Antisense oligonucleotide for inhibiting metastasis of esophageal squamous cell carcinoma and application of antisense oligonucleotide
By designing a targeted antisense oligonucleotide sequence for exon 19 of ACTN1 gene, the metastasis ability of esophageal squamous cell carcinoma cells is regulated, and the inadequacy of lymph node metastasis treatment in the prior art has been solved, and the effect of inhibiting metastasis and improving the treatment effect has been achieved.
Patent Information
- Application Number
- CN202510133678.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-06
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to effectively inhibit lymph node metastasis of esophageal squamous cell carcinoma, and conventional treatment methods have major side effects and are not effective.
A highly targeted antisense oligonucleotide sequence was designed to target exon No. 19 of the ACTN1 gene, and inhibit metastasis and invasion of esophageal cancer cells by regulating the expression level of the ACTN1 gene.
It significantly inhibits the metastatic ability of esophageal squamous cell carcinoma cells, improves the prognosis and treatment effect of esophageal squamous cell carcinoma, and has a small impact on normal cells and is low in toxicity.
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Figure CN120060249A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and particularly relates to an antisense oligonucleotide for inhibiting the metastasis of esophageal squamous cell carcinoma and its application. Background Art
[0002] Esophageal squamous cell carcinoma (ESCC) is a type of tumor with a high incidence and poor prognosis in China. Lymph node metastasis is the most common metastasis mode of esophageal squamous cell carcinoma and is an independent factor affecting the prognosis of esophageal squamous cell carcinoma. However, for the current treatment of esophageal squamous cell carcinoma metastasis, due to the unclear molecular mechanism, the main treatment methods are still conventional radiotherapy, chemotherapy, surgery, etc.
[0003] Conventional radiotherapy, chemotherapy, surgery and other methods have large damage and many side effects to the patient itself due to the lack of targeting, and the curative effect is not good; for some new drugs developed by frontier research, due to the lack of sufficient clinical evidence, their curative effects are difficult to determine.
[0004] Antisense oligonucleotides (ASO) are short, chemically synthesized single-stranded oligonucleotides. By modifying their backbones and glycosyl groups, their stability, pharmacological properties and binding to targets can be increased, and they usually have low toxicity. They are commonly used in gene function research in cells and animals and the development of ASO nucleic acid drugs, etc. Research shows that the activity of ASO is related to the level of RNase H. The level of RNase H in the cell nucleus is relatively high, and the degradation rate of nuclear target mRNAs is higher than that in the cytoplasm. It is often more suitable for situations where it is necessary to affect the function of pre-mRNA or RNAs located in the cell nucleus than siRNA which mainly acts in the cytoplasm. In addition to gene silencing, ASO can also be used for the regulation of gene alternative splicing, affecting the splicing of precursor RNA by splicing factors through the occupancy effect. Therefore, carrying out research on esophageal squamous cell carcinoma lymph node metastasis with the help of ASO technology will help improve the treatment effect of esophageal squamous cell carcinoma metastasis. Summary of the Invention
[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an antisense oligonucleotide for inhibiting the metastasis of esophageal squamous cell carcinoma and its application.
[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0007] In the first aspect, the present invention provides an antisense oligonucleotide for inhibiting the metastasis of esophageal squamous cell carcinoma, and the nucleotide sequence of the antisense oligonucleotide is 5'-CATAACCCAAGCTGATGAGGCAGGC-3' (SEQ ID NO.1).
[0008] By performing full-length transcriptome sequencing and qPCR detection on 5 pairs of self-matched esophageal squamous cell carcinoma tissues, it was found that exon 19 of ACTN1 frequently undergoes alternative splicing in esophageal squamous cell carcinoma (with a splicing frequency of approximately 71%), thereby generating different transcripts (transcripts with / without exon 19). Based on AlphaFold prediction and CoIP verification, the protein encoded by the transcript containing exon 19 can specifically bind to certain metastasis-promoting cytoskeletal proteins (such as vimentin), and is involved in the metastasis of esophageal squamous cell carcinoma. Exon 19 of ACTN1 encodes an important calcium-binding domain, which has an important impact on the metastasis of esophageal cancer cells, and the proportion of transcripts containing this exon is significantly increased in esophageal cancer. The present invention designed a highly targeted oligonucleotide sequence based on exon 19 of the ACTN1 gene, which can reduce the proportion of transcripts containing exon 19 without affecting the overall expression of ACTN1, thus targeting and inhibiting the metastatic ability of tumor cells with less impact on normal cells. Therefore, the present invention designed an antisense oligonucleotide with the sequence of SEQ ID NO.1 using exon 19 of ACTN1 as the target sequence to regulate the expression level of the ACTN1 gene in esophageal cancer cells, thereby inhibiting the metastasis and invasion of esophageal cancer cells, achieving inhibition of lymph node metastasis of esophageal squamous cell carcinoma, and improving the prognosis and treatment effect of esophageal squamous cell carcinoma.
[0009] The reason why the present invention chose to use antisense oligonucleotides to regulate the alternative splicing of the ACTN1 gene is that antisense oligonucleotides are different from other ways of regulating target genes: for example, the way shRNA regulates target genes is by using RNAi (RNA interference). shRNA regulates the expression of the entire gene at the transcriptional level, can only knockdown the level of the entire gene, and cannot regulate a specific exon of a gene. Therefore, it is impossible to perform fine regulation on a specific exon. In contrast, the antisense oligonucleotides of the present invention regulate the proportion of gene transcripts (i.e., reduce the proportion of transcripts containing exon 19) without affecting the overall expression of the entire gene, and inhibit the alternative splicing of transcripts post-transcriptionally. This not only ensures that the ACTN1 gene in normal cells is not affected, but also can precisely splice exon 19 of the ACTN1 gene in esophageal cancer cells, so that normal cells are not affected, while at the same time achieving inhibition of the metastasis of esophageal squamous cell carcinoma and improving the prognosis and treatment effect of esophageal squamous cell carcinoma.
[0010] For example, for certain diseases caused by genetic mutations, such as DMD (Duchenne muscular dystrophy), there is a mutation at exon 51 of the DMD gene in patients, resulting in no protein expression (premature termination). Eteplirsen (an antisense oligonucleotide preparation) can directly delete exon 51 during the splicing process of the DMD gene, skip the mutated part, and enable normal protein expression. Although the re-spliced protein is smaller than the normal protein, it retains the basic functions of the normal DMD protein. In contrast, shRNA cannot achieve such a precise therapeutic effect. Although the CRISPR-cas9 gene editing technology can achieve this effect by editing the genome, its cost is expensive, and the side effects and off-target effects caused by genome editing are also difficult to predict.
[0011] As a preferred embodiment of the present invention, the ribose of 5 bases at both the head and tail ends of the sequence of the antisense oligonucleotide is introduced with 2'-methoxy modification.
[0012] In a second aspect, the present invention provides a recombinant expression vector, a transgenic cell line or a recombinant virus containing a sequence of an antisense oligonucleotide encoding exon 19 of the above-mentioned targeted ACTN1 gene.
[0013] In a third aspect, the present invention provides a kit of the above-mentioned antisense oligonucleotide targeting exon 19 of the ACTN1 gene.
[0014] As a preferred embodiment of the present invention, the kit further includes a primer set for detecting the expression of exon 19 of the ACTN1 gene.
[0015] In a fourth aspect, the present invention provides the application of the antisense oligonucleotide described in the first aspect, the recombinant expression vector, the transgenic cell line or the recombinant virus described in the second aspect, and the kit described in the third aspect in the identification of lymph node metastasis and distant metastasis of esophageal squamous cell carcinoma.
[0016] In a fifth aspect, the present invention provides a drug for treating esophageal squamous cell carcinoma metastasis, and the drug includes the antisense oligonucleotide described in the first aspect and a pharmaceutically acceptable excipient.
[0017] In a sixth aspect, the present invention provides a conjugate, including the antisense oligonucleotide described in the first aspect and at least one conjugate moiety covalently linked to the oligonucleotide.
[0018] In a seventh aspect, the present invention provides a composition, including the antisense oligonucleotide described in the first aspect or the conjugate described in the sixth aspect and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.
[0019] In an eighth aspect, the present invention provides a method for regulating the proportion of ACTN1 gene transcripts in target cells in vitro, the method comprising administering to the target cells, in an effective amount, the antisense oligonucleotide described in the first aspect, the conjugate described in the sixth aspect, or the composition described in the seventh aspect.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0021] 1. Significantly inhibiting metastasis: In cell experiments, the metastasis and invasion ability of esophageal squamous carcinoma cells transfected with the antisense oligonucleotide drug was significantly weakened; in animal experiments, among the 7 mice injected with esophageal squamous carcinoma cells in the control group, all developed metastases, and the lymph node volume was significantly larger than that of the mice injected with the antisense oligonucleotide drug; only 2 out of the 7 mice injected with the antisense oligonucleotide drug developed metastases, and the lymph node volume was significantly smaller than that of the control group.
[0022] 2. High specificity: The proportion of metastasis-promoting transcripts of the ACTN1 gene in esophageal squamous carcinoma is significantly higher than that in normal tissues. The antisense oligonucleotide drug has a stronger effect on tumor cells and less impact on normal tissues. After injecting the ASO in animal experiments, the normal organs of the mice were basically not affected.
[0023] 3. Low toxicity: In animal experiments, no obvious pathological changes were found in the main organs of the mice injected with the antisense oligonucleotide drug, and no abnormal conditions (such as cyanosis, emaciation, death, etc.) occurred in the mice during the entire animal experiment.
[0024] The present invention is different from the prior art that uses gene silencing of ACTN1 in the following aspects:
[0025] 1) Different ways of regulating target genes: The way of regulating target genes by shRNA is to use RNAi (RNA interference), which regulates gene expression at the transcriptional level, while the antisense oligonucleotide of the present invention regulates the proportion of gene transcripts without affecting the overall gene expression and inhibits alternative splicing of transcripts post-transcriptionally. Therefore, the present invention is completely different from other ways of regulating target genes by silencing (RNA interference).
[0026] 2) Exon 19 of ACTN1 encodes an important calcium-binding domain, which has an important impact on the metastasis of esophageal cancer cells, and the proportion of transcripts containing this exon is significantly increased in esophageal cancer. The present invention designs a highly targeted oligonucleotide sequence based on exon 19 of ACTN1, which can change and reduce the proportion of transcripts containing exon 19 without affecting the overall expression of ACTN1, thereby targeting and inhibiting the metastasis ability of tumor cells with less impact on normal cells. Description of the Drawings
[0027] Figure 1Schematic diagram of the splicing of exon 19 of the ACTN1 gene in KYSE410 after transfection with antisense oligonucleotides in cell experiments (where CTR represents the control group and ASO represents the group injected with antisense oligonucleotides);
[0028] Figure 2 Schematic diagram of the decreased migration and invasion ability of KYSE410 after transfection with antisense oligonucleotides in cell transwell experiments;
[0029] Figure 3 Schematic diagram of the decreased migration and invasion ability of KYSE410 after transfection with antisense oligonucleotides in cell scratch experiments;
[0030] Figure 4 Schematic diagram of the obvious destruction of the cytoskeleton in tumor cells after transfection with antisense oligonucleotides;
[0031] Figure 5 Schematic diagram of the alternative splicing of the ACTN1 gene changing with the dose and concentration of oligonucleotides;
[0032] Figure 6 Schematic diagram of the splicing of exon 19 of the ACTN1 gene caused by the antisense oligonucleotide drug in animal experiments;
[0033] Figure 7 Schematic diagram of the comparison of lymph node sizes between the antisense oligonucleotide drug group and the control group in animal experiments;
[0034] Figure 8 Schematic diagram of the comparison of lymph node metastasis rates between the antisense oligonucleotide drug group and the control group in animal experiments;
[0035] Figure 9 Schematic diagram of the comparison of lung metastases between the antisense oligonucleotide drug group and the control group in animal experiments. Detailed implementation manners
[0036] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0037] Example 1
[0038] Preparation method of antisense oligonucleotides
[0039] The antisense oligonucleotide sequence 5’-CATAACCCAAGCTGATGAGGCAGGC-3’ (SEQ ID NO.1) was synthesized by Tsingke. The specific steps include using nucleotides as raw materials to chemically synthesize the oligonucleotides of the target sequence, followed by ammonolysis, HPLC purification and other steps, and the preparation is completed after quality inspection (sequencing verification).
[0040] Example 2: Cell experiment
[0041] 1) In this invention, the KYSE410 cell line (human esophageal squamous cell carcinoma cells) was used to test the effect of the antisense oligonucleotides of this invention on human esophageal squamous cell carcinoma cells.
[0042] 2) Lipo3000 was used as the transfection reagent. According to the instructions, the antisense oligonucleotides (the specific concentration was determined by cells and experimental conditions) were mixed with Lipo3000 (at the default ratio in the instructions) and incubated for about 20 minutes to form a transfection complex. The transfection complex was added to the cell culture medium and treated for 6 - 12 hours. Then, the complete medium (DMEM) was replaced. After 48 hours, RNA was extracted. Through agarose gel electrophoresis verification, as Figure 1 , after transfection with the antisense oligonucleotides, exon 19 of the ACTN1 gene in KYSE410 cells was spliced, the ratio of long and short transcripts changed, the ratio of short transcripts (without exon 19) increased, while the ratio of long transcripts (containing exon 19) decreased, indicating that the antisense oligonucleotides were successfully transfected into the cells and exerted their effects, promoting the skipping of exon 19 of the ACTN1 gene.
[0043] 3) The transwell experiment and scratch experiment were used to verify the effect of the antisense oligonucleotides on inhibiting the metastasis and invasion of esophageal squamous cell carcinoma cells. As Figure 2 , 3 , after transfection with the antisense oligonucleotides, the metastasis ability of KYSE410 significantly decreased.
[0044] 4) The cytoskeleton of tumor cells (represented by F - actin) was stained with phalloidin. It was observed that the cytoskeleton of tumor cells was significantly damaged after transfection with the antisense oligonucleotides, while the normal cytoskeleton was not damaged. As Figure 4 , which was consistent with the above experiments, indicating that the motility of tumor cells significantly decreased.
[0045] 5) By changing the action concentration (0, 20, 50, 100 nM respectively) and action time (0, 24, 48, 72 hours) of the antisense oligonucleotides, as Figure 5 , it could be observed that the alternative splicing of the ACTN1 gene changed with the dose and concentration of the oligonucleotides, indicating that this antisense oligonucleotide had a dose - and time - dependent effect on the splicing of the ACTN1 gene and had good druggability.
[0046] Example 3: Animal experiment (plantar popliteal lymph node metastasis model)
[0047] 1) Fourteen nude mice were randomly divided into two groups, the antisense oligonucleotide drug group (ASO) and the control group (CTR). 4×10 6 K410 cells were injected into the plantar of each mouse.
[0048] 2) Two weeks later, mice in the antisense oligonucleotide drug group were selected. Each mouse was injected with 5 nmol of the antisense oligonucleotide drug into the primary tumor on the plantar surface and 5 nmol of the antisense oligonucleotide drug via the tail vein each time. The control group was injected with an equal amount of the control drug (a nonsense RNA sequence with a similar length to the antisense oligonucleotide, and its sequence was 5'-TTCTCCGAACGTGTCACGTT-3', and other treatments were the same as those for the production of the antisense oligonucleotide), and the injection was carried out twice a week.
[0049] 3) Four weeks later, the mice were sacrificed, the popliteal lymph nodes were removed, RNA was extracted, and RT-qPCR was used to detect the expression of human HPRT (because human cells were injected, if metastasis occurred, the presence of human RNA could be detected in the mice, and the human HPRT gene was used as the detection index for human RNA). If the CT value ≤ 35, it was considered that there was lymph node metastasis.
[0050] 4) As Figure 6 , exon 19 of the ACTN1 gene in the antisense oligonucleotide drug group was spliced, indicating that the antisense oligonucleotide drug played a role successfully in the animal experiment.
[0051] 5) As Figure 7 、 8 , the lymph node size in the antisense oligonucleotide drug group was significantly smaller than that in the control group, and the lymph node metastasis rate was significantly lower than that in the control group, indicating that the antisense oligonucleotide drug had the effect of inhibiting the metastasis of esophageal cancer.
[0052] Example 4 Animal experiment (tail vein lung metastasis model)
[0053] 1) Twenty-four nude mice were randomly divided into two groups, the antisense oligonucleotide drug group (ASO) and the control group (CTR). Each mouse was injected with 5×10 6 K30 cells via the tail vein.
[0054] 2) Two weeks later, mice in the antisense oligonucleotide drug group were selected. Each mouse was injected with 5 nmol of the antisense oligonucleotide drug via the tail vein each time. The control group was injected with an equal amount of the control drug (a nonsense RNA sequence with a similar length to the antisense oligonucleotide, and its sequence was 5'-TTCTCCGAACGTGTCACGTT-3', and other treatments were the same as those for the production of the antisense oligonucleotide), and the injection was carried out twice a week.
[0055] 3) Four weeks later, the mice were sacrificed, the lung tissues were removed, and HE staining was performed to check for lung metastasis.
[0056] 4) As Figure 9 , after adding the antisense oligonucleotide, the lung metastasis of the mice was significantly inhibited, indicating that the antisense oligonucleotide drug had the effect of inhibiting the distant metastasis of esophageal cancer.
[0057] Comparative Example 1
[0058] In Comparative Example 1, shRNA of ACTN1 was used to knockdown the overall transcription level of ACTN1. The results showed that due to the lack of precise targeting of shRNA, the overall transcription level of ACTN1 was reduced. The amount of ACTN1 prometastatic transcripts decreased with the reduction of the overall transcription level. However, due to the reduction of the overall transcription level of ACTN1, normal cells could not perform physiological functions such as movement and proliferation, and even apoptosis of normal cells might be induced. Therefore, using shRNA to knockdown the ACTN1 gene would produce significant side effects and toxicity.
[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. An antisense oligonucleotide for inhibiting metastasis of esophageal squamous cell carcinoma, characterized in that: The sequence of the antisense oligonucleotide is shown in SEQ ID NO.
1.
2. The antisense oligonucleotide according to claim 1, characterized in that The sequence of the antisense oligonucleotide introduces 2' methoxy modification into the ribose of 5 bases at both ends.
3. A recombinant expression vector, transgenic cell line or recombinant virus containing a sequence encoding the antisense oligonucleotide targeting exon 19 of the ACTN1 gene as claimed in claim 1 or 2.
4. A kit, characterized in that: The kit comprises the antisense oligonucleotide targeting exon 19 of the ACTN1 gene as claimed in claim 1 or 2.
5. The kit according to claim 4, characterized in that Also included is a primer set for detecting the expression of exon 19 of the ACTN1 gene.
6. Use of the antisense oligonucleotide according to claim 1 or 2, the recombinant expression vector, transgenic cell line or recombinant virus according to claim 3, or the kit according to claim 4 or 5 in the preparation of a drug for treating lymph node metastasis and distant metastasis of esophageal squamous cell carcinoma.
7. A drug for treating metastasis of esophageal squamous cell carcinoma, characterized in that: The drug comprises the antisense oligonucleotide according to claim 1 or 2 and a medically acceptable excipient.
8. A conjugate, characterized in that The invention comprises the antisense oligonucleotide of claim 1, and at least one conjugate part covalently linked to the oligonucleotide.
9. A composition, characterized in that The method comprises the antisense oligonucleotide according to claim 1 or the conjugate according to claim 8 and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.
10. An in vitro method for regulating the ratio of ACTN1 transcripts in target cells expressing ACTN1, the method comprising administering an effective amount of the antisense oligonucleotide of claim 1 or 2, the conjugate of claim 8 or the composition of claim 9 to the target cells.