A dual fluorescence reporter recombinant plasmid for synchronously monitoring hypoxia and ferroptosis core gene activity and a construction method and application thereof

Abstract

The application discloses a construction method of a double-fluorescent reporter recombinant plasmid for synchronously monitoring hypoxia and ferroptosis core gene activity and application thereof, and belongs to the technical field of biotechnology.The recombinant plasmid comprises a hypoxia response expression box and a ferroptosis core gene promoter expression box, the hypoxia response expression box comprises a hypoxia response element and a first fluorescent protein coding gene, and the ferroptosis core gene promoter expression box comprises a promoter of a ferroptosis core gene ATG7 or KDM5C and a second fluorescent protein coding gene.The double-fluorescent reporter system capable of synchronously monitoring a hypoxia signal and ferroptosis core gene activity is constructed by combining the hypoxia response element and the ferroptosis core gene promoter.Further, a stable-transfected hepatoma cell line is established based on the recombinant plasmid, and the stable-transfected hepatoma cell line can be applied to screening and pharmacodynamic evaluation of a candidate drug targeting the 'hypoxia-ferroptosis' axis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing a dual-fluorescent reporter gene recombinant plasmid that can be used to simultaneously monitor the activation levels of hypoxia and ferroptosis core genes, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is characterized by high mortality and a high susceptibility to treatment resistance, severely limiting patient survival. In recent years, ferroptosis, an iron-dependent programmed cell death pathway, has played an increasingly prominent role in overcoming tumor drug resistance. Numerous studies have shown that the antitumor efficacy of various clinical intervention strategies, such as sorafenib, radiotherapy, and immunotherapy, is closely related to their ability to induce lipid peroxidation in tumor cells, thereby triggering ferroptosis. Therefore, activating the ferroptosis pathway has become an important mechanistic basis for HCC treatment.

[0003] However, the typical hypoxic microenvironment formed during the malignant progression of solid tumors severely limits the effectiveness of therapeutic strategies based on ferroptosis induction. Under hypoxic partial pressure, hypoxia-inducible factor-1α (HIF-1α) is stably expressed and enriched, inducing ferroptosis resistance in tumor cells through multiple mechanisms. Although solute carrier family 7 member 11 (SLC7A11) is known to be an important effector molecule in the hypoxia-HIF-1α-ferroptosis regulatory axis, this regulatory network is highly complex, and the existence of other key regulatory pathways independent of SLC7A11 remains to be explored. To systematically elucidate the complex interaction network between HIF-1α and ferroptosis and develop novel combination therapy strategies, a cell research platform capable of reflecting the dynamic changes of this network in real time is urgently needed. However, existing detection methods are mostly endpoint detection methods targeting single pathways, and cannot achieve simultaneous visual monitoring of "hypoxia signal activation" and "ferroptosis core gene response" at the live cell level. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a dual-fluorescent reporter gene recombinant plasmid that can simultaneously monitor the activity of hypoxia and ferroptosis core genes, its construction method, and its application.

[0005] This invention first screened two core hypoxia-related ferroptosis genes—autophagy-related gene 7 (ATG7) and lysine-specific demethylase 5C (KDM5C)—through bioinformatics analysis and experimental verification. Subsequently, two dual-fluorescent reporter gene recombinant plasmids were designed and constructed to simultaneously detect the activities of ATG7 and KDM5C under hypoxia, respectively. Transiently transfected and stably transfected cell lines were established using liposome-mediated transient transfection and lentiviral infection. Laser scanning confocal microscopy was used to track changes in the activation levels of HIF-1α and ATG7 or HIF-1α and KDM5C in a hypoxic microenvironment. This provides a visualized cellular platform for visualizing the molecular network of HIF-1α-related ferroptosis at the cellular level and for high-throughput screening and evaluation of related antitumor drugs.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A dual-fluorescent reporter gene recombinant plasmid for simultaneously monitoring the activity of hypoxia and ferroptosis core genes, the recombinant plasmid comprising: A hypoxia-responsive expression cassette, comprising a hypoxia-responsive element and a gene encoding a first fluorescent protein; and The ferroptosis core gene promoter expression cassette contains the promoter of the ferroptosis core gene ATG7 or KDM5C and the second fluorescent protein encoding gene.

[0007] Preferably, the hypoxia-responsive element is five tandemly repeated HREs.

[0008] Preferably, the first fluorescent protein is enhanced green fluorescent protein EGFP, and the second fluorescent protein is near-infrared fluorescent protein miRFP670.

[0009] Preferably, the promoter of the ferroptosis core gene ATG7 is ATG7p2, and its nucleic acid sequence is SEQ ID NO.2; the promoter of the ferroptosis core gene KDM5C is KDM5Cp3, and its nucleic acid sequence is SEQ ID NO.6.

[0010] Preferably, the recombinant plasmid further contains the neomycin resistance gene NeoR selection marker for screening stable cell lines.

[0011] In some embodiments, the above-described dual-fluorescent reporter gene recombinant plasmid was transiently transfected into HEK293T cells, and detected by laser scanning confocal microscopy under normoxic and hypoxic conditions. A dual-channel response was successfully achieved: in a hypoxic environment, the expression of 5HRE-EGFP, activated by the HIF-1α signaling pathway, was enhanced, while the fluorescence intensity of miRFP670 induced by the ferroptosis core gene promoter decreased.

[0012] This invention also provides a method for constructing the above-mentioned dual-fluorescent reporter gene recombinant plasmid, comprising the following steps: (1) Constructing a red fluorescent reporter gene recombinant plasmid driven by the promoter of the ferroptosis core gene: Insert the truncated promoter fragment of the ferroptosis core gene ATG7 or KDM5C into a linearized fluorescent protein expression vector to obtain a red fluorescent reporter gene recombinant plasmid. (2) Constructing a dual fluorescent reporter gene recombinant plasmid: The hypoxia response element and the fragment of the gene encoding the first fluorescent protein are inserted into the red fluorescent reporter gene recombinant plasmid obtained in step (1) by homologous recombination technology to obtain a dual fluorescent reporter gene recombinant plasmid.

[0013] Preferably, in step (1), the promoter truncated fragment of the ferroptosis core gene ATG7 is ATG7p2, and its nucleic acid sequence is SEQ ID NO.2; the promoter truncated fragment of the ferroptosis core gene KDM5C is KDM5Cp3, and its nucleic acid sequence is SEQ ID NO.6.

[0014] Preferably, in step (2), the homologous recombination technology is ClonExpress seamless cloning technology, the hypoxia response element is a tandem repeat of HRE, the first fluorescent protein is EGFP, and the second fluorescent protein is miRFP670.

[0015] The present invention also provides a stable cell line for simultaneously monitoring the activity of hypoxia and ferroptosis core genes. The stable cell line is constructed by introducing the above-mentioned dual fluorescent reporter gene recombinant plasmid into host cells through lentiviral infection or liposome transfection.

[0016] Preferably, the host cell is a liver cancer cell, HepG2.

[0017] The present invention also provides the application of the above-mentioned stable cell lines in screening candidate drugs that regulate hypoxia signaling or ferroptosis pathways.

[0018] In some embodiments, this stable cell line is used in in vitro experiments with anticancer compounds such as PX-478 to visualize and verify its ability to simultaneously reflect changes in cellular hypoxia response and ATG7 / KDM5C expression, providing an evaluation tool for targeted microenvironment intervention.

[0019] Compared with the prior art, the present invention has the following beneficial effects: Precise target selection and optimal promoter truncated versions exhibit extremely high transcriptional activity and specificity. The dual fluorescence response exhibits high sensitivity, low background noise, and independent emission wavelengths. It enables simultaneous and visual monitoring of the dynamic interaction between the hypoxic microenvironment and the ferroptosis pathway at the cellular level; Stable cell lines have stable genetic properties and can serve as a standardized platform for screening and evaluating the efficacy of high-throughput anti-tumor targeted drugs in vitro. Attached Figure Description

[0020] Figure 1 To screen and validate core genes related to hypoxia-related ferroptosis, the following data are presented: A) Scatter plot of consistent clustering of 6 groups in the TCGA-LIHC sample based on PCA dimensionality reduction; B) Bar chart of average hypoxia score of the 6 clustered samples; C) Venn diagram of differentially expressed genes related to hypoxia-related ferroptosis obtained from screening based on TCGA and GEO datasets; D) Survival difference analysis of high and low expression groups of core candidate genes in the LIHC sample; E) Expression difference of candidate core genes in the GSE18494 dataset under normoxic and hypoxic conditions; F) In vitro qPCR validation of changes in expression levels of core genes under hypoxic treatment. P < 0.05 P < 0.01 P < 0.001, ns: no significant difference.

[0021] Figure 2 The sequencing alignment results are for the red fluorescent reporter gene recombinant plasmids induced by the truncated ATG7 promoter. Among them, A is the sequencing alignment result of the full-length third-generation sequencing of the ATG7p1-miRFP670-G418 plasmid; B is the sequencing alignment result of the full-length third-generation sequencing of the ATG7p2-miRFP670-G418 plasmid; and C is the sequencing alignment result of the full-length third-generation sequencing of the ATG7p3-miRFP670-G418 plasmid.

[0022] Figure 3 The sequencing alignment results are for the red fluorescent reporter gene recombinant plasmids induced by the truncated KDM5C promoter. Among them, A is the sequencing alignment result of the full-length KDM5Cp1-miRFP670-G418 plasmid; B is the sequencing alignment result of the full-length KDM5Cp2-miRFP670-G418 plasmid; and C is the sequencing alignment result of the full-length KDM5Cp3-miRFP670-G418 plasmid.

[0023] Figure 4 The plasmid structure map of ATG7p1-miRFP670-G418.

[0024] Figure 5 The plasmid structure map of ATG7p2-miRFP670-G418.

[0025] Figure 6 The plasmid structure map of ATG7p3-miRFP670-G418 is shown.

[0026] Figure 7 The plasmid structure map of KDM5Cp1-miRFP670-G418.

[0027] Figure 8 The plasmid structure map of KDM5Cp2-miRFP670-G418.

[0028] Figure 9 The plasmid structure map of KDM5Cp3-miRFP670-G418.

[0029] Figure 10 This study aims to construct and identify recombinant red fluorescent reporter gene plasmids induced by the promoter of the ferroptosis core gene and to screen for the optimal promoter truncated form. A shows agarose gel electrophoresis of the single fluorescent recombinant plasmid after PCR amplification and restriction endonuclease digestion; B shows laser confocal imaging of HEK293T cells after transient transfection with different recombinant plasmids; C shows a comparison of quantitative analysis of fluorescence intensity in laser confocal imaging after transient cell transfection; and D shows a quantitative analysis of miRFP670 expression activity induced by promoter truncated forms of different lengths, detected by qPCR.

[0030] Figure 11 To verify the sequence of the red-green dual fluorescent reporter gene recombinant plasmid in response to hypoxia and ferroptosis, A represents the third-generation full-length sequencing alignment result of ATG7p2-miRFP670-5HRE-EGFP; B represents the third-generation full-length sequencing alignment result of KDM5Cp3-miRFP670-5HRE-EGFP.

[0031] Figure 12 The plasmid structure map of ATG7p2-miRFP670-5HRE-EGFP.

[0032] Figure 13 The plasmid structure map of KDM5Cp3-miRFP670-5HRE-EGFP.

[0033] Figure 14 To illustrate the in vitro functional identification and cellular visualization applications of the red-green dual-fluorescent reporter gene recombinant plasmid responding to hypoxia and ferroptosis, the following images are presented: A) Agarose gel electrophoresis image of the dual-fluorescent recombinant plasmid after PCR amplification and restriction endonuclease digestion; B) Laser confocal imaging image of HEK293T cells after transient transfection of the dual-fluorescent recombinant plasmid in response to normoxic / hypoxic dynamic changes; C) Laser confocal imaging image of the HepG2-ARHE / KRHE stable transfected cell line expressing the dual-fluorescent system under hypoxia and intervention with the targeted drug PX-478. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0035] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purpose of more clearly describing the invention, the following terms are defined as follows: Hypoxia-responsive elements (HREs): Hypoxia-responsive elements are specific DNA sequences located in the promoter region of genes and are binding sites for hypoxia-inducible factor-1α (HIF-1α). When cells are in a hypoxic (hypoxic) environment, HIF-1α is stably expressed and enters the cell nucleus, where it binds to HREs, thereby initiating or enhancing the transcription of downstream genes. In this application, the hypoxia-responsive element is a core sequence of five tandemly repeated HREs.

[0037] 5HRE: refers to five hypoxia response elements that are repeated in series. The sensitivity and intensity of the response to hypoxia signals can be enhanced by repeating them in series.

[0038] Ferrocyte core gene promoter: refers to the DNA regulatory sequence that drives the transcription of ferroptosis core genes.

[0039] ATG7: Autophagy-related gene 7 is a core hypoxia-related ferroptosis gene that was verified by bioinformatics and experiments in this application, and its expression is regulated by the hypoxia microenvironment.

[0040] KDM5C: Lysine-specific demethylase 5C is a core gene of hypoxia-associated ferroptosis that has been bioinformatically and experimentally verified in this application, and its expression is regulated by the hypoxic microenvironment.

[0041] EGFP: Enhanced Green Fluorescent Protein, is a commonly used reporter gene protein that emits green fluorescence upon excitation by light of a corresponding wavelength. In this application, EGFP is used as the first fluorescent protein, and its expression is driven by the hypoxia-responsive element (5HRE) for visual monitoring of cellular hypoxia signals.

[0042] miRFP670: a near-infrared fluorescent protein, is a fluorescent protein with an emission peak around 670 nm. In this application, miRFP670 is used as a second fluorescent protein, and its expression is driven by the promoter of the ferroptosis core gene (ATG7p2 or KDM5Cp3) for visual monitoring of the activity of the ferroptosis core gene.

[0043] NeoR: Neomycin resistance gene, encoding an aminoglycoside phosphotransferase that confers resistance to G418 (Genzyme), thus enabling the screening of positive cell clones.

[0044] Stable cell lines: These are cell lines in which exogenous genes are integrated into the genome of host cells via lentiviruses or other vectors, enabling the exogenous genes to be expressed stably over a long period. This application specifically refers to the HepG2 liver cancer cell line constructed from the aforementioned dual-fluorescent reporter gene recombinant plasmid, which stably expresses EGFP and miRFP670.

[0045] Unless otherwise specified, all units used in this specification are international standard units (SI). Numerical values ​​and ranges appearing in this invention should be understood to include unavoidable systematic errors in industrial production.

[0046] The primer information used for PCR amplification in the following examples is shown in Table 1, and the primer information used for qRT-PCR analysis is shown in Table 2.

[0047] Table 1 Primer information used for PCR amplification in the examples

[0048] Table 2 Primer information used in the qRT-PCR analysis in the examples

[0049] Example 1: Screening and validation of core genes for hypoxia-associated ferroptosis This embodiment aims to precisely identify the core genes of ferroptosis regulated by the hypoxic microenvironment through combined cross-validation of cancer big data screening and in vitro physical hypoxia cell models. The specific steps are as follows: 1. Bioinformatics screening of core genes for hypoxia-associated ferroptosis (1) Data acquisition and grouping: Extract the gene expression matrix of hepatocellular carcinoma samples from the TCGA-LIHC dataset in TCGA and combine it with the gene expression profile of HepG2 cells from the GSE18494 dataset in GEO.

[0050] (2) Consistent clustering and hypoxia score: Based on the ferroptosis-related gene set provided by the FerrDb database, ConsensusClusterPlus was used to perform consistent clustering analysis on the TCGA-LIHC samples (the optimal number of clusters K = 6, see...). Figure 1 A). Hypoxia-related gene sets were obtained from the MsigDB database, and hypoxia scores were calculated for each clustered group using the Seurat software package. The results showed that group 3 had the highest hypoxia score, and group 4 had the lowest (see...). Figure 1 B).

[0051] (3) Differential expression and prognostic analysis screening: Differentially expressed genes (P<0.05) were extracted from the hypoxia score groups with the highest and lowest scores, and their intersection with the ferroptosis core genes were obtained, yielding 109 preliminary candidate genes; simultaneously, differential expression profiles of normoxic / hypoxic HepG2 cells in the GSE18494 dataset were analyzed, yielding 45 candidate genes. The intersection of the two was used to obtain 14 hypoxia-related ferroptosis core genes (see...). Figure 1 C). Further survival analysis of these 14 genes using the GSCA database ultimately identified six core candidate genes significantly associated with prognostic indicators in liver cancer patients, including disease-free interval (DFI), disease-specific survival (DSS), overall survival (OS), and progression-free survival (PFS). These six genes are ACADSB, ATG7, ATG13, KDM5C, PRKCA, and SQSTM1 (see [link to relevant documentation]). Figure 1 D). Analysis of differential expression between normoxic and hypoxic conditions using the GSE18494 dataset revealed that ACADSB, ATG7, and KDM5C showed a significant downregulation trend under hypoxic conditions, while SQSTM1 showed a significant upregulation trend (see...). Figure 1 E).

[0052] 2. Cross-validation of qRT-PCR in vitro for cellular hypoxia response To verify the above bioinformatics analysis results, the transcriptional activity of candidate genes was confirmed in vitro using a physically hypoxic environment.

[0053] (1) Cell treatment: HepG2 cells in the logarithmic growth phase were seeded in culture plates and placed in a special hypoxia chamber containing 0.6% O2 for 24 h of hypoxia culture. At the same time, a normoxic control group was set up.

[0054] (2) qRT-PCR analysis: Total RNA was extracted from cells in the normoxic and hypoxic groups, reverse transcribed into cDNA, and then the selected core candidate genes were quantitatively detected using the SYBR Green chimeric fluorescence method.

[0055] (3) Verification results and accurate target identification: qPCR data confirmed that after 24 h of hypoxic stress treatment, the expression levels of ATG7 and KDM5C were significantly downregulated compared with the normoxic control group (see Figure 1 F). This in vitro experimental trend is perfectly consistent with the gene expression change trend in the aforementioned GSE18494 dataset and shows a highly significant difference. Based on this rigorous joint validation, this invention formally identifies ATG7 and KDM5C as target genes for ferroptosis surveillance in a dual-fluorescence reporter system.

[0056] Example 2: Construction and identification of a red fluorescent reporter gene recombinant plasmid induced by the ferroptosis core gene promoter. This embodiment aims to obtain the optimal promoter sequences of the target genes ATG7 and KDM5C, and to construct a red fluorescent reporter gene recombinant plasmid with extremely high specificity and a low signal-to-noise ratio. The specific steps are as follows: 1. Target gene promoter sequence analysis and truncated design (1) Promoter region acquisition: The human ATG7 and KDM5C genes were retrieved from the NCBI database, and the nucleic acid sequences from 2000 bp upstream to 99 bp downstream of their gene transcription start sites were extracted as potential promoter analysis regions.

[0057] (2) Core promoter prediction and truncation: Multiple prediction tools, including BDGP, ​​Promoter 2.0, and Softberry (containing FPROM, TSSW, TSSP, and TSSG algorithms), were used to cross-validate the above sequences. Based on the core regions of the prediction results, three promoter truncated variants of different lengths were designed for each target gene. The truncated variants of ATG7 were: ATG7p1 (2083 bp, nucleotide sequence as shown in SEQ ID NO.1), ATG7p2 (890 bp, nucleotide sequence as shown in SEQ ID NO.2), and ATG7p3 (423 bp, nucleotide sequence as shown in SEQ ID NO.3); the truncated variants of KDM5C were: KDM5Cp1 (2097 bp, nucleotide sequence as shown in SEQ ID NO.4), KDM5Cp2 (634 bp, nucleotide sequence as shown in SEQ ID NO.5), and KDM5Cp3 (380 bp, nucleotide sequence as shown in SEQ ID NO.6).

[0058] 2. Seamless Cloning Construction of Red Fluorescent Reporter Gene Recombinant Plasmid (1) Vector linearization: Using the red fluorescent reporter gene vector pLenti6.2_miRFP670_G418 carrying neomycin resistance (NeoR) as the backbone, specific primers were designed and linearized (size 8490bp) using reverse PCR technology.

[0059] (2) Amplification of target fragments: Genomic DNA of HepG2 cells was extracted as a template, and primers with 15-20 bp vector terminal homologous sequences were used to amplify the above 6 promoter truncated fragments respectively.

[0060] (3) Homologous recombination: The purified linearized vector was mixed with each promoter fragment at a molar ratio of 1:2, and reacted at 37 °C for 30 min using ClonExpress seamless cloning technology. Then it was transformed into DH5α competent cells and plated on ampicillin-resistant plates for overnight culture.

[0061] 3. Identification of positive clones of recombinant plasmids Single colonies from transformation plates were picked, cultured extensively, and plasmids were extracted for the following rigorous identification: (1) Plasmid PCR and restriction endonuclease digestion: Plasmid PCR amplification was performed using promoter-specific primers; simultaneously, the extracted recombinant plasmids were identified by single-enzyme digestion with XhoI restriction enzyme. Agarose gel electrophoresis results confirmed that the PCR amplification bands of each group of recombinant plasmids and the molecular weight of the linearized DNA after enzyme digestion were completely consistent with theoretical expectations (see...). Figure 10 A, the recombinant plasmid map is shown below. Figures 4 to 9 ).

[0062] (2) Confirmation by third-generation full-length sequencing: The correctly identified plasmids were sent for KBSeq third-generation full-length sequencing. The alignment results between the sequencing sequence and the theoretical sequence confirmed that the core element sequences of the recombinant plasmids ATG7p-miRFP670-G418 and KDM5Cp-miRFP670-G418 were basically correct, and the cloning was successful (see sequencing alignment results). Figure 2 and Figure 3 (The nucleic acid sequence is shown in SEQ ID NO. 42-47).

[0063] 4. Screening for transcriptional activity of optimal promoter truncated forms Since the transcriptional driving activity of promoter fragments of different lengths varies, this step uses transient transfection experiments to quantitatively evaluate the expression efficiency of the six recombinant plasmids.

[0064] (1) Transcriptional level assessment: Recombinant plasmids were transiently transfected into HEK293T cells using liposome transfection reagent. RNA was extracted from cells 48 h after transfection, and the mRNA expression level of promoter-driven miRFP670 was detected by qPCR. The results showed that the expression levels of miRFP670 driven by ATG7p2 and ATG7p3 were significantly higher than those driven by ATG7p1; while there was no significant difference among the three promoter truncated variants of KDM5C (see...). Figure 10 D).

[0065] (2) Fluorescence intensity assessment: 48 h after transfection of HEK293T cells, the cells were fixed and stained with DAPI. The fluorescence expression of miRFP670 was directly observed using a laser confocal microscope. Fluorescence imaging and quantitative analysis confirmed that plasmids ATGp2-miRFP670-G418 and KDM5Cp3-miRFP670-G418 had the highest fluorescence expression efficiency (see...). Figure 10 B, C).

[0066] (3) Optimal conclusion: Based on the combined results of transcription and translation level detection, the present invention finally screened and identified ATG7p2 and KDM5Cp3 as the optimal promoter sequences with the strongest transcriptional activity, and used them for the subsequent construction of dual fluorescent reporter gene system.

[0067] Example 3: Construction and Identification of Recombinant Plasmids for Red-Green Dual-Fluorescent Reporter Genes Responding to Hypoxia and Ferrocyte Death This embodiment aims to introduce a hypoxia-responsive element into an optimal red fluorescent reporter gene recombinant plasmid to construct a red-green dual fluorescent reporter gene recombinant plasmid that can simultaneously respond to hypoxia and ferroptosis. The specific steps are as follows: 1. Preparation of linearized carriers Based on the spectral analysis of the optimal red fluorescent reporter gene recombinant plasmids (ATG7p2-miRFP670-G418 and KDM5Cp3-miRFP670-G418), their sequences contain specific KpnI and XhoI restriction enzyme sites, respectively, with only 5 base pairs between the two restriction sites, which does not affect the original functional elements of the plasmids. Therefore, the two optimized plasmids were double-digested with KpnI and XhoI restriction enzymes to obtain the complete linearized vector backbone after removing the very short sequence, and then purified using an agarose gel extraction kit.

[0068] 2. Amplification and recombination of hypoxia-responsive element fragments (1) Insertion fragment amplification: Using the 5HRE / GFP plasmid as a template, the 5HRE-EGFP target fragment (theoretical size 1186 bp, nucleic acid sequence see SEQ ID NO.7) containing 5 tandem repeats of HRE, Minimal CMV promoter and EGFP coding sequence was amplified by high-fidelity DNA polymerization. A 15-20 bp vector terminal homologous sequence and corresponding restriction enzyme site were pre-introduced at the 5' end of the amplification primers.

[0069] (2) Homologous recombination construction: The concentrations of the purified linearized vector and the 5HRE-EGFP insert were determined. The ClonExpress seamless cloning recombination reaction system was prepared on ice at a molar ratio of 1:2. After reacting at 37 °C for 30 min, the reaction was transformed into DH5α competent cells and plated on ampicillin-resistant plates for positive clone screening.

[0070] 3. Identification of dual-fluorescent reporter gene recombinant plasmids (1) Physical structure identification: Positive clones were selected and plasmids were extracted. First, plasmid PCR amplification was performed using primers for the 5HRE-EGFP fragment, followed by single-enzyme digestion with KpnI restriction enzyme for verification. Agarose gel electrophoresis imaging results showed that the target fragment had been successfully integrated, and dual-fluorescent reporter gene recombinant plasmids (named ATG7p2-miRFP670-5HRE-EGFP and KDM5Cp3-miRFP670-5HRE-EGFP, respectively; see graphs below) were successfully constructed. Figure 12 and 13 Enzyme digestion identification is shown in Figure 14 A).

[0071] (2) Non-destructive sequence confirmation: The plasmid that was initially identified was sent to KBSeq third-generation full-length sequencing. The sequencing alignment results showed that 5HRE-EGFP and the promoter fragment were accurately inserted, and the core element sequence of the plasmid was complete (see sequencing alignment results). Figure 11 Its nucleic acid sequence is shown in SEQ ID NO. 8-9.

[0072] Example 4: Transient transfection and hypoxia-responsive functional verification of recombinant plasmids containing red-green dual-fluorescent reporter genes in response to hypoxia and ferroptosis. This embodiment aims to verify the dual-channel dynamic response capability of the above-mentioned dual-fluorescent reporter gene recombinant plasmid under hypoxic microenvironment conditions through live cell in vitro transfection experiments. The specific steps are as follows: 1. Cell processing and transient transfection Human embryonic kidney cells HEK293T were seeded in glass-bottom confocal culture dishes pretreated with complete culture medium. When the cell confluence reached about 70%, the purified dual-fluorescent reporter gene recombinant plasmid (ATG7p2-miRFP670-5HRE-EGFP or KDM5Cp3-miRFP670-5HRE-EGFP) was transiently transfected into the target cells using transfection reagents.

[0073] 2. Physical hypoxia induction and sample pretreatment 24 h after transfection, the experimental group culture dishes were transferred to a dedicated hypoxia chamber containing 0.6% O2 for continuous induction for 24 h, while a normoxic control group was set up. After the hypoxia treatment, the culture medium was discarded, and the cells were gently washed with PBS buffer preheated to 37 °C. Cells were then fixed with 4% paraformaldehyde for 15 min, washed again, and then stained with DAPI for nuclear staining.

[0074] 3. Laser confocal imaging The fixed cells were subjected to dual-channel fluorescence signal detection using laser confocal microscopy (green channel to excite EGFP, near-infrared channel to excite miRFP670). Imaging and quantitative fluorescence analysis revealed a typical characteristic dynamic response: in a hypoxic environment, the green fluorescence signal of 5HRE-EGFP activated by HIF-1α was significantly enhanced, while the near-infrared fluorescence signal of miRFP670, driven by the promoter of the ferroptosis core gene suppressed by the hypoxic microenvironment, was significantly attenuated (see...). Figure 14 B). This result confirms that the recombinant plasmid possesses highly sensitive interactive network monitoring capabilities at the living cell level.

[0075] Example 5: Establishment of stable hepatocellular carcinoma cell lines and application of drug intervention targeting the hypoxia-ferroptosis axis This embodiment aims to construct a genetically stable dual-channel visualization cell platform, and uses natural antitumor compounds as an example to verify the industrial practical value of this platform in high-throughput drug screening and efficacy evaluation of anti-drug resistance microenvironments. The specific steps are as follows: 1. Lentiviral packaging and screening of stable cell lines (1) Lentiviral packaging and concentration: Using a second-generation lentivirus packaging system, transfer plasmids (ATG7p2-miRFP670-5HRE-EGFP or KDM5Cp3-miRFP670-5HRE-EGFP), packaging plasmid psPAX2, and envelope plasmid pCMV-VSV-G were co-transfected into HEK293T cells. After 48 h and 72 h, virus-rich culture supernatants were collected, filtered through a 0.22 μm filter, and treated with a concentration kit to obtain high-titer lentivirus suspensions.

[0076] (2) Construction of stable hepatocellular carcinoma cell lines: HepG2 hepatocellular carcinoma cells were infected with the above-mentioned lentivirus with the assistance of polybrene. The culture medium was changed 24 h after infection, and then multiple rounds of resistance screening were carried out using G418 selection medium with the optimal lethal concentration (5 mg / mL) determined in advance. Finally, stable cell lines with stable genetic properties were successfully established and named HepG2-ARHE and HepG2-KRHE, respectively.

[0077] 2. In vitro intervention experiments with the targeted compound (PX-478) Visual evaluation of efficacy: HepG2-ARHE and HepG2-KRHE cells stably expressing the dual fluorescence system were seeded in confocal culture dishes and divided into three groups: normoxic control group, hypoxia treatment group, and hypoxia + PX-478 combined intervention group. After 24 h of treatment, the cells were fixed, stained with DAPI nuclei, and observed under a laser confocal microscope in a dual-channel manner.

[0078] Confocal imaging results showed that under hypoxia alone, intracellular EGFP fluorescence was enhanced while miRFP670 fluorescence was weakened; however, under hypoxia combined with PX-478 intervention, 5HRE-EGFP expression was significantly reduced (indicating successful blockade of the hypoxia signaling pathway), while miRFP670 expression was significantly increased (indicating that the transcriptional activity of the hypoxia-inhibited core ferroptosis gene ATG7 / KDM5C was reversed and restored) (see... Figure 14 C). This embodiment fully demonstrates that the stable cell line platform constructed in this invention has the technical capability to perform high-throughput, visualized screening of candidate drugs that can reverse tumor hypoxia-resistant drug resistance and restore ferroptosis sensitivity at the cellular microenvironment level.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual-fluorescent reporter gene recombinant plasmid for simultaneously monitoring the activity of hypoxia and ferroptosis core genes, characterized in that, The recombinant plasmid comprises: The hypoxia-responsive expression cassette contains a hypoxia-responsive element and a first fluorescent protein encoding gene; and the ferroptosis core gene promoter expression cassette contains a promoter of the ferroptosis core gene ATG7 or KDM5C and a second fluorescent protein encoding gene.

2. The recombinant plasmid according to claim 1, characterized in that, The hypoxia-responsive element is five tandemly repeated HREs.

3. The recombinant plasmid according to claim 1, characterized in that, The first fluorescent protein is enhanced green fluorescent protein EGFP, and the second fluorescent protein is near-infrared fluorescent protein miRFP670.

4. The recombinant plasmid according to claim 1, characterized in that, The promoter of the ferroptosis core gene ATG7 is ATG7p2, and its nucleic acid sequence is SEQ ID NO.2; the promoter of the ferroptosis core gene KDM5C is KDM5Cp3, and its nucleic acid sequence is SEQ ID NO.

6.

5. The recombinant plasmid according to claim 1, characterized in that, The recombinant plasmid also contains the Neomycin resistance gene NeoR selection marker for screening stable cell lines.

6. A method for constructing the dual-fluorescent reporter gene recombinant plasmid according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Constructing a red fluorescent reporter gene recombinant plasmid driven by the promoter of the ferroptosis core gene: Insert the truncated promoter fragment of the ferroptosis core gene ATG7 or KDM5C into a linearized fluorescent protein expression vector to obtain a red fluorescent reporter gene recombinant plasmid. (2) Constructing a dual fluorescent reporter gene recombinant plasmid: The hypoxia response element and the fragment of the gene encoding the first fluorescent protein are inserted into the red fluorescent reporter gene recombinant plasmid obtained in step (1) by homologous recombination technology to obtain a dual fluorescent reporter gene recombinant plasmid.

7. The method according to claim 6, characterized in that, In step (1), the promoter truncated fragment of the ferroptosis core gene ATG7 is ATG7p2, and its nucleic acid sequence is SEQ ID NO.2; the promoter truncated fragment of the ferroptosis core gene KDM5C is KDM5Cp3, and its nucleic acid sequence is SEQ ID NO.6; In step (2), the homologous recombination technology is ClonExpress seamless cloning technology, the hypoxia response element is a tandem repeat of HRE, the first fluorescent protein is EGFP, and the second fluorescent protein is miRFP670.

8. A stable cell line for simultaneously monitoring the activity of hypoxia and ferroptosis core genes, characterized in that, The stable cell line is constructed by introducing the dual fluorescent reporter gene recombinant plasmid as described in any one of claims 1-5 into host cells via lentiviral infection or liposome transfection.

9. The stable cell line according to claim 8, characterized in that, The host cell was a liver cancer cell, HepG2.

10. The use of the stable cell line of claim 8 or 9 in screening candidate drugs that regulate hypoxia signaling or ferroptosis pathway.

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