Application of ALKBH5 in inhibition of expression of colorectal cancer PHF20

Abstract

The invention provides application of ALKBH5 in inhibition of expression of colorectal cancer PHF20, which comprises the following steps: step 1, selecting a proper ALKBH5 overexpression vector, knocking down PHF20, and regulating the activity of ALKBH5 by using an ALKBH5 micromolecule activator or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and a Cas9 system, so as to obviously inhibit proliferation and metastasis of colorectal cancer cells; 2, the regulatory element is delivered to colorectal cancer cells or tumor tissues in a delivery mode including viral vectors, lipidosome, local injection or intravenous injection, so that the treatment accuracy and effectiveness are improved; and 3, detecting the change of the expression levels of ALKBH5 and PHF20, and evaluating the inhibition effect of tumor proliferation, metastasis or drug resistance. The core of the invention is to confirm the effect of ALKBH5 in inhibiting expression of PHF20 in colorectal cancer cells, and proliferation, metastasis and drug resistance of the colorectal cancer cells are inhibited through various regulation and control means and delivery systems. In addition, the invention further provides a corresponding pharmaceutical composition and a diagnosis evaluation method, and a new strategy is provided for treatment and prognosis of colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor biology, and particularly relates to a use of ALKBH5 in inhibiting the expression of PHF20 in colorectal cancer. Background Art

[0002] With the rapid development of molecular biology techniques, researchers have begun exploring the mechanisms of colorectal cancer development and progression at the genetic level. The interaction between ALKBH5 and PHF20, genes closely associated with colorectal cancer, has become a research hotspot, providing new insights and directions for colorectal cancer treatment.

[0003] ALKBH5 is an important DNA repair enzyme and a member of the AlkB family. It specifically recognizes and repairs DNA damage caused by alkylating agents, playing a key role in maintaining genetic stability and normal cellular function. Studies have shown that abnormal ALKBH5 expression in colorectal cancer is closely associated with tumor development and progression. ALKBH5 may inhibit PHF20 expression, providing a potential new target for colorectal cancer treatment.

[0004] Given the critical role of ALKBH5 in DNA repair and its potential impact on PHF20 expression, it is hoped that by regulating the activity or expression level of ALKBH5, the expression of PHF20 in colorectal cancer cells can be effectively reduced, thereby inhibiting tumor cell proliferation, invasion, and metastasis, providing more effective and safe treatment options for colorectal cancer patients. This may also provide new ideas and methods for the treatment of other related cancers. Summary of the Invention

[0005] The present invention proposes a use of ALKBH5 in inhibiting the expression of PHF20 in colorectal cancer, which solves the problems in the prior art.

[0006] The technical solution of the present invention is achieved as follows: A use of ALKBH5 in inhibiting the expression of PHF20 in colorectal cancer comprises the following steps: Step 1: Select an appropriate ALKBH5 overexpression vector, knock down PHF20, and use an ALKBH5 small molecule activator or CRISPR / Cas9 system to regulate ALKBH5 activity, thereby significantly inhibiting the proliferation and metastasis of colorectal cancer cells; Step 2: Delivering the regulatory elements to colorectal cancer cells or tumor tissues using methods including viral vectors, liposomes, local injection, or intravenous injection to improve the precision and effectiveness of treatment; Step 3: Detect changes in ALKBH5 and PHF20 expression levels and evaluate the inhibitory effects on tumor proliferation, metastasis, or drug resistance to verify the feasibility of the treatment strategy.

[0007] As a preferred embodiment, the regulatory elements of ALKBH5 include: An adenoviral overexpression vector comprising the ALKBH5 coding sequence of SEQ ID NO: 1, which can effectively enhance the expression level of ALKBH5 in cancer cells; The CRISPR activation system, which includes dCas9 protein and sgRNA targeting the ALKBH5 promoter, can precisely regulate the activity of ALKBH5; Small molecule activators, identified through a high-throughput screening system, act on the m6A demethylation active site of ALKBH5 to enhance therapeutic efficacy.

[0008] The above scheme uses ALKBH5 overexpression vector, CRISPR, Cas9 activation system, and high-throughput screening of small molecule activators to achieve multi-dimensional regulation of ALKBH5 activity, combined with the strategy of knocking down PHF20 to synergistically inhibit the expression of colorectal cancer-related genes.

[0009] As a preferred embodiment, the colorectal cancer cells are tumor cell lines cultured in vitro or primary tumor cells derived from patients, and the tumor tissue is obtained by biopsy or surgical resection.

[0010] As a preferred embodiment, the detection means include: real-time fluorescence quantitative PCR, immunohistochemistry or RNA sequencing, and the evaluation index is the change in the ALKBH5 / PHF20 expression ratio or the suppression of the tumor malignant phenotype.

[0011] The above scheme was adopted, and real-time fluorescence quantitative PCR, immunohistochemistry, RNA sequencing and other technologies were used to quantitatively evaluate the changes in ALKBH5 and PHF20 expression and the inhibitory effect on tumor malignant phenotype, so as to achieve dynamic monitoring of the treatment process.

[0012] A pharmaceutical composition for treating colorectal cancer, comprising a regulatory element, mixed with a pharmaceutically acceptable carrier, and combined with at least one of the following treatments: (1) Oxaliplatin, to enhance the killing effect on cancer cells; (2) Immune checkpoint inhibitors to enhance the patient's immune system's ability to recognize and eliminate tumors; (3) siRNA or inhibitors targeting PHF20 to further inhibit tumor growth and metastasis; As a preferred embodiment, the immune checkpoint inhibitor is a PD-L1 inhibitor.

[0013] Using the above scheme, ALKBH5 regulatory elements are combined with multiple treatment methods (oxaliplatin chemotherapy, PD-L1 immune checkpoint inhibitors, siRNA / inhibitors targeting PHF20) to form a synergistic anti-tumor effect, enhance efficacy and overcome drug resistance.

[0014] As a preferred embodiment, the preparation method thereof comprises: S1. Co-encapsulate the ALKBH5 overexpression vector and chemotherapy drugs into pH-sensitive nanoparticles to improve drug targeting and release efficiency; S2. Design a bispecific antibody that couples an ALKBH5 activator and a PD-L1 inhibitor to achieve multiple therapeutic effects.

[0015] After adopting the above technical solution, the beneficial effects of the present invention are: through the direct inhibition of PHF20 expression by ALKBH5, the proliferation, invasion and metastasis ability of colorectal cancer cells are effectively blocked, and the malignancy of the tumor is reduced; delivery systems such as viral vectors, nanoparticles and bispecific antibodies can achieve local precise drug delivery to the tumor, reduce systemic drug side effects, and improve treatment safety; a multi-strategy combination of chemotherapy, immunotherapy and targeting PHF20 can break the tumor resistance mechanism and enhance the durability of the overall treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0017] Figure 1 Graph A shows the clinical pathological characteristics of ALKBH5 patients analyzed based on mRNA levels in the present invention; Figure 2 Bar graph B shows the clinical pathological characteristics of ALKBH5 patients analyzed based on mRNA levels in the present invention; Figure 3 Bar graph C shows the clinical pathological characteristics of ALKBH5 patients analyzed by protein level in the present invention; Figure 4 The bar graph D is the clinical pathological characteristics of ALKBH5 patients analyzed by protein level in the present invention; DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention. Example

[0019] like Figure 1-4 As shown, a use of ALKBH5 in inhibiting the expression of PHF20 in colorectal cancer comprises the following steps: Step 1: Select an appropriate ALKBH5 overexpression vector, knock down PHF20, and use an ALKBH5 small molecule activator or CRISPR / Cas9 system to regulate ALKBH5 activity, thereby significantly inhibiting the proliferation and metastasis of colorectal cancer cells; Step 2: Delivering the regulatory elements to colorectal cancer cells or tumor tissues using methods including viral vectors, liposomes, local injection, or intravenous injection to improve the precision and effectiveness of treatment; Step 3: Detect changes in ALKBH5 and PHF20 expression levels and evaluate the inhibitory effects on tumor proliferation, metastasis, or drug resistance to verify the feasibility of the treatment strategy.

[0020] Among them, the regulatory elements of ALKBH5 include: An adenoviral overexpression vector comprising the ALKBH5 coding sequence of SEQ ID NO: 1, which can effectively enhance the expression level of ALKBH5 in cancer cells; The CRISPR activation system, which includes dCas9 protein and sgRNA targeting the ALKBH5 promoter, can precisely regulate the activity of ALKBH5; Small molecule activators, identified through a high-throughput screening system, act on the m6A demethylation active site of ALKBH5 to enhance therapeutic efficacy.

[0021] Furthermore, ALKBH5 overexpression vectors, CRISPR, Cas9 activation systems, and high-throughput screened small molecule activators were used to achieve multi-dimensional regulation of ALKBH5 activity, combined with the strategy of knocking down PHF20 to synergistically inhibit the expression of colorectal cancer-related genes.

[0022] Among them, colorectal cancer cells are tumor cell lines cultured in vitro or primary tumor cells from patients, and tumor tissues are obtained by biopsy or surgical resection.

[0023] Among them, the detection methods include: real-time fluorescence quantitative PCR, immunohistochemistry or RNA sequencing, and the evaluation indicators are changes in the ALKBH5 / PHF20 expression ratio or the inhibition of tumor malignant phenotype.

[0024] Furthermore, real-time fluorescence quantitative PCR, immunohistochemistry, RNA sequencing and other technologies were used to quantitatively evaluate the changes in ALKBH5 and PHF20 expression and the inhibitory effect on tumor malignant phenotypes, thereby achieving dynamic monitoring of the treatment process. Example

[0025] A pharmaceutical composition for treating colorectal cancer, comprising an ALKBH5 overexpression vector mixed with pharmaceutically acceptable nanoparticles, and combined with at least one of the following treatments: (1) Oxaliplatin, to enhance the killing effect on cancer cells; (2) Immune checkpoint inhibitors to enhance the patient's immune system's ability to recognize and eliminate tumors; (3) siRNA or inhibitors targeting PHF20 to further inhibit tumor growth and metastasis; Among them, the immune checkpoint inhibitor is PD-L1 inhibitor.

[0026] Furthermore, ALKBH5 regulatory elements are combined with multiple therapeutic approaches to form a synergistic anti-tumor effect, enhance efficacy and overcome drug resistance.

[0027] The preparation method includes: S1. Co-encapsulate the ALKBH5 overexpression vector and chemotherapy drugs into pH-sensitive nanoparticles to improve drug targeting and release efficiency; S2. Design a bispecific antibody that couples an ALKBH5 activator and a PD-L1 inhibitor to achieve multiple therapeutic effects.

[0028] The core of this invention is to demonstrate the role of ALKBH5 in inhibiting PHF20 expression in colorectal cancer cells. Through various regulatory approaches and delivery systems, the invention suppresses the proliferation, metastasis, and drug resistance of colorectal cancer cells. Furthermore, corresponding pharmaceutical compositions and diagnostic evaluation methods are provided, offering new strategies for the treatment and prognosis of colorectal cancer.

[0029] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A use of ALKBH5 in inhibiting the expression of PHF20 in colorectal cancer, characterized in that: The following steps are involved: Step 1: Select an appropriate ALKBH5 overexpression vector, knock down PHF20, and use an ALKBH5 small molecule activator or CRISPR / Cas9 system to regulate ALKBH5 activity; Step 2: delivering the regulatory element to colorectal cancer cells or tumor tissues using methods including viral vectors, liposomes, local injection, or intravenous injection; Step 3: Detect changes in ALKBH5 and PHF20 expression levels to evaluate the inhibitory effects on tumor proliferation, metastasis, or drug resistance.

2. The use of ALKBH5 in inhibiting the expression of PHF20 in colorectal cancer according to claim 1, characterized in that: The regulatory elements of ALKBH5 include: An adenoviral overexpression vector comprising the ALKBH5 coding sequence of SEQ ID NO: 1; CRISPR activation system, consisting of dCas9 protein and sgRNA targeting the ALKBH5 promoter; Small molecule activators, identified by a high-throughput screening system, act on the m6A demethylation active site of ALKBH5.

3. The use of ALKBH5 in inhibiting the expression of PHF20 in colorectal cancer according to claim 1, characterized in that: The colorectal cancer cells are tumor cell lines cultured in vitro or primary tumor cells derived from patients, and the tumor tissue is obtained by biopsy or surgical resection.

4. The use of ALKBH5 in inhibiting the expression of PHF20 in colorectal cancer according to claim 1, characterized in that: The detection means include: real-time fluorescence quantitative PCR, immunohistochemistry or RNA sequencing, and the evaluation index is the change in the ALKBH5 / PHF20 expression ratio or the inhibition of the tumor malignant phenotype.

5. A pharmaceutical composition for treating colorectal cancer, comprising the regulatory element of claim 1 or 2, mixed with a pharmaceutically acceptable carrier, and combined with at least one of the following treatments: (1) Oxaliplatin; (2) immune checkpoint inhibitors; (3) siRNA or inhibitors targeting PHF20.

6. A pharmaceutical composition for treating colorectal cancer according to claim 5, characterized in that: The immune checkpoint inhibitor is a PD-L1 inhibitor.

7. The pharmaceutical composition for treating colorectal cancer according to claim 5, characterized in that: The preparation method thereof comprises: S1, co-encapsulating the ALKBH5 overexpression vector and chemotherapy drugs into pH-sensitive nanoparticles; S2. Design a bispecific antibody that couples an ALKBH5 activator and a PD-L1 inhibitor.

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