Rnf130 gene, encoded protein and application responsive to bmaa signal

Abstract

The application discloses a kind of RNF130 gene responding BMAA signal, the protein coded and application, the present application is found by high-throughput screening, RNF130 is a gene that responds BMAA signal strongly.Knock out RNF130 gene, mammalian cell reduces the cell proliferation inhibition caused by BMAA.In addition, RNF 130 transduces BMAA signal to downstream GSK-3 beta protein, thereby with Wnt signal path produces association.GSK-3 beta inhibitor is used to inhibit GSK-3 beta activity after using small molecule, the reaction of RNF130 to BMAA signal changes, and cell can also remove the influence of cell proliferation inhibition caused by BMAA.So the knock-out of RNF130 gene and the inhibition of GSK-3 beta activity can remove the cell proliferation inhibition caused by BMAA, so the present application provides the key response protein and molecular mechanism of algal toxin BMAA to cause cell proliferation toxicity, provides important reference target for developing antidote for preventing or treating BMAA toxicity or developing drug for RNF130 related domain in the future.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the RNF130 gene, the encoded protein, and its applications in response to BMAA signaling, and more specifically to the cell proliferation inhibition effect caused by the response of the RNF130 gene, the encoded protein, and downstream signaling pathways to treatment with the algal toxin β-methylamino-L-alanine (BMAA). Background Technology

[0002] The algal toxin β-methylamino-L-alanine (BMAA), isolated from cycads in 1967, is believed to be a potential cause of Guam-type amyotrophic lateral sclerosis-Parkinson's syndrome-dementia complex (ALS-PDC). Treatment of mice, rats, chickens, and monkeys with BMAA via feeding and injection resulted in varying degrees of neuronal damage and neurodegenerative disease symptoms. Therefore, ingestion of BMAA carries a potential risk of inducing neurodegenerative diseases.

[0003] There are many hypotheses about the molecular mechanisms by which BMAA causes neuronal damage, including the formation of glutamate analogs by BMAA that cause neuronal excitotoxicity, and the erroneous incorporation of serine sites during protein synthesis. However, no receptor for BMAA has been found in animals so far, meaning that no receptor or protein has been found that responds to BMAA signals in the first instance.

[0004] In fact, BMAAs are widely distributed in the environment. Most algae, such as cyanobacteria, dinoflagellates, and diatoms, which constitute a large proportion of the biosphere, produce BMAAs. Furthermore, BMAAs can accumulate in large quantities in fish, shellfish, and even chickens through the food chain, causing contamination of human food. Ingesting these BMAAs may also lead to some degree of neurological damage in humans.

[0005] At the cellular level, BMAA treatment leads to inhibition of proliferation in both neural and non-neuronal cells. Cell proliferation inhibition is a manifestation of cellular damage. Using this phenotype, we identified a BMAA-responsive protein on the cell membrane through high-throughput screening. This protein responds to BMAA signaling in response to BMAA treatment; its absence essentially neutralizes the BMAA-induced inhibition of cell proliferation.

[0006] GSK-3β and the Wnt signaling pathway are crucial signaling pathways in cell proliferation. The discovery of the RNF130 gene linked BMAA signaling to the Wnt signaling pathway via GSK-3β, elucidating the mechanism by which BMAA induces cell proliferation inhibition.

[0007] Based on existing research at home and abroad, this invention focuses on the molecular mechanism of the lack of algal toxin BMAA toxicity. It has discovered that the cell surface protein RNF130 is highly responsive to BMAA signaling and identified the downstream signaling pathways related to the RNF130 response to BMAA signaling. This is of great significance for the development of related drugs and the prevention or treatment of acute or long-term nerve damage and related neurodegenerative diseases caused by BMAA. Summary of the Invention

[0008] The purpose of this invention is to provide an RNF130 gene that responds to BMAA signaling, the protein it encodes, and its applications.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] The RNF130 gene, whose nucleotides are shown in SEQ ID NO.1, consists of 1260 bases and encodes a protein including an extracellular domain, a transmembrane sequence, and an intracellular signal transduction domain. The protein is 419 amino acids in total, and its amino acid sequence is shown in SEQ ID NO.2.

[0011] The extracellular domain is encoded by the sequence SEQ ID NO.3, and the intracellular signal transduction domain is encoded by the sequence SEQ ID NO.4.

[0012] The sequence of the extracellular domain of the RNF130 protein is shown in SEQ ID NO. 5, and the sequence of the intracellular signal transduction domain is shown in SEQ ID NO. 6.

[0013] Due to post-transcriptional modifications, the RNF130 protein has multiple protein isoforms, including one or more isoforms that do not contain the complete extracellular sequence of SEQ ID NO.3, in addition to the full-length isoform. Furthermore, the protein encoded by RNF130 may also include RNF130-derived proteins with BMAA-responsive function formed by substitution, deletion, or addition of one or more (e.g., 1-30; preferably 1-20; more preferably 1-10, such as 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO.2; or RNF130-derived proteins with 80% (preferably more than 90%, such as 95%, 98%, 99% or higher) homology to the protein sequence of SEQ ID NO.2 and with BMAA-responsive function.

[0014] The most important inventive point of this invention is the disclosure of the response function of RNF130 to the algal toxin BMAA, as well as the downstream proteins and signaling pathways involved in this response. This invention obtains an RNF130 gene knockout mutant through gene editing. Utilizing the phenotype of BMAA-induced cell proliferation inhibition, cell proliferation counting revealed that the RNF130 gene knockout mutant reduces BMAA-induced cell proliferation inhibition. Truncating experiments confirmed that the extracellular structure of RNF130 includes the sequence portion of SEQ ID NO:3, which directly or indirectly contacts BMAA and responds to it; the intracellular structure of RNF130 includes the sequence portion of SEQ ID NO:4, which directly or indirectly interacts with GSK-3β, and this interaction affects the degradation of both RNF130 itself and conventional substrates of GSK-3β.

[0015] Therefore, this invention primarily protects the application of the RNF130 gene or protein and the cell lines expressing the gene or protein in the inhibition of cell proliferation induced by the algal toxin BMAA signal, wherein the cells include 293T cells and nerve cells. The response to the algal toxin BMAA signal is specifically manifested as follows: under the stimulation of the BMAA signal (i.e., when BMAA toxicity is present in the cells), the proliferation rate of cells containing the RNF130 gene slows down; after inhibiting the expression of the RNF130 gene, inhibiting the activity of the RNF130 protein, or disrupting the structure of the RNF130 protein, the cell proliferation rate is no longer inhibited, and the toxicity of BMAA to the cells is reduced.

[0016] During high-throughput screening and validation, mutations in multiple Wnt signaling pathway-related genes were observed, all of which weakened BMAA-induced proliferation inhibition. This established the association between RNF130 and the Wnt signaling pathway. Furthermore, the invention also discovered that GSK-3β acts as an intermediary protein in the BMAA pathway, transducing the signal to the Wnt signaling pathway after receiving an RNF130 gene response, effectively reducing BMAA-induced proliferation inhibition in 293T cells and neurons. Specifically:

[0017] (1) GSK-3β responds to the BMAA signal in the presence of RNF130;

[0018] (2) To receive the signal transmission of RNF130 after it comes into contact with BMAA;

[0019] (3) After receiving BMAA signals from RNF130, it undertakes intracellular degradation functions and affects the Wnt signaling pathway.

[0020] Based on the conclusions of the above studies, the present invention also protects a method for reducing the cytotoxicity of BMAA, the method comprising the following (1) or (2) or (3):

[0021] (1) Reduce the activity and content of RNF130 protein in normal cells, thereby reducing the proliferation inhibition caused by BMAA;

[0022] (2) Inhibit the expression of RNF130 gene in normal cells, thereby reducing the proliferation inhibition caused by BMAA;

[0023] (3) Inhibit GSK-3β activity and inhibit the downstream of intracellular BMAA signaling, thereby reducing the proliferation inhibition caused by BMAA on cells;

[0024] Among the methods for inhibiting the expression of the RNF130 gene in normal cells are:

[0025] (1) The RNF130 gene was knocked out to obtain the RNF130 gene knockout mutant;

[0026] (2) Use methods such as small RNA interference to reduce the expression level of RNF130 gene;

[0027] (3) Mutate the sequence of the RNF130 gene to change the structure of RNF130 and affect its response to BMAA signal.

[0028] This invention demonstrates through experiments that the response to BMAA signaling requires the RNF130 protein, which contains both extracellular and intracellular domains. If the complete structure of RNF130 is disrupted, RNF130 will not respond to BMAA signaling.

[0029] One way to disrupt the complete structure of RNF130 is to truncate the part shown in SEQ ID NO.5 or SEQ ID NO.6 of the RNF130 sequence.

[0030] Therefore, by responding to extracellular BMAA signals through RNF130 on the cell membrane, the signals are transduced to GSK-3β and affect the Wnt signaling pathway. Drugs developed targeting RNF130 and its downstream signaling pathways, including GSK-3β, to alleviate or prevent cell damage caused by BMAA can relieve acute or long-term cell damage caused by BMAA. Drugs or compounds developed targeting RNF130 to regulate GSK-3β activity are all inspired by this invention and therefore fall within the protection scope of this invention.

[0031] Advantages of this invention:

[0032] This invention discloses for the first time a novel function of the RNF130 gene, which can respond to the phenotype of cell proliferation inhibition caused by BMAA signaling. It also reveals for the first time that the RNF130 protein, as a BMAA-responsive protein, transduces signals to the GSK-3β and Wnt signaling pathways, playing a crucial role in BMAA-induced cell proliferation toxicity.

[0033] This invention, through high-throughput screening, discovered that RNF130 is a gene that strongly responds to BMAA signaling. Knocking out the RNF130 gene reduced BMAA-induced cell proliferation inhibition in mammalian cells. Furthermore, RNF130 transduces BMAA signals to the downstream GSK-3β protein, thereby associating with the Wnt signaling pathway. Inhibition of GSK-3β activity using a small-molecule GSK-3β inhibitor altered the response of RNF130 to BMAA signaling, and cells could partially alleviate the cell proliferation inhibition caused by BMAA. Moreover, this invention experimentally demonstrated that the response to BMAA signaling requires the RNF130 protein to contain both extracellular and intracellular domains, and that it interacts with intracellular GSK-3β upon signaling, thus influencing cellular activity. Therefore, knockout of the RNF130 gene and inhibition of GSK-3β activity can relieve cell proliferation inhibition caused by BMAA. Thus, this invention provides key response proteins and molecular mechanisms of cell proliferation toxicity caused by algal toxin BMAA, providing important reference targets for the future development of antidotes to prevent or treat BMAA toxicity or for the development of drugs targeting RNF130-related domains. Attached Figure Description

[0034] Figure 1 Evidence of RNF130 gene knockout;

[0035] Figure 2 Evidence that BMAA causes inhibition of 293T cell proliferation in non-neuronal cells;

[0036] Figure 3 Evidence that BMAA causes inhibition of nerve cell proliferation;

[0037] Figure 4 Evidence that RNF130 knockout relieves BMAA-induced cell proliferation inhibition;

[0038] Figure 5 Evidence that GSK-3β inhibitors relieve BMAA-induced inhibition of 293T cell proliferation;

[0039] Figure 6 Evidence of interaction between the full-length RNF130 protein and GSK-3β under BMAA treatment. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0041] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0043] At the cellular level, BMAA treatment leads to inhibition of proliferation in both neural and non-neuronal cells. Cell proliferation inhibition is a manifestation of cellular damage. Utilizing this phenotype, we identified the BMAA-related protein RNF130 on the cell membrane through high-throughput screening. This protein responds to BMAA signals in response to BMAA treatment; the inhibition of cell proliferation caused by BMAA signals is relieved when this protein receptor is absent. Therefore, in our experiments, we first obtained an RNF130 gene knockout mutant and compared the cell numbers of the RNF130 gene knockout mutant and the control group to verify the function of the RNF130 gene in responding to the cell proliferation inhibition induced by the algal toxin BMAA signal. Furthermore, this invention also utilized GSK-3β inhibitor treatment, which showed that it could increase cell proliferation and relieve BMAA-induced cell proliferation inhibition. Through the construction of the RNF130 gene and its truncated overexpression vector and subcellular co-localization experiments with GSK-3β, we concluded that a response to BMAA signals requires an RNF130 protein containing both extracellular and intracellular domains. The specific experimental procedures are as follows:

[0044] Example 1: Obtaining the RNF130 gene knockout mutant

[0045] The RNF130 gene contains 1260 nucleotides in its full length, as shown in SEQ ID NO.1. The translated protein has a total of 419 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2.

[0046] 1. Construction of RNF130 gene knockout vector

[0047] Primers were designed based on sgRNA (Table 1). After annealing, double-stranded DNA with sticky ends was obtained. The DNA was then ligated into the expression plasmid using T4 ligase and expressed by the U6 promoter.

[0048] Table 1. Primers for RNF130 mutant knockout

[0049]

[0050] 2. Construction of Cas9 stably expressing cell lines

[0051] The Cas9 protein coding sequence was cloned into a lentiviral expression vector, along with a nuclear localization signal and an antibiotic resistance gene. This vector, along with a lentiviral packaging helper vector, was then transformed into the 293T cell line. After 48 hours, cells and culture medium containing lentivirus were obtained. The mixture was filtered through a 0.45 μm pore size membrane, and only the supernatant containing the virus was harvested.

[0052] The virus-containing supernatant was then added to the culture medium of the target cell line to allow for sufficient viral infection. After 48 hours, the appropriate antibiotic was added. Cells exhibiting antibiotic resistance, i.e., those with the lentiviral expression vector integrated into their genome, survived the antibiotic treatment, while cells without the integrated expression vector were killed by the antibiotic. This resulted in a stable Cas9-expressing cell population.

[0053] To obtain a stable cell line, the cell population needs to be processed by flow cytometry or infinite dilution to obtain individual cells, which are then cultured into cell lines to obtain monoclonal cell lines.

[0054] 3. Obtaining RNF130 gene knockout cell population through liposome transfection and antibiotic screening.

[0055] RNF130 knockout cell populations were obtained through two methods: One method involved transfecting an sgRNA expression vector into a Cas9-stable target cell line, using an empty expression plasmid as a control, and obtaining an RNF130 knockout cell population through highly efficient plasmid transfection. The other method involved introducing a lentivirus containing the sgRNA sequence into a Cas9-stable target cell line via infection, using cells infected with the lentivirus packaged in the empty vector plasmid as a control group, and obtaining an RNF130 knockout cell population through highly efficient viral infection. These RNF130 knockout cell populations can then be further modified by adding appropriate antibiotics to kill cells that do not express sgRNA, resulting in RNF130 gene knockout mutants with even higher knockout efficiency.

[0056] The RNF130 gene knockout mutant obtained by the above method was used to verify the expression of RNF130 protein in cells by Western blotting. Compared with wild-type 293T cells, using GAPDH protein expression level as a reference, the intracellular RNF130 protein in the CRISPR knockout 293T cell line decreased by more than 75%. Figure 1 This indicates that the RNF130 gene knockout mutant was successfully constructed.

[0057] Example 2: Effects of RNF130 gene and GSK-3β inhibitor on cell proliferation.

[0058] 1. Cell counting and spreading

[0059] 293T cells: 293T cells were digested into single cells using 0.25% trypsin-EDTA digestion solution, then stained with 0.4% trypan blue solution, and counted using a hemocytometer. 10,000-20,000 cells were then divided into 24-well plates and allowed to grow.

[0060] Neural cells: Mouse pluripotent stem cell line E14 was digested into single cells using 0.25% trypsin-EDTA digestion solution. Cells were diluted to a density of 500 cells / 20 μl using differentiation medium (15% fetal bovine serum, high-glucose DMEM, β-mercaptoethanol, L-glutamine, antibiotics, and sodium pyruvate) and cultured as hanging drops on culture dish lids for 2 days. Droplets were then collected to obtain uniformly sized embryoid bodies, which were cultured in differentiation medium for 2 days. Embryoid bodies were collected by centrifugation at 200 g and cultured in differentiation medium containing 5 μM retinoic acid (RA) for 4 days. These pre-differentiated neurocytes were then collected by centrifugation at 200 g. The collected neurocytes were digested with trypsin and passed through a 40 μm nylon sieve to obtain digested cells. Cells were then cultured at 1.5*103 5 / em 2 The cells were cultured at a density in N2 medium (DMEM / F12 medium, N2 supplement, 10 ng / ml bFGF, L-glutamine, and antibiotics). After 1 day, the N2 medium was replaced with N2B27 medium (50% DMEM / F12 medium, 50% Neural Basal medium, 1 / 2 N2 supplement, 1 / 2 B27 supplement, L-glutamine, and antibiotics). After 1 day, in vitro differentiated neural cells were obtained as material for the next stage of experiments.

[0061] 2. Treatment with BMAA and GSK-3β inhibitors

[0062] BMAA powder was diluted with sterile deionized water to obtain a BMAA solution with a final concentration of approximately 100 mM. The BMAA solution was added to the cell culture medium, with sterile deionized water added as a control group.

[0063] The GSK-3β inhibitor (purchased from the market) was diluted with DMSO and added to the cell culture medium, with the same amount of DMSO added as a control group.

[0064] 3. Cell counting

[0065] After sufficient processing time, 293T cells and nerve cells were digested into single cells using 0.25% trypsin-EDTA digestion solution, and then stained with 0.4% trypan blue solution. Unstained live cells were counted using a hemocytometer.

[0066] BMAA significantly inhibited cell growth. Under bright-field microscopy, a marked reduction in the number of 293T cells and nerve cells treated with BMAA compared to the control group treated with deionized water was clearly observed, indicating that BMAA inhibits cell proliferation. For 293T cells, treatment with 1 mM BMAA resulted in a significant cell reduction, and the reduction was even more pronounced with a higher concentration of 3 mM BMAA, demonstrating that higher concentrations of BMAA have a stronger inhibitory effect on cell proliferation. Figure 2 In nerve cells, a concentration of only 0.1 mM BMAA can cause very significant inhibition, indicating that nerve cells are more sensitive to BMAA concentrations. Figure 3 In different cell lines, BMAAs can inhibit cell proliferation, indicating that the mechanism by which BMAAs inhibit cell growth is quite widespread.

[0067] The RNF130 gene in 293T cells was knocked out using the method described in Example 1, followed by treatment with BMAA added to the cell culture medium as described in Example 2. Compared to the control group transfected with the empty vector plasmid, the RNF130 gene knockout mutant was no longer affected by BMAA, i.e., it no longer exhibited the phenotype of proliferation inhibition. Figure 4 This indicates that the RNF130 gene knockout mutant no longer responds to BMAA signals, thus demonstrating that the RNF130 gene plays a crucial role in the BMAA signal response.

[0068] In Example 2, cells were treated with either BMAA or GSK-3β inhibitors in the cell culture medium. BMAA was treated with the same amount of deionized water as a control group (marked as no BMAA), and GSK-3β inhibitor was treated with the same amount of DMSO as a control group (marked as no GSK-3β). A group receiving both BMAA and GSK-3β inhibitors was treated with the same amounts of deionized water and DMSO as a control group (marked as no GSK-3β). The results showed that BMAA significantly inhibited 293T cells, with the high-concentration (3 mM BMAA) group exhibiting a more pronounced inhibitory effect than the low-concentration (1 mM BMAA) group. GSK-3β inhibitors increased 293T cell proliferation, with the high-concentration (50 nM CHIR-99021) group showing a more significant increase than the low-concentration (20 nM CHIR-99021) group compared to the control group. When both were added simultaneously, compared to the control group receiving the same amounts of deionized water and DMSO, 293T cells no longer exhibited a significant inhibitory phenotype, indicating they were no longer sensitive to BMAA signals. Figure 5 Experiments have shown that GSK-3β inhibitors can reduce BMAA-induced cell proliferation inhibition, indicating that GSK-3β activity is also crucial for the response to BMAA signaling.

[0069] Example 3: Construction of RNF130 protein and its truncated overexpression vector and subcellular co-localization with GSK-3β

[0070] 1. Construction of overexpression vectors

[0071] Cellular RNA was extracted using Trizol, and eDNA was obtained through reverse transcription. The RNF130 gene was amplified by polymerase chain reaction (PCR) to obtain the full-length sequence SEQ ID NO.1, and a FLAG tag (DYKDDDDK) was added to the C-terminus using primers. Similarly, the sequences encoding the extracellular domain of RNF130 (SEQ ID NO.3) and the intracellular domain (SEQ ID NO.4) were amplified by PCR, and FLAG tags were added to the C-terminus of the proteins using primers. The resulting sequences were then loaded into the pcDNA3.1 vector via recombination.

[0072] 2. Protein expression

[0073] The recombinant vector was delivered into 293T cells via liposome transfection, and cells overexpressing the gene were enriched using G418 screening.

[0074] 3. Immunofluorescence staining

[0075] Cells overexpressing the gene were fixed with 4% PFA for 10 minutes, followed by three washes with PBS. Then, the fixed cells were perforated with perforation buffer (PBS, 0.1% Triton X-100, 0.1% Tween-20) for 15 minutes, and washed three times with PBS. The perforated cells were then treated with blocking buffer (PBS, 0.1% Tween-20, 3% BSA) to block non-specific sites. The primary antibody was diluted with blocking buffer and incubated overnight at 4°C. The next day, the cells were washed three times with PBS containing 0.1% Tween-20, and the secondary antibody was diluted with blocking buffer and incubated at room temperature for at least 2 hours, followed by three washes with PBS containing 0.1% Tween-20. The cell nuclei were then stained with DAPI and washed three times with PBS. Finally, the cells were mounted with a mounting medium and observed using a fluorescence confocal microscope.

[0076] Taking advantage of the FLAG tag at the C-terminus of the overexpressed protein, FLAG-tagged antibodies were used to identify the full-length and truncated overexpressed RNF130 proteins and labeled them with Alexa Fluor 594 fluorescence (red), while GSK-3β was labeled with Alexa Fluor 488 fluorescence (green), and the nuclear region labeled with DAPI was labeled in blue.

[0077] Full-length RNF130 is localized in the cytoplasm. In the absence of BMAA, GSK-3β is also localized in the cytoplasm, but co-localization between the two is rare. However, after the addition of BMAA, significant co-localization (yellow) of red RNF130 and green GSK-3β was clearly observed in the cytoplasm, forming a large intracytoplasmic complex. Simultaneously, the RNF130 signal decreased significantly. This indicates that when BMAA signaling is present, full-length RNF130 interacts with intracytoplasmic GSK-3β, thereby affecting downstream cell function. The extracellular domain of RNF130 is localized on the cytoplasmic membrane. After the addition of BMAA, there were no significant changes in co-localization signaling with intracytoplasmic GSK-3β, and no significant degradation of the signal on the cell membrane was observed. This suggests that simply expressing the extracellular domain of RNF130 is insufficient for a complete response to BMAA signaling. Similarly, the RNF130 intracellular domain is located in the cytoplasm rather than on the cell membrane. When BMAA was added, the RNF130 intracellular domain protein in the cytoplasm did not show significant changes, indicating that the presence of the RNF130 intracellular domain alone is insufficient to respond to BMAA signaling. Figure 6 ).

[0078] In summary, the response to BMAA signaling requires the RNF130 protein, which contains both extracellular and intracellular domains. Furthermore, it interacts with intracellular GSK-3β upon signaling (i.e., when BMAA toxicity is present in the cell), thus influencing the cell. Knockout of the RNF130 gene and inhibition of GSK-3β activity can both relieve BMAA-induced cell proliferation inhibition, providing important information for the future development of drugs to prevent or treat BMAA toxicity.

[0079] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A method of reducing the cytotoxicity of BMAA in vitro, characterized in that, The method comprises the following (1) or (2): (1) reducing the content of RNF130 protein in normal in-vitro cells, thereby reducing the proliferation inhibition of BMAA on in-vitro cells; (2) inhibiting expression of the gene in normal in vitro cells, thereby reducing the proliferation inhibition caused by BMAA on in vitro cells; RNF130 gene, thereby reducing the proliferation inhibition caused by BMAA on in vitro cells; The RNF130 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, the amino acid sequence of the RNF130 protein is shown as SEQ ID NO. 2, and the in vitro cell is a 293T cell.

2. The method of reducing BMAA cytotoxicity in vitro of claim 1, wherein, Inhibit normal cells in vitro RNF130 Gene expression occurs in the following ways: (1) Knockout RNF130 Gene acquisition RNF130 Gene knockout mutant; (2) reducing the expression of a gene using small RNA interference methods RNF130 gene expression (3) to mutate the sequence of the gene, changing the structure of RNF130. RNF130 the sequence of the gene, changing the structure of RNF130.

3. The method of reducing BMAA cytotoxicity in vitro of claim 2, wherein, Changing the structure of RNF130 is to destroy the complete structure of RNF130, affect its response to the signal of BMAA or affect its signal transduction to GSK-3β.

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