Antisense oligonucleotide targeting FXR1 protein and application thereof
By designing antisense oligonucleotides targeting FXR1 protein, the problem of limited therapeutic effect of existing targeted drugs on mid- and late-stage liver cancer was solved, the effect of inhibiting the proliferation and metastasis of liver cancer cells was achieved, and it has the potential to prepare anti-liver cancer drugs.
Patent Information
- Application Number
- CN202510985741.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-17
- Publication Date
- 2025-10-17
AI Technical Summary
Existing targeted drugs have limited therapeutic effects on advanced liver cancer, and new therapeutic targets and anti-tumor drugs are needed to improve patient prognosis.
An antisense oligonucleotide targeting FXR1 protein was designed, with a nucleotide sequence of 5'-CATAAGCATCAACTTCGTACG-3' modified by 2'-O-methyl, to inhibit the expression of FXR1 to prevent the proliferation and metastasis of liver cancer cells.
It significantly inhibits the proliferation of liver cancer cells and reduces subcutaneous tumor formation in mice, and has potential clinical application value for the preparation of anti-liver cancer drugs.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and relates to an antisense oligonucleotide targeting FXR1 protein and application thereof. BACKGROUND
[0002] Primary liver cancer is one of the most common malignant tumors. Primary liver cancer mainly includes hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and mixed type. Among them, hepatocellular carcinoma (HCC) has insidious onset, and most patients are in the middle and late stages when they seek medical treatment. In addition, HCC has high malignancy, patients often have chronic hepatitis and cirrhosis background, and early metastasis and transfer are easy to occur, resulting in a high postoperative recurrence rate, which seriously affects the prognosis and survival rate of patients. In recent years, although some targeted drugs have entered clinical trials for the treatment of advanced liver cancer, their effect on prolonging the survival period of patients is still very limited. Therefore, it is particularly urgent to find new therapeutic targets and anti-tumor drugs.
[0003] RNA binding proteins (RBPs) regulate gene expression processes such as polyadenylation, RNA splicing, export, stability, translation, etc. by interacting with specific RNAs to form ribonucleoprotein (RNP) compounds, and play an important role in various stages of gene regulation. Changes in RBPs often affect various pathological and physiological processes leading to diseases. Fragile X-related protein (FXR) is a highly conserved RNA-binding protein family, including Fragile X mental retardation 1 (FMR1), Fragile X mental retardation 1 (FXR1), and Fragile X mental retardation 2 (FXR2). Among them, FXR1 is more likely to induce cancer than the other two proteins in the family. Many studies have found that overexpression of FXR1 at the RNA and protein levels is closely related to the poor prognosis of various cancers, and high expression of FXR1 in liver cancer tissues is closely related to poor prognosis of liver cancer patients. The above studies suggest that FXR1 may be a potential therapeutic target.
[0004] Antisense oligonucleotide (ASO) is a kind of short-chain nucleic acid molecule that can specifically bind to target RNA and regulate its function. It can target the mRNA of oncogenes to inhibit their expression, thereby preventing the proliferation, invasion and metastasis of cancer cells. SUMMARY
[0005] The application aims to provide an antisense oligonucleotide targeting FXR1 protein and its pharmaceutical use.
[0006] The application provides the following technical solutions through research:
[0007] 1. An antisense oligonucleotide targeting FXR1 protein, the nucleotide sequence of which is shown in SEQ ID No. 1.
[0008] Further, the antisense oligonucleotide targeting FXR1 protein is 2'-O-methyl modified on the ribose of each nucleotide.
[0009] 2. The antisense oligonucleotide targeting FXR1 protein is used in the preparation of an anti-liver cancer drug.
[0010] Further, the anti-liver cancer drug is a drug for inhibiting the proliferation of liver cancer cells.
[0011] The application has the beneficial effect that the application provides an antisense oligonucleotide targeting FXR1 protein, which can significantly inhibit the proliferation of liver cancer cells when transfected into human liver cancer cells, and can also inhibit the subcutaneous tumor formation in mice, indicating that the antisense oligonucleotide specifically targeting FXR1 protein has the function of inhibiting the proliferation of liver cancer cells, and can be used for the preparation of an anti-liver cancer drug for clinical treatment. The application discloses the application value of the antisense oligonucleotide in improving the prognosis of liver cancer by targeting and blocking FXR1. BRIEF DESCRIPTION OF DRAWINGS
[0012] Figure 1 The figure shows the comparison of the expression of FXR1 in liver cancer tissue (T) and the corresponding para-cancer tissue (NT) in the TCGA database; wherein the left graph shows that the RNA expression level of FXR1 is high in liver cancer patients, and the right graph shows that the expression level of FXR1 in liver cancer tissue progressing to T3-T4 stage is higher than that in T1-T2 stage with lower tumor malignancy.
[0013] Figure 2 The figure shows that knocking out FXR1 inhibits the proliferation of liver cancer cells in the colony formation experiment.
[0014] Figure 3 The figure shows that the expression level of FXR1 and the cell proliferation ability are significantly inhibited after the human liver cancer cells are transfected with the antisense oligonucleotide targeting FXR1 protein; wherein A is the Western Blot experiment result, and B is the colony formation experiment result.
[0015] Figure 4 The figure shows that the antisense oligonucleotide targeting FXR1 protein inhibits the tumor formation in mice in vivo. DETAILED DESCRIPTION
[0016] In order to make the purposes, technical solutions and beneficial effects of the present application clearer, preferred embodiments of the present application are described in detail below.
[0017] The sequence of the antisense oligonucleotide targeting FXR1 protein used in the preferred embodiments is 5'-CATAAGCATCAACTTCGTACG-3' (SEQ ID No. 1), and each nucleotide sequence has a 2'-O-methyl modification on the ribose.
[0018] Example 1
[0019] The expression of FXR1 in liver cancer tissues and corresponding para-cancer tissues in The Cancer Genome Atlas (TCGA) was compared. The method is as follows: the RNA-Seq expression matrix and clinical data of liver cancer (LIHC) were downloaded through UCSC Xena, the data was pre-processed and tumor / para-cancer samples were screened using R language (DESeq2), then differential expression analysis was performed to obtain the log2FC and p value of the target gene (FXR1), and finally the results were analyzed using Graphpad Prism10.
[0020] The results are shown in Figure 1 Compared with para-cancer tissues, the RNA expression level of FXR1 in liver cancer patients showed high expression; and compared with T1-T2 stage with lower tumor malignancy, the expression level of FXR1 in liver cancer tissues progressing to T3-T4 stage was higher.
[0021] Example 2
[0022] In order to further verify the physiological function of FXR1, first, an online CRISPR design tool was used to screen 2 specific sgRNA target sequences (sgFXR1-1# and sgFXR1-2#) on the exons of the FXR1 gene, after chemical synthesis of the sgRNA target sequence, the sgRNA plasmid was co-transfected with the packaging plasmid (psPAX2, pMD2.G) into HEK293T cells to produce lentivirus, and the culture supernatant containing the virus was collected, SNU449 cells and MHCC97H cells were infected respectively, and puromycin was used for screening to obtain cell strains stably knocking out FXR1. Next, the successfully constructed FXR1 knockout cells (sgFXR1-1# group and sgFXR1-2# group) and control cells (FXR1 unknocked out cells, sgControl group) were used for colony formation experiment: the cells were inoculated in 6-well plates at an appropriate density, 3 replicate wells were set for each group, and incubated at 37℃ for 10-14 days, then fixed with polyformaldehyde and stained with crystal violet, the number of cell colonies with ≥50 cells was counted, and the colony formation rate (%) was calculated as (number of cell colonies / number of inoculated cells) x 100%.
[0023] The results are shown inFigure 2 Compared with the sgControl group, the proliferation ability of liver cancer cells in the sgFXR1-1# group and the sgFXR1-2# group was significantly weakened, indicating that knocking out FXR1 can inhibit the proliferation of liver cancer cells.
[0024] Example 3
[0025] Using lipo8000 as a transfection reagent, according to the reagent instructions, the ASO group incubated the antisense oligonucleotide targeting FXR1 protein with lipo8000 and Opti-MEM for about 20 minutes to form a transfection complex. The control (NC) group used a scrambled control oligonucleotide of similar length to the antisense oligonucleotide targeting FXR1 protein to incubate with lipo8000 and Opti-MEM to form a transfection complex. The transfection complex was added to the cell culture medium to treat PLC / PRF / 5 cells, and after 48 hours, cell proteins were extracted for Western Blot to verify the expression levels of FXR1 and its downstream target genes (p-GSK3β, β-catenin, CyclinD-1, c-Myc). At the same time, the transfection complex was added to the cell culture medium to treat PLC / PRF / 5 cells and MHCC97H cells, and a clonogenicity experiment was performed.
[0026] The results are shown in Figure 3 , and the Western Blot experiment results confirmed that the expression levels of FXR1 and its downstream target genes in the ASO group were down-regulated compared with the NC group, as shown in Figure 3 A; the clonogenicity experiment results confirmed that the proliferation ability of liver cancer cells in the ASO group was significantly weakened compared with the NC group, as shown in Figure 3 B.
[0027] Example 4
[0028] Twelve nude mice were randomly divided into two groups: the ASO group and the control (NC) group. Each nude mouse was subcutaneously injected with 2x10 6 MHCC97H cells. When the subcutaneous tumor diameter of the nude mice reached 5mm, the ASO group was treated with intratumoral injection of antisense oligonucleotide targeting FXR1 protein, and the NC group was injected with an equal amount of control drug (scrambled control oligonucleotide of similar length to the antisense oligonucleotide targeting FXR1 protein), twice a week. After 4 weeks, the nude mice were sacrificed for observation.
[0029] The results are shown in Figure 4 , the tumor in the ASO group was significantly smaller than that in the NC group, indicating that the antisense oligonucleotide targeting FXR1 protein can inhibit tumor formation in vivo and can be used to prepare anti-liver cancer drugs.
[0030] Finally, it should be noted that the above preferred embodiments are merely intended to illustrate the technical solutions of the present application but not to limit the present application. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various modifications can be made in form and details without departing from the scope of the present application defined by the claims.
Claims
1. An antisense oligonucleotide targeting FXR1 protein, characterized in that: Its nucleotide sequence is shown in SEQ ID No.
1.
2. The antisense oligonucleotide targeting FXR1 protein according to claim 1, wherein: Each nucleotide has a 2'-O-methyl modification on the ribose sugar.
3. Use of the antisense oligonucleotide targeting FXR1 protein according to claim 1 or 2 in the preparation of anti-liver cancer drugs.
4. The use according to claim 3, characterized in that: The anti-liver cancer drug is a drug that inhibits the proliferation of liver cancer cells.