SiRNA molecule of targeted human SLC25A17 gene and application of siRNA molecule

Abstract

The invention discloses a siRNA molecule targeting a human SLC25A17 gene, the siRNA molecule comprises a positive-sense strand and an antisense strand, the specific sequence is as follows: the positive-sense strand: 5 '-CCUUGGAUGUGUUCAUCAUNn-3', the antisense strand: 5 '-AUGAUGAACACAUCAAGGNn-3', 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 SLC25A17 gene, and the siRNA molecule is applied to liver cancer treatment by efficiently inhibiting the expression level of SLC25A17.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to siRNA molecules targeting the human SLC25A17 gene. This invention also relates to the application of the above-mentioned siRNA molecules in the preparation of drugs for treating liver cancer. Background Technology

[0002] Liver cancer, one of the most common malignant tumors worldwide, ranks among the top in both incidence and mortality among malignant tumors, seriously threatening human health. Most patients are diagnosed at an advanced stage, losing the opportunity for surgery, and traditional treatments have limited effectiveness. The emergence of molecularly targeted drugs has significantly improved the prognosis of patients with advanced liver cancer and has gradually become an important means of clinical oncology treatment. The advantage of molecularly targeted drugs lies in their high specificity, acting by precisely intervening in key signaling pathways of tumor growth and spread. For example, sorafenib, the first approved multi-kinase inhibitor for advanced liver cancer, inhibits vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR), thereby blocking tumor angiogenesis. However, due to the high heterogeneity of tumors and the diversity of molecular targets, single-target drugs cannot cover all patient populations, urgently requiring the search for new specific molecular targets.

[0003] The solute carrier (SLC) superfamily, the largest class of membrane transport proteins in cells, mediates the transmembrane transport of various solutes (carbohydrates, amino acids, fatty acids, vitamins, nucleic acids, ions, minerals, drugs, etc.) between the cell and the external environment or within the cell, thereby participating in important physiological activities such as intercellular transport, energy transfer, nutrient metabolism, and signal transduction. The largest family within this superfamily is the SLC25 family, also known as the mitochondrial carrier family (MCF), comprising 53 members named SLC25A1–SLC25A53. These members mediate the transmembrane transport of substrates (such as nucleotides, coenzyme A, carboxyl-containing metabolites, amino acids, etc.) between the mitochondrial intermembrane / cytoplasm and the mitochondrial matrix. Recent studies have found that the SLC25 family plays a crucial role in tumorigenesis and development by regulating metabolic reprogramming.

[0004] SLC25A17 (mitochondrial solute carrier family 25, member 17) is a member of the SLC25 family located on the peroxisome membrane. The human SLC25A17 gene is located on chromosome 22q13.2, with a full-length transcript of 2226 bp encoding 307 amino acids. The main physiological function of SLC25A17 is to facilitate the exchange of free coenzyme A (CoA), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD) from the cytoplasm via a reverse exchange mechanism. + SLC25A17 is transported to the peroxisome matrix. Studies have found that upregulated expression of SLC25A17 in head and neck squamous cell carcinoma is closely related to poor patient prognosis, the level of immune infiltration in the tumor microenvironment, and immunotherapy response. A recent study found that SLC25A17 expression was also significantly increased in breast cancer and associated with poor prognosis; SLC25A17 can promote tumor progression by regulating reactive oxygen species (ROS) generation and autophagy. Therefore, SLC25A17 is a potential target for cancer therapy. However, whether SLC25A17 expression is abnormal in liver cancer and whether it participates in the development and progression of liver cancer remains unclear.

[0005] Small interfering RNA (siRNA) is a double-stranded RNA molecule approximately 21-25 nucleotides in length that precisely regulates gene expression through RNA interference (RNAi) mechanisms. Once exogenous siRNA enters the cell, it can specifically recognize and degrade target homologous messenger RNA (mRNA), thereby inhibiting the synthesis of pathogenic proteins and ultimately achieving the goal 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, six siRNA drugs have been marketed globally. Currently, although several anti-tumor siRNA therapies have entered clinical trials, no siRNA drugs for the treatment of liver cancer have been marketed. Summary of the Invention

[0006] The first objective of this invention is to provide an siRNA molecule that targets the human SLC25A17 gene, which can be used to treat liver cancer by efficiently inhibiting the expression level of SLC25A17.

[0007] A second objective of this invention is to provide the use of the above-mentioned siRNA molecule in the preparation of drugs for inhibiting SLC25A17 expression.

[0008] A third objective of this invention is to provide a drug for treating liver cancer.

[0009] A fourth objective of this invention is to provide a pharmaceutical formulation for treating liver cancer.

[0010] The first technical solution adopted in this invention is: an siRNA molecule targeting the human SLC25A17 gene, composed of a sense strand and an antisense strand, with the specific sequence as follows: Chain of Justice: 5'-CCUUGGAUGUGUUCAUCAUNn-3' Antonym chain: 5'-AUGAUGAACACAUCCAAGGNn-3' In this chain, the N in the sense chain and the antisense chain are 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.

[0011] The first technical solution adopted in this invention is further characterized by: Furthermore, siRNA molecules targeting the human SLC25A17 gene, 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.

[0012] Furthermore, the siRNA molecule targeting the human SLC25A17 gene, where N is dT and n is 2, has the following specific sequence: Chain of Justice: 5'-CCUUGGAUGUGUUCAUCAUdTdT-3' Antonym chain: 5'-AUGAUGAACACAUCCAAGGdTdT-3'.

[0013] The second technical solution adopted in this invention is the application of siRNA molecules targeting the human SLC25A17 gene in the preparation of a drug for inhibiting SLC25A17 expression, which is a drug for treating liver cancer.

[0014] The third technical solution adopted in this invention is: a drug for treating liver cancer, comprising siRNA molecules as described above as the active pharmaceutical ingredient.

[0015] The third technical solution adopted in this invention is further characterized by: Furthermore, the drug is a composition comprising a therapeutically effective amount of siRNA molecules as described above as an active ingredient, and also comprising a pharmaceutically acceptable carrier.

[0016] Furthermore, the drug is a composition comprising a therapeutically effective amount of the siRNA molecule as described above, and also comprising one or more other pharmaceutical ingredients that inhibit SLC25A17 gene expression.

[0017] Furthermore, the drug is a composition comprising a therapeutically effective amount of siRNA molecules as described above, and also comprising one or more other drug components for treating liver cancer.

[0018] Furthermore, the drug dosage form is either an injection or a gel.

[0019] The fourth technical solution adopted in this invention is: 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 the siRNA molecule as described above with one or more of the following modifications: ribose modification, base modification, and phosphate backbone modification of any nucleotide; The vector is selected from viruses, nanoparticles, cholesterol, or liposomes.

[0020] The beneficial effects of this invention are: The siRNA molecule of this invention can significantly inhibit the expression level of SLC25A17, and can effectively inhibit the malignant progression of liver cancer cells by inhibiting the proliferation, colony formation, migration and invasion of liver cancer cells and inducing apoptosis of liver cancer cells, thus providing a new target for the development of liver cancer therapeutic drugs. Attached Figure Description

[0021] Figure 1 This diagram illustrates the expression levels of SLC25A17 in normal human hepatocytes and five hepatocellular carcinoma cell lines. Figure 2 This is a schematic diagram illustrating the interference efficiency of siRNA targeting SLC25A17 in hepatocellular carcinoma cells Bel-7402 and HepG2. Figure 3 A schematic diagram illustrating the effect of siRNA targeting SLC25A17 on the proliferation ability of liver cancer cells; Figure 4 A schematic diagram illustrating the effect of siRNA targeting SLC25A17 on the clonogenic ability of liver cancer cells; Figure 5 A schematic diagram illustrating the effect of siRNA targeting SLC25A17 on apoptosis in liver cancer cells; Figure 6a A schematic diagram illustrating the effect of siRNA targeting SLC25A17 on the migration ability of liver cancer cells; Figure 6b This is a schematic diagram illustrating the effect of siRNA targeting SLC25A17 on the invasive ability of liver cancer cells. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0023] This invention provides an siRNA molecule capable of efficiently inhibiting SLC25A17 gene expression, which consists of a sense strand and an antisense strand with the following sequence: Chain of Justice: 5'-CCUUGGAUGUGUUCAUCAUNn-3' Antonym chain: 5'-AUGAUGAACACAUCCAAGGNn-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, which is 0, 1, or 2.

[0024] The expression level of SLC25A17 in hepatocellular carcinoma (HCC) cells and normal hepatocytes was detected using quantitative real-time PCR (qPCR). The results showed that SLC25A17 was highly expressed in various HCC cell lines compared to normal hepatocytes. Furthermore, siRNA specifically targeting SLC25A17 was designed and synthesized based on the SLC25A17 gene sequence. After transfection of HCC cells using liposome-mediated transfection, the efficiency of siRNA in inhibiting SLC25A17 expression was detected by qPCR. Simultaneously, the effects of siRNA on cell proliferation, apoptosis, migration, and invasion were investigated using CCK-8 assays, colony formation assays, Annexin-V / PI staining flow cytometry, and Transwell assays. The results indicated that siRNA had a high silencing efficiency for SLC25A17, significantly inhibiting the proliferation, migration, and invasion of HCC cells, while simultaneously promoting apoptosis, thus significantly inhibiting HCC cell progression.

[0025] The present invention also provides the use of the above-mentioned siRNA molecules or mixtures thereof in the preparation of a medicament for inhibiting SLC25A17 expression.

[0026] Optionally, the above-mentioned drugs are anti-tumor drugs.

[0027] Preferably, the above-mentioned drug is a drug for treating liver cancer.

[0028] siRNA molecules can be used as an effective component to inhibit the proliferation, migration, and invasion of liver cancer cells, or to promote apoptosis of liver cancer cells.

[0029] The present invention also provides a medicament for treating liver cancer, which comprises the above-mentioned siRNA molecule as a pharmaceutical active ingredient.

[0030] Optionally, the above-mentioned drug is a pharmaceutical composition that, in addition to containing a therapeutically effective amount of the above-mentioned siRNA molecule as an active ingredient, also contains a pharmaceutically acceptable carrier.

[0031] The present invention also provides a pharmaceutical formulation for treating liver cancer, comprising a nucleic acid sequence modifier and a vector, wherein the nucleic acid sequence modifier is obtained by modifying the above-mentioned siRNA molecule by one or more of the following modifications: ribose modification, base modification, and phosphate backbone modification of any nucleotide.

[0032] Preferably, the carrier is selected from viruses, nanoparticles, cholesterol, or liposomes.

[0033] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments: Example 1 In one implementation, n=0, that is Chain of Justice: 5'-CCUUGGAUGUGUUCAUCAU-3' (SEQ ID NO: 1) Antisense chain: 5'-AUGAUGAACACAUCCAAGG-3' (SEQ ID NO: 2); This siRNA molecule is the backbone sequence of this group of siRNA molecules.

[0034] Example 2 In another preferred embodiment, N is dT, n=2, that is... Chain of Justice: 5'-CCUUGGAUGUGUUCAUCAUdTdT-3', Antonym chain: 5'-AUGAUGAACACAUCCAAGGdTdT-3'.

[0035] Example 3 In one embodiment, the above-mentioned drug is a pharmaceutical composition that, in addition to containing a therapeutically effective amount of the above-mentioned siRNA molecule, also contains one or more other pharmaceutical components for inhibiting SLC25A17 expression.

[0036] Example 4 In another embodiment, the above-mentioned drug is a pharmaceutical composition that, in addition to containing a therapeutically effective amount of the above-mentioned siRNA molecule, also contains one or more other pharmaceutical ingredients for treating liver cancer.

[0037] Example 5 In one embodiment, the drug is an injectable dosage form or a gel. The injectable dosage form is suitable for subcutaneous injection, intramuscular injection, intravenous injection, or intravenous infusion.

[0038] Example 6 Detection of SLC25A17 expression in human hepatocellular carcinoma cell lines and normal hepatocyte cell lines 1. Materials Cells: Human hepatocellular carcinoma cell lines Bel-7402, HepG2, SMMC-7721, MHCC97H, MHCC97L, and normal hepatocyte line L-O2. All were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0039] Reagents: DMEM culture medium was purchased from Hyclone Pharmaceuticals (USA); fetal bovine serum was purchased from Corning Pharmaceuticals (USA); RNA extraction reagent RNAiso Plus, reverse transcription kit PrimeScript™ RT reagent Kit with gDNA Eraser, and real-time quantitative PCR kit TB Green® Premix Ex Taq™ II were all purchased from TAKARA Pharmaceuticals (Japan). qRT-PCR specific primers were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0040] 2. Method Human hepatocellular carcinoma cell lines and normal hepatocyte lines were cultured in DMEM containing 10% fetal bovine serum in a constant temperature incubator at 37°C and 5% CO2.

[0041] Total RNA was extracted according to the RNAiso Plus instruction manual from TAKARA. The concentration and quality of RNA were assessed using an ultra-micro spectrophotometer (Shanghai Unico Instruments Co., Ltd., UPT100) and agarose gel electrophoresis. The following two steps were performed using the PrimeScript™ RT reagent Kit with gDNA Eraser from TAKARA: 1) Removal of genomic DNA from total RNA: Prepare the reaction solution (containing 1 μg total RNA, 2 μL 5 × gDNA Eraser Buffer, 1 μL gDNA Eraser, and ddH2O to 10 μL), incubate at 42°C for 2 min in a PCR instrument, and keep on ice until needed; 2) Reverse transcription reaction: Based on the reaction solution obtained in the previous step, further prepare the reverse transcription reaction solution (containing 10 μL of the reaction solution from the previous step, 4 μL 5 × Primescript Buffer, 1 μL Primescript RT Enzyme Mix I, 1 μL RT Primer Mix, 4 μL ddH2O, for a total volume of 20 μL), incubate at 37°C for 15 min in a PCR instrument, and inactivate the reverse transcriptase at 85°C for 5 s to obtain cDNA.

[0042] Using TB Green from TAKARA ®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 SLC25A17 gene are as follows: upstream primer 5'-AGCACTCTGGGTCAAAGGTC-3' (SEQ ID NO: 3) and downstream primer 5'-TTCCATAAAGCCGAGATTCCTTC-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 SLC25A17 mRNA was calculated using analytical methods.

[0043] 3. Results Figure 1 The expression levels of SLC25A17 in normal human hepatocyte cell lines and five hepatocellular carcinoma cell lines (Bel-7402, HepG2, SMMC-7721, MHCC97H, and MHCC97L) are shown below: P <0.01, the difference is statistically significant.

[0044] The results are as follows Figure 1 As shown, compared with the normal human hepatocyte cell line L-O2, SLC25A17 was upregulated in all five human hepatocellular carcinoma cell lines, with statistically significant differences. P The result showed a fold increase of <0.01, suggesting that SLC25A17 may play a pro-cancer role in liver cancer. The results showed that SLC25A17 was upregulated most significantly in the Bel-7402 and HepG2 cell lines. Therefore, Bel-7402 and HepG2 cell lines were selected as target cells for the knockdown experiment in subsequent functional experiments.

[0045] Example 7 Effects of siRNA inhibition of SLC25A17 expression on liver cancer cell function 1. Materials Cells: Human hepatocellular carcinoma lines Bel-7402 and HepG2, purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0046] Reagents: Lipofectamine 3000 transfection reagent was purchased from Thermo Fisher Scientific; CCK-8 kit was purchased from Dojindo, Japan; Transwell chambers (suitable for 24-well plates, PET membrane, 8 µm) were purchased from Corning, USA; Crystal violet was purchased from Beijing Solarbio Science & Technology Co., Ltd.; Apoptosis kit was purchased from Dojindo, Japan.

[0047] 2. Method 2.1 Design and synthesis of siRNA sequence targeting SLC25A17 Three different splice variant cDNA sequences of the SLC25A17 gene (NM_001282726.2, NM_006358.4, and NM_001282727.2) were obtained from the NCBI database. Using the BLOCK-iT RNAi Designer software (Thermo Fisher Scientific), a siRNA specifically targeting SLC25A17 (siSLC25A17) was designed based on the common exon regions of the different splice variants. The specific siRNA sequence consists of the sense and antisense strands of the following sequence: siRNA positive strand: 5'-CCUUGGAUGUGUUCAUCAU-3' (SEQ ID NO: 1) siRNA antisense strand: 5'-AUGAUGAACACAUCCAAGG-3' (SEQ ID NO: 2); The aforementioned siRNA targeting SLC25A17 was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

[0048] The negative control sequence (siNC, which has no target on the human genome) used in the experiment was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

[0049] 2.3 Cell transfection Human hepatocellular carcinoma cell lines were seeded into 6-well plates and cultured overnight at 37°C with 5% CO2 to achieve approximately 30% cell confluence before transfection. Transfection was then performed according to the Lipofectamine 3000 (Thermo Fisher Scientific) instructions. The specific steps are as follows: 1) Add 5 μL of siSLC25A17 / siNC (final concentration 50 nm) and 3.75 μL of Lipofectamine 3000 transfection reagent to 125 μL of serum-free DMEM culture medium, mix well, then add the DMEM culture medium containing siRNA to the DMEM culture medium containing Lipofectamine 3000 transfection reagent, mix carefully, and let stand for 5 min to obtain the transfection solution; 2) Transfection: Add the above transfection solution to the above 6-well plate (containing 2 mL of culture medium) and incubate at 37 ℃ in a 5% CO2 incubator; 3) Collect cells 24 h after transfection and use qRT-PCR to detect the interference effect of siRNA on SLC25A17, or perform cell proliferation, colony formation, apoptosis, migration and invasion experiments.

[0050] 2.4 qRT-PCR detection of the interference effect of siRNA on SLC25A17 The transfected experimental group cells (hepatocellular carcinoma cells transfected with siSLC25A17) and control group cells (hepatocellular carcinoma cells transfected with siNC) were collected, and total RNA was extracted, reverse transcribed, and qRT-PCR was performed, using the same methods as in Example 6.

[0051] 2.5 CCK-8 cell proliferation activity assay Cells from the experimental and control groups transfected with siSLC25A17 and siNC (50 nm) for 24 h were collected, resuspended in complete culture medium, counted, and seeded at a density of 3000 cells / well in 96-well plates. 100 μL of PBS was added to each well in the outermost wells. The plates were incubated at 37°C with 5% CO2. At set time points (0 h, 24 h, 48 h, 72 h), 10 μL of CCK-8 reagent was added to each well, and incubation continued for another 1 h 30 min. The absorbance of each well was then measured at 450 nm using a multi-mode microplate reader. Complete culture medium was added to cell-free wells as zero-adjustment wells. 2.6 Clonogenesis Assay Cells from the experimental and control groups transfected with siSLC25A17 and siNC (50 nm) for 24 h were collected, resuspended in complete culture medium, counted, and seeded at a density of 500 cells / well in 6-well plates with 3 replicates per group. The plates were incubated at 37°C in a 5% CO2 incubator for 7 days until clear cell colonies were visible to the naked eye. The culture medium in each well was removed, and the plates were washed twice with PBS. 1 mL of 4% paraformaldehyde was added to each well for fixation for 25 min. The paraformaldehyde was removed, and the plates were washed twice with PBS. 1 mL of 0.1% crystal violet was added to each well for staining. After 10 min, the staining was removed. The 6-well plates were rinsed under running tap water, dried, photographed, and colonies were counted.

[0052] 2.7 Apoptosis Experiment Cells were seeded in 6-well plates and transfected with siSLC25A17 and siNC (50 nm). After 48 h, cells from the experimental and control groups were collected. After washing and centrifugation with pre-cooled PBS, 195 μL of Annexin V-FITC binding buffer was added to resuspend the cells. Then, 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) were added. The cells were incubated at room temperature for 15 min in the dark, and apoptosis results were detected by flow cytometry within 1 h.

[0053] 2.8 Transwell test Cells from the experimental and control groups were collected 24 h after transfection with siSLC25A17 and siNC (50 nm). Cells were resuspended in complete culture medium, counted, and the cell suspension was 200 µl (containing approximately 1 × 10⁻⁶ cells / mL). 4 Cells were seeded into the upper chambers of 8 µm well Transwell chambers (either uncoated or pre-coated with Matrigel). Separately, in a 24-well plate, 500 µl of DMEM medium containing 20% ​​serum was added to form the lower chamber. The Transwell chambers were then placed into the 24-well plate and incubated at 37°C with 5% CO2 for 24-48 h. After incubation, the chambers were removed, washed three times with PBS, fixed with methanol, and air-dried. The chambers were then stained with 600 µl of 0.1% crystal violet for 10 min, followed by rinsing three times with PBS. The chambers were then inverted and air-dried. Using an inverted microscope at low magnification, five fields of view were randomly selected from each chamber for observation and photography, and the number of cells penetrating the lower layer of the microporous membrane was counted.

[0054] 3 Results 3.1 Interference efficiency of siSLC25A17 against SLC25A17 liver cancer cells Figure 2 The interference efficiency of siRNA targeting SLC25A17 in Bel-7402 and HepG2 liver cancer cells is shown, where:** P <0.01, the difference is statistically significant.

[0055] like Figure 2 As shown, compared with the control group transfected with siNC, the expression of SLC25A17 was significantly inhibited in liver cancer cells transfected with siRNA molecules that specifically target SLC25A17, with inhibition efficiencies of 85.7% and 78.6% in Bel-7402 and HepG2 cell lines, respectively.

[0056] 3.2 Effects of siSLC25A17 on the proliferation and colony formation ability of hepatocellular carcinoma cells Figure 3 The effect of SLC25A17-targeting siRNA on the proliferation ability of liver cancer cells, including: ** P <0.01, the difference is statistically significant.

[0057] like Figure 3 As shown, compared with the siNC control group, the proliferation activity of liver cancer cells transfected with siRNA was significantly inhibited. P All values ​​were <0.01, indicating statistical significance.

[0058] Figure 4 The effect of SLC25A17-targeting siRNA on the clonogenic ability of liver cancer cells, including:** P <0.01, the difference is statistically significant.

[0059] like Figure 4 As shown, the results of the plate colony formation assay were consistent with those of the CCK-8 assay, indicating that the plate colony formation ability of liver cancer cells transfected with siRNA was significantly weakened. P All values ​​were <0.01, indicating statistical significance. In summary, transfection with siRNA targeting SLC25A17 significantly inhibited the proliferation of liver cancer cells.

[0060] 3.3 Effect of siSLC25A17 on apoptosis in liver cancer cells Figure 5 The effect of SLC25A17-targeting siRNA on apoptosis in liver cancer cells, including:* P <0.05;** P <0.01, the difference is statistically significant.

[0061] like Figure 5 As shown, compared with the control group transfected with siNC, the apoptosis rate of liver cancer cells in the siRNA transfected group was significantly increased. P (All values ​​<0.05, indicating statistical significance) This suggests that transfection with siRNA targeting SLC25A17 can significantly induce apoptosis in liver cancer cells.

[0062] 3.4 Effects of siSLC25A17 on the migration and invasion abilities of liver cancer cells like Figure 6a and Figure 6b As shown, compared with the control group transfected with siNC, the number of liver cancer cells that migrated and invaded was significantly reduced in the siRNA transfected group. P (All values ​​<0.01, indicating statistical significance) This suggests that transfection with siRNA targeting SLC25A17 can significantly inhibit the migration and invasion of liver cancer cells.

[0063] 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.

[0064] sequence list sequence list <110> Xi'an Peihua University <120> siRNA molecules targeting the human SLC25A17 gene and their applications <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> RNA <213> Artificial sequence <400> 1 ccuuggaugu guucaucau 19 <210> 2 <211> 19 <212> RNA <213> Artificial sequence <400> 2 augaugaaca cauccaagg 19 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 agcactctgg gtcaaaggtc 20 <210> 4 <211> twenty three <212> DNA <213> Artificial sequence <400> 4 ttccataaag ccgagattcc ttc 23 <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 SLC25A17 gene, characterized in that, It consists of a justice chain and an antithesis chain, with the specific sequence as follows: Chain of Justice: 5'-CCUUGGAUGUGUUCAUCAUNn-3' Antonym chain: 5'-AUGAUGAACACAUCCAAGGNn-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 SLC25A17 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 SLC25A17 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'-CCUUGGAUGUGUUCAUCAUdTdT-3' Antonym chain: 5'-AUGAUGAACACAUCCAAGGdTdT-3'.

4. The use of the siRNA molecule targeting the human SLC25A17 gene according to any one of claims 1-3 in the preparation of a drug for inhibiting SLC25A17 expression, wherein the drug 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 SLC25A17 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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