CN114525281B - ESRP1 promoter reporter gene transcriptional activity visualization and application thereof in demethylation anticancer drug screening - Google Patents
ESRP1 promoter reporter gene transcriptional activity visualization and application thereof in demethylation anticancer drug screening Download PDFInfo
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Abstract
The invention provides visualization of ESRP1 promoter reporter gene transcription activity and application thereof in demethylation anticancer drug screening, wherein an ESRP1 promoter reporter gene vector is constructed as shown in SEQ ID NO.3, and after a tumor cell is transfected with a reporter gene and a stable transfected cell strain is established, an exogenous ESRP1 gene promoter is integrated into a genome and is synchronously methylated with an endogenous promoter. After stably transfected cell lines are treated by the demethylated drugs, the expression of the reporter gene is up-regulated due to demethylation of the promoter, and the fluorescence signal and the luminous intensity of cells are enhanced, so that the method can be used for monitoring the action effect of the DNA methylation inhibitor anticancer drugs under in-vitro and in-vivo conditions. The reporter gene can be used for early evaluation of the curative effect of anticancer drugs in a living animal tumor model in real time and noninvasively, and provides a powerful tool for screening anticancer drugs with DNA methylation as a target point.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an ESRP1 promoter reporter gene transcriptional activity visualization and application thereof in demethylation anticancer drug screening.
Background
A Reporter gene is a gene encoding a protein or enzyme that can be detected, and the expression product is very easily identified. The coding sequence of the reporter gene and the expression regulating sequence of a certain gene are fused to form a chimeric gene or fused with other target genes, and the chimeric gene is expressed under the control of the regulating sequence, so that the effect of marking the regulation and control of the target gene expression by using the expression product of the reporter gene can be realized. The reporter gene technology is widely applied to the fields of gene promoter transcriptional activity analysis, drug screening and the like because of the characteristics of high sensitivity, convenient detection and the like. The fluorescent enzyme gene is used as a reporter gene, has the advantages of high detection speed, high sensitivity, low cost, no need of using radioactive isotopes and the like, and is widely adopted.
Epigenetics (Epigenetics) primarily study heritable changes in gene expression without alterations in the nucleotide sequence of the gene. In recent years the role of epigenetic modification mechanisms in cancer has been increasingly emphasized by researchers, where DNA methylation is considered one of the important mechanisms of tumor formation. Studies show that methylation of promoters of tumor suppressor genes causes gene inactivation to play an important role in the tumorigenesis process, and demethylated drugs can up-regulate the expression of the genes by reversing the methylation state of the promoters so as to play a role in inhibiting tumors. The use of epigenetic drugs, such as DNA methylase inhibitors, has become a new approach to tumor treatment. Currently, epigenetic drug therapy has achieved significant effects in a variety of tumors, and many epigenetic anticancer drugs are in clinical trials, and have been partially put into clinical use. In the development process of the anticancer drugs, the traditional animal experiment method needs to kill animal resected tumor after the tumor grows to a certain extent, and then the detection index can be obtained through the technologies such as immunohistochemistry and the like. The invasive research method needs to kill a large number of animals, can not be repeated in the same group of animals, is difficult to visually, living and dynamically observe the growth and metastasis conditions of tumors continuously, and limits the research and evaluation of the tumor, especially early tumor occurrence, development, metastasis, diffusion and treatment effects. Therefore, the establishment of the anti-tumor drug model can be used for researching the anti-tumor drug model in real time, repeatedly and noninvasively in living small animals, so that the development of drugs can be accelerated, a new treatment scheme can be rapidly optimized, the number of animals used for developing the drugs can be reduced, the change of cancer cells in cancer treatment can be observed and evaluated in real time, and the anti-tumor drug model has important significance in screening the anti-tumor drugs and researching the action mechanism of the anti-tumor drugs. Molecular optical imaging technology is used as a non-invasive dynamic imaging technology, is increasingly applied to life science, medical research, drug development and other aspects with high resolution and high sensitivity, and becomes the most important progress of animal model research in recent years, and the perfection and development of the technology enable the non-invasive research on the molecular mechanism of antitumor drugs. Up to now, domestic and foreign scientific research institutions have developed a plurality of bioluminescent reporter genes for screening anticancer drugs based on the technology, but bioluminescent reporter genes for DNA methylase inhibitor anticancer drugs are not reported, which limits the development process of the drugs to a certain extent.
Epithelial splice regulatory proteins (Epithelial splicing regulatoryproteins, ESRPs) are epithelial cell-specific splicing factors found in recent years, and can act as oncogenes by inhibiting tumor cell proliferation and the occurrence of EMT. Although studies have reported that the ESRP family is regulated by ZEB1/2 transcription factors in breast cancer cells, the mechanism of expression regulation of ESRP1 in most tumor cells (including kidney cancer cells) is currently unclear. This study found that ESRP1 gene is expressed in kidney cancer cells, its gene promoter region is in hypermethylation state, and DNA methylase inhibitor can up regulate its expression by down regulating its promoter methylation level. Therefore, if the ESRP1 gene promoter sequence is fused with the luciferase gene by a molecular cloning technique, a bioluminescence reporter gene reflecting the transcriptional activity of the ESRP1 promoter can be constructed. After the reporter gene is transfected into the kidney cancer cells, the reporter gene can be used for visualizing the methylation state of ESRP1 gene promoters in tumor cells and monitoring the action effect of DNA methylation inhibitors on anticancer drugs under in-vitro and in-vivo conditions, and can be helpful for preclinical research and development process acceleration of the innovative drugs. The working principle of the reporter gene is shown in figure 1.
Disclosure of Invention
The invention aims to determine that ESRP1 gene low expression and promoter thereof are in hypermethylation state in kidney cancer cells, and DNA methylation inhibitor drug treatment can up-regulate ESRP1 gene expression by down-regulating promoter methylation level.
The invention also aims at constructing a recombinant plasmid containing an ESRP1 gene promoter sequence and a reporter gene aiming at the defects of the prior art, namely the lack of in-vitro and in-vivo screening tools of DNA methylation inhibitor anticancer drugs at present: the recombinant plasmid containing the ESRP1 gene promoter sequence and the reporter gene is obtained by taking pGL4.19-Luc2 plasmid containing the luciferase reporter gene (Luc 2) as a vector and inserting the ESRP1 gene promoter sequence between restriction enzyme cleavage sites of Kpn I and Xho I of the pGL4.19-Luc2 plasmid. The recombinant plasmid is named as pGL4.19-E1P-Luc2, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO.3.
The invention also aims to utilize the reporter gene to visualize the methylation state of the ESRP1 gene promoter in kidney cancer cells and monitor the action of the DNA demethylation anticancer drugs under in-vitro and in-vivo conditions.
Specifically, to achieve the above object, the solution of the present invention is:
an ESRP1 gene promoter having the nucleotide sequence: CTGCGTGGCCCGATTTAAGAAAATTTTTATTAGGCATGCGGATAGATGCTTTTCATTTGGGGCCCAAGGCAAATAAAAGAAATGGGCCCCAGCTCTGGAGGATGTTCCAGCTTCGAACTAGCAGCCAGGACCAAACCAGACATAGCATTCAGAGTTGACAGAAAAAGAAGCGGGCTCCATTGTGCCTGAGTTTCCTTGCGGGGTCCCCCAGCTGCTTTGCGCGCGATAACTGCGTCCTGGGGAGTGACTAGGTGGTTGGGCGTCCAGCCCCTGGCGTCCGGCTCTGCCGCGCGCGGGGTTCCTCTCCGGAAGGTGGGCAGCGCGGCGGGTTTGGGACGAGCGTGCACCCGGGCTCCGGCCCGGAGAAGGGGGGGCTCGCAGGATTTCTCCTGCTGTTTGCACTGAAAGTTGTGTTGGCTCAGGAGCTGCTTTTCCGGGGATCTGCAGTTGCCCCCGCCACCTCCTGGCTGCGGTTGGCAGGTCCCTCCCTCAGCAGTTCGTCCTCCGCCTGCGCCGCGCCCTGGGCAGCTCCGCGCCCCGGGCCTCACCTCTGGCCTCCTTTCGCCTCCCTGCACCTGGCCTTTTCGCTTGA CCGTTCAGAACCTTCGGCTTCGTTCCCTGCAGCCGGTATTCTCCGAGCCCCCCTGCACCTCTCAGTTACCTCCAGTGGGAACCCCTCCCCCCAGCGACTCCGAGCCCTTTACCTCTCTGAGCCCTTTCCCCGTCTGGCCTCGTCTACCCTCTGGGTGTCAACCGCCTCTTCCCTGCCCCTCACGTCTCCCCCTCCTCCTCCCCTGCCCTCGCCTCTCCATTCATCCAGCCATTGTCTCCCGCCCCTTCCTCCCCCTCCCGAAGCGGCCTCCTCCCCCACCGCTGCCACGTCGTGGTTTGAAGGAGCCAATGGGCCGGCGCCGCCAGGTGTCTCTTACCTGCACCACGTGGGGGAGGGGGAAGGGGCGGGCAGGTAAAGCCACATCCCAAAACAGAAAAGCTTTCAGCCATTGCGTGCCTCCCGGGGGGGGCAGCCTTGCTCCAGGCTTTTTGCATAGACGCCCGGGCAACTGAATACAAAAAGGGCAGGCCTCCTGCGCCCTCCTTCCCACCCCCCTTCCTGCCCTGGGCGTGGAGCACCGACCAGGTGTGGGCTTGGGTGGTTGGTTACCGCCTTTTGCACTAGCAGTAGCAAGGAAGGGGGGTGGGCGCTCTTTCTTTTTCTCTTAGAAGAGGGTTTAGCACAGGTTTTTTCGTTCTCACTTCCACACCACCTTACCGCCT (SEQ ID NO. 1).
A recombinant plasmid comprising the above ESRP1 gene promoter and a plasmid.
Preferably, the plasmid is pGL4.19-Luc2 containing a luciferase reporter gene.
Preferably, pGL4.19-Luc2 has the nucleotide sequence of ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACG ACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGA (SEQ ID NO. 2).
Preferably, the recombinant plasmid is an ESRP1 promoter reporter gene, the nucleotide sequence of which is shown as CTGCGTGGCCCGATTTAAGAAAATTTTTATTAGGCATGCGGATAGATGCTTTTCATTTGGGGCCCAAGGCAAATAAAAGAAATGGGCCCCAGCTCTGGAGGATGTTCCAGCTTCGAACTAGCAGCCAGGACCAAACCAGACATAGCATTCAGAGTTGACAGAAAAAGAAGCGGGCTCCATTGTGCCTGAGTTTCCTTGCGGGGTCCCCCAGCTGCTTTGCGCGCGATAACTGCGTCCTGGGGAGTGACTAGGTGGTTGGGCGTCCAGCCCCTGGCGTCCGGCTCTGCCGCGCGCGGGGTTCCTCTCCGGAAGGTGGGCAGCGCGGCGGGTTTGGGACGAGCGTGCACCCGGGCTCCGGCCCGGAGAAGGGGGGGCTCGCAGGATTTCTCCTGCTGTTTGCACTGAAAGTTGTGTTGGCTCAGGAGCTGCTTTTCCGGGGATCTGCAGTTGCCCCCGCCACCTCCTGGCTGCGGTTGGCAGGTCCCTCCCTCAGCAGTTCGTCCTCCGCCTGCGCCGCGCCCTGGGCAGCTCCGCGCCCCGGGCCTCACCTCTGGCCTCCTTTCGCCTCCCTGCACCTGGCCTTTTCGCTTGACCGTTCAGAACCTTCGGCTTCGTTCCCTGCAGCCGGTATTCTCCGAGCCCCCCTGCACCTCTCAGTTACCTCCAGTGGGAACCCCTCCCCCCAGCGACTCCGAGCCCTTTACCTCTCTGAGCCCTTTCCCCGTCTGGCCTCGTCTACCCTCTGGGTGTCAACCGCCTCTTCCCTGCCCCTCACGTCTCCCCCTCCTCCTCCCCTGCCCTCGCCTCTCCATTCATCCAGCCATTGTCTCCCGCCCCTTCCTCCCCCTCCCGAAGCGGCCTCCTCCCCCACCGCTGCCACGTCGTGGTTTGAAGGAGCCAATGGGCCGGCGCCGCCAGGTGTCTCTTACCTGCACCACGTGGGGGAGGGGGAAGGGGCGGGCAGGTAAAGCCACATCCCAAAACAGAAAAGCTTTCAGCCATTGCGTGCCTCCCGGGGGGGGCAGCCTTGCTCCAGGCTTTTTGCATAGACGCCCGGGCAACTGAATACAAAAAGGGCAGGCCTCCTGCGCCCTCCTTCCCACCCCCCTTCCTGCCCTGGGCGTGGAGCACCGACCAGGTGTGGGCTTGGGTGGTTGGTTACCGCCTTTTGCACTAGCAGTAGCAAGGAAGGGGGGTGGGCGCTCTTTCTTTTTCTCTTAGAAGAGGGTTTAGCACAGGTTTTTTC GTTCTCACTTCCACACCACCTTACCGCCTCTCGAGGATATCAAGATCTGGCCTCGGCGGCCAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGA (SEQ ID NO. 3).
Use of a DNA methylation inhibitor for the preparation of a method for reducing the methylation level of an ESRP1 gene promoter in renal cancer cells.
The ESRP1 gene promoter is the ESRP1 gene promoter.
Preferably, the DNA methylation inhibitor is 5-Aza-CdR.
Preferably, when methylation of the ESRP1 gene promoter in kidney cancer cells is detected, the upstream primer sequence of the PCR amplified fragment of the ESRP1 gene promoter is shown as SEQ ID NO.4, and the downstream primer sequence is shown as SEQ ID NO. 5;
the upstream primer sequence of the internal reference fragment is shown as SEQ ID NO.6, and the downstream primer sequence is shown as SEQ ID NO. 7.
Preferably, the use of a DNA methylation inhibitor in the preparation of a demethylated anti-cancer drug screen.
By adopting the scheme, the invention has the beneficial effects that:
the invention verifies for the first time that the ESRP1 promoter is in a hypermethylation state in kidney cancer cells, and constructs an ESRP1 promoter reporter gene vector. After transfection of the reporter gene into tumor cells and establishment of a stably transfected cell line, the exogenous ESRP1 gene promoter will integrate into the genome and be methylated synchronously with the endogenous promoter. After stably transfected cell lines are treated by the demethylated drugs, the expression of the reporter gene is up-regulated due to demethylation of the promoter, and the fluorescence signal and the luminous intensity of cells are enhanced, so that the method can be used for visualizing the methylation state of the ESRP1 gene promoter and monitoring the action effect of the DNA methylation inhibitor on the anticancer drugs under in-vivo and in-vitro conditions. The reporter gene has the greatest advantage of being capable of being used for early evaluation of the curative effect of anticancer drugs in real time and noninvasively under the in-vivo condition, and provides a powerful tool for screening anticancer drugs targeting DNA methylation.
Drawings
FIG. 1 is a schematic diagram of the operational principle of ESRP1 promoter transcriptional activity visualization reporter gene in demethylation drug screening.
FIG. 2 is a graph showing the determination of ESRP1 under-expression in each of the kidney cancer cell lines in example 1 of the present invention.
FIG. 3 is a graph showing the abnormal hypermethylation of ESRP1 promoter in kidney cancer cells according to example 2 of the present invention.
FIG. 4 is a graph showing that methylase inhibitors can reduce the methylation level of ESRP1 promoters in kidney cancer cells as determined in example 3 of the present invention.
FIG. 5 is a schematic and sequencing diagram of ESRP1 promoter bioluminescence reporter vector in example 4 of the present invention.
FIG. 6 is a diagram of a kidney cancer cell line in which a stably expressed reporter gene was established in example 5 of the present invention.
FIG. 7 is a graph showing the effect of determining that the ESRP1 promoter reporter gene can reflect a DNA demethylating agent in example 6 of the present invention.
Detailed Description
The invention provides an ESRP1 promoter reporter gene transcriptional activity visualization and application thereof in demethylation anticancer drug screening.
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: determination of ESRP1 expression levels in renal cancer cells
1. Analysis of ESRP1 expression levels in renal cancer tissues and cells
mRNA expression differential analysis was performed using the UALCAN database. The results showed that ESRP1 expression levels in kidney cancer tissue (KIRC) were significantly lower than that in paracancerous tissue, and that the differences were statistically significant (FIG. 2A). The CCLE database was used to analyze the expression level of ESRP1 in kidney cancer cells. The results showed that ESRP1 was under-expressed in renal cancer cells using HCT116 cells as a control (B in FIG. 2).
2. Determination of ESRP1 mRNA expression levels in renal cancer cells
The experimental method is as follows:
(1) Cell culture
A498 cells and HEK293 cells (Wuhanplaxol) were cultured in MEM medium, and 10% fetal bovine serum and 1% penicillin were added. Cells at 37℃with 5% CO 2 Culturing in a constant temperature incubator.
(2) Extraction and reverse transcription of total RNA of cells
Extraction of total RNA from cells according to RNA extractionThe kit (Beijing tiansu) was used for the procedure. Reverse transcription was performed according to All-in-One TM First-Strand cDNA Synthesis Kit (GeneCopoeia) instructions.
(3) qPCR detection
The experiment was performed using SYBR GreenI, using 2 -△△Ct The method is used for analyzing results, and the specific procedures and steps are as follows:
the reaction system: 2. Mu.L of cDNA template, 2. Mu.L of upstream and downstream mixed primer (2. Mu.M), 2 XPCRMix 10. Mu.L, ddH2O make up to 20. Mu.L.
The reaction conditions are as follows: pre-denaturation at 95℃for 30s, 5s,63, 20s,72, 20s,40 cycles.
ESRP1 certified upstream and downstream mixed primer (GeneCopoeia, HSQRP 013766), GAPDH certified upstream and downstream mixed primer (GeneCopoeia, HSQRP 20026).
Experimental results:
the qPCR detection result shows that compared with the normal embryo kidney cell HEK293, the expression level of ESRP1 mRNA level in the A498 cells is obviously reduced, and the difference has statistical significance (C in figure 2).
Example 2: determination of aberrant hypermethylation of ESRP1 promoter in renal cancer cells
1. Analysis of ESRP1 methylation level at the promoter of renal cancer tissue
ESRP1 promoter methylation differential analysis was performed using UALCAN online database. The results showed that the methylation level in KIRC was significantly higher than that in the paracancerous tissue, and the differences were statistically significant (fig. 3 a).
BSP clone sequencing method for detecting promoter methylation level of ESRP1 in kidney cancer cells
The ESRP1 promoter CpG islands were predicted using the online tool MethPrimer, and three CpG islands were found within the ESRP1 gene promoter sequence (B in FIG. 3). Methylation of the CpG island "221bp" fragment was detected using BSP clone sequencing.
The experimental method is as follows:
(1) Cell culture: as above.
(2) Cell DNA extraction: the extraction of cellular RNA was performed according to the DNA extraction kit (Beijing Tian Gen).
(3) BSP clone sequencing: methylation at each site in the CpG island "221bp" fragment of the ESRP1 gene promoter in HEK293 cells and A498 cells was detected by BSP clone sequencing (Bisulfite SequencingPCR, BSP) (the sequencing experiment was completed by Shanghai).
Experimental results:
the BSP clone sequencing experiment result shows that the total methylation rate of 23 CpG sites in the 221bp fragment of the ESRP1 gene promoter in the kidney cancer cell line A498 is up to 100%, and the HEK293 cell is 91%, which indicates that the ESRP1 gene promoter is in a hypermethylation state in the kidney cancer cell. In FIG. 3, C is the sequence before and after bisulphite treatment of the fragment, the upper panel is the sequence of the promoter fragment of the gene before treatment, and the lower panel is the sequence after treatment. In FIG. 2, D is a bead diagram of the BSP clone assay, wherein the upper panel shows A498 cells and the lower panel shows HEK293 cells.
Example 3: DNA methylation inhibitor treatment can reduce the methylation level of ESRP1 promoters in kidney cancer cells 1. MSRE-qPCR method was used to detect methylation of ESRP1 gene promoters (methylation island "221bp" fragment) in HEK293 cells.
The experimental method is as follows:
(1) Cell culture: as above.
(2) DNA extraction: as above.
(3) Enzyme digestion and agarose gel electrophoresis
The DNA samples were digested with methylation sensitive hhai, digestion system: the total system was 20 μl, comprising: 3 μLDNA,2 μL of 10 XBuffer, 1 μLHhaI enzyme, 15 μL of sterile water. And (3) carrying out enzyme digestion at 37 ℃ after gently mixing, wherein the enzyme digestion time is respectively 0h, 1h, 8h and 12h. Agarose gel electrophoresis was performed on each sample after the cleavage.
(4) qPCR detection
The experiment was performed using SYBR GreenI, using 2 -△△Ct The method is used for analyzing results, and the specific procedures and steps are as follows:
the reaction system was 20. Mu.L, comprising: 2 mu LDNA template, 2 mu L upstream and downstream Mix primer (2 mu M), 10 mu L2 XPCR Mix, ddH 2 O was made up to 20. Mu.L.
Reaction conditions: pre-denaturation at 95℃for 30s, 5s,63, 20s,72, 20s,40 cycles. Fragment sequence primer of ESRP1 promoter order (Shanghai, ind.), upstream primer: GCTCCATTGTGCCTGAGTTTCC (SEQ ID NO. 4), downstream primer: CACGCTCGTCCCAAACCCG (SEQ ID NO. 5). Internal reference fragment sequence primer, upstream primer: AGGCAAATAAAAGGAAATGGG (SEQ ID NO. 6), downstream primer: GCAAGGAAACTCAGGCACAAT (SEQ ID NO. 7).
Experimental results:
the distribution of CpG island methylation sites of ESRP1 gene promoter is shown in FIG. 4A, which includes the positions (primer design sites) of selected PCR amplified target fragment and internal reference fragment. The cleavage results showed that the amount of DNA fragments was gradually decreased with the extension of the cleavage time, indicating that the HhaI enzyme can function as a restriction enzyme (B in FIG. 4). The qPCR detection result shows that the amount of the amplified target fragment contained in the HEK293 cell genome gradually decreases along with the extension of the digestion time, and the difference has statistical significance, which suggests that the methylation state in the ESRP1 promoter '221 bp' fragment can be determined by using the MSRE-qPCR method (C in FIG. 4).
Dna methylation inhibitors can reduce the level of methylation of the ESRP1 promoter (methylation island "221bp" fragment) in kidney cancer cells.
The experimental method is as follows:
(1) Cell culture: as above.
(2) The cells were treated with DNA methylation inhibitors 5-Aza-CdR (0. Mu.M, 1. Mu.M and 5. Mu.M) at various concentrations for 48h.
(3) DNA extraction: as above.
(4) And (3) enzyme cutting: the treatment time is 24 hours, and the method is the same.
(5) qPCR detection: the detection method is the same as that described above.
Experimental results:
qPCR results showed that the amount of the selected target fragment in the promoter "221bp" fragment was significantly reduced after treatment of A498 cells with 5-Aza-CdR, indicating that 5-Aza-CdR treatment reduced the methylation level of the target fragment. The experimental results are shown in fig. 4D.
Example 4: construction of ESRP1 promoter bioluminescence reporter gene vector
The vector was purchased from Nanjing Public Protein/Plasmid Library (PPL) and designated "pGL4.19-E1P-Luc2". The vector pattern and sequencing results are shown in FIG. 5. After the vector is successfully constructed, transformation and plasmid extraction are carried out for subsequent experimental analysis.
The experimental method is as follows:
(1) Transformation
(1) 100. Mu.L of competent cells (GeneCopoeia, guangzhou) were taken.
(2) Add 2. Mu.L (3 ng/. Mu.L) of plasmid, gently shake and place on ice for 30min.
(3) The mixture was heat-shocked in a water bath at 42℃for 90s.
(4) Cooling on ice for 5min.
(5) The bacterial liquid is coated on a flat plate containing Amp, the flat plate is vertically placed for 30min, and a culture dish is inverted after the bacterial liquid is completely absorbed by a culture medium, and the culture dish is cultivated for 16h at 37 ℃.
(6) 5 colonies were picked and cultured overnight in 2mL LB medium containing Amp with shaking.
(2) Plasmid extraction
Plasmid extraction was performed according to the instructions of the plasmid extraction kit (Beijing apathy).
Example 5: establishment of cell lines stably expressing reporter genes
The experimental method is as follows:
(1) Cell culture: a498 cells were cultured as above.
(2) Cell transfection:
(1) a498 cells were cultured in six well plates to a confluency of about 70%.
(2) After 24h, according to the instructions of transfection reagent HighGeneplusTransfectionreagent (ABclonal), 4.0. Mu.g pGL4.19-E1P-Luc2 plasmid was added to a 200. Mu.L serum-free MEM basal medium centrifuge tube, and was stirred and mixed well, followed by addition of 8. Mu.L of the LHighGENEplus transfection reagent.
(3) After 48 hours, the culture medium in the dish was changed to a conventional MEM culture medium containing G418 for selection. After the control cells (untransfected plasmid) were all killed (about two weeks), the cells were cultured in a growth environment with a relatively low drug concentration (800. Mu.g/mL). Under the above conditions, the cells were able to continue to grow, indicating that a stably transfected cell line was established, designated "A498-E1P-Luc2", for use in subsequent experiments.
(4) A portion of the cells collected were lysed using PLB lysate and analyzed for luciferase activity.
(5) Another portion of the cells was collected and transferred to a 96-well plate, and luciferase substrate D-Luciferin (50. Mu.g/mL) was added for detection of the luminescence intensity of the cells in a luminescence imaging experiment. The correlation of the luminous intensity of cells with the number of cells was determined by the dilution of the multiple ratio method.
Experimental results:
the luciferase activity analysis results show that the luciferase activity of the stably transfected cell strain A498-E1P-Luc2 is obviously higher than that of the control cell A498, and the cells are indicated to express luciferase (A in FIG. 6). The results of the cell luminescence imaging experiments show that the cell luminescence intensity gradually increases with the increase of the cell number, and the cell luminescence intensity are in a straight line (B in FIG. 6). These results indicate that the reporter gene is integrated into the cell genome, and that the A498 cells can stably express the reporter gene, i.e., the stably transfected cells A498-E1P-Luc2 are successfully constructed and can be used for the next experiment.
Example 6: determining that the reporter gene can reflect the action effect of DNA methylation inhibitor drugs
DNA methylation inhibitor treatment can up-regulate the ESRP1 mRNA expression levels of A498 cells.
The experimental method is as follows:
(1) Cell culture: a498 cells were cultured as above.
(2) The cells were treated with DNA methylation inhibitors 5-Aza-CdR (0. Mu.M, 1. Mu.M and 5. Mu.M) at various concentrations for 48h.
(3) Extraction of total RNA from cells, reverse transcription and qPCR detection were as above.
Experimental results:
qPCR detection results show that the expression level of ESRP1 mRNA is obviously up-regulated after the DNA methylation inhibitor 5-Aza-CdR is treated on A498 cells for 48 hours, and the dosage dependency is achieved (A in FIG. 7).
DNA methylation inhibitor treatment can up-regulate the luminous intensity of A498-E1P-Luc2 cells.
(1) A six-well plate cell culture dish was taken, and a certain amount of A498-E1P-Luc2 cells was added thereto, and cultured at 37℃until about 70% confluence was achieved.
(2) 5-Aza-CdR (0. Mu.M, 1. Mu.M and 5. Mu.M) A498-E1P-Luc2 cells were treated separately and cell samples were collected after 48 hours.
(3) A part of the cells collected was lysed by PLB lysate, and the protein concentration was measured by BCA method, followed by luciferase activity analysis.
(4) Another portion of the cells was transferred to a 96-well plate and luciferase substrate D-Luciferin (50. Mu.g/mL) was added for detection of the luminescence intensity of the cells in a luminescence imaging experiment.
Experimental results:
the results of luciferase activity analysis showed that luciferase activity was up-regulated after treatment of A498-E1P-Luc2 cells with 5-Aza-CdR, and was dose-dependent (B in FIG. 7). The results of the chemiluminescence imaging experiments showed that the intensity of luminescence of the cells increases and the cells are dose dependent after the cells A498-E1P-Luc2 are treated by 5-Aza-CdR (C in FIG. 7). The experimental result suggests that the luminous intensity of the cells indicated by the reporter gene can reflect the action effect of the DNA methylase inhibitor drugs.
Taken together, the present invention demonstrates that ESRP1 is low expressed in kidney cancer cells and that the ESRP1 promoter is in a hypermethylated state in kidney cancer cells. DNA methylation inhibitor 5-Aza-CdR can down-regulate ESRP1 promoter methylation status and up-regulate ESRP1 molecule expression levels after treatment of kidney cancer cells. In addition, the invention constructs an ESRP1 promoter luciferase reporter gene vector pGL4.19-E1P-Luc2, and after transfecting tumor cells with the reporter gene plasmid, a stable transfected cell strain is established, and the change of the transcription activity of the ESRP1 gene promoter is reflected by detecting the luminous intensity of the luminous cells. After the DNA methylation inhibitor 5-Aza-CdR is treated to stably transfect the cell strain, the luminous intensity of the cell is increased and the cell is dose-dependent. Therefore, the optical image reporter gene can be used for monitoring the action effect of the DNA methylation inhibitor anticancer drugs under in-vitro and in-vivo conditions.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Sequence listing
<110> national medical college of Right river
<120> ESRP1 promoter reporter gene transcriptional activity visualization and application thereof in demethylation anticancer drug screening
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1275
<212> DNA
<213> Artificial sequence (Artficial Sequence)
<400> 1
ctgcgtggcc cgatttaaga aaatttttat taggcatgcg gatagatgct tttcatttgg 60
ggcccaaggc aaataaaaga aatgggcccc agctctggag gatgttccag cttcgaacta 120
gcagccagga ccaaaccaga catagcattc agagttgaca gaaaaagaag cgggctccat 180
tgtgcctgag tttccttgcg gggtccccca gctgctttgc gcgcgataac tgcgtcctgg 240
ggagtgacta ggtggttggg cgtccagccc ctggcgtccg gctctgccgc gcgcggggtt 300
cctctccgga aggtgggcag cgcggcgggt ttgggacgag cgtgcacccg ggctccggcc 360
cggagaaggg ggggctcgca ggatttctcc tgctgtttgc actgaaagtt gtgttggctc 420
aggagctgct tttccgggga tctgcagttg cccccgccac ctcctggctg cggttggcag 480
gtccctccct cagcagttcg tcctccgcct gcgccgcgcc ctgggcagct ccgcgccccg 540
ggcctcacct ctggcctcct ttcgcctccc tgcacctggc cttttcgctt gaccgttcag 600
aaccttcggc ttcgttccct gcagccggta ttctccgagc ccccctgcac ctctcagtta 660
cctccagtgg gaacccctcc ccccagcgac tccgagccct ttacctctct gagccctttc 720
cccgtctggc ctcgtctacc ctctgggtgt caaccgcctc ttccctgccc ctcacgtctc 780
cccctcctcc tcccctgccc tcgcctctcc attcatccag ccattgtctc ccgccccttc 840
ctccccctcc cgaagcggcc tcctccccca ccgctgccac gtcgtggttt gaaggagcca 900
atgggccggc gccgccaggt gtctcttacc tgcaccacgt gggggagggg gaaggggcgg 960
gcaggtaaag ccacatccca aaacagaaaa gctttcagcc attgcgtgcc tcccgggggg 1020
ggcagccttg ctccaggctt tttgcataga cgcccgggca actgaataca aaaagggcag 1080
gcctcctgcg ccctccttcc cacccccctt cctgccctgg gcgtggagca ccgaccaggt 1140
gtgggcttgg gtggttggtt accgcctttt gcactagcag tagcaaggaa ggggggtggg 1200
cgctctttct ttttctctta gaagagggtt tagcacaggt tttttcgttc tcacttccac 1260
accaccttac cgcct 1275
<210> 2
<211> 1651
<212> DNA
<213> Artificial sequence (Artficial Sequence)
<400> 2
atggaagatg ccaaaaacat taagaagggc ccagcgccat tctacccact cgaagacggg 60
accgccggcg agcagctgca caaagccatg aagcgctacg ccctggtgcc cggcaccatc 120
gcctttaccg acgcacatat cgaggtggac attacctacg ccgagtactt cgagatgagc 180
gttcggctgg cagaagctat gaagcgctat gggctgaata caaaccatcg gatcgtggtg 240
tgcagcgaga atagcttgca gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg 300
gctgtggccc cagctaacga catctacaac gagcgcgagc tgctgaacag catgggcatc 360
agccagccca ccgtcgtatt cgtgagcaag aaagggctgc aaaagatcct caacgtgcaa 420
aagaagctac cgatcataca aaagatcatc atcatggata gcaagaccga ctaccagggc 480
ttccaaagca tgtacacctt cgtgacttcc catttgccac ccggcttcaa cgagtacgac 540
ttcgtgcccg agagcttcga ccgggacaaa accatcgccc tgatcatgaa cagtagtggc 600
agtaccggat tgcccaaggg cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt 660
catgcccgcg accccatctt cggcaaccag atcatccccg acaccgctat cctcagcgtg 720
gtgccatttc accacggctt cggcatgttc accacgctgg gctacttgat ctgcggcttt 780
cgggtcgtgc tcatgtaccg cttcgaggag gagctattct tgcgcagctt gcaagactat 840
aagattcaat ctgccctgct ggtgcccaca ctatttagct tcttcgctaa gagcactctc 900
atcgacaagt acgacctaag caacttgcac gagatcgcca gcggcggggc gccgctcagc 960
aaggaggtag gtgaggccgt ggccaaacgc ttccacctac caggcatccg ccagggctac 1020
ggcctgacag aaacaaccag cgccattctg atcacccccg aaggggacga caagcctggc 1080
gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg tggacttgga caccggtaag 1140
acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg gccccatgat catgagcggc 1200
tacgttaaca accccgaggc tacaaacgct ctcatcgaca aggacggctg gctgcacagc 1260
ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc 1320
ctgatcaaat acaagggcta ccaggtagcc ccagccgaac tggagagcat cctgctgcaa 1380
caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg acgacgatgc cggcgagctg 1440
cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga ccgagaagga gatcgtggac 1500
tatgtggcca gccaggttac aaccgccaag aagctgcgcg gtggtgttgt gttcgtggac 1560
gaggtgccta aaggactgac cggcaagttg gacgcccgca agatccgcga gattctcatt 1620
aaggccaaga agggcggcaa gatcgccgtg a 1651
<210> 3
<211> 2993
<212> DNA
<213> Artificial sequence (Artficial Sequence)
<400> 3
ctgcgtggcc cgatttaaga aaatttttat taggcatgcg gatagatgct tttcatttgg 60
ggcccaaggc aaataaaaga aatgggcccc agctctggag gatgttccag cttcgaacta 120
gcagccagga ccaaaccaga catagcattc agagttgaca gaaaaagaag cgggctccat 180
tgtgcctgag tttccttgcg gggtccccca gctgctttgc gcgcgataac tgcgtcctgg 240
ggagtgacta ggtggttggg cgtccagccc ctggcgtccg gctctgccgc gcgcggggtt 300
cctctccgga aggtgggcag cgcggcgggt ttgggacgag cgtgcacccg ggctccggcc 360
cggagaaggg ggggctcgca ggatttctcc tgctgtttgc actgaaagtt gtgttggctc 420
aggagctgct tttccgggga tctgcagttg cccccgccac ctcctggctg cggttggcag 480
gtccctccct cagcagttcg tcctccgcct gcgccgcgcc ctgggcagct ccgcgccccg 540
ggcctcacct ctggcctcct ttcgcctccc tgcacctggc cttttcgctt gaccgttcag 600
aaccttcggc ttcgttccct gcagccggta ttctccgagc ccccctgcac ctctcagtta 660
cctccagtgg gaacccctcc ccccagcgac tccgagccct ttacctctct gagccctttc 720
cccgtctggc ctcgtctacc ctctgggtgt caaccgcctc ttccctgccc ctcacgtctc 780
cccctcctcc tcccctgccc tcgcctctcc attcatccag ccattgtctc ccgccccttc 840
ctccccctcc cgaagcggcc tcctccccca ccgctgccac gtcgtggttt gaaggagcca 900
atgggccggc gccgccaggt gtctcttacc tgcaccacgt gggggagggg gaaggggcgg 960
gcaggtaaag ccacatccca aaacagaaaa gctttcagcc attgcgtgcc tcccgggggg 1020
ggcagccttg ctccaggctt tttgcataga cgcccgggca actgaataca aaaagggcag 1080
gcctcctgcg ccctccttcc cacccccctt cctgccctgg gcgtggagca ccgaccaggt 1140
gtgggcttgg gtggttggtt accgcctttt gcactagcag tagcaaggaa ggggggtggg 1200
cgctctttct ttttctctta gaagagggtt tagcacaggt tttttcgttc tcacttccac 1260
accaccttac cgcctctcga ggatatcaag atctggcctc ggcggccaag cttggcaatc 1320
cggtactgtt ggtaaagcca ccatggaaga tgccaaaaac attaagaagg gcccagcgcc 1380
attctaccca ctcgaagacg ggaccgccgg cgagcagctg cacaaagcca tgaagcgcta 1440
cgccctggtg cccggcacca tcgcctttac cgacgcacat atcgaggtgg acattaccta 1500
cgccgagtac ttcgagatga gcgttcggct ggcagaagct atgaagcgct atgggctgaa 1560
tacaaaccat cggatcgtgg tgtgcagcga gaatagcttg cagttcttca tgcccgtgtt 1620
gggtgccctg ttcatcggtg tggctgtggc cccagctaac gacatctaca acgagcgcga 1680
gctgctgaac agcatgggca tcagccagcc caccgtcgta ttcgtgagca agaaagggct 1740
gcaaaagatc ctcaacgtgc aaaagaagct accgatcata caaaagatca tcatcatgga 1800
tagcaagacc gactaccagg gcttccaaag catgtacacc ttcgtgactt cccatttgcc 1860
acccggcttc aacgagtacg acttcgtgcc cgagagcttc gaccgggaca aaaccatcgc 1920
cctgatcatg aacagtagtg gcagtaccgg attgcccaag ggcgtagccc taccgcaccg 1980
caccgcttgt gtccgattca gtcatgcccg cgaccccatc ttcggcaacc agatcatccc 2040
cgacaccgct atcctcagcg tggtgccatt tcaccacggc ttcggcatgt tcaccacgct 2100
gggctacttg atctgcggct ttcgggtcgt gctcatgtac cgcttcgagg aggagctatt 2160
cttgcgcagc ttgcaagact ataagattca atctgccctg ctggtgccca cactatttag 2220
cttcttcgct aagagcactc tcatcgacaa gtacgaccta agcaacttgc acgagatcgc 2280
cagcggcggg gcgccgctca gcaaggaggt aggtgaggcc gtggccaaac gcttccacct 2340
accaggcatc cgccagggct acggcctgac agaaacaacc agcgccattc tgatcacccc 2400
cgaaggggac gacaagcctg gcgcagtagg caaggtggtg cccttcttcg aggctaaggt 2460
ggtggacttg gacaccggta agacactggg tgtgaaccag cgcggcgagc tgtgcgtccg 2520
tggccccatg atcatgagcg gctacgttaa caaccccgag gctacaaacg ctctcatcga 2580
caaggacggc tggctgcaca gcggcgacat cgcctactgg gacgaggacg agcacttctt 2640
catcgtggac cggctgaaga gcctgatcaa atacaagggc taccaggtag ccccagccga 2700
actggagagc atcctgctgc aacaccccaa catcttcgac gccggggtcg ccggcctgcc 2760
cgacgacgat gccggcgagc tgcccgccgc agtcgtcgtg ctggaacacg gtaaaaccat 2820
gaccgagaag gagatcgtgg actatgtggc cagccaggtt acaaccgcca agaagctgcg 2880
cggtggtgtt gtgttcgtgg acgaggtgcc taaaggactg accggcaagt tggacgcccg 2940
caagatccgc gagattctca ttaaggccaa gaagggcggc aagatcgccg tga 2993
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence (Artficial Sequence)
<400> 4
gctccattgt gcctgagttt cc 22
<210> 5
<211> 19
<212> DNA
<213> Artificial sequence (Artficial Sequence)
<400> 5
cacgctcgtc ccaaacccg 19
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence (Artficial Sequence)
<400> 6
aggcaaataa aaggaaatgg g 21
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (Artficial Sequence)
<400> 7
gcaaggaaac tcaggcacaa t 21
Claims (2)
1. A renal cancer cell strain for stably expressing ESRP1 promoter luciferase reporter gene comprises the following construction methods: constructing a recombinant plasmid containing an ESRP1 promoter luciferase reporter gene, taking pGL4.19-Luc2 plasmid containing the luciferase reporter gene Luc2 as a vector, and inserting an ESRP1 gene promoter sequence SEQ ID NO.1 between restriction enzyme cutting sites of Kpn I and Xho I of the pGL4.19-Luc2 plasmid to obtain the recombinant plasmid containing the ESRP1 promoter luciferase reporter gene, wherein the nucleotide sequence of the ESRP1 promoter luciferase reporter gene is shown as SEQ ID NO. 3;
the recombinant plasmid containing ESRP1 promoter luciferase reporter gene is transfected into human kidney cancer cells A498, and a kidney cancer cell strain for stably expressing ESRP1 promoter luciferase reporter gene is established.
2. Use of a renal cancer cell line stably expressing an ESRP1 promoter luciferase reporter gene of claim 1 in vitro screening of demethylated anti-renal cancer drugs.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104066851A (en) * | 2011-09-30 | 2014-09-24 | 基因泰克公司 | Diagnostic methylation markers of epithelial or mesenchymal phenotype and response to EGFR kinase inhibitor in tumours or tumour cells |
Non-Patent Citations (4)
| Title |
|---|
| Cheemala Ashok等.E2F1 and epigenetic modifiers orchestrate breast cancer progression by regulating oxygen-dependent ESRP1 expression.Oncogenesis.2021,第10卷文章编号58第1-10页. * |
| E2F1 and epigenetic modifiers orchestrate breast cancer progression by regulating oxygen-dependent ESRP1 expression;Cheemala Ashok等;Oncogenesis;第10卷;文章编号58第1-10页 * |
| Transcriptome-wide Landscape of Pre-mRNA Alternative Splicing Associated with Metastatic Colonization;Zhi-xiang Lu等;Mol Cancer Res;第13卷(第2期);第305-318页 * |
| 上皮剪接调节蛋白1的研究进展;贾静;马百涛;朱盛兰;王少帅;冯玲;;现代生物医学进展(第03期) * |
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