A screening method for RNA-dependent RNA polymerase truncation, truncation and application thereof

Abstract

This invention relates to the field of molecular and cellular biotechnology, specifically to a method for screening truncated RNA polymerase variants, the truncated variants themselves, and their applications. The method truncates the amino acid sequence of a full-length RNA-dependent RNA polymerase and screens for the truncated variants with the most significant cell growth inhibitory effects. These truncated variants contain RdRP enzyme active sites and conserved amino acid sites, and possess the complete secondary structure of the RdRP region, exhibiting low toxicity to healthy cells. The truncated variants are suitable for various delivery methods, enabling more efficient and rapid inhibition of tumor cell proliferation and improving the inhibitory effect to a certain extent. These truncated variants offer significant advantages for subsequent drug delivery, novel drug reagent development, and clinical translation.

Description

Technical Field

[0001] This invention relates to the field of molecular and cellular biotechnology, and more specifically, to a method for screening RNA-dependent RNA polymerase truncated forms, the truncated forms, and their applications. Background Technology

[0002] Cancer is one of the leading causes of death worldwide. According to statistics from the World Health Organization, nearly ten million people died from cancer in 2020 alone. This equates to one in six deaths that year being caused by cancer. There are many types of cancer, with breast, lung, colorectal, and prostate cancers being the most common. Besides lifestyle factors (such as smoking, being overweight, alcohol consumption, low fruit and vegetable intake, and lack of exercise), cancer-causing infections are also a cause of cancer in 30% of cases in low- and lower-middle-income countries. Therefore, cancer is a major threat to life for people worldwide and a disease that the scientific community has been working to address. However, due to limitations in diagnostic methods and the difficulty in detecting early-stage cancer symptoms, most cancer patients are diagnosed at an advanced stage. Therefore, how to treat advanced cancer has long been one of the most critical issues in cancer treatment.

[0003] Currently, existing cancer treatment methods include radiotherapy, chemotherapy, CAR-T (chimeric antigen receptor T-cell immunotherapy) as the mainstream immunotherapy, and various emerging small molecule therapies. However, the high heterogeneity of tumors and the gradual emergence of chemotherapy-resistant tumor types mean that we lack a "panacea" for broad-spectrum cancer treatment.

[0004] RDR1 is a novel drug molecule targeting tumor-specific miRNA (microRNA) abnormalities, precisely and broadly inhibiting the proliferation of many tumors. In existing technologies, researchers have found in in vitro experiments that RDR1 protein can broadly inhibit tumor growth while exhibiting low toxicity to non-cancer cells. In vivo experiments have also shown excellent inhibitory activity against tumor growth. Therefore, utilizing full-length RDR1 to inhibit tumor cell proliferation has attracted increasing attention.

[0005] However, due to the size of the RDR1 molecule itself, its ability to be expressed heterologously in human cells, its retention in blood, and its requirements for drug delivery are all somewhat unsatisfactory.

[0006] The full-length RDR1 protein is 1107 amino acid residues long, with a molecular weight of approximately 130 kDa after Western blotting. Such a large exogenous protein undoubtedly poses a significant obstacle in the drug development process. Whether it is gene therapy aimed at ectopic expression or conventional delivery via nanoparticle encapsulation and injection, there are considerable issues with efficiency and stability. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for screening RNA-dependent RNA polymerase truncated forms, the truncated forms, and their applications.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0009] This invention provides a method for screening RNA-dependent RNA polymerase truncated variants, comprising the following steps:

[0010] S1. Predict the position of the RdRP region in the amino acid sequence of the RNA-dependent RNA polymerase, as well as the complete secondary structure of the RdRP region, the RdRP enzyme active site, and the positions and corresponding amino acid sequences of multiple conserved RdRP amino acid sites.

[0011] S2. The amino acid sequence of the full-length RNA-dependent RNA polymerase is truncated in the first round to obtain the amino acid sequences of various first truncated forms;

[0012] Each of the first truncated amino acid sequences contains the RdRP enzyme active site and the conserved amino acid site, and has the complete secondary structure of the RdRP region;

[0013] S2. Each of the first truncated segments is truncated in a second round to obtain the amino acid sequences of various second truncated segments;

[0014] The second round of truncation occurs at the conserved amino acid sites of the RdRP.

[0015] S3. Based on the amino acid sequence of each of the second truncated variants, bioengineering methods were used to detect the expression level of each second truncated variant in tumor cells, its inhibition rate on tumor cell proliferation, and its inhibition rate on healthy cell proliferation, and target truncated variants were screened.

[0016] Furthermore, in step S3, the tumor cells are HepG2 cells, H1299 cells, or HeLa cells; the healthy cells are RPE-1 cells.

[0017] Furthermore, in step S3, firstly, a second truncated variant that can be well expressed is screened, then two second truncated variants with high inhibition rates against tumor cells are screened, and finally, the second truncated variant with low inhibition rate against healthy cell proliferation is selected as the target truncated variant.

[0018] Furthermore, the target truncated variant, in tumor cells, has a relative expression level of 100% relative to each of the second truncated variants, and exhibits an inhibition rate of 70% on the proliferation of HepG2 cells and 10% on the proliferation of RPE-1 cells.

[0019] Furthermore, in step S3, the bioengineering method includes the following steps:

[0020] S3-1. Obtain the nucleotide sequence encoding the full-length RNA-dependent RNA polymerase;

[0021] S3-2. Determine the truncation position based on the amino acid sequence of each of the second truncated variants, and use a site-directed mutagenesis kit to truncate the nucleotide sequence of the full-length RNA-dependent RNA polymerase to obtain the nucleotide sequence encoding each of the second truncated variants;

[0022] S3-3. Establish expression vectors containing the nucleotide sequences of each of the second truncated variants, and then use each of the expression vectors to establish cell lines that can be inducibly expressed for each of the second truncated variants;

[0023] S3-4. The cell lines obtained in step S3-3 are induced respectively.

[0024] Furthermore, in step S3-3, the expression vector is constructed by viral embedding, and then the nucleotide sequence encoding each of the second truncated variants is introduced into cells by transfection to obtain the cell line that can be inducibly expressed for each of the second truncated variants;

[0025] Specifically, when detecting the expression level and the inhibition rate on tumor cell proliferation, the introduced cells are tumor cells; when detecting the inhibition rate on healthy cell proliferation, the introduced cells are healthy cells.

[0026] Furthermore, the active site of the RdRP enzyme is the DXDGD sequence.

[0027] Furthermore, the RNA-dependent RNA polymerase is RDR1.

[0028] Furthermore, the RDR1 is derived from plants or protozoa.

[0029] Furthermore, the RDR1 is derived from Arabidopsis thaliana, rice, or tobacco.

[0030] The present invention also provides an RNA-dependent RNA polymerase truncated form, which is obtained by the screening method described above.

[0031] Furthermore, the target truncated form is derived from Arabidopsis thaliana; the truncated form removes amino acid residues from position 814 onwards to the C-terminus of the full-length Arabidopsis thaliana RDR1 protein.

[0032] Furthermore, the nucleotide sequence encoding the truncated form is shown in SEQ ID NO.1.

[0033] This invention also provides the application of the above-mentioned RNA-dependent RNA polymerase truncated form in the preparation of broad-spectrum cancer drugs.

[0034] The beneficial effects of this invention are as follows:

[0035] (1) The screening method for RNA-dependent RNA polymerase truncated variants of the present invention can screen out truncated variants with good tumor growth inhibition function, and the screened truncated variants have no obvious toxicity to healthy cells and can be applied to various delivery methods, thereby enabling more efficient and rapid inhibition of tumor cell proliferation.

[0036] (2) The screening method for RNA-dependent RNA polymerase truncated variants of the present invention can screen RNA-dependent RNA polymerase truncated variants from different species and has a wide range of applications.

[0037] (3) The screening method for RNA-dependent RNA polymerase truncated variants of the present invention roughly calculates the location of the functional domains, secondary structures and hydrophilic and hydrophobic regions of the RDR1 protein through the algorithm of the protein database; then, from the perspective of evolution, it finds sites or peptides with specific characteristics or highly conserved amino acids, and finds the core enzyme active site; combining the above two sets of information, it is possible to truncate RDR1 without interrupting independent working units as much as possible.

[0038] (4) The RNA-dependent RNA polymerase truncated form of the present invention has a molecular weight reduced by one-third, making it more suitable for preparing anti-tumor drugs;

[0039] (5) The RNA-dependent RNA polymerase truncated form of the present invention has a significantly reduced molecular weight, which improves the delivery efficiency and thus enhances its inhibitory effect to a certain extent.

[0040] (6) The RNA-dependent RNA polymerase truncated form of the present invention provides great advantages for subsequent drug delivery, development of novel drug reagents and clinical translation. Attached Figure Description

[0041] Figure 1In the screening method for RNA-dependent RNA polymerase truncated variants of the present invention, in the examples, the domain prediction results of the AtRDR1 amino acid sequence were obtained using the SMART website;

[0042] Figure 2 In the method for screening RNA-dependent RNA polymerase truncated variants of the present invention, the amino acid sequence diagram of each truncated variant is shown in the examples.

[0043] Figure 3 In the screening method for RNA-dependent RNA polymerase truncated variants of the present invention, in the examples, Western blotting was used to detect the electrophoretic images of the first to fifth truncated variants in HepG2 cells.

[0044] Figure 4 In the screening method for RNA-dependent RNA polymerase truncated variants of the present invention, in the examples, Dox induces cell counting of HepG2 cells expressing T1 truncated variants to T5 truncated variants; Figure 4 In figure A, the cell count of each truncated body is shown on day 2. Figure 4 B represents the cell count of each truncated body on day 4. Figure 4 C represents the cell count of each truncated body on day 6;

[0045] Figure 5 In the screening method for RNA-dependent RNA polymerase truncated variants of the present invention, the proliferation curves of HepG2 cells expressing T1 truncated variants to T5 truncated variants induced by Dox are shown in the examples.

[0046] Figure 6 In the screening method for RNA-dependent RNA polymerase truncated variants of the present invention, an electrophoretic image of cell cycle proteins in HepG2 cells expressing Dox-induced T4 truncated variants is shown in the examples.

[0047] Figure 7 In the screening method for RNA-dependent RNA polymerase truncated variants of the present invention, in the examples, the up- and down-regulated gene distribution in HepG2 cells expressing Dox-induced T4 truncated variants compared to the control group;

[0048] Figure 8 In the screening method for RNA-dependent RNA polymerase truncated variants of the present invention, the GO enrichment map of relatively downregulated genes in HepG2 cells expressing Dox-induced T4 truncated variants is shown in the examples. Figure 8 In the diagram, A represents the enrichment of upregulated genes, and B represents the enrichment of downregulated genes.

[0049] Figure 9In the screening method for RNA-dependent RNA polymerase truncated variants of the present invention, heatmaps of cell cycle-related core genes in the transcriptomes of T4 truncated variants and full-length protein HepG2 cell lines are shown in the examples.

[0050] Figure 10 In the RNA-dependent RNA polymerase truncated variant screening method of the present invention, in the example, the electrophoretic image of the T4 truncated variant in RPE-1 cells detected by Western Blot is shown. Figure 10 In the method for screening RNA-dependent RNA polymerase truncated variants of the present invention, in example B, the cell proliferation curve of RPE-1 cells expressing T4 truncated variants induced by Dox is shown.

[0051] Figure 11 In the screening method for RNA-dependent RNA polymerase truncated variants of the present invention, in the examples, Western blotting was used to detect the electrophoretic images of T4 truncated variants in H1299 and HeLa cells;

[0052] Figure 12 In the method for screening RNA-dependent RNA polymerase truncated variants of the present invention, in example, the cell proliferation curve of H1299 cells expressing Dox-induced T4 truncated variants is shown. Figure 12 In the method for screening RNA-dependent RNA polymerase truncated variants of the present invention, B represents the cell proliferation curve of HeLa cells expressing Dox-induced T4 truncated variants in the examples. Detailed Implementation

[0053] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0054] The screening method for RNA-dependent RNA polymerase truncated variants of the present invention can truncate and screen RNA-dependent RNA polymerases from various species. Therefore, the truncated variants obtained by screening using the method of the present invention can be applied to more delivery methods.

[0055] Specifically, the following steps are included:

[0056] S1. Predict the location of the RdRP region in the amino acid sequence of RNA-dependent RNA polymerase, as well as the complete secondary structure of the RdRP region, the RdRP enzyme active site, and the locations and corresponding amino acid sequences of multiple conserved RdRP amino acid sites.

[0057] S2. The amino acid sequence of the full-length RNA-dependent RNA polymerase is truncated in the first round to obtain the amino acid sequences of various first truncated forms;

[0058] Each of the first truncated amino acid sequences contains the RdRP enzyme active site and conserved amino acid sites, and has the complete secondary structure of the RdRP region;

[0059] S2. Each of the first truncated segments is truncated in a second round to obtain the amino acid sequences of various second truncated segments;

[0060] The second round of truncation occurs at RdRP conserved amino acid sites;

[0061] S3. Based on the amino acid sequences of each second truncated variant, bioengineering methods were used to detect the expression level of each second truncated variant in tumor cells, its inhibition rate on tumor cell proliferation, and its inhibition rate on healthy cell proliferation, and target truncated variants were screened.

[0062] In step S3, the tumor cells are HepG2 cells, H1299 cells, or HeLa cells; the healthy cells are RPE-1 cells.

[0063] In step S3, firstly, the second truncated variant that can be well expressed is screened, then two second truncated variants with high inhibition rates against tumor cells are screened, and finally, the second truncated variant with low inhibition rate against healthy cell proliferation is selected as the target truncated variant.

[0064] In step S3, the bioengineering method includes the following steps:

[0065] S3-1. Obtain the nucleotide sequence encoding the full-length RNA-dependent RNA polymerase.

[0066] S3-2. Determine the truncation position based on the amino acid sequence of each of the second truncated variants, and truncate the nucleotide sequence of the full-length RNA-dependent RNA polymerase using a site-directed mutagenesis kit to obtain the nucleotide sequence encoding each of the second truncated variants.

[0067] S3-3. Establish expression vectors containing the nucleotide sequence of each of the second truncated variants, and then use each of the expression vectors to establish cell lines that can be inducibly expressed for each of the second truncated variants.

[0068] The expression vector was constructed by viral embedding, and then the nucleotide sequences encoding each of the second truncated variants were introduced into cells by transfection to obtain the cell lines that can be inducibly expressed for each of the second truncated variants.

[0069] Specifically, when detecting the expression level and the inhibition rate on tumor cell proliferation, the introduced cells are tumor cells; when detecting the inhibition rate on healthy cell proliferation, the introduced cells are healthy cells.

[0070] S3-4. The cell lines obtained in step S3-3 are induced respectively.

[0071] Furthermore, the active site of the RdRP enzyme is the DXDGD sequence.

[0072] In the method of this invention, the RNA-dependent RNA polymerase is RDR1.

[0073] In the method of the present invention, the RDR1 used is derived from plants or protozoa; preferably, the RDR1 is derived from Arabidopsis thaliana, rice, or tobacco.

[0074] The present invention also provides an RNA-dependent RNA polymerase truncated form, which is a target truncated form obtained by the screening method described above.

[0075] The truncated form of this invention, compared to the full-length RDR1, has a smaller molecular weight while retaining its function of inhibiting tumor cell proliferation; it also exhibits a more efficient and rapid inhibitory effect. Furthermore, it significantly reduces toxic side effects on healthy cells.

[0076] Preferably, the truncated form is derived from Arabidopsis thaliana, and the truncated form removes the amino acid residues from position 814 onwards to the C-terminus of the full-length Arabidopsis thaliana RDR1 protein; the truncated form corresponds to the T4 truncated form in the example, which has 814 amino acid residues, a molecular weight of approximately 85 kDa, and a gene length of 2442 bp encoding it.

[0077] The T4 truncated variant demonstrated significant tumor cell inhibition at both the cellular and transcriptional levels, indicating that while its molecular weight was significantly reduced, it met the requirements of most delivery methods and still maintained an inhibitory effect equivalent to that of the full-length Arabidopsis RDR1. Furthermore, compared to the full-length Arabidopsis RDR1, it exhibited lower toxicity to healthy cells due to the deletion of some potentially toxic sequences.

[0078] Among the T1-T5 truncated variants, T4 is the shortest and has the highest potential expression level. The T4 truncated variant, as the target truncated variant, showed 100% relative expression in tumor cells, inhibiting HepG2 cell proliferation by 70% and RPE-1 cell proliferation by 10%.

[0079] The nucleotide sequence encoding the T4 truncated form is shown in SEQ ID NO.1.

[0080] This invention provides the application of RNA-dependent RNA polymerase truncated derivatives in the preparation of broad-spectrum cancer drugs.

[0081] The technical solution of the present invention will be explained and described below through specific embodiments.

[0082] Example

[0083] The RDR1 derived from Arabidopsis thaliana has a strong function; therefore, this embodiment uses Arabidopsis-derived RDR1 (AtRDR1) as an example for truncation. This embodiment develops a truncation scheme by analyzing protein domains and secondary structures. The truncation conditions in this embodiment mainly involve preserving conserved sites or peptides in the protein's amino acid sequence and retaining the enzyme active site (DXDGD sequence). In this way, the truncated form still possesses independent working units and a complete secondary structure, thus retaining the function of AtRDR1 itself.

[0084] The full-length AtRDR1 protein (WT) has 1107 amino acid residues and a molecular weight of approximately 130 kDa. The gene encoding the full-length AtRDR1 is 3321 bp in length.

[0085] The specific process of analyzing, screening, and verifying the truncated body function in this embodiment is as follows:

[0086] (1) Prediction of the domains of the AtRDR1 protein sequence

[0087] In this embodiment, the domains in the AtRDR1 protein sequence were predicted using SMART software (http: / / smart.embl.de, released in 1997 and keeping updating until now) to resolve the amino acid sequences corresponding to the major functional sites of AtRDR1.

[0088] Prediction results are as follows Figure 1 As shown. According to Figure 1 It can be seen that the AtRDR1 protein mainly has two major regions: the RRM (RNA Recognition Motif) region and the RdRP region. At the same time, there are some irregularly structured regions between the two and at the C-terminus of the RdRP.

[0089] Based on existing research on the full-length RDR1 protein, regions with irregular structures do not significantly affect the function of RDR1; therefore, these regions can be removed.

[0090] (2) Prediction of the secondary structure of the AtRDR1 protein sequence

[0091] Based on the analysis of the structural domains, this embodiment further identified several conserved amino acid sites and the most critical RdRp enzyme active site (DXDGD sequence).

[0092] The conserved amino acid sites and DXDGD sequence in the RdRP region were determined in this embodiment based on the literature "Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases" (Lakshminarayan M Iyer, Eugene V Koonin and L Aravind et al.).

[0093] For more precise cut sites, this embodiment uses PSIPRED webpage developed by UCL Department of Computer Science: Bioinformatics Group to predict the secondary structure of the RdRp Domain.

[0094] (3) Determine the amino acid sequence of the AtRDR1 truncated form and truncate the nucleotide sequence encoding the full-length AtRDR1 truncated form.

[0095] Although existing studies of the full-length AtRDR1 protein have shown that non-RdRP regions are not related to its protein function, this embodiment requires truncating the RdRP region, and it is unclear whether these non-functional regions will affect the truncated RdRP region. Therefore, this embodiment divides the truncating process into two rounds. For the various truncated variants obtained after the first round of truncating, this embodiment only screens for truncated variants containing conserved amino acid sites in the RdRP region and the DXDGD sequence. After screening and verifying truncated variants with good function, a second round of truncating is performed to further examine whether the function of the truncated variants after removing the non-RdRP region is affected.

[0096] First, leaves of wild-type Arabidopsis thaliana were taken, flash-frozen in liquid nitrogen, and then ground to extract RNA. The extracted RNA was used to obtain cDNA using a reverse transcription kit, and then the cDNA was used as a template to clone into the full-length RDR1 gene.

[0097] Subsequently, based on the above conclusions, this embodiment first determined five truncated forms that met the above conditions based on the analysis results of the domains, secondary structures, conserved sites, and enzyme active sites, and then adopted NEB's... The Site-Directed Mutagenesis Kit performs a first round of truncation on the gene encoding the full-length AtRDR1 protein, resulting in gene sequences of various different truncated forms.

[0098] In this embodiment, the specific truncation positions of the five truncated sections are as follows: Figure 2 As shown.

[0099] T1 truncated form: The 425th amino acid T of the full-length AtRDR1 protein and all the amino acids at its C-terminus; the truncated form contains a truncated RdRP region and an unstructured region at its C-terminus, and the truncated RdRP region is partially truncated at the N-terminus.

[0100] T2 truncated form: The 527th amino acid S and all amino acids at the C-terminus of the full-length AtRDR1 protein; this truncated form contains a truncated RdRP region and an unstructured region at the C-terminus, and the truncated RdRP region is partially shortened at the N-terminus, and this truncated RdRP region is shorter than the truncated RdRP region in the first truncated form.

[0101] T3 truncated form: The 608th amino acid Y of the full-length AtRDR1 protein and all amino acids at its C-terminus; this truncated form contains a truncated RdRP region and its C-terminal unstructured region, and the truncated RdRP region is partially shortened at the N-terminus, and this truncated RdRP region is shorter than the truncated RdRP region in the second truncated form.

[0102] T4 truncated form: amino acids 1 to 814 of the full-length AtRDR1 protein; this truncated form contains a truncated RdRP region, an N-terminal unstructured region, and an RRM region, and the truncated RdRP region is partially truncated at the C-terminus.

[0103] T5 truncated form: amino acids 1 to 895 of the full-length AtRDR1 protein; this truncated form contains a truncated RdRP region, an N-terminal unstructured region, and an RRM region, and the truncated RdRP region is partially shortened at the C-terminus, and this truncated RdRP region is longer than the RdRP region of the T4 truncated form.

[0104] (4) Establish a cell line that can inducibly express the RDR1 truncated protein

[0105] The genes encoding the five truncated variants were embedded with viruses, and expression vectors and inducible expression cell lines were established separately.

[0106] In the prior art, HepG2 cells are generally used to verify the expression of the full-length AtRDR1 protein. Therefore, this embodiment also uses this cell to verify the expression of the truncated form.

[0107] The specific steps for viral embedding, expression vector preparation, and establishment of inducible expression cell lines for each truncated variant are as follows:

[0108] Virus embedding was performed on 293T cells using Lipofectamine 3000 transfection reagent. The embedding was performed in tube A containing 500 μL OptiMEM and 31 μL lipo3000, and in tube B containing 500 μL OptiMEM, 40 μL P3000, 10 μg pOs / AtRDR1-T2A-EGFP, 2.5 μg pMD2G, and 7.5 μg pSPAX2.

[0109] After embedding, the cell culture supernatant was collected 48 hours later. Cell debris was removed by centrifugation at 1500 rpm for 5 minutes at room temperature, and the virus was collected. In addition to the expression vector containing the RDR1 truncated variant, the TetOn-3G expression vector was also embedded with the virus.

[0110] Because the full-length AtRDR1 protein inhibits the cell cycle of tumor cells, this characteristic can cause tumor cells stably expressing RDR1 protein to stop growing during routine culture. Therefore, this embodiment uses a Dox (polytetracycline) induction expression system. When expression comparison is needed, normal DMEM medium containing 4 μg / ml Dox is added to the culture dish to induce the expression of the truncated RDR1 protein in cells.

[0111] Subsequently, the virus embedded in the truncated expression vector, along with the virus containing the TetOn-3G expression vector, was added to the culture medium of the target cells. The viruses containing the truncated expression vector and the TetOn-3G expression vector were transfected at a ratio of 3:1 or 4:1. During transfection, polybrene was added to achieve a final concentration of 6 μg / mL in the medium. Twelve hours after viral infection, the medium was replaced with ordinary DMEM medium and cultured for one day. The medium was then changed daily with 100x G418 to screen for TetOn-3G-containing cells. After two weeks of continuous screening with G418, the medium was changed 24 hours in advance to DMEM medium containing 100 ng / mL and cultured at 37°C in a 5% CO2 incubator. EGFP-positive cells were then sorted using an Aria III flow cytometer. Once the cells had separated and reached a certain size, cell experiments were initiated.

[0112] For the expression induction of the RDR1 truncated variant, this embodiment used DMEM medium containing 4 μg / mL Dox for 6 days, changing the medium daily until the final sample collection. This embodiment used RIPA cell lysis buffer to lyse the cell membrane and obtain the protein samples. Appropriate amounts of PMSF, DTT, and other additives required for different experiments, such as RNase inhibitors, were also added.

[0113] The collected proteins were analyzed to detect truncated expression levels and various protein levels related to cell cycle.

[0114] This embodiment uses Western blotting to verify the expression of each truncated variant in HepG2 cells. The results are as follows: Figure 3 As shown. According to Figure 3 The electrophoresis results show that each truncated variant can be well expressed in HepG2 cells. Therefore, the above experiment yielded a cell line that can be expressed by Dox-induced RDR1 truncated variant protein.

[0115] (5) Effects of RDR1 truncated variant overexpression on tumor cell growth cycle

[0116] For the cell lines obtained by expressing truncated RDR1 protein using Dox, this section directly conducted a cell counting experiment to determine whether the cell proliferation efficiency was affected by the truncated RDR1 protein.

[0117] The specific experimental steps were as follows: 10,000 cells were seeded into each well of a 12-well plate as the starting material. The experimental group was given DMEM complete medium containing a high concentration of Dox (4 μg / mL) and changed daily, while the control group was given DMEM complete medium without Dox as a control. Each experiment was designed with three to four replicates, and samples were collected for six consecutive days.

[0118] After digesting the cells, they were thoroughly mixed by pipetting and staining with trypan blue. The cell density was then calculated using a cell counter, and the total cell count was determined based on the resuspended volume. A stable HepG2 cell line transfected with Mut-AtRDR1 protein, which was completely inactivated due to a mutation at the enzyme's active site, was included as a negative control. This demonstrates that the truncated variant indeed exhibits stronger cell cycle inhibition compared to the mock control.

[0119] according to Figure 4 The results of cell counting and Figure 5The cell growth curves show that, compared with the control group, both the T4 truncated variant (Truncation 4) and the T5 truncated variant (Truncation 5) exhibit significant cell cycle inhibition.

[0120] (6) Validation experiments on the inhibition of T4 truncated cells at various levels of the cell cycle

[0121] 1. At the protein level, the inhibitory effect of the T4 truncated variant on the core cell cycle proteins of HepG2 cells.

[0122] The following proteins are the target proteins to verify the cell cycle inhibition ability of the T4 truncated variant:

[0123] CCND1 (cell cyclin D1): mainly plays a regulatory role in the transition from G1 phase to S phase of the cell cycle;

[0124] CCNE2 (cell cyclin E2): mainly plays a regulatory role in the G1-S phase of the cell cycle;

[0125] CDK6 (Cell-cycle dependent kinase 6): mainly plays a regulatory role in the G1-S phase of the cell cycle;

[0126] MCM2 (minichromosome maintenance complex component 2): mainly plays a regulatory role in the S phase of the cell cycle;

[0127] PLK1 (polo-like kinase 1): mainly plays a regulatory role in the G2-M phase of the cell cycle.

[0128] In this part of the experiment, the WT group was used as the negative control, HepG2 cells cultured for 6 days without Dox were used as the control group, and samples of cells collected after culturing with high Dox concentration (4μg / mL) for 6 days were used as the experimental group.

[0129] from Figure 6 As we can see, compared to the negative control and control groups, the expression levels of cyclin were significantly downregulated in the experimental group cells at the same β-Actin concentration. This indicates that the cell cycle was significantly inhibited in HepG2 cells expressing the T4 truncated variant, resulting in a reduced proportion of cells in the cell cycle and consequently a decrease in core cell cycle-related proteins.

[0130] Therefore, at the protein expression level, the T4 truncated form can significantly inhibit the cell cycle of HepG2 cells.

[0131] 2. The inhibitory effect of T4 truncated variant expression on HepG2 cells at the cellular transcriptome level.

[0132] This section uses RNA-seq for detection. The experiment was divided into three groups: wild-type HepG2 cells (WT group), a stable cell line with HepG2 truncation 4 induced expression (without treatment) (-Dox group), and a stable cell line with HepG2 truncation 4 induced expression (with treatment) (+Dox group), which are the experimental groups. The first two groups served as control groups, and the average of the two control groups was used as a reference to ensure the stability of the control groups.

[0133] like Figure 7 As shown, after standardizing the number of reads for different samples, the number of reads in the experimental group (+Dox) and the control group (Ave.) were compared. It was found that, compared with the control group, the drug-treated group had a total of 649 genes upregulated and 382 genes downregulated.

[0134] like Figure 8 As shown, GO enrichment analysis was performed on the downregulated genes in the experimental group compared to the control group. The results indicate that most of the downregulated genes in the experimental group were enriched in cell cycle-related biological processes. Conversely, the upregulated genes did not show significant enrichment in specific pathways related to cell proliferation and cancer.

[0135] Since this part of the experiment mainly focuses on the transcriptomic regulation of genes related to the cell cycle, core genes related to the cell cycle were also analyzed. Compared with the control group, the transcriptome of HepG2 cells induced by Dox to express RDR1 truncated variants showed relatively weaker expression of core genes related to the cell cycle.

[0136] 3. Comparison of transcriptome expression levels of core cell cycle genes between truncated T4 and full-length AtRDR1 proteins.

[0137] This section still uses RNA-seq for detection. The results are as follows: Figure 9 As shown, the truncated T4 protein appears to exhibit a slightly enhanced repression of cell cycle genes at the transcriptome level compared to the full-length AtRDR1 protein. This may be because the shorter protein length results in a higher expression level compared to the full-length AtRDR1 protein, leading to a stronger repression of cell cycle genes in tumor cells.

[0138] Additionally, it should be noted that this section compared the expression profiles of HepG2 wild-type cells from the two studies and found that cell cycle-related genes showed high reproducibility in both library preparations. This indicates that the two library preparations are very stable and can be used for comparative data analysis.

[0139] In summary, both the T4 and T5 truncated variants exhibit growth inhibition of tumor cells at the cellular level, and their whole-transcriptome gene enrichment results show that the intracellular molecular functions of the two variants are extremely similar. Because the T4 truncated variant has a shorter nucleotide sequence than the T5 truncated variant, and because it significantly inhibits cell cycle-related proteins and genes at both the protein and transcriptome levels, with an inhibitory effect comparable to that of the full-length AtRDR1 protein, this indicates that the T4 truncated variant can rely on globally increasing miRNA expression to rescue cancer cell miRNA defects, thereby interfering with the cell cycle progression of tumor cells and leading to a significant downregulation of cell cycle-related genes at both the protein and transcriptome levels.

[0140] (7) Verify the safety of the T4 truncated formulation.

[0141] Following the experimental methods described in Part (4) above, a cell line containing the T4 truncated variant and inducibly expressed RDR1 truncated variant protein was established. Here, we used the healthy human retinal epithelial cell line RPE-1 for construction.

[0142] After the construction and screening of stable cell lines were completed, the cells were cultured in medium containing a high concentration of Dox daily for six days. Proteins were extracted and analyzed by Western blotting to verify the expression of truncated somatic proteins and the stability of the induced expression system. The control group consisted of wild-type RPE-1 cells and truncated RPE-1 somatic cells cultured in Dox-free medium. The Western blotting results are as follows: Figure 10 As shown, compared to the control group, the T4 truncated variant was expressed in Dox-induced RPE-1 cells.

[0143] To verify whether the truncated T4 cell line has an inhibitory effect on healthy cells, this section designed a control group with and without the drug, and conducted cell counting experiments. A 12-well plate was used, with 10,000 cells initially seeded in each well. 1 ml of culture medium containing or without Dox was added each time, with the medium changed daily. Three bioreplicated wells were collected daily. After digesting the cells with 100 μL of trypsin, the cells were resuspended in 500 μL of culture medium, centrifuged at 500g for 3 minutes, and the supernatant was removed. The cells were then resuspended in 100 μL of culture medium, and cell density was counted using a cell counting chamber and a cell counter. The total cell count was calculated by multiplying 100 μL by the cell density. This process of collecting cells and counting cell proliferation was repeated daily for six days.

[0144] like Figure 10 As shown, after six days of cell counting experiments, the T4 truncated form of RDR1 showed no significant inhibitory effect on the human healthy cell line RPE-1. Microscopic observation of cell density and morphology showed no difference compared to the wild-type control and the (-Dox) group. This significant reduction in toxicity may be due to the removal of potentially toxic sequences through truncation, resulting in low toxicity of the truncated RDR1 form to healthy cells and tissues.

[0145] (8) Verify the broad-spectrum efficacy of the T4 truncated derivative.

[0146] To further verify the broad-spectrum antitumor activity of the screened RDR1 truncated variant, in addition to HepG2 liver cancer cells, we also selected H1299 lung cancer cells and HeLa cervical cancer cells to construct stable cell lines.

[0147] After the construction and screening of stable cell lines were completed, the cells were cultured in medium containing a high concentration of Dox daily for six days. Proteins were extracted and analyzed by Western blotting to verify the expression of truncated somatic proteins and the stability of the induced expression system. The control group consisted of HeLa and H1299 truncated somatic cells cultured in medium without Dox. The Western blotting results are as follows: Figure 11 As shown, compared to the control group, both stable cell lines expressed truncated forms of RDR1 under Dox induction.

[0148] To investigate the inhibitory effect of T4 truncated cells on other cancer cells, this section designed a control group with and without drug administration, and conducted cell counting experiments. A 12-well plate was used, with 10,000 cells initially seeded in each well. 1 ml of culture medium containing or without Dox was added each time, with the medium changed daily. Three bioreplicated wells were collected daily. After digesting the cells with 100 μL of trypsin, the cells were resuspended in 500 μL of culture medium, centrifuged at 500g for 3 minutes, and the supernatant was removed. The cells were then resuspended in 100 μL of culture medium, and cell density was counted using a cell counting chamber and a cell counter. The total cell count was calculated by multiplying 100 μL by the cell density. This process of collecting cells and counting cell proliferation was repeated daily for six days.

[0149] like Figure 12 As shown, after six days of cell counting experiments, the truncated T4 protein, AtRDR1, exhibited significant inhibitory effects on cell proliferation in both lung cancer and cervical cancer cells, with this effect generally becoming significant after 3-4 days. This result indicates that the truncated T4 protein may possess inhibitory capabilities on cell cycle and cell proliferation at different cellular levels. Similarly, compared to the full-length AtRDR1 protein, the shorter T4 truncated protein results in a higher expression level, leading to a stronger inhibitory effect on tumor cell proliferation.

[0150] (9) RNA silencing occurs in almost all major eukaryotic lineages, and evolutionary analysis shows that the main components of the RNA silencing mechanism can be traced back to the ancestors of eukaryotes. Comparison of RDR1 sequences from different plants and microorganisms reveals that these RDR1 sequences all possess conserved enzyme activation sites (DXDGD) and other similar conserved sequences. This indicates that, while preserving key enzyme activation sites and conserved sequences, redundant RDR1 sequences from other species can be artificially edited to shorten protein length, further optimizing their application in translational medicine. It is also noteworthy that RDR1 sequences from different plants, such as those from rice and tobacco, also possess similar antiviral functions. Therefore, it is not difficult to infer that RDR1 sequences from different species, due to their important structures, conserved sequences, and consistent antiviral functions, may have excellent effects in treating proliferative diseases such as cancer.

[0151] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An RNA-dependent RNA polymerase truncated variant, characterized in that, The nucleotide sequence of the truncated form is shown in SEQ ID NO.

1.

2. The use of the RNA-dependent RNA polymerase truncated form as described in claim 1 in the preparation of a drug for treating cancer, characterized in that, The cancer in question is liver cancer, lung cancer, or cervical cancer.

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