve as a potential biomarker for distinguishing patient types. A recent study in bladder cancer found that the RBPMS (RNA-binding protein with multiple splicing) gene can inhibit the migration and invasion of bladder cancer cells by regulating the alternative splicing of ANKRD10. The mechanism is that RBPMS can inhibit the expression of ANKRD10-2 splice variant, thereby inhibiting the metastasis of bladder cancer cells by reducing the activity of the MYC pathway. Currently, it is not clear whether ANKRD10 expression is abnormal in hepatocellular carcinoma and whether it is involved in the occurrence and development of HCC.

[0004] Small interfering RNA (siRNA) is a double-stranded RNA molecule with a length of about 21-25 nucleotides, which can achieve precise regulation of gene expression through the RNA interference (RNAi) mechanism. Exogenous siRNA can specifically recognize and degrade target homologous messenger RNA (mRNA) after entering the cell, thereby inhibiting the synthesis of pathogenic proteins and achieving the purpose of treating diseases. It is an ideal molecular targeted therapy. Studies have shown that siRNA drugs have great potential in the treatment of tumors. As of June 2025, there have been six siRNA drugs on the market worldwide. Currently, although several anti-tumor siRNA therapies have entered the clinical trial stage, there is no siRNA drug for liver cancer treatment on the market. SUMMARY

[0005] The first object of the present application is to provide siRNA molecules targeting the human ANKRD10 gene, which can be used for liver cancer treatment by efficiently inhibiting the expression level of ANKRD10.

[0006] A second object of the present application is to provide use of the siRNA molecule as described above in the preparation of a medicament for inhibiting expression of ANKRD10.

[0007] A third object of the present application is to provide a medicament for treating liver cancer.

[0008] A fourth object of the present application is to provide a pharmaceutical preparation for treating liver cancer.

[0009] The first technical solution adopted by the present application is: a siRNA molecule targeting human ANKRD10 gene, composed of a sense strand and an antisense strand, and the specific sequences are as follows: the sense strand: 5'-GGAGUUUGCUGUUGUAACANn-3', the antisense strand: 5'-UGUUACAACAGCAAACUCCNn-3'; wherein N in the sense strand and the antisense strand is the same or different, and each independently is cytosine C, uracil U, guanine G, adenine A, deoxycytidine dC, deoxyguanosine dG, deoxyadenine dA or deoxythymidine dT; n represents the number of N, n = 0, 1 or 2.

[0010] The first technical solution adopted by the present application is further characterized in that: Further, the siRNA molecule targeting human ANKRD10 gene, n = 0; the sequence of the sense strand is shown as SEQ ID NO: 1, and the sequence of the antisense strand is shown as SEQ ID NO: 2.

[0011] Further, the siRNA molecule targeting human ANKRD10 gene, N is dT, n is 2, and the specific sequence of the siRNA molecule is as follows: the sense strand: 5'-GGAGUUUGCUGUUGUAACAdTdT-3', the antisense strand: 5'-UGUUACAACAGCAAACUCCdTdT-3'.

[0012] The second technical solution adopted by the present application is: use of the siRNA molecule in the preparation of a medicament for inhibiting expression of ANKRD10, and the medicament is a medicament for treating liver cancer.

[0013] The third technical solution adopted by the present application is: a medicament for treating liver cancer, comprising the siRNA molecule as described above as a pharmaceutically active ingredient.

[0014] The third technical solution adopted by the present application is further characterized in that: Further, the medicine is a composition comprising a therapeutically effective amount of the siRNA molecule as described above as an active ingredient, and further comprising a pharmaceutically acceptable carrier.

[0015] Further, the medicine is a composition comprising a therapeutically effective amount of the siRNA molecule as described above, and further comprising one or more other medicine ingredients for inhibiting the expression of the ANKRD10 gene.

[0016] Further, the medicine is a composition comprising a therapeutically effective amount of the siRNA molecule as described above, and further comprising one or more other medicine ingredients for treating liver cancer.

[0017] Further, the medicine is in the form of an injection or a gel.

[0018] The fourth technical solution adopted in the present application is: a medicine preparation for treating liver cancer, comprising a nucleic acid sequence modifier and a carrier, wherein the nucleic acid sequence modifier is a siRNA molecule as described above, which is obtained by one or more of the following modifications: ribose modification, base modification and phosphate skeleton modification of any nucleotide. The carrier is selected from a virus, a nanoparticle, cholesterol or a liposome.

[0019] The present application has the following beneficial effects: The siRNA molecule of the present application can significantly inhibit the expression level of ANKRD10, and can effectively inhibit the malignant progression of liver cancer cells by inhibiting the proliferation, clonogenicity, migration and invasion ability of liver cancer cells and inducing apoptosis of liver cancer cells, thereby providing a new target for the research and development of liver cancer treatment drugs. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 4 is a schematic diagram of the expression level of ANKRD10 in human normal liver cell lines and five liver cancer cell lines; Figure 2 Figure 5 is a schematic diagram of the interference efficiency of siRNA targeting ANKRD10 in liver cancer cells Bel-7402 and Hep3B; Figure 3 Figure 6 is a schematic diagram of the effect of siRNA targeting ANKRD10 on the proliferation ability of liver cancer cells Bel-7402; Figure 4 Figure 7 is a schematic diagram of the effect of siRNA targeting ANKRD10 on the clonogenicity of liver cancer cells Bel-7402; Figure 5 Figure 8 is a schematic diagram of the effect of siRNA targeting ANKRD10 on the apoptosis of liver cancer cells Bel-7402; Figure 6a Figure 9 is a schematic diagram of the effect of siRNA targeting ANKRD10 on the migration ability of liver cancer cells Bel-7402. Figure 6b The schematic diagram of the influence of siRNA targeting ANKRD10 on the invasion ability of liver cancer cells Bel-7402. DETAILED DESCRIPTION

[0021] The application will be described in detail below in combination with the drawings and specific embodiments.

[0022] The application provides an siRNA molecule capable of efficiently inhibiting the expression of ANKRD10 gene, which consists of a sense strand and an antisense strand of the following sequences: the sense strand: 5'-GGAGUUUGCUGUUGUAACANn-3', the antisense strand: 5'-UGUUACAACAGCAAACUCCNn-3'; wherein N in the sense strand and the antisense strand is the same or different, and each independently is cytosine C, uracil U, guanine G, adenine A, deoxycytidine dC, deoxyguanine dG, deoxyadenine dA or deoxythymidine dT; n represents the number of N, and n is 0, 1 or 2.

[0023] The expression levels of ANKRD10 in liver cancer cell lines / normal liver cells were detected by the method of fluorescent quantitative PCR, and it was found that ANKRD10 was highly expressed in a plurality of liver cancer cell lines compared with the normal liver cell line; secondly, siRNA specifically targeting ANKRD10 was designed and synthesized according to the sequence of ANKRD10 gene, and the liver cancer cells were transfected by the method of liposome mediation, and the efficiency of siRNA in inhibiting the expression of ANKRD10 was detected by the method of fluorescent quantitative PCR, and at the same time, the influence of siRNA on cell proliferation, apoptosis, migration and invasion was detected by the experimental methods of CCK-8, plate colony formation test, Annexin-V / PI staining flow cytometry analysis and Transwell, and the results showed that the siRNA molecule had a high silencing efficiency on ANKRD10, and could significantly inhibit the proliferation, migration and invasion ability of liver cancer cells, and promote the apoptosis of liver cancer cells, that is, significantly inhibit the progression of liver cancer cells.

[0024] The application also provides the use of the above-mentioned siRNA molecule or mixture of the two in the preparation of a medicine for inhibiting the expression of ANKRD10.

[0025] Optionally, the above-mentioned medicine is an antitumor medicine.

[0026] Preferably, the above-mentioned medicine is a liver cancer treatment medicine.

[0027] The siRNA molecule can be used as an effective component to inhibit the proliferation, migration and invasion of liver cancer cells, or to promote the apoptosis of liver cancer cells.

[0028] The present application also provides a medicament for treating liver cancer, comprising the above-mentioned siRNA molecule as a pharmaceutically active ingredient.

[0029] Optionally, the above-mentioned medicament is a pharmaceutical composition, in addition to comprising a therapeutically effective amount of the above-mentioned siRNA molecule as an active ingredient, further comprising a pharmaceutically acceptable carrier.

[0030] The present application also provides a pharmaceutical preparation for treating liver cancer, comprising a nucleic acid sequence modifier and a carrier, wherein the nucleic acid sequence modifier is obtained by modifying one or more of a ribose modification, a base modification and a phosphate backbone modification of any nucleotide of the above-mentioned siRNA molecule.

[0031] Preferably, the carrier is selected from a virus, a nanoparticle, a cholesterol or a liposome.

[0032] The technical solutions of the present application will be described in further detail below in conjunction with the accompanying drawings and examples: Example 1 In one embodiment, n = 0, i.e. Sense strand: 5'-GGAGUUUGCUGUUGUAACA-3' (SEQ ID NO: 1), Antisense strand: 5'-UGUUACAACAGCAAACUCC-3' (SEQ ID NO: 2); The siRNA molecule is the backbone sequence of the group of siRNA molecules.

[0033] Example 2 In another preferred embodiment, N is dT and n = 2, i.e. Sense strand: 5'-GGAGUUUGCUGUUGUAACAdTdT-3', Antisense strand: 5'-UGUUACAACAGCAAACUCCdTdT-3'.

[0034] Example 3 In one embodiment, the above-mentioned medicament is a pharmaceutical composition, in addition to comprising a therapeutically effective amount of the above-mentioned siRNA molecule, further comprising one or more other pharmaceutical ingredients for inhibiting the expression of ANKRD10.

[0035] Example 4 In another embodiment, the above-mentioned medicament is a pharmaceutical composition, in addition to comprising a therapeutically effective amount of the above-mentioned siRNA molecule, further comprising one or more other pharmaceutical ingredients for treating liver cancer.

[0036] Example 5 In one embodiment, the above-mentioned medicament is in the form of an injection or a gel. The injection is suitable for subcutaneous injection, intramuscular injection, intravenous injection or intravenous drip.

[0037] Example 6 Detection of expression of ANKRD10 in human hepatocellular carcinoma cell lines and normal hepatocyte strains 1. Materials Cells: Human hepatocellular carcinoma cell lines HepG2, Hep3B, Bel-7402, Bel-7404, MHCC97H and normal hepatocyte strain L-O2 were purchased from the Cell Resource Center of Shanghai Life Science Research Institute, Chinese Academy of Sciences.

[0038] Reagents: DMEM culture medium was purchased from Hyclone, USA, fetal bovine serum was purchased from Corning, USA, RNA extraction reagent RNAiso Plus, reverse transcription kit PrimeScript™ RT reagent Kit with gDNA Eraser and real-time fluorescent quantitative PCR TB Green® Premix Ex Taq™ II kit were all purchased from TAKARA, Japan. qRT-PCR specific primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0039] 2. Methods Human hepatocellular carcinoma cell lines and normal hepatocyte strains were cultured in DMEM culture medium containing 10% fetal bovine serum in a constant temperature incubator at 37°C and 5% CO2.

[0040] Total RNA was extracted according to the instruction of RNAiso Plus from TAKARA. The concentration and quality of RNA were evaluated by ultramicro spectrophotometer (UPT100 from Shanghai Unico Instrument Co., Ltd.) and agarose gel electrophoresis. The following operations were carried out in two steps using the PrimeScript™ RT reagent Kit with gDNA Eraser kit from TAKARA according to the instruction: 1) removing genomic DNA in total RNA: preparing reaction solution (containing total RNA 1 μg, 5 × gDNA Eraser Buffer 2 μL, gDNA Eraser 1 μL, ddH2O to 10 μL), incubating in PCR instrument at 42℃ for 2 min, and standing on ice for use; 2) reverse transcription reaction: further preparing reverse transcription reaction solution (containing reaction solution of step 1) 10 μL, 5 × Primescript Buffer 4 μL, Primescript RT Enzyme Mix I 1 μL, RT Primer Mix 1 μL, ddH2O 4 μL, total volume 20 μL) based on the reaction solution obtained in step 1), incubating in PCR instrument at 37℃ for 15 min, and incubating at 85℃ for 5 s to inactivate reverse transcriptase, to obtain cDNA.

[0041] TB Green Premix Ex Taq II (TliRNase H-) ®The Premix Ex Taq™ II kit was used with an ABI Q3 Real-time PCR instrument for real-time quantitative PCR detection. The qRT-PCR reaction system was as follows: 10 μL TB Green Premix Ex Taq II (2×), 1 μL forward primer (10 μM), 1 μL cDNA template, 0.4 μL ROX ReferenceDye II, 5.6 μL ddH2O, for a total volume of 20 μL. The PCR reaction conditions were as follows: 95 ℃ pre-denaturation for 30 s; 95 ℃ denaturation for 5 s, 60 ℃ annealing and extension for 30 s, for 40 cycles. Human Beta-actin gene was used as an internal control. The primers for the ANKRD10 gene were as follows: upstream primer 5'-AGTGTGGGAACAAATCGAAAGAG-3' (SEQ ID NO: 3) and downstream primer 5'-TGGCACGGCAGAATCCAAG-3' (SEQ ID NO: 4). The primer sequences for the internal reference gene Beta-actin are as follows: upstream primer 5'-TGGCACCCAGCACAATGAA-3' (SEQ ID NO: 5) and downstream primer 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3' (SEQ ID NO: 6). Using 2... -ΔΔCt The expression abundance of ANKRD10 mRNA was calculated using analytical methods.

[0042] 3. Results Figure 1 The expression levels of ANKRD10 in normal human hepatocyte cell lines and five hepatocellular carcinoma cell lines (HepG2, Hep3B, Bel-7402, Bel-7404, and MHCC97H) are shown below: P <0.01, the difference is statistically significant.

[0043] The results are as follows Figure 1 As shown, compared with the normal human hepatocyte cell line L-O2, the expression of ANKRD10 was significantly upregulated in all five human hepatocellular carcinoma cell lines, with statistically significant differences (all...). P The result showed a fold increase of <0.01%, suggesting that ANKRD10 may play a pro-cancer role in liver cancer. The results showed that ANKRD10 was upregulated most significantly in the Bel-7402 cell line, so Bel-7402 cell line was selected as the target cell for the knockdown experiment in subsequent functional experiments.

[0044] Example 7 Effects of siRNA inhibition of ANKRD10 expression on malignant phenotypes of liver cancer cells 1. Materials Cell: Human hepatocellular carcinoma cell line Bel-7402 was obtained from the Cell Resource Center of Shanghai Life Science Research Institute, Chinese Academy of Sciences.

[0045] Reagents: Transfection reagent Lipofectamine 3000 was purchased from Thermo Fisher Scientific, CCK-8 kit was purchased from Dojindo, Japan; Uncoated and pre-coated Matrigel Transwell chamber (for 24-well plate, PET membrane, 8 μm) were purchased from CORNING, USA; Crystal violet staining solution was purchased from Solabio Technology Co., Ltd., Beijing, China; Apoptosis kit was purchased from Dojindo, Japan.

[0046] 2. Method 2.1 Design and synthesis of siRNA sequence targeting ANKRD10 Two different splice variants of ANKRD10 cDNA sequences (NM_017664.4 and NM_001286721.3) were obtained from the NCBI database. The BLOCK-iT RNAi Designer software (Thermo Fisher Scientific) was used to design siRNA (siANKRD10) targeting ANKRD10 based on the common exon region of different splice variants. The specific sequence of siRNA was composed of the following sense and antisense strands: siRNA sense strand: 5'-GGAGUUUGCUGUUGUAACA-3' (SEQ ID NO: 1), siRNA antisense strand: 5'-UGUUACAACAGCAAACUCC-3' (SEQ ID NO: 2); The above siRNA targeting ANKRD10 was synthesized by Guangzhou Ribobio Biotech Co., Ltd.

[0047] The negative control sequence (siNC, no active target site on human genome) used in the experiment was purchased from Guangzhou Ribobio Biotech Co., Ltd.

[0048] 2.3 Cell transfection The human hepatocellular carcinoma cell line was inoculated in a 6-well plate and cultured at 37°C, 5% CO2 overnight to achieve a cell confluence of about 30% before transfection. The transfection was performed according to the instructions of Lipofectamine 3000 (Thermo Fisher Scientific). The specific steps were as follows: 1) 5 μL siANKRD10 / siNC (final concentration 50 nm) and 3.75 μL Lipofectamine 3000 transfection reagent were added into 125 μL serum-free DMEM culture solution respectively, mixed well, then the siRNA-containing DMEM culture solution was added into the DMEM culture solution containing Lipofectamine 3000 transfection reagent, mixed well, and incubated for 5 min to obtain the transfection solution; 2) Transfection: the transfection solution was added into the above 6-well plate (containing 2 mL culture solution) and incubated in a 37 °C, 5% CO2 incubator; 3) After 24 h of transfection, the cells were collected, and the interference effect of siRNA on ANKRD10 was detected by qRT-PCR, or cell proliferation, colony formation, apoptosis, migration and invasion experiments were performed.

[0049] 2.4 qRT-PCR detection of the interference effect of siRNA on ANKRD10 The transfected experimental group cells (liver cancer cells transfected with siANKRD10) and control group cells (liver cancer cells transfected with siNC) were collected, total RNA was extracted, reverse transcription and qRT-PCR were performed, and the method was the same as in Example 6.

[0050] 2.5 CCK-8 cell proliferation activity detection experiment The experimental group and control group cells transfected with siANKRD10 and siNC (50 nm) for 24 h were collected, resuspended in complete culture solution, counted, and seeded in a 96-well plate at a density of 3000 cells / well. 100 μL / well of PBS was added to the peripheral wells, and the plate was incubated in a 37 °C, 5% CO2 incubator. At the set time points (0 h, 24 h, 48 h, 72 h and 96 h), 10 μL of CCK-8 reagent was added to the test wells, and the plate was incubated for another 1 h 30 min. Then, the absorbance value of each well was measured at 450 nm wavelength using a multifunctional enzyme label instrument. Complete culture medium was added to the wells without cells as zero adjustment wells. 2.6 Plate colony formation test The experimental group and control group cells transfected with siANKRD10 and siNC (50 nm) for 24 h were collected, resuspended in complete culture solution, counted, and seeded in a 6-well plate at a density of 200 cells / well, with 3 replicate wells for each group. The plate was incubated in a 37 °C, 5% CO2 incubator for 7 days. After visible cell colonies were formed, the culture medium in each well was removed, the wells were washed with PBS twice, 1 mL of 4% paraformaldehyde was added to each well for fixation for 25 min, the paraformaldehyde was removed, the wells were washed with PBS twice, and 1 mL of 0.1% crystal violet was added to each well for staining. After 10 min, the crystal violet was removed, the 6-well plate was rinsed under tap water, and then dried, photographed and the colonies were counted.

[0051] 2.7 Apoptosis experiment Cells were seeded in 6-well plates, and after 48 h of transfection with siANKRD10 and siNC (50 nm), the experimental and control cells were collected. After washing and centrifugation with pre-cooled PBS, 195 μL of Annexin V-FITC binding solution was added to resuspend the cells, followed by the addition of 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI). After incubation at room temperature for 15 min in the dark, the apoptosis results were detected by flow cytometry within 1 h.

[0052] 2.8 Transwell experiment After 24 h of transfection with siANKRD10 and siNC (50 nm), the experimental and control cells were collected, resuspended in complete culture medium, counted, and 200 μl of cell suspension (containing about 1 × 10 4 ) was seeded in the upper chamber of a Transwell chamber with an 8-μm pore size without or with pre-coated Matrigel. Another 24-well plate was prepared by adding 500 μl of DMEM medium containing 20% serum to form the lower chamber. Then, the Transwell chamber was placed in the 24-well plate and incubated at 37 °C in a 5% CO2 incubator for 24-48 h. After incubation, the chamber was removed, washed with PBS for 3 times, and then the cells were fixed with methanol. After air-drying, the chamber was stained with 600 μL of 0.1% crystal violet staining solution for 10 min, followed by washing with PBS for 3 times. The chamber was inverted and air-dried. Using an inverted microscope, 5 random fields were selected for observation and photography in each chamber, and the number of cells that passed through the microporous membrane was counted.

[0053] 3 Results 3.1 Interference efficiency of siANKRD10 on ANKRD10 in liver cancer cells Figure 2 The interference efficiency of siRNA targeting ANKRD10 in liver cancer cells Bel-7402 and Hep3B was determined. P P < 0.01, the difference was statistically significant.

[0054] As shown in Figure 2 compared with the control group transfected with siNC, the expression of ANKRD10 in liver cancer cells transfected with siRNA specifically targeting ANKRD10 was significantly inhibited, and the inhibition efficiency in Bel-7402 and Hep3B cell lines was 75.6% and 70.5%, respectively.

[0055] 3.2 Effect of siANKRD10 on the proliferation and clonogenicity of liver cancer cells Figure 3 Effect of siRNA targeting ANKRD10 on proliferation of hepatoma cell Bel-7402, wherein: P <0.01, the difference was statistically significant.

[0056] As shown in Figure 3 , compared with the siNC control group, the proliferation activity of hepatoma cells in the siRNA transfection group was significantly inhibited ( P <0.01, the difference was statistically significant).

[0057] Figure 4 Effect of siRNA targeting ANKRD10 on clonogenicity of hepatoma cell Bel-7402, wherein: P <0.01, the difference was statistically significant.

[0058] As shown in Figure 4 , the results of the plate colony formation test were consistent with the results of the CCK-8 test, and the plate colony formation ability of hepatoma cells in the siRNA transfection group was significantly weakened ( P <0.01, the difference was statistically significant). In summary, transfection of siRNA targeting ANKRD10 can significantly inhibit the proliferation of hepatoma cells.

[0059] 3.3 Effect of siANKRD10 on apoptosis of hepatoma cells Figure 5 Effect of siRNA targeting ANKRD10 on apoptosis of hepatoma cell Bel-7402, wherein: P <0.05, the difference was statistically significant.

[0060] As shown in Figure 5 , compared with the control group transfected with siNC, the apoptosis rate of hepatoma cells in the siRNA transfection group was significantly increased ( P <0.05, the difference was statistically significant), indicating that transfection of siRNA targeting ANKRD10 can significantly induce apoptosis of hepatoma cells.

[0061] 3.4 Effect of siANKRD10 on migration and invasion ability of hepatoma cells As shown in Figure 6a and Figure 6b , compared with the control group transfected with siNC, the number of migrated and invaded cells in the siRNA transfection group was significantly reduced ( P both <0.01, the difference was statistically significant), indicating that transfection of siRNA targeting ANKRD10 can significantly inhibit the migration and invasion ability of hepatoma cells.

[0062] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

[0063] sequence list sequence list <110> Xi'an Peihua University <120> siRNA molecules targeting the human ANKRD10 gene and their applications <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> RNA <213> Artificial sequence <400> 1 ggaguuugcu guuguaaca 19 <210> 2 <211> 19 <212> RNA <213> Artificial sequence <400> 2 uguuacaaca gcaaacucc 19 <210> 3 <211> twenty three <212> DNA <213> Artificial sequence <400> 3 agtgtgggaa caaatcgaaa gag 23 <210> 4 <211> 19 <212> DNA <213> Artificial sequence <400> 4 tggcacggca gaatccaag 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <400>5 tggcacccag cacaatgaa 19 <210>6 <211> 25 <212> DNA <213> Artificial Sequence <400>6 ctaagtcata gtccgcctag aagca 25

Claims

1. A siRNA molecule targeting the human ANKRD10 gene, characterized in that, It consists of a justice chain and an antithesis chain, with the specific sequence as follows: Chain of Justice: 5'-GGAGUUUGCUGUUGUAACANn-3' Antonym chain: 5'-UGUUACAACAGCAAACUCCNn-3'; In this chain, N in the sense chain and the antisense chain may be the same or different, and each is independently cytosine C, uracil U, guanine G, adenine A, deoxycytosine dC, deoxyguanine dG, deoxyadenine dA, or deoxythymidine dT; n represents the number of N, n=0, 1, or 2.

2. The siRNA molecule targeting the human ANKRD10 gene according to claim 1, characterized in that, Where n=0; The sense chain sequence is shown in SEQ ID NO: 1, and the antisense chain sequence is shown in SEQ ID NO:

2.

3. The siRNA molecule targeting the human ANKRD10 gene according to claim 1, characterized in that, Where N is dT, n is 2, and the specific sequence of the siRNA molecule is as follows: Chain of Justice: 5'-GGAGUUUGCUGUUGUAACAdTdT-3' Antonym chain: 5'-UGUUACAACAGCAAACUCCdTdT-3'.

4. The use of the siRNA molecule according to any one of claims 1-3 in the preparation of a medicament for inhibiting ANKRD10 expression, wherein the medicament is a drug for treating liver cancer.

5. A drug for treating liver cancer, characterized in that, Includes as claimed in claim 1 The siRNA molecule described in any one of the three options is used as the active pharmaceutical ingredient.

6. The drug according to claim 5, characterized in that, The drug is a composition comprising a therapeutically effective amount as claimed in claim 1. The siRNA molecule described in any one of the 3 is an active ingredient and also includes a pharmaceutically acceptable carrier.

7. The drug according to claim 5, characterized in that, The drug is a composition comprising a therapeutically effective amount as claimed in claim 1. The siRNA molecule described in any one of the three methods further includes one or more other pharmaceutical components that inhibit the expression of the ANKRD10 gene.

8. The drug according to claim 5, characterized in that, The drug is a composition comprising a therapeutically effective amount as claimed in claim 1. The siRNA molecule described in any one of the three methods further includes one or more other pharmaceutical ingredients for treating liver cancer.

9. The drug according to claim 5, characterized in that, The drug dosage form is an injection or a gel.

10. A pharmaceutical preparation for treating liver cancer, characterized in that, It includes a nucleic acid sequence modifier and a vector, wherein the nucleic acid sequence modifier is as described in claim 1. The siRNA molecule described in any one of the following 3 is obtained by performing one or more modifications of any nucleotide ribose modification, base modification, and phosphate backbone modification; The carrier is selected from viruses, nanoparticles, cholesterol, or liposomes.

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