Cell model for high-throughput screening of 2-DG anti-kidney cancer drugs as well as construction method and application of cell model

Abstract

The invention discloses a cell model for high-throughput screening of 2-DG anti-kidney cancer drugs as well as a construction method and application of the cell model. The construction method of the model comprises the following steps: cloning a TXNIP (Transmit X Nuclear Induced Protein) promoter onto a pGL4.19-Luc2 vector, so as to obtain a luciferase reporter gene expression vector 'pGL4.19-TXNIP-P-Luc2' regulated and controlled by the TXNIP promoter; the vector is transfected to kidney cancer cells, and a cell line capable of stably expressing the reporter gene is obtained through G418 screening. According to the application, the obtained cell model is used for high-throughput screening of 2-DG anti-kidney cancer drugs. After the 2-DG anti-kidney cancer drug acts on the cell model, the cell fluorescence signal is enhanced, and the luminous intensity is increased. The cell model is combined with a living small animal imaging technology, so that the curative effect of 2-DG anti-cancer drugs can be noninvasively and early evaluated in real time. The invention provides an effective tool for research and development of high-throughput screening of 2-DG anti-kidney cancer drugs.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly relates to a cell model for high-throughput screening of 2-DG anti-renal cancer drugs, a construction method thereof, and an application thereof. Background Art

[0002] Renal cell carcinoma (RCC) is one of the common malignant tumors of the urinary system, accounting for about 3% of all adult malignant tumors. Clear cell renal cell carcinoma (ccRCC) is the most common pathological type of RCC, accounting for 70% - 85% of the total number of RCC. In recent years, the incidence of primary RCC has been increasing year by year, but targeted treatment methods for RCC are still lacking. Therefore, the development of new targeted anti-renal cancer drugs is of great significance for the prevention and treatment of RCC.

[0003] Energy metabolism reprogramming is one of the important characteristics of tumors, among which glucose metabolism reprogramming is the most prominent feature of tumor metabolism. In normal cells, glucose uptake is maintained at a certain level, that is, glucose homeostasis is maintained. In order to adapt to the environment, tumor cells uptake a large amount of glucose through metabolic reprogramming for glycolysis to obtain energy to meet the needs of rapid cell proliferation. Therefore, inhibiting the glycolysis pathway of tumor cells can effectively inhibit the proliferation of tumor cells and even kill tumor cells. Targeting the glycolysis pathway is considered a potential anti-tumor drug with broad application prospects. At present, anti-cancer drugs of glycolysis inhibitors have achieved obvious effects in various tumors, and many are in clinical trials, such as glucose analog 2-deoxy-D-glucose (2-DG) drugs. 2-DG is a glucose analog that can competitively inhibit the activity of hexokinase 2 (HK2). 2-DG is phosphorylated by HK2 in cells to 2-deoxyglucose-6-phosphate (2-DG-6P), and 2-DG-6P cannot be metabolized further, thus affecting the glucose metabolism pathway. Appropriate treatment with 2-DG can inhibit tumor cell growth and induce apoptosis by affecting the glucose metabolism process. A number of recent studies have shown that various 2-DG derivatives, including 2-deoxy-2-fluoro-D-glucose (2-FG) and 2-deoxy-D-glucose-d (2-DG-d), also have good anti-tumor activities. As potential targeted glycolysis inhibitor anti-cancer drugs, 2-DG and its derivatives show good clinical application prospects. Since the modified 2-DG derivatives need to verify whether they retain the anti-tumor biological activity of the drug through in vitro and in vivo experiments, an effective tool is needed to achieve high-throughput screening to accelerate the R & D process of such drugs, but there is no report on such a tool at home and abroad.

[0004] Cell models are important tools for drug research. In recent years, cell models based on bioluminescence molecular imaging technology have played an important role in the screening of anti-cancer drugs. By constructing cell lines stably expressing reporter genes and inoculating them into animals or culturing them in vitro, cell models for anti-cancer drug screening can be established. Thioredoxin-interacting protein (TXNIP) is a key regulator involved in glucose metabolism. It can inhibit the reprogramming of tumor cell glucose metabolism by regulating glucose transporters to control cell glucose uptake, thereby playing its anti-tumor role. Our research results show that TXNIP is lowly expressed in renal cancer cells, and 2-DG can up-regulate the expression level of TXNIP in renal cancer cells at the transcriptional level through MLXIP (the transcription factor of TXNIP). Therefore, if the promoter sequence of the TXNIP gene is fused with the luciferase gene by molecular cloning technology, a bioluminescence reporter gene reflecting the transcriptional activity of the TXNIP promoter can be constructed. After transfecting this reporter gene into renal cancer cells, a cell model can be established to monitor the effects of 2-DG anti-renal cancer drugs in vivo and in vitro. The successful construction of this cell model will contribute to preclinical research of innovative drugs and accelerate the R & D process. The working principle of the reporter gene is as Figure 1 shown. Summary of the Invention

[0005] The object of the present invention is to provide a cell model for high-throughput screening of 2-DG anti-renal cancer drugs, its construction method and application, so as to solve the problems existing in the above-mentioned prior art. Based on the principle that 2-DG can up-regulate the expression of TXNIP at the transcriptional level, the present invention develops a cell model that can reflect the effects of 2-DG anti-renal cancer drugs and can be used to screen this type of anti-cancer drugs under in vivo conditions.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A renal cancer cell line stably expressing the TXNIP promoter luciferase reporter gene. The construction method of the renal cancer cell line includes: constructing a plasmid containing the TXNIP promoter luciferase reporter gene, using pGL4.19-Luc2 as the vector, and inserting the TXNIP gene promoter sequence SEQ ID NO.1 between the KpnI and XhoI restriction enzyme cleavage sites of the pGL4.19-Luc2 plasmid to obtain a recombinant plasmid containing the TXNIP promoter luciferase reporter gene. The nucleotide sequence of the TXNIP promoter luciferase reporter gene is as shown in SEQ ID NO.2; transfecting the recombinant plasmid containing the TXNIP promoter luciferase reporter gene into human renal cancer cells A498 to establish a renal cancer cell line stably expressing the TXNIP promoter luciferase reporter gene.

[0008] Technical solution 2: Application of the described renal cancer cell line in screening 2-DG anti-renal cancer drugs in vitro.

[0009] The present invention discloses the following technical effects:

[0010] The present invention fuses the TXNIP gene promoter sequence with the luciferase gene through molecular cloning technology to construct a bioluminescent reporter gene reflecting the transcriptional activity of the TXNIP promoter. After transfection of this reporter gene into renal cancer cells, a cell model can be established for monitoring the effects of 2-DG anti-renal cancer drugs in vitro and in vivo conditions. The cell model provided by the present invention, combined with in vivo small animal imaging technology, can evaluate the efficacy of anti-cancer drugs in real-time and non-invasively at an early stage. The present invention provides an effective tool for high-throughput screening of 2-DG anti-renal cancer drug R & D. Description of the drawings

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0012] Figure 1 Schematic diagram of the working principle of the reporter gene;

[0013] Figure 2 For determining the low expression of TXNIP in renal cancer cells in Example 1; among them, A is the analysis of TXNIP expression levels in renal clear cell carcinoma (KIRC) and adjacent tissues by the TCGA online tool UALCAN, and B is the analysis of TXNIP mRNA level expression in normal embryonic kidney cells HEK293 and renal cancer cells A498 by RT-qPCR;

[0014] Figure 3 For the inhibitory effects of 2-DG and its derivatives 2-FG and 2-DG-d on the proliferation and glycolysis of A498 cells in Example 2; among them, A-C are the CCK-8 experimental results of the effects of different concentrations of 2-DG (A), 2-FG (B), and 2-DG-d (C) on the proliferation of A498 cells; D-F are the experimental results of the effects of different concentrations of 2-DG (D), 2-FG (E), and 2-DG-d (F) on the glucose consumption of A498; G-I are the experimental results of the effects of different concentrations of 2-DG (D), 2-FG (E), and 2-DG-d (F) on the lactate production of A498;

[0015] Figure 4RT-qPCR and Western blot experimental results of the upregulation of TXNIP expression by 2-DG and its derivatives 2-FG and 2-DG-d through the transcription factor MLXIP in Example 3; among them, A shows the effect of treatment with different concentrations of 2-DG on the expression of TXNIP mRNA in A498 cells detected by RT-qPCR; B shows the effect of treatment with different concentrations of 2-DG on the expression of TXNIP protein in A498 cells detected by Western blot; C shows the effect of treatment with different concentrations of 2-DG on the expression of MLXIP protein in A498 cells detected by Western blot; D shows the effect of 2-DG treatment on the nuclear entry of MLXIP protein in A498 cells detected by Western blot; E shows the effect of overexpressing MLXIP on the expression of TXNIP mRNA in A498 cells detected by RT-qPCR; F shows the effect of overexpressing MLXIP on the expression of TXNIP protein in A498 cells detected by Western blot;

[0016] Figure 5 Schematic diagram of the vector (A) and sequencing results (B);

[0017] Figure 6 Results of luciferase activity analysis and cell luminescence imaging experiments in Example 4; among them, A shows the relative luciferase intensity detected by luciferase activity analysis in A498 cells, A498-TXNIP-Luc2 and A498-Luc2 cells; B shows the luminescence intensity of A498 cells, A498-TXNIP-Luc2 and A498-Luc2 cells detected by a small animal in vivo imaging device (note: the left side is the cell luminescence image, and the right bar graph is the quantification result of luminescence); C and D show the correlation between the luminescence intensity and the cell number of A498-Luc2 cells (C) and A498-TXNIP-Luc2 cells (D) detected by a small animal in vivo imaging device;

[0018] Figure 7 Results of luciferase activity analysis in Example 5; among them, A-C are the statistical results of luciferase activity analysis experiments after treating A498-TXNIP-Luc2 cells with drugs of different concentration gradients of 2-DG (A), 2-FG (B) and 2-DG-d (C); D-F are the statistical results of luciferase activity analysis experiments after treating A498-Luc2 cells with drugs of different concentration gradients of 2-DG (D), 2-FG (E) and 2-DG-d (F);

[0019] Figure 8Results of the cell luminescence imaging experiment in Example 5; among them, A, C, and E are the changes in the luminescence intensity of A498-TXNIP-Luc2 cells after treatment with 2-DG (A), 2-FG (C), and 2-DG-d (E) and the statistical analysis results; B, D, and F are the changes in the luminescence intensity of A498-Luc2 cells after treatment with 2-DG (B), 2-FG (D), and 2-DG-d (F) and the statistical analysis results (Note: The left side is the cell luminescence image, and the right bar graph is the quantification result of cell luminescence). Detailed implementation manners

[0020] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0023] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0024] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0025] Studies have shown that Thioredoxin-Interacting Protein (TXNIP) belongs to the thioredoxin superfamily and is a key regulatory factor involved in glucose metabolism. It can inhibit the reprogramming of tumor cell glucose metabolism by regulating the glucose transporter 1 (GLUT1) to control cell glucose uptake. Since the TXNIP gene is lowly expressed in renal cancer cells, based on the basic principle that 2-DG can upregulate the expression of TXNIP at the transcriptional level, the present invention constructs a TXNIP promoter reporter gene vector, transfects this reporter gene vector into renal cancer cells and establishes a stable transfected cell line. The exogenous TXNIP gene promoter and reporter gene sequence will be integrated into the genome, and the reporter gene will be synchronously expressed with endogenous TXNIP. When the 2-DG-based anti-renal cancer drugs (2-DG and its derivatives 2-FG and 2-DG-d) are used to treat the cells stably expressing the reporter gene, the transcriptional activity of the TXNIP gene promoter is enhanced, resulting in an upregulation of the reporter gene expression, an increase in fluorescence signal and cell luminescence intensity. Therefore, it can be used to reflect the effect of 2-DG-based anti-renal cancer drugs.

[0026] In the present invention, the TXNIP gene promoter sequence is fused with the luciferase gene through molecular cloning technology to construct a bioluminescent reporter gene reflecting the transcriptional activity of the TXNIP promoter. After this reporter gene is transfected into renal cancer cells, it can be used to visualize the transcriptional activity of the TXNIP gene promoter in tumor cells, thereby realizing the monitoring of the effect of 2-DG-based anti-cancer drugs in vitro and in vivo (for the working principle of the reporter gene, see Figure 1 ).

[0027] Example 1 Determination of the expression level of TXNIP in renal cancer cells

[0028] 1. Analysis of the expression difference of TXNIP in clear cell renal cell carcinoma and adjacent tissues using the TCGA online tool

[0029] The UALCAN online tool of TCGA was used for mRNA expression difference analysis. The results showed that the expression level of TXNIP in clear cell renal cell carcinoma (KIRC) was significantly lower than that in adjacent tissues, and the difference was statistically significant ( Figure 2 A).

[0030] 2. Detection of the expression difference of TXNIP in HEK293 and A498 cells by RT-qPCR

[0031] Experimental method:

[0032] (1) Cell culture

[0033] A498 cells and HEK293 cells (Wuhan Punosai) were cultured in MEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixture. The cells were cultured in a 2 constant temperature incubator at 37 °C and 5% CO

[0034] (2) Extraction and reverse transcription of total cellular RNA

[0035] Total cellular RNA was extracted according to the instructions of the RNA extraction kit (Beijing Tianmo). Reverse transcription was carried out according to the instructions of the All-in-One TM First-Strand cDNA Synthesis Kit (GeneCopoeia);

[0036] (3) qPCR detection

[0037] SYBR Green I was used for detection, and the 2 -△△Ct -ΔΔCt method was used for result analysis. The specific procedures and steps are as follows:

[0038] Reaction system: 2 μL cDNA template, 2 μL upstream and downstream mixed primers (2 μM), 10 μL 2×PCR Mix, and ddH 2 2O was added to make up to 20 μL. Reaction conditions: pre-denaturation at 95 °C for 30 s, 95 °C for 5 s, 63 °C for 20 s, 72 °C for 20 s, for 40 cycles. Verification primers for TXNIP (upstream and downstream mixture) (Guangzhou Funeng, Cat. No.: HQP090834); The sequences of the verification upstream and downstream primers for GAPDH are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0039] Nucleotide sequence of SEQ ID NO.3: AACGGATTTGGTCGTATTG

[0040] Nucleotide sequence of SEQ ID NO.4: GGAAGATGGTGATGGGATT

[0041] Experimental results:

[0042] The results of RT-qPCR detection showed that compared with normal embryonic kidney cells HEK293, the expression level of TXNIP mRNA in A498 cells was significantly decreased, and the difference was statistically significant ( Figure 2 P<0.05).

[0043] Example 2 2-DG and its derivatives inhibit glycolysis and cell proliferation of renal cancer cells

[0044] Experimental method:

[0045] (1) Cell culture: The culture of A498 cells was the same as in Example 1;

[0046] (2) Drug treatment: Cells were treated with 2-DG (0, 5, 10, and 15 mM), 2-FG (0, 1, 2, 5, and 10 mM), and 2-DG-d (0, 1, 2, 5, and 10 mM) for 48 h;

[0047] (3) CCK-8 cell proliferation assay, glucose consumption, and lactate content detection were performed according to the instructions in the CCK-8 kit (Biosharp), lactate assay kit (Nanjing Jiancheng), and glucose kit (Nanjing Jiancheng), respectively.

[0048] Experimental results:

[0049] Treatment with 2-DG and its derivatives 2-FG and 2-DG-d could inhibit the proliferation and glycolysis of A498 cells. As the drug concentrations of 2-DG and its derivatives 2-FG and 2-DG-d increased, the cell number, glucose consumption, and lactate production decreased, showing a concentration gradient dependence ( Figure 3 ).

[0050] Example 3 2-DG and its derivatives can upregulate TXNIP through the transcription factor MLXIP

[0051] 1. 2-DG and its derivatives can upregulate the expression levels of MLXIP and TXNIP in A498 cells

[0052] Experimental method:

[0053] (1) Cell culture: The culture of A498 cells was the same as in Example 1;

[0054] (2) Extraction and reverse transcription of total cellular RNA: The extraction of total cellular RNA was performed according to the instructions of the RNA extraction kit (Tianmo). Reverse transcription was performed according to the instructions of the All-in-One TM First-Strand cDNA Synthesis Kit (GeneCopoeia);

[0055] (3) Protein extraction and concentration determination: Cells were lysed with RLB lysis buffer, and then the protein concentration was determined by the BCA method;

[0056] (4) qPCR and Western blot detection: Detection was performed using SYBR Green I, and the 2 -△△Ct -method was used for result analysis. The specific procedures and steps were as follows:

[0057] Reaction system: 2 μL cDNA template, 2 μL upstream and downstream mixed primers (2 μM), 10 μL of 2×PCR Mix, supplemented with ddH 2 O to 20 μL. Reaction conditions: pre-denaturation at 95°C for 30 s, 95°C for 5 s, 63°C for 20 s, 72°C for 20 s, for 40 cycles. The upstream and downstream verification primers for TXNIP and the upstream and downstream verification primers for GAPDH are the same as in Example 1.

[0058] Western blot detection was performed according to the conventional operation. Rabbit anti-human TXNIP monoclonal antibody was purchased from NatureBiosciences; rabbit anti-human MLXIP monoclonal antibody was purchased from Proteintech.

[0059] Experimental results:

[0060] The results of qPCR and Western blot detection showed that treatment with 2-DG and its derivatives 2-FG and 2-DG-d could up-regulate the expression levels of MLXIP protein, TXNIP mRNA and protein in A498 cells ( Figure 4 A-C in).

[0061] 2. Overexpression of MLXIP can up-regulate the expression level of TXNIP in A498 cells

[0062] Experimental method:

[0063] (1) Cell culture: A498 cells were cultured as above;

[0064] (2) Cell transient transfection: A498 cells were seeded in a six-well cell culture plate, and according to the instructions of the transfection reagent HighGeneplus Transfection reagent (ABclonal), the eukaryotic expression vector of MLXIP (PPL company) (0, 4, and 8 μg) was transfected into A498 cells for 48 h;

[0065] (3) Protein extraction, concentration determination, and Western blot detection were the same as above.

[0066] Experimental results:

[0067] The results of RT-qPCR and Western blot detection showed that overexpression of MLXIP could up-regulate the expression levels of TXNIP mRNA and protein in A498 cells ( Figure 4 D-F in).

[0068] Example 4 Establishment of a cell line stably expressing a luciferase reporter gene

[0069] 1. Construction of a TXNIP promoter bioluminescence reporter gene vector

[0070] This vector was purchased from PPL Company and named "pGL4.19-TXNIP-P-Luc2".

[0071] Specifically: between the Kpn I and Xho I restriction enzyme cleavage sites of the pGL4.19-Luc2 plasmid, the promoter sequence of the TXNIP gene SEQ ID NO.1 was inserted to obtain a recombinant plasmid containing the TXNIP promoter luciferase reporter gene, and the nucleotide sequence of the TXNIP promoter luciferase reporter gene is as shown in SEQ ID NO.2;

[0072] Nucleotide sequence of SEQ ID NO.1:

[0073]

[0074] Nucleotide sequence of SEQ ID NO.2:

[0075]

[0076] The vector pattern diagram and sequencing results are shown in Figure 5 the figure. After the vector was successfully constructed, transformation and plasmid extraction were carried out for subsequent experimental analysis.

[0077] 2. Establishment of a cell line stably expressing a reporter gene

[0078] Experimental method:

[0079] (1) Cell culture: The same as in Example 1;

[0080] (2) Cell transfection: Transfect the pGL4.19-TXNIP-P-Luc2 or pcDNA3.1-Luc2 plasmid (stored in this laboratory, only used as a control) into A498 cells.

[0081] (3) Establishment of a stably expressing cell line: 48 hours after transfection, replace the culture medium in the culture dish with normal MEM medium containing G418 for screening. The established cell lines were named "A498-TXNIP-P-Luc2" and "A498-Luc2" for subsequent experiments;

[0082] (4) Luciferase activity analysis: Collect a part of the cells and perform luciferase activity analysis after using PLB lysis buffer;

[0083] (5) Cell luminescence experiment: Collect another part of the cells and transfer them to a 96-well plate, add the luciferase substrate D-Luciferin (50 μg / mL) for cell luminescence imaging experiment to detect the cell luminescence intensity. The correlation between the cell luminescence intensity and the cell number was determined by the serial dilution method.

[0084] Experimental results:

[0085] The results of luciferase activity analysis showed that the luciferase activity of the stably transfected cell A498-TXNIP-P-Luc2 was significantly higher than that of the control cell A498, indicating that the cells expressed luciferase. The results of the cell luminescence imaging experiment showed that as the cell number increased, the cell luminescence intensity gradually increased, and the two were linearly correlated. These results indicated that the reporter gene was integrated into the cell genome, and A498 cells could stably express the reporter gene, that is, the stably transfected cells were successfully constructed ( Figure 6 ).

[0086] Example 5 determined that A498-TXNIP-P-Luc2 cells could reflect the effects of 2-DG and its derivatives

[0087] Experimental method:

[0088] (1) Cell culture: The same as in Example 1.

[0089] (2) Drug treatment: A498-TXNIP-P-Luc2 cells were treated with different concentrations of 2-DG (0, 5, 10, and 15 mM), 2-FG (0, 1, 2, and 5 mM), and 2-DG-d (0, 1, 2, and 5 mM). After 48 h, cell samples were collected.

[0090] (3) Luciferase activity assay: The same as in Example 4.

[0091] (4) Cell luminescence experiment: The same as in Example 4.

[0092] Experimental results:

[0093] The results of luciferase activity assay showed that after treatment of A498-TXNIP-P-Luc2 cells with 2-DG, 2-FG, and 2-DG-d, the luciferase activity was up-regulated in a dose-dependent manner ( Figure 7 ). The results of cell luminescence imaging experiment showed that after treatment of A498-TXNIP-P-Luc2 cells with 2-DG glycolysis inhibitors, the cell luminescence intensity increased in a dose-dependent manner ( Figure 8 ).

[0094] The above experimental results suggest that the cell model established based on the transcriptional activity of the TXNIP promoter can reflect the effects of 2-DG drugs.

[0095] The embodiments described above are only for describing the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A renal cancer cell line stably expressing a TXNIP promoter luciferase reporter gene, characterized in that: The method for constructing the renal cancer cell line comprises: constructing a plasmid containing a TXNIP promoter luciferase reporter gene, using pGL4.19-Luc2 as a vector, inserting a TXNIP gene promoter sequence SEQ ID NO.1 between the restriction enzyme cutting sites of KpnI and XhoI of the vector, and obtaining a recombinant plasmid containing a TXNIP promoter luciferase reporter gene, wherein the nucleotide sequence of the TXNIP promoter luciferase reporter gene is shown in SEQ ID NO.2; and transfecting the recombinant plasmid containing the TXNIP promoter luciferase reporter gene into human renal cancer cell A498 to establish a renal cancer cell line stably expressing the TXNIP promoter luciferase reporter gene.

2. Use of the renal cancer cell line according to claim 1 in in vitro screening of 2-deoxy-D-glucose (2-DG) anti-renal cancer drugs.

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