Anti-cancer small interfering RNA (Ribonucleic Acid) capable of simultaneously targeting cancer genes PTTG1 and STMN1 and application of anti-cancer small interfering RNA

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a cancer suppression small interfering RNA (Ribonucleic Acid) capable of simultaneously targeting cancer genes PTTG1 and STMN1 and application of the cancer suppression small interfering RNA, and the technical key points are as follows: the positive-sense strand sequence of the small interfering RNA is GGGAGAUCUCAAGUUUCAATT, and the antisense strand sequence of the small interfering RNA is UUGAAACUUGAGAUCUCCCTT. The small interfering RNA provided by the invention can specifically silence the expression of PTTG1 and STMN1 at the same time. Compared with siRNA (such as siRNA for ASCC3 or TRAPPC4) targeting a single gene in the prior art, the siRNA provided by the invention has the advantages that synergistic inhibition on tumor proliferation and metastasis pathways is realized through double-targeting design, and the inhibition effect on tumor cell growth is more remarkable; compared with a method of physically mixing two single siRNAs, the method provided by the invention has higher cell apoptosis induction efficiency and better in-vivo tumor inhibition effect; the small interfering RNA is effective in various liver cancer cells, has a lasting effect in an animal model, and provides a core molecular entity for developing novel antitumor drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to tumor suppressor small interfering RNAs that simultaneously target oncogenes PTTG1 and STMN1 and their applications. Background Technology

[0002] Cancer is a malignant disease caused by the abnormal proliferation of malignant cells, characterized by its invasiveness and metastasis. With high incidence and mortality rates, cancer is a major disease that seriously threatens national health. The abnormal activation of oncogenes in normal cells leads to their transformation into malignant cells with unlimited proliferative capacity and the ability to invade and metastasize, which is the main cause of cancer. Malignant tumor cells are characterized by unlimited proliferation and invasion / metastasis; this series of malignant phenotypes is mainly driven by the synergistic activation of multiple oncogenes. Therefore, inhibiting the abnormal upregulation of these key oncogenes is an effective strategy to reduce the proliferation and metastatic ability of malignant tumor cells.

[0003] Among numerous oncogenes, pituitary tumor transforming gene 1 (PTTG1) and microtubule depolymerization protein (STMN1) have attracted significant attention. PTTG1, an oncogene initially isolated from pituitary tumor cells, is abnormally highly expressed in various cancers, including liver cancer, and its high expression level is significantly associated with poor patient prognosis. Studies have shown that tumor cells expressing high levels of PTTG1 have stronger proliferative capacity, while reducing PTTG1 expression can effectively inhibit tumor cell proliferation. STMN1 is a pro-cancer phosphorylated protein that participates in tumor invasion and migration by regulating microtubule stability. STMN1 is also abnormally highly expressed in various tumors, and its high expression is significantly positively correlated with metastasis and recurrence of tumors such as liver cancer. Knocking down STMN1 expression can significantly inhibit the migration and invasion capabilities of malignant tumor cells.

[0004] Small interfering RNA (siRNA) technology provides a powerful tool for targeting and inhibiting the expression of specific genes. siRNAs are double-stranded RNA molecules approximately 20-25 base pairs long. Through RNA interference (RNAi) mechanisms, they specifically degrade target messenger RNA (mRNA) complementary to their sequence, thereby efficiently and specifically silencing the expression of target genes. Using siRNA to inhibit the expression of oncogenes has proven to be an effective tumor intervention strategy. Existing technologies include numerous studies on the design of siRNAs targeting single oncogenes. A search reveals that Chinese patent CN115820632A discloses siRNA targeting the ASCC3 gene and its application in inhibiting bladder cancer proliferation, migration, and invasion; Chinese patent CN102899325A discloses siRNA targeting the TRAPPC4 gene and its application in treating colorectal cancer by inhibiting the ERK-MAPK pathway. These existing technologies demonstrate the potential of siRNAs targeting single oncogenes in the treatment of specific cancers.

[0005] However, the occurrence and development of malignant tumors are the result of the interaction and joint driving force of multiple oncogenes. Traditional siRNA blocking strategies targeting single oncogenes often fail to completely inhibit or reverse tumor growth, especially in addressing the two key biological processes of tumor proliferation and metastasis simultaneously. Tumor cells not only possess unlimited proliferative potential but also have the ability to invade and migrate; even after surgical resection of the lesion in situ, the risk of tumor metastasis and recurrence remains high. Therefore, inhibiting only a single oncogene (such as PTTG1 or STMN1) cannot achieve a synergistic therapeutic effect of simultaneously and effectively inhibiting tumor proliferation and metastasis.

[0006] In summary, there is an urgent need in this field to develop a novel siRNA molecule that can simultaneously and efficiently silence the two key and functionally complementary oncogenes PTTG1 and STMN1, in order to overcome the limitations of existing single-target siRNA technologies and achieve more effective synergistic inhibition of tumor proliferation and metastasis.

[0007] To address these issues, this application proposes a tumor suppressor small interfering RNA that simultaneously targets the oncogenes PTTG1 and STMN1 and its application. Summary of the Invention

[0008] The purpose of this invention is to solve the technical problems mentioned in the background section above, and to provide a tumor suppressor small interfering RNA that simultaneously targets the oncogenes PTTG1 and STMN1 and its applications.

[0009] The above-mentioned objective of the present invention is achieved as follows: The present invention provides a tumor suppressor small interfering RNA that simultaneously targets the oncogenes PTTG1 and STMN1. The nucleotide sequence of the sense strand of the tumor suppressor small interfering RNA is shown in SEQ ID NO: 1 (sense (5'-3'): GGGAGAUCUCAAGUUUCAATT), and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 2 (antisense (5'-3') UUGAAACUUGAGAUCUCCCTT).

[0010] The present invention also provides the application of small interfering RNA in the preparation of drugs for inhibiting tumors.

[0011] Furthermore, the tumor is liver cancer.

[0012] Furthermore, the drug is used to inhibit the proliferation, migration, and / or invasion of tumor cells.

[0013] Furthermore, the drug exerts its effect by simultaneously inhibiting the expression of the PTTG1 and STMN1 genes in tumor cells.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention designs and verifies for the first time a single-sequence siRNA (SEQ ID NO: 1 / 2) that can simultaneously, efficiently and specifically silence PTTG1 and STMN1, two key oncogenes that dominate the proliferation and metastasis processes in tumor development. This design concept is fundamentally different from the existing siRNAs that target only a single gene, and solves the core problem of limited efficacy of single-target therapy in the context of multi-gene synergistic pathogenesis.

[0015] 2. The solution of the present invention produces significant synergistic effects. Experiments have shown that the small interfering RNA of the present invention has a significantly stronger inhibitory effect on the growth of liver cancer cells than a single siRNA that targets only PTTG1 or only STMN1, demonstrating the synergistic anti-proliferative effect brought about by dual targeting.

[0016] 3. The small interfering RNA of the present invention is significantly better than the scheme of physically mixing two siRNAs that target PTTG1 and STMN1 respectively in terms of inhibiting cell growth and inducing cell apoptosis. This indicates that the scheme of the present invention brings significantly improved pharmacokinetic properties and efficacy.

[0017] 4. In various liver cancer cell lines with different biological characteristics (such as LM3, Huh7, and PLC5), the small interfering RNA of the present invention can effectively reduce the expression of PTTG1 and STMN1 and significantly inhibit cell colony formation and growth, demonstrating broad applicability and potential clinical application value.

[0018] 5. The solution of the present invention has a long-lasting and stable effect in vivo. In animal models, the small interfering RNA of the present invention exhibits a sustained tumor inhibition effect for up to 4 weeks, overcoming the shortcomings of traditional siRNA, which is easily and rapidly cleared in vivo and has a short duration of action, thus providing a favorable basis for the formulation of clinical dosing regimens. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the hypothetical binding sites of the small interfering RNA (siRNA-PTTG1 & STMN1) targeting PTTG1 and STMN1 mRNA in this invention; Figure 2 In this embodiment of the invention, siRNA-PTTG1&STMN1 simultaneously inhibits the expression of PTTG1 and STMN1 (A: RT-qPCR detection of the relative expression levels of PTTG1 and STMN1 in PLC5 cells transfected with siRNA-PTTG1&STMN1 for 72 hours; B: RT-qPCR detection of the relative expression levels of PTTG1 and STMN1 in PLC5 cells transfected with siRNA-PTTG1-1 for 72 hours; C: RT-qPCR detection of the relative expression levels of PTTG1 and STMN1 in PLC5 cells transfected with siRNA-PTTG1-2 for 72 hours; D: RT-qPCR detection of the relative expression levels of PTTG1 and STMN1 in PLC5 cells transfected with siRNA-STMN1-1 for 72 hours; E: RT-qPCR detection of the relative expression levels of PTTG1 and STMN1 in PLC5 cells transfected with siRNA-STMN1-2 for 72 hours). Figure 3 In this embodiment of the invention, siRNA-PTTG1&STMN1 exhibits a more efficient tumor-suppressive effect than other siRNAs (A: CCK-8 assay growth curves of PLC5 cells transfected with siRNA-PTTG1&STMN1, siRNA-PTTG1-1, and siRNA-PTTG1-2 at 0h, 24h, 48h, and 72h; B: CCK-8 assay growth curves of PLC5 cells transfected with siRNA-PTTG1&STMN1, siRNA-STMN1-1, and siRNA-STMN1-2 at 0h, 24h, 48h, and 72h). Figure 4In the embodiments of this invention, siRNA-PTTG1&STMN1 showed a more efficient inhibitory effect on liver cancer growth and a higher effect on promoting liver cancer cell apoptosis than the combination of two siRNA-PTTG1 and siRNA-STMN1 (A: LM3 cells transfected with siRNA-PTTG1&STMN1 and transfected with the combination of siRNA-PTTG1-1 and siRNA-STMN1-1 at 0, 24, ...). A: Growth at 48 and 72 hours; B: Statistical graph of the growth and fusion rate of LM3 cells transfected with siRNA-PTTG1&STMN1 and transfected with superimposed siRNA-PTTG1-1 and siRNA-STMN1-1 at 0, 24, 48, and 72 hours; C: Statistical graph of apoptosis at 72 hours of LM3 cells transfected with siRNA-PTTG1&STMN1 and transfected with superimposed siRNA-PTTG1-1 and siRNA-STMN1-1; D: Apoptosis rate of LM3 cells transfected with siRNA-PTTG1&STMN1 and transfected with superimposed siRNA-PTTG1-1 and siRNA-STMN1-1 detected by flow cytometry at 72 hours. Figure 5 In this embodiment of the invention, siRNA-PTTG1 & STMN1 reduced the expression of PTTG1 and STMN1 in various cell types and inhibited the growth of tumor cells (A: RT-qPCR detection of the relative expression levels of PTTG1 and STMN1 in LM3 cells transfected with siRNA-PTTG1 & STMN1 for 72 hours; B: RT-qPCR detection of the relative expression levels of PTTG1 and STMN1 in Huh7 cells transfected with siRNA-PTTG1 & STMN1 for 72 hours; C: RT-qPCR detection of the relative expression levels of PTTG1 and STMN1 in PLC5 cells transfected with siRNA-PTTG1 & STMN1 for 72 hours; D: Western blotting detection of the relative expression levels of PTTG1 and STMN1 in PLC5 cells transfected with siRNA-PTTG1 & STMN1). E: Protein expression levels of PTTG1 and STMN1 in LM3, Huh7, and PLC5 cells after 72 hours; F: Colony formation ability of LM3, Huh7, and PLC5 cells after transfection with siRNA-PTTG1 & STMN1, as determined by plate colony formation assay; G: Growth of LM3, Huh7, and PLC5 cells after 72 hours of transfection with siRNA-PTTG1 & STMN1; G: Statistics on the growth and fusion rate of LM3, Huh7, and PLC5 cells after 72 hours of transfection with siRNA-PTTG1 & STMN1. Figure 6This invention demonstrates that siRNA-PTTG1 & STMN1 significantly inhibited tumor growth in vivo for an extended period (A: Tumor bearing in mice with LM3 cells transfected with siRNA-PTTG1 & STMN1 for 4 weeks; B: Tumor size in mice with LM3 cells transfected with siRNA-PTTG1 & STMN1 for 4 weeks; C: Tumor weight statistics in mice with LM3 cells transfected with siRNA-PTTG1 & STMN1 for 4 weeks). Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0021] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0022] This invention provides a tumor-suppressing small interfering RNA that simultaneously targets the oncogenes PTTG1 and STMN1, and its applications, aiming to overcome the limitations of existing single-target siRNA technologies and achieve more effective synergistic inhibition of tumor proliferation and metastasis. Through its unique sequence design, this siRNA molecule can simultaneously target and efficiently inhibit the expression of the oncogenes PTTG1 and STMN1, thereby producing a synergistic anti-tumor effect. Its efficacy in inhibiting tumor growth, metastasis, and inducing apoptosis is significantly superior to that of a single-target siRNA or a simple physical mixture of two siRNAs.

[0023] In this invention, small interfering RNAs (siRNA-PTTG1 & STMN1) with specific sequences are constructed to achieve simultaneous targeted inhibition of the oncogenes PTTG1 and STMN1. The sequences of the specific small interfering RNAs (siRNA-PTTG1 & STMN1) constructed in this invention are as follows: name sense (5'-3') antisense (5'-3') siRNA-PTTG1&STMN1 GGGAGAUCUCAAGUUUCAATT UUGAAACUUGAGAUCUCCCTT The small interfering RNA (siRNA-PTTG1 & STMN1) constructed in this invention can simultaneously target the oncogene PTTG1, which is associated with tumor proliferation, and the oncogene STMN1, which is associated with tumor metastasis. Its specific binding sites are as follows: Figure 1 As shown, it can significantly inhibit the expression levels of PTTG1 and STMN1 ( Figure 2 The small interfering RNAs (siRNA-PTTG1 & STMN1) constructed in this invention have higher tumor suppression efficiency and can significantly inhibit the growth of tumor cells. Figure 3Furthermore, compared to the method of superimposing two small interfering RNAs, the small interfering RNA of this invention has a better inhibitory effect on liver cancer growth and a higher efficiency in inducing tumor apoptosis. Figure 4 ).

[0024] Furthermore, the small interfering RNAs (siRNA-PTTG1 & STMN1) constructed in this invention can significantly inhibit the proliferation and tumorigenicity of tumor cells in multiple cell types. Figure 5 The small interfering RNAs (siRNA-PTTG1 & STMN1) constructed in this invention also exhibit significant inhibitory effects on tumor growth in vivo. Figure 6 The following is a specific implementation of the present invention.

[0025] Example 1: siRNA-PTTG1 & STMN1 can simultaneously target the oncogenes PTTG1 and STMN1 Small interfering RNA (siRNA-PTTG1 & STMN1) was chemically synthesized using the sequence “sense (5'-3'): GGGAGAUCUCAAGUUUCAATT; antisense (5'-3') UUGAAACUUGAGAUCUCCCTT” constructed in this invention. The target binding sites of the synthesized siRNA (siRNA-PTTG1 & STMN1) for PTTG1 and STMN1 are as follows: Figure 1 As shown, siRNA-PTTG1 & STMN1 can be used for tumor intervention therapy. Chemically synthesized small interfering RNA (siRNA-PTTG1 & STMN1) was mixed with transfection reagents to prepare a mixture. This mixture was then added to the culture medium of PLC5 liver cancer cells plated 24 hours in advance. After culturing for 72 hours, total RNA was extracted from the liver cancer cells, and the expression of oncogenes PTTG1 and STMN1 was detected by real-time quantitative PCR. The results showed that the small interfering RNA (siRNA-PTTG1 & STMN1) constructed in this invention can significantly inhibit the expression of oncogenes PTTG1 and STMN1 simultaneously. Figure 2 (A) and other small interfering RNAs (siRNA-PTTG1-1, siRNA-PTTG1-2, siRNA-STMN1-1, siRNA-STMN1-2) that have sequences different from those of this invention cannot simultaneously inhibit the expression of PTTG1 and STMN1. Figure 2 (B, C, D, E). siRNA-PTTG1-1 and siRNA-PTTG1-2 can only inhibit PTTG1 expression, but cannot inhibit STMN1 expression. Figure 2(B, C). siRNA-STMN1-1 and siRNA-STMN1-2 can only inhibit STMN1 expression, not PTTG1 expression. Only the siRNA-PTTG1&STMN1 constructed in this invention can simultaneously target PTTG1 and STMN1. Figure 1 ), and significantly suppressed the expression levels of both PTTG1 and STMN1 simultaneously. Figure 2 A) Example 2: siRNA-PTTG1 & STMN1 have a more efficient effect in inhibiting tumor growth. siRNA-PTTG1 & STMN1 were synthesized according to the method in Example 1 and prepared into a mixture. This mixture was added to the culture medium of PLC5 liver cancer cells. Cell growth curves were plotted using CCK-8 reagent at 0 h, 24 h, 48 h, and 72 h of culture. The results showed that the siRNA-PTTG1 & STMN1 constructed in this invention had a more efficient tumor cell growth inhibition effect. Figure 3 (A, B), and siRNA-PTTG1-1 and siRNA-PTTG1-2 that target PTTG1 only. Figure 3 (A) and siRNA-STMN1-1 and siRNA-STMN1-2 that target STMN1 only. Figure 3 Compared to (B) in this invention, the siRNA-PTTG1&STMN1 constructed in this invention simultaneously targets PTTG1 and STMN1, resulting in slower growth of liver cancer cells and a more significant inhibitory effect on liver cancer cell growth. Figure 3 (A, B) Example 3: Small interfering RNA siRNA-PTTG1 & STMN showed higher anti-cancer effects than the combination of two siRNAs. Chemically synthesized small interfering RNAs (siRNA-PTTG1 & STMN1) were mixed with transfection reagents to prepare a mixture named siRNA-PTTG1 & STMN, which was then added to the culture medium of LM3 liver cancer cells plated 24 hours prior. Simultaneously, two siRNAs, PTTG1-1 and siRNA-STMN1-1, were mixed with transfection reagents to prepare a mixture of two superimposed siRNAs, which was also added to the culture medium of LM3 liver cancer cells plated 24 hours prior. The growth of LM3 liver cancer cells was photographed and recorded at 0, 24, 48, and 72 hours post-transfection, and LM3 cells were collected at 72 hours for apoptosis detection. The results showed that the siRNA-PTTG1 & STMN1 constructed in this invention had a more efficient inhibitory effect on liver cancer cell growth compared to the method of superimposing multiple siRNAs. Figure 4 (A and B in the text), and also has a more efficient effect in promoting apoptosis of liver cancer cells ( Figure 4 (C, D in the original text). Compared with the simultaneous superposition of two (siRNA-PTTG1-1, siRNAASTMN1-1), the siRNA-PTTG1&STMN constructed in this invention has a more significant inhibitory effect on the growth of liver cancer cells. Figure 4 In cases A and B), the apoptosis rate of liver cancer cells was higher, reaching over 30%, while the apoptosis rate was only 20% with the combination of two siRNAs. Figure 4 (C, D in the original text). This invention constructs an siRNA that simultaneously targets two oncogenes, exhibiting a significant inhibitory effect on the growth of liver cancer cells. Compared to methods that simultaneously superimpose two siRNAs, this method demonstrates higher tumor suppression efficiency and fewer side effects. The method of superimposing two siRNAs suffers from competitive binding during cellular absorption, resulting in lower tumor growth inhibition efficiency and liver cancer cell apoptosis-promoting efficiency compared to the tumor-suppressing small interfering RNA constructed in this invention.

[0026] Example 4: siRNA-PTTG1 & STMN1 can inhibit the expression of PTTG1 and STMN1 in various liver cancer cells and suppress the growth of various liver cancer cells. siRNA-PTTG1 & STMN1 were synthesized according to the method in Example 1 and prepared into a mixture. This mixture was added to the culture medium of different hepatocellular carcinoma cells (LM3, Huh7, PLC5). 72 hours after the addition of siRNA-PTTG1 & STMN1, quantitative real-time PCR and Western blotting were performed to detect the expression of PTTG1 and STMN1 in the three types of hepatocellular carcinoma cells. After adding the siRNA-PTTG1 & STMN1 mixture to the cells, a plate colony assay was performed on the three types of hepatocellular carcinoma cells. 72 hours after adding the siRNA-PTTG1 & STMN1 mixture, the growth of the three types of hepatocellular carcinoma cells was observed and statistically analyzed. The results showed that the siRNA-PTTG1 & STMN1 constructed in this invention can target and reduce the expression levels of PTTG1 and STMN1 in various cell types. Figure 5 (A, B, C, D in the original text). The siRNA-PTTG1 & STMN1 constructed in this invention can inhibit the clonogenic ability of liver cancer cells in various cell types. Figure 5 The siRNA-PTTG1 & STMN1 constructed in this invention significantly inhibited the growth of liver cancer cells in various cell types. Figure 5 (F, G).

[0027] Example 5: siRNA-PTTG1 & STMN1 can inhibit tumor growth in vivo for a long time. siRNA-PTTG1 & STMN1 were synthesized according to the method in Example 1 and prepared into a mixture. This mixture was added to the culture medium of LM3 liver cancer cells. After 48 hours, the cells were digested and subcutaneously inoculated into nude mice, and tumor growth was recorded. At week 4, the tumors were removed, photographed, weighed, and their growth was compared. The results showed that compared with the control group (NC), siRNA-PTTG1 & STMN1 significantly inhibited tumor growth. Figure 6 (A, B, C in the original text).

[0028] Through the above embodiments of the present invention, the present invention has innovatively designed and verified a single siRNA molecule that can simultaneously and efficiently silence two key and functionally complementary oncogenes (PTTG1 and STMN1); the above embodiments demonstrate that the technical solution of the present invention produces a significant synergistic anti-tumor effect, which is superior to single-target strategies and simple physical mixing strategies, and has broad applicability and long-lasting effect in vivo.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tumor suppressor small interfering RNA that simultaneously targets oncogenes PTTG1 and STMN1, characterized in that, The nucleotide sequence of the sense strand of the tumor suppressor small interfering RNA is shown in SEQ ID NO: 1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:

2.

2. The use of the small interfering RNA according to claim 1 in the preparation of a drug for inhibiting tumors.

3. The application according to claim 2, characterized in that, The tumor is liver cancer.

4. The application according to claim 2 or 3, characterized in that, The drug is used to inhibit the proliferation, migration, and / or invasion of tumor cells.

5. The application according to any one of claims 2-4, characterized in that, The drug works by simultaneously inhibiting the expression of the PTTG1 and STMN1 genes in tumor cells.

Images