A nfe2l1 protein ntd polypeptide and use thereof in the preparation of an anti-tumor drug

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an NTD polypeptide of NFE2L1 protein and application thereof in preparation of an antitumor drug. The amino acid sequence of the NTD polypeptide monomer is shown as SEQ ID NO:1. The application constructs the polypeptide of the NTD amino acid sequence of NFE2L1, and it is unexpectedly found that the NTD polypeptide has good anticancer function, and it is verified through a tumor-bearing experiment of an immunodeficient mouse that the NTD polypeptide provided by the application has a significant effect of inhibiting tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an NTD polypeptide of NFE2L1 protein and its use in the preparation of antitumor drugs. Background Technology

[0002] NFE2L1 (Nuclear Factor, Erythroid 2-Like 1) is a transcription factor belonging to the NFE2L family, involved in important biological processes such as intracellular oxidative stress, cell cycle, and insulin homeostasis. The full-length NFE2L1 protein is approximately 120-140 kDa and contains 772 amino acids. The NFE2L1 protein contains key functional structural regions including the N-terminal region (NTD), transcription activation domain 1 (AD1), transcription activation domain 2 (AD2), DNA-binding domain (DBD), and C-terminal region (CTD).

[0003] The NTD domain of NFE2L1 is its anchoring structure in the cell membrane system. Removing the NTD facilitates the entry of NFE2L1 protein into the cell nucleus, while the function of the remaining NTD structure is currently unknown. The NFE2L1 gene is widely expressed in various human tissues and organs and is highly sensitive to environmental factors. Radiation, nutrients, metabolites, various drugs or heavy metal oxidative stimulation, and even environmental temperature can induce differential expression of the NFE2L1 gene. The molecular functions of NFE2L1 involve regulating redox homeostasis, glucose and lipid metabolism homeostasis, and protein turnover. Its physiological functions include antioxidant detoxification, cell proliferation and apoptosis, tissue and organ development and functional specialization, cancer development, inflammatory responses, and immune responses. Its function in chronic systemic diseases such as chronic inflammation, cancer, diabetes, obesity, and neurodegenerative diseases suggests that this protein has potential clinical application value. However, because transcription factors directly regulate the expression of a large number of downstream genes and involve a wide range of biological functions, they are often not suitable as target molecules for clinical application research. Similarly, simply overexpressing or knocking down (activating or inhibiting) NFE2L1 can lead to systemic diseases such as organ dysplasia, neurodegenerative diseases, and diabetes.

[0004] Existing molecular theoretical models such as the NFE2L1 protein modification and cleavage regulation theory and the multi-subtype model are all based on its antioxidant transcription factor function. No research on the possible non-transcription factor functions of NFE2L1 has been found in the available literature. Summary of the Invention

[0005] To address the aforementioned technical problems, the inventors of this invention constructed a polypeptide of the NTD amino acid sequence of NFE2L1 and verified that the NTD polypeptide could be normally expressed by using Western blotting. Consequently, they unexpectedly discovered that the polypeptide represented by SEQ ID NO:1 of the NTD domain of NFE2L1 could significantly inhibit tumor growth.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] In a first aspect, the present invention provides an NTD polypeptide of NFE2L1 protein, wherein the amino acid sequence of the NTD polypeptide monomer is shown in SEQ ID NO:1.

[0008] In this patent, the inventors took a unique approach, constructing a polypeptide with an NTD amino acid sequence (equivalent to amino acid regions 1-130 of the NFE2L1 protein, i.e., the NTD domain of the full-length NFE2L1 protein). They then verified the normal expression of the NTD polypeptide using Western blotting, thus innovatively discovering the anti-cancer function of the NTD domain of NFE2L1. The inventors conducted tumor-bearing experiments in immunodeficient mice using HepG2 cell lines stably overexpressing the NTD polypeptide, and the results showed that the NTD polypeptide significantly inhibited tumor growth.

[0009] Furthermore, the NTD polypeptide also includes a polypeptide derived from SEQ ID NO:1 that has antitumor function by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO:1.

[0010] Derived NTD peptides can maintain or even enhance their activity and specificity against tumor cells by modifying, substituting, deleting, or adding amino acid sequences of the NTD peptide with the amino acid sequence SEQ ID NO:1, making them suitable as potential anti-tumor drugs or treatments.

[0011] Secondly, the present invention also provides the encoding gene of the above-mentioned polypeptide.

[0012] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:2.

[0013] Thirdly, the present invention provides a recombinant vector, transgenic cell line or recombinant bacteria encoding the above-mentioned gene.

[0014] Further, the recombinant vector is obtained by inserting the coding gene of the NTD polypeptide with the above-mentioned amino acid sequence SEQ ID NO:1 into the pLVX-EGFP vector to obtain a recombinant vector expressing the polypeptide.

[0015] Furthermore, the transgenic cell line is obtained by introducing the recombinant vector into a host cell, wherein the host cell is a liver cancer tissue cell.

[0016] Furthermore, the liver cancer tissue cells are HepG2 cells.

[0017] Through experimental research, the inventors discovered that by inserting the gene encoding the NTD polypeptide described in this invention into the pLVX-EGFP vector to prepare a recombinant vector expressing the polypeptide, and then infecting HepG2 liver cancer cells with this vector, the NTD polypeptide was found to be expressed normally in the cells. Furthermore, when the infected HepG2 cells were implanted into mice to form artificial hepatocellular carcinoma, the expressed NTD polypeptide was found to significantly inhibit tumor growth.

[0018] Fourthly, the present invention also provides the use of the polypeptide, the gene encoding the polypeptide, the recombinant vector or transgenic cell line in the preparation of antitumor drugs.

[0019] Furthermore, the tumor is liver cancer.

[0020] Fourthly, the present invention also provides an antitumor drug, the active ingredient of which is the polypeptide, the encoding gene, the recombinant vector or the transgenic cell line.

[0021] Furthermore, the tumor is a liver cancer cell, and the liver cancer cell is particularly preferably HepG2 cells.

[0022] Furthermore, the antitumor drug is a lyophilized powder or a liquid formulation.

[0023] Furthermore, the drug also includes a pharmaceutically acceptable carrier. Those skilled in the art can routinely select a pharmaceutically acceptable carrier based on the drug's dosage form, etc.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention, through the construction of a polypeptide with the NTD amino acid sequence of NFE2L1, unexpectedly discovered that the NTD polypeptide has good anti-cancer function. This was confirmed by tumor-bearing experiments in immunodeficient mice. The NTD polypeptide provided by this invention has a significant inhibitory effect on tumor growth. Therefore, this NTD polypeptide may become a good anti-tumor drug with low side effects. Attached Figure Description

[0026] Figure 1 This is a graph showing the NTD peptide expression effect of the NFE2L1 protein in this invention.

[0027] Figure 2 shows the effect of stable overexpression of the NTD of the NFE2L1 protein in this invention on the subcutaneous tumorigenicity of tumor cells; wherein... Figure 2A This is a comparison image of tumor volume; Figure 2B The tumor-bearing tissue was removed and photographed for comparison at 36 days after tumor formation. The scale bar in the image is 1 cm. Figure 2C This is a statistical chart showing tumor quality after 36 days of tumor bearing. Detailed Implementation

[0028] The following detailed embodiments illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments.

[0029] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0030] Example 1

[0031] This embodiment provides the process of constructing and validating the NTD peptide expression plasmid for the NFE2L1 protein. The specific steps are as follows:

[0032] 1. Total RNA was extracted from human cell lines using a total RNA extraction kit, and the total RNA was reverse transcribed into cDNA using a reverse transcription kit as a template for PCR reaction;

[0033] 2. The nucleic acid sequence encoding the NTD domain in the NFE2L1 gene was amplified using NTD amplification primers (NTD-F: 5'-CTCGAGGAATTCATGCTTTCTCTGAAGA AATAC-3'; NTD-R: 5'-TGTCACGGATCCGAGGCCACTGGAAC-3') and inserted into the pLVX-EGFP vector to obtain the NTD overexpression plasmid pLVX-NTD::EGFP.

[0034] 3. pLVX-EGFP and pLVX-NTD::EGFG plasmids were transfected into HepG2 cells. Total protein was collected 24 hours later and detected by Western blotting.

[0035] The results are as follows Figure 1 As shown, the nucleic acid sequence encoding the NTD domain in the NFE2L1 gene amplified by this invention can be normally expressed to generate NTD polypeptide.

[0036] Example 2

[0037] This embodiment investigated the effect of NTD overexpression on tumor cells bearing tumors subcutaneously in nude mice. The specific steps are as follows:

[0038] 1. Lentiviral cells overexpressing NTD and empty vector lentivirus were packaged using a lentiviral packaging system and infected with HepG2 cells. The control cell line pLVX and the NTD-overexpressing cell line pNTD were screened by culturing in puromycin-containing medium.

[0039] 2. After digesting the pLVX and pNTD cell lines, wash them twice with PBS, and then resuspend them in PBS to a density of 10 million cells per milliliter.

[0040] 3. Eight-week-old nude mice were randomly divided into two groups: pLVX and pNTD, with four mice in each group. Each mouse was subcutaneously injected with 1.5 million cells. Starting from the second day after tumor implantation, the length (a) and width (b) of the tumor-bearing tissue were recorded every other day. The changes in tumor volume were calculated and recorded according to the following formula. The results are as follows: Figure 2A As shown:

[0041] V (体积) = (a×b) 2 ) / 2

[0042] 4. When the largest tumor reaches a size of 1cm 3 At approximately 10:00 AM, the tumor-bearing mice were euthanized, and the tumors were removed and photographed (e.g., ...). Figure 2B (as shown) and weigh (e.g.) Figure 2C (As shown).

[0043] Figure 2A The -C results fully demonstrate that the NTD polypeptide generated by overexpression of the nucleic acid sequence encoding the NTD domain in the NFE2L1 gene can significantly inhibit tumor growth.

[0044] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An NTD polypeptide of NFE2L1 protein, characterized in that, The amino acid sequence of the NTD polypeptide monomer is shown in SEQ ID NO:

1.

2. A gene encoding a gene, characterized in that, The gene encoding the NTD polypeptide of claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

4. A recombinant vector, transgenic cell line, or recombinant bacterium containing the encoding gene as described in claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector is obtained by inserting the coding gene of the NTD polypeptide of claim 1 into the pLVX-EGFP vector to obtain a recombinant vector expressing the polypeptide.

6. The transgenic cell line according to claim 4, characterized in that, The transgenic cell line is obtained by introducing the recombinant vector of claim 5 into a host cell, wherein the host cell is a liver cancer tissue cell.

7. The use of the polypeptide as described in claim 1, the encoding gene as described in claim 2 or 3, the recombinant vector or transgenic cell line as described in claim 4 in the preparation of an antitumor drug, characterized in that, The tumor is liver cancer.

8. An antitumor drug, characterized in that, Its active ingredients are the polypeptide of claim 1, the encoding gene of claim 2 or 3, the recombinant vector or transgenic cell line of claim 4; the tumor is liver cancer.

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