Gene dose effect target for improving rapamycin resistance and application

Abstract

The invention relates to a gene dose effect target spot for improving rapamycin resistance and application of the gene dose effect target spot. A target spot gene is YCL039W and / or YCL037C, or human WDR26 and / or LARP1; the YCL039W and / or YCL037C are / is overexpressed in the saccharomycetes, so that the rapamycin resistance of the saccharomycetes can be improved, and the constructed recombinant function can better grow under the pressure condition of the rapamycin; and the sensitivity of the rapamycin can be improved by knocking down the WDR26 and / or the LARP1 in human cells, so that the application of the rapamycin drug resistance in clinical treatment is widened.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to gene dose-effect targets and applications for enhancing rapamycin resistance. Background Technology

[0002] Rapamycin (RPM) is a macrolide antibiotic with immunosuppressive activity and is an FDA-approved anti-rejection drug for kidney transplantation. Currently, rapamycin has been successfully developed as a novel and potent immunosuppressant for organ transplantation anti-rejection; and as a cardiovascular coating stent. Drug synthesis research using rapamycin as a basic structural unit for derivatives can also be used to treat various malignant tumors. However, due to low fermentation levels and high production costs, the application of rapamycin is somewhat limited.

[0003] Yeast is an important "cellular factory" used to produce fuels, chemicals, and drugs (such as insulin). Rapamycin targets the TOR protein (called TOR in yeast and mTOR in mammals). This is a central controller integrating nutrients, energy, growth factors, and stress signals, determining whether the cell engages in anabolism (growth and proliferation) or catabolism (survival). The TOR pathway is central to sensing nutrient status and controlling growth. In nutrient-rich conditions, TOR is activated, leading to cell growth; in nutrient-deficient conditions, TOR is inhibited, triggering stress and survival mechanisms. By exploring the target of rapamycin resistance, we can not only decode the core code of cell growth regulation; but also, by adding rapamycin to inhibit TOR activity after reaching a certain cell density, switch the metabolic mode from "growth" to "production," improving the robustness of the strain and constructing more efficient industrial microbial cell factories. Furthermore, this can lay a solid theoretical foundation for understanding disease mechanisms and developing new drugs. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides gene dose-effect targets for enhancing rapamycin resistance and their applications.

[0005] The technical solution adopted in this invention is: to enhance the gene dose-effect target of rapamycin resistance, and the target gene is... YCL039W and / or YCL037C , or for WDR26 and / or LARP1 .

[0006] Preferably, YCL039W Gene ID: 850318 YCL037C Gene ID: 850320 WDR26 GeneID: 80232 LARP1 Gene ID: 23367.

[0007] Preferably, the target gene or target genome enhances the Ras signaling pathway through dose-dependent antagonism, thereby increasing yeast rapamycin resistance.

[0008] A rapamycin-resistant engineered yeast strain, overexpressing YCL039W and / or YCL037C Yeast.

[0009] Application of rapamycin-resistant engineered yeast strains in yeast cell factories.

[0010] One method to improve rapamycin sensitivity is to knock down... WDR26 and / or LARP1 .

[0011] Application of gene dose-effect targets that enhance rapamycin resistance in the preparation of anticancer drugs.

[0012] Application of methods to improve rapamycin sensitivity in the preparation of anticancer drugs.

[0013] The advantages and positive effects of this invention are: it provides a target that can enhance yeast rapamycin resistance. YCL039W (GID7) and / or YCL037C (SRO9), and human homologs WDR26 and / or LARP1 This target gene can enhance rapamycin resistance;

[0014] By analyzing yeast YCL039W and / or YCL037C Overexpression of this substance can switch the metabolic mode of microorganisms from "growth" to "production", improve production efficiency, regulate cell growth, and provide key modification targets and regulatory strategies for building efficient and robust industrial microbial cell factories.

[0015] Through human homologs that enhance rapamycin sensitivity WDR26 and LARP1 The research and application of this technology have deepened our understanding of drug resistance mechanisms and provided new insights for understanding disease mechanisms, developing new drugs, and clinical treatment. Attached Figure Description

[0016] Figure 1 overexpression YCL039W and / or YCL037C haploid strain dilution spotting experiment;

[0017] Figure 2 overexpression YCL039W and / or YCL037C Transcriptional analysis of haploid strains under rapamycin conditions;

[0018] Figure 3 overexpression YCL039W strains and overexpression YCL039W and YCL037C Protein levels of the strain under rapamycin conditions;

[0019] Figure 4 BY4741、BY4741+ YCL039W BY4741+ YCL037C and BY4741+ YCL039W + YCL037C Z-scores of genes involved in the Ras signaling pathway;

[0020] Figure 5 YCL039W and YCL037C Models driving rapamycin resistance;

[0021] Figure 6 Based on the SGD database, homologs of ID7 and SRO9 were identified in different species;

[0022] Figure 7 Detection of targeted antibodies using qPCR analysis WDR26 and LARP1 The knockdown efficiency of shRNAs in HEK293T cells was determined. Each strain had n = 3 independent biological replicates; each biological replicate was technically repeated three times, and each point represents the mean within the biological replicate. Data are expressed as mean ± standard deviation. Data were analyzed using an unpaired two-tailed t-test.

[0023] Figure 8 CCK-8 cell viability assay WDR26 or LARP1 Dose-response curves of the sensitivity of knockdown HeLa cells to rapamycin. Detailed Implementation

[0024] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0025] This invention relates to gene dose-effect targets for enhancing rapamycin resistance and their applications. Firstly, it provides a target that can improve yeast rapamycin resistance, specifically... YCL039W and / or YCL037C This study focuses on fundamental research into rapamycin resistance in yeast. Through individual and combined overexpression of the two genes, the necessity and interaction of the two genes were systematically verified. Furthermore, by analyzing the dose-response effect between the genes, it was discovered that the gene combination containing these two targets confers rapamycin resistance in yeast through a dose-dependent antagonistic effect, demonstrating potential industrial application value.

[0026] YCL039W Gene ID: 850318 YCL037C Gene ID: 850320. The target gene or target genome enhances the Ras signaling pathway through dose-dependent antagonism, increasing yeast rapamycin resistance. Based on this characteristic, it can be used to improve resistance by overexpressing [the target gene / genome] in yeast cells. YCL039W and / or YCL037C Constructing engineered yeast strains resistant to rapamycin. This was achieved by targeting yeast... YCL039W (GID7) and / or YCL037C Overexpression of SRO9 can switch the metabolic mode of microorganisms from "growth" to "production," improving production efficiency and regulating cell growth. This provides a key modification target and regulatory strategy for constructing efficient and robust industrial microbial cell factories. For example, overexpression of the transcription factor repressor activator protein 1 (Rap1) can enhance the production of triterpenoid saponins in Saccharomyces cerevisiae. Rapamycin-resistant yeast may be more conducive to maintaining normal cell metabolism and growth under Rap1 overexpression, thereby increasing the yield of chemicals such as triterpenoid saponins.

[0027] In addition, with YCL039W (GID7) and YCL037C (SRO9) Human homology WDR26 and LARP1 It also has the effect of regulating rapamycin resistance. WDR26 Gene ID: 80232 LARP1 Gene ID: 23367. Knockdown WDR26 and / or LARP1 It can increase the sensitivity of human cells to rapamycin. Based on this property, research on cancer treatment targets can be further applied to the development and manufacture of anti-cancer drugs.

[0028] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described are performed according to the corresponding product instructions. Unless otherwise specified, all instruments, reagents, and consumables used in the examples can be purchased from commercial companies. The genes screened in the following examples can all have their specific sequence information obtained by searching for the gene name on NCBI.

[0029] Example 1: Validation of enhancing rapamycin resistance genes

[0030] Will YCL039W and YCL037C We investigated the effects of individual and combined overexpression of the two genes on enhancing yeast rapamycin resistance.

[0031] (1) Construction of co-overexpression strains

[0032] Will YCL039W and YCL037C The recombinant plasmid was constructed by ligating the plasmid into the vector PRS413 using Gibson assembly. This plasmid was then transformed into BY4741 cells and cultured in SC selective medium at 30°C. Single clones were picked for sequencing verification. Correctly sequenced BY4741+ cells were successfully sequenced. YCL039W + YCL037C The strain was subjected to dilution spot plate experiments.

[0033] SC medium: 20 g / L glucose, 6.7 g / L deaminized yeast nitrogen source (YNB), 2 g / L drop-out (deficient in L-leucine (Leu), L-histidine (His), L-tryptophan (Trp), and uracil (Ura); adjust pH to 6.0 based on the amount of amino acid stock solution added). For solid medium, add an additional 20 g / L agar powder and adjust pH to 6.5. Under rapamycin conditions, add an additional 1 mM rapamycin. Incubate at 30°C.

[0034] Dilution plate assay: Yeast cells were cultured in selective medium at 30°C and shaken overnight at 220 rpm. Then, 200 μL of the culture was transferred to 5 mL of selective medium and cultured at 30°C until OD600 = 1. The solution was serially diluted 5 times in distilled water in 10-fold increments, and 3-4 μL of the diluted solution was dropped onto plates sequentially from the lowest to the highest concentration. The culture was incubated at 30°C for 2–6 days. Images were acquired using a PhenoBooth + Colony Counter instrument (SingerInstruments, UK).

[0035] BY4741+ YCL039W + YCL037C Results of the strain dilution spot test are as follows Figure 1 As shown, this strain exhibits some resistance to rapamycin.

[0036] (2) Construction of single-gene overexpression strains

[0037] Will YCL039W and YCL037C The recombinant plasmids were assembled and ligated into the vector PRS413 using Gibson assembly, and constructed according to the method in step (1). The recombinant plasmids were then transformed into BY4741 to construct BY4741+ plasmids. YCL039W and BY4741+ YCL037C Two strains obtained from the multiple constructions were subjected to dilution plate experiments, and the results are as follows: Figure 1 As shown, it was found that overexpression alone... YCL039W or YCL037C Both significantly promoted rapamycin tolerance, and overexpression YCL039WThe strains exhibit better growth fitness.

[0038] Comparison with individual overexpression YCL039W or YCL037C strains, and simultaneously overexpressing YCL039W and YCL037C Dilution spotting experiments of the strains showed that strains that simultaneously overexpressed two genes exhibited decreased rapamycin tolerance compared to strains that overexpressed a single gene.

[0039] Example 2: Exploring the Genetic Mechanisms that Enhance Rapamycin Resistance

[0040] overexpression YCL039W and / or YCL037C The strain was analyzed by qPCR, and the results are as follows: Figure 2 As shown, when both genes were overexpressed simultaneously, their transcriptional levels were lower than those in the single-gene overexpression group, indicating a dose-dependent antagonistic effect. The GFP tag was fused to... YCL039W Above, construct overexpression YCL039W strains and overexpression YCL039W and YCL037C The strains were identified, and their protein levels were studied. The results are as follows: Figure 3 As shown, the results are consistent with qPCR results and with those of individual... YCL039W Compared to strains with increased copy number, simultaneous overexpression of both genes resulted in a significant reduction in GFP signal.

[0041] Known YCL039W (GID7) encodes a subunit of the glucose-induced degradation defect (GID) complex. YCL037C (SRO9) encodes a cytoplasmic RNA-binding protein, and experiments have shown that both are associated with rapamycin resistance. RNA-seq analysis revealed significant alterations in genes related to the Ras signaling pathway. Overexpression of SRO9... YCL039W and / or YCL037C Analysis of proteins in the strain related to the Ras signaling pathway, such as Figure 4 As shown, compared to strain BY4741, YCL039W or YCL037C Overexpression significantly upregulated the expression of genes related to the Ras signaling pathway, including RAS2, BCY1, TPK1 and TPK2 Conversely, Ras signaling activates negative regulators. MSN2, RIM15 and ATG1 The expression was significantly reduced. For example... Figure 5 As shown, activation of the Ras signaling pathway compensates for rapamycin-induced TOR deficiency in yeast. This indicates... YCL039W and YCL037CIt may enhance the Ras signaling pathway through dose-dependent antagonism, compensate for the inhibitory effect of rapamycin on the TOR signaling pathway, and ultimately confer rapamycin resistance in yeast strains.

[0042] Example 3: Verification of the effect of homologous genes in enhancing rapamycin resistance

[0043] Rapamycin, as a TOR inhibitor, often suffers from resistance limitations in its clinical efficacy. YCL039W (GID7) and YCL037C Increased (SRO9) copy number has been shown to contribute to yeast resistance to rapamycin, specifically by selection of [specific components]. YCL039W (GID7) and YCL037C (SRO9) homologous genes, such as Figure 6 As shown, further research is needed on their human homologs. WDR26 and LARP1 Whether it possesses conserved function. To mimic the reduction in gene dosage in mammalian cells, a shRNA-mediated knockdown strategy was employed to target and knock down [genes]. WDR26 and LARP1 .

[0044] WDR26 and LARP1 The knockdown cell line was generated as follows: Two shRNA sequences targeting each gene and a non-targeting shRNA sequence were cloned into a lentiviral vector. Using Lipofectamine 3000 (Thermo Fisher Scientific), the shRNA plasmid was co-transfected with psPAX2 (additional gene packaging) and pcMV-VSV-G (additional gene packaging) into HEK293T cells to generate lentiviral particles. Viral supernatants were collected at 48 h and 72 h. HeLa cells were infected with the resulting lentivirus in the presence of polystyrene (MCE). After 48 h, the medium was replaced with fresh medium containing puromycin for selection, establishing stable lentiviral cells. WDR26 or LARP1 Knockdown of cell lines. Detection of targeted [cell lines] using qPCR analysis. WDR26 and LARP1 The knockdown efficiency of shRNAs in HEK293T cells was as follows: Figure 7 As shown.

[0045] Effective shRNA constructs were packaged into lentiviral vectors to establish stable HeLa cell lines, and human homologs were knocked down. WDR26 or LARP1 Cell viability was determined by CCK-8 assay. WDR26 or LARP1 Dose-response curves of the sensitivity of knockdown HeLa cells to rapamycin.

[0046] Will WDR26 or LARP1 Knockdown HeLa cells and non-targeted control cells were seeded in 96-well plates at 5 × 10⁶ cells per well. 3 Cells were seeded in 100 µL of intact DMEM medium. Cells were incubated overnight at 37°C with 5% CO2 to allow for attachment. The next day, different concentrations (0.1 µM to 50 µM) of rapamycin were added to the wells to generate dose-response curves. Rapamycin was dissolved in DMSO, and the control contained the same percentage of DMSO as the final solution. Concentrations were determined in triplicate for each biological replicate, with blank wells (medium + CCK-8) used as controls. After 48 hours of treatment, 10 µL of CCK-8 solution (MCE) was added to each well, and the cells were incubated at 37°C in the dark for 2 hours. The absorbance was measured at 450 nm, and the percentage of viable cells relative to the untreated control was calculated. The IC50 value was determined by fitting a nonlinear regression curve in GraphPad Prism v8.

[0047] The results are as follows Figure 8 As shown, knockdown reduced the half-maximal inhibitory concentration (IC50) of rapamycin in cells, indicating that... WDR26 and LARP1 It plays a conserved role in rapamycin resistance and is expected to become a potential target for combination therapy with rapamycin.

[0048] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A gene dose-response target for enhancing rapamycin resistance, characterized by: Target gene is YCL039W and / or YCL037C , or for WDR26 and / or LARP1 .

2. The gene dose-response target for enhancing rapamycin resistance according to claim 1, characterized in that: YCL039W Gene ID: 850318 YCL037C Gene ID: 850320 WDR26 Gene ID: 80232 LARP1 Gene ID: 23367.

3. The gene dose-response target for enhancing rapamycin resistance according to claim 1 or 2, characterized in that: The target gene enhances the Ras signaling pathway through dose-dependent antagonism, thereby increasing yeast resistance to rapamycin.

4. A engineered yeast strain resistant to rapamycin, characterized in that: overexpression YCL039W and / or YCL037C Yeast.

5. The application of the rapamycin-resistant engineered yeast strain according to claim 4 in a yeast cell factory.

6. A method for improving rapamycin sensitivity, characterized in that: Knock down WDR26 and / or LARP1 .

7. The application of the gene dose-effect target for enhancing rapamycin resistance as described in any one of claims 1-3 in the preparation of anticancer drugs.

8. The application of the method for improving rapamycin sensitivity as described in claim 7 in the preparation of anticancer drugs.

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