CN112553331B - Application of TXDDC 16 gene in preparation of detection kit for detecting drug resistance of lung cancer chemotherapeutic drug - Google Patents
Application of TXDDC 16 gene in preparation of detection kit for detecting drug resistance of lung cancer chemotherapeutic drug Download PDFInfo
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Abstract
The invention discloses an application of a TXNDC16 gene in preparation of a detection kit for detecting drug resistance of a lung cancer chemotherapeutic drug, belongs to the technical field of gene mutation, and aims to probe the relation between the TXNDC16 gene and the drug resistance of the lung cancer chemotherapeutic drug, the invention provides an application of the TXNDC16 gene in preparation of the detection kit for detecting the drug resistance of the lung cancer chemotherapeutic drug, wherein a downstream sequence of the TXNDC16 gene is subjected to insertion mutation and then used for detecting the drug resistance of the lung cancer chemotherapeutic drug; the insertion mutation is characterized in that an exogenous gene is inserted at the position 30,655bp downstream of the TXNDC16 gene, and the nucleotide sequence of the exogenous gene is shown as SEQ ID No. 1. The invention can be used for detecting the drug resistance of lung cancer chemotherapeutic drugs.
Description
Technical Field
The invention particularly relates to application of a TXDDC 16 gene in preparation of a detection kit for detecting drug resistance of lung cancer chemotherapeutic drugs, belonging to the technical field of gene mutation.
Background
Lung cancer is the first cause of death of cancer worldwide, most patients have metastasis at the time of diagnosis, and since surgical treatment is not suitable, chemotherapy becomes the most important means for treating cancer patients. However, the problem most afflicting clinicians in chemotherapy is poor or even ineffective chemotherapeutic drug treatment resulting from cancer cell chemotherapy resistance. The most fundamental reason for this problem is that the mechanism of resistance of human beings to cancer cell chemotherapy is unclear, thus causing no good response strategy. In recent years, the concept of synthetic lethality has been proposed, the basic principle of which is that two genes have a lethal effect on cells when mutated at the same time, but neither gene alone causes cell death. It has now been found in cancer research that the interaction of several genes can promote apoptosis or inhibit growth of tumor cells. Therefore, screening and identifying key genes for controlling the drug resistance of the lung cancer chemotherapy and further researching the molecular mechanism of the drug resistance of the lung cancer chemotherapy so as to develop the drug combination of novel lung cancer treatment products is a key step of treatment and is a breakthrough for researching and treating the drug resistance of the lung cancer chemotherapy.
In recent years, with the development of human genomics, transcriptomics and proteomics, many methods have been used to screen cancer chemotherapy drug resistance related genes, including gene chip, microRNA chip, small RNA interference (siRNAs), chemical mutagenesis DNA and retrovirus insertion mutation. For example, key genes CDK10 for drug resistance in breast cancer treatment, BRCA and P53 signal pathway genes related to cisplatin chemotherapy drug resistance in lung cancer have been screened by RNA interference (RNAi) technology, and PARP inhibitor sensitive genes CDK5, MAPK12, PLK3, PNKP, STK22c, STK36 and the like have been screened by using CAL51 cells as a model. However, the technology needs to know the sequence of a target gene in advance to design dsRNA for silencing the target gene, so that the range of gene screening is reduced, and some unknown genes are usually missed; in addition, RNAi technology can only knock out a target gene, but cannot over-express a certain gene, and needs to be matched with cDNA library technology, which limits the application of the technology, but still cannot screen unknown genes. In addition, Whiteside and the like respectively compare the gene expression difference of lung cancer cell lines before and after receiving cisplatin treatment by utilizing a microarray technology, and find out 44 gene expression differences, thereby laying a certain foundation for the clinical individualized treatment of lung cancer patients. However, the gene expression difference between different tumor cell drug-resistant strains is analyzed by using the gene chip, the obtained gene does not have a definite causal relationship with the chemotherapy drug resistance, and can only be understood as a chemotherapy drug-resistant related gene, because the chemotherapy drug resistance of the tumor cell is a very complex and dynamic process, wherein a plurality of key genes may play a role, but when the expression level of the genes returns to the original level after the drug-resistant strains are formed, the important genes are probably ignored as a result. In addition, after Microarray comparison, data are huge, and thousands of gene expressions are different, which brings great difficulty to subsequent analysis work. Therefore, a critical gene for controlling the drug resistance of lung cancer chemotherapy is found, and a research method for identifying important genes which control the drug resistance of lung cancer chemotherapy and have a causal relationship with the occurrence of the drug resistance is urgently needed.
The insertion mutation is caused by using a known exogenous DNA insertion sequence as a marker and destroying the structure of a gene, can directly verify the relation between individual genes and screened traits, and is an ideal functional genomics research method. At present, the genes related to tumorigenesis and metastasis are screened by utilizing the Technology, but the application problem of the method is that the efficiency of insertion mutation is high, and the Random Gene mutation regulation Technology (CRGP) is a functional genomics Technology invented by the applicant based on the integrated transposon insertion mutation Technology, the antisense RNA Technology and the eukaryotic Gene expression regulation Technology. CRGP can generate a whole genome homozygous gene mutation; providing whole genome gene screening and gene function analysis; simultaneously, the relationship between gene mutation and functional expression is discovered and confirmed; systemic gene function localization and analysis of its functional characteristics in genetic and biochemical pathways; quickly separating disease-related genes and rheostat gene expression regulation. Genes and signal transduction pathways related to a plurality of major diseases, including tumor formation, tumor drug-resistant molecular mechanism, cancer invasion and metastasis, influenza virus pathological mechanism and senile dementia pathological mechanism research, have been successfully screened and identified by using the technology or based on the technical principle.
Disclosure of Invention
In order to explore the relationship between the TXDDC 16 gene and the drug resistance of the lung cancer chemotherapeutic drug, the invention provides application of the TXDDC 16 gene in preparing a detection kit for detecting the drug resistance of the lung cancer chemotherapeutic drug.
Further defined, the chemotherapeutic agent is cisplatin.
Further defined, the nucleotide sequence of the TXNDC16 gene is GenBank accession No. NM-020784.
In a further limitation, the application refers to that the downstream sequence of the TXDDC 16 gene is subjected to insertion mutation and then is used for preparing a detection kit for detecting the drug resistance of the lung cancer chemotherapeutic drug.
Further defined, the insertion mutation is that a foreign gene is inserted at 30,655bp downstream of the TXDDC 16 gene.
Further limited, the nucleotide sequence of the exogenous gene is shown as SEQ ID No. 1.
Advantageous effects
The invention screens the key gene for controlling the cisplatin tolerance of the lung cancer cells by using a random gene mutation regulation method, and can be applied to the preparation of a detection kit for detecting the drug resistance of lung cancer chemotherapy.
Drawings
FIG. 1 shows a cell growth curve with drug dose on the abscissa and absorbance value OD450 on the ordinate;
FIG. 2 shows the screening of Tet-off clones, the luciferase reporter gene detection is relative to the change multiple of light units, the abscissa is the Tet-off clone number, and the ordinate is the regulation multiple;
FIG. 3 shows that the DOX-regulated characteristics of the candidate 4 Tet-off clone cell strains are compared;
FIG. 4 IC50 detection of a candidate CIS-platin resistant mutant clone, CIS4, on the abscissa of drug dose and on the ordinate of absorbance value, OD 450;
FIG. 5 DNA sequencing results of both ends of GSV insertions.
Detailed Description
The technical principle of the invention is as follows: a Gene Search Vector (GSV) is constructed on the basis of a piggyBac transposon (GI: 226433913) which can be efficiently integrated in a eukaryotic cell and randomly inserted into a genome, wherein a tetracycline response element (TRE, GI: 76667907) carried by the GSV is preferably spliced by two tetracycline response elements, so that a transcription activator has stronger activation effect on the gene search vector, the transcription initiation distance is farther, the gene search vector can be activated by the transcription activator tTA, the piggyBac transposon can be efficiently integrated in the eukaryotic cell, the integration site is biased to a coding gene, and traceless excision can be performed by a transposase after the gene search vector is inserted into the genome, and the requirements of an ideal gene search vector are met. Under the action of tTA, 14Tet in GSV can strongly start the transcription of nearby DNA sequences; if the transcribed RNA is in the reverse direction to the upstream gene, antisense RNA can be obtained, and the antisense RNA binds to the mRNA of the upstream gene to prevent the translation expression of the gene; if the orientation is the same, the downstream gene can be overexpressed, so that the gene near the insertion site can be mutated by the vector.
The GSV can also contain a neomycin resistance gene, the resistance gene can be used for screening positive clones by utilizing G418 in eukaryotic cells, and the resistance gene shows kanamycin resistance in prokaryotic cells and is used for screening positive colonies. Of course, the resistance gene in the GSV may be any element suitable for allowing resistance screening in eukaryotic and prokaryotic cells, respectively. In addition, the combination of the transcription activator tTA and the TRE is also regulated and controlled by tetracycline drugs such as doxycycline (doxycycline), and whether the tTA activates the TRE to start transcription can be artificially regulated and controlled by adding or not adding DOX, so that the relationship between gene mutation and functional expression can be simultaneously discovered and confirmed, and the technology is a mutation regulation technology. It is well known to those skilled in the art that, in order to achieve or better achieve the objects of the present invention, the gene search vector of the present invention may further comprise a plasmid origin of replication, which is not particularly required, so long as it is effective in initiating the replication of the plasmid in the corresponding host, such as the p15A replicon; a multiple cloning site facilitating insertion of the foreign fragment; a tetracycline responsive element, such as a TRE, that regulates transcriptional translation of a target gene; transposons capable of random integration in eukaryotic cells, such as PiggyBac; the ampicillin resistance gene of the positive bacterial colony is screened in a prokaryotic system; promoters effective to initiate transcription and translation of gene search vectors in host cells, such as P-CMV; and other common elements of plasmids, such as enhancers, fluorescent protein genes to facilitate detection of gene expression, terminators, and the like.
It is well known to those skilled in the art that the gene search vector may further comprise other functional elements, all of which are operably linked to achieve the purpose of being replicated, amplified, detected, screened, isolated or purified in a prokaryotic host cell and/or replicated, amplified, integrated with the host genome, transcribed, translated, detected, screened, isolated or purified in a eukaryotic host cell, in advance of ensuring the purpose of gene search. Meanwhile, it is well known to those skilled in the art that the order of the constituent elements of the gene search vector may be appropriately adjusted, for example, the positions of the prokaryotic resistance gene and the eukaryotic resistance gene may be exchanged with each other, while the purpose of gene search is ensured. Such variations are also included within the scope of the present invention.
It will be appreciated by those skilled in the art that the various components used in the gene search vectors of the invention are known in the art and that the particular nucleotide sequences may be conveniently found and applied in databases known in the art.
The whole genome random mutation library obtained by the method of the invention is randomly generated, the mutation character can be inherited, the included mutation gene covers the whole genome in probability, and the method can be applied to screening the control gene or signal path to be detected and acted on the target character, such as but not limited to accelerating tumor cell transfer, reducing tumor cell transfer, screening tumorigenesis, drug resistance and the like, and can also be used for screening cells expressing a certain specific molecular marker on the cell surface by flow cytometry FACS.
The invention will now be further illustrated by the following non-limiting examples, and it will be apparent to those skilled in the art that many modifications can be made without departing from the spirit of the invention, such modifications also falling within the scope of the invention.
The following experimental methods are all conventional methods unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.
The main experimental materials:
restriction enzymes, T4DNA ligase, were purchased from NEB, primers, sequencing, Taq polymerase from Invitrogen, plasmid extraction kit, gel recovery kit from OMEGA, fetal bovine serum, DMEM medium, PBS from Hyclone, FugenHD from Roche, various cell culture consumables from Corning, puromycin (puromycin), Luciferin substrates from Invivo, neomycin (Neomyicn, G418) from Merck.
The gene search vector GSV, the plasmid (CAG-tTA) and the MPB plasmid (the plasmid expresses mPB enzyme) based on PiggyBac are recorded in the published patent CN102747096A entitled gene search vector, random gene mutation regulation and control method and application, and can be obtained by the third medical center of the general hospital of people Release force in China.
plasmid pUHD13-3, described in Gossen, M., A.L.Bonin, and H.Bujard.1994.control of gene activity in high her eukarstic cells by prokarstic regulation elements, trends biochem. Sci.18: 471-475.16. gosssen, m., and h.bujard.1992.light control expression in mammalian cells by tetracyline-responsive promoters, proc.natl.acad.sci.usa 89: 5547 and 5551, the public can be obtained from the third medical center of the general hospital of people's liberation army in China.
Example 1 determination of lung cancer cell line a549 on median lethal dose IC50 of cisplatin:
inoculating 5000 cells per well into 96-well plate, 100ul per well, adding cisplatin 24 hr later, repeating 3 wells per group according to final concentration of 200uM, 100uM, 50uM, 25uM, 12.5uM and 0uM, culturing for 48 hr, adding WST-1 reagent 5ul according to WST-1 kit (Roche) instruction, and introducing CO at 37 deg.C2After incubation in the incubator for 4 hours, the light absorbance of each well was measured on an enzyme linked immunosorbent instrument at a wavelength of 450nm, the results were recorded, a cell growth curve was plotted with the drug dose as abscissa and the light absorbance as ordinate, and IC50 was calculated (see fig. 1).
Example 2. study of the relationship of TXNDC16 to resistance to lung cancer chemotherapeutic drugs.
Firstly, establishing a Tet-off human lung cancer A549 cell strain with stable expression.
1) Installation of gene expression regulation switch: digestion, centrifugation, cell collection, resuspension and cell counting, inoculation of A549 cells into 6-well plates (3X 10)5Hole), placing the culture solution in a 5% CO2 incubator at 37 ℃ for overnight culture; the next day, 2. mu.g of plasmid containing tetracycline transcriptional activator protein (tTA) (CAG-tTA) was transfected into A549 cells according to FugeneHD (Roche) instructions, and 24h later the cells were digested, centrifuged, resuspended and divided into 3 10cm dishes in a single well, and screened with 1. mu.g/ml puromycin in DMEM medium, with medium changed every 2-3 days. After 2 weeks, the formation of clones was visually observed, and after the clones had grown to a certain size, the clones were picked up and cloned into a 96-well plate, and then sequentially transferred to a 48-well plate, a 24-well plate, and a 6-well plate for expanded culture for examining tetracycline transcriptional activator protein (tTA) activity.
2) And (3) detecting the activity of the gene expression regulation switch: digesting, centrifuging, resuspending and counting the puromycin-resistant cell clones obtained in step 1) as 104Inoculating cells/well into 96-well plate, dividing into two groups when cell density reaches 80% the next day, adding 1 μ g/ml tetracycline (DOxCyline, DOX) into one group, and adding 0.05 μ g plasmid (pUHC13-3) containing luciferase reporter gene (tTA) into the selected cell clone by FuGeneHD instruction transfection, and splitting according to luciferase reporter gene kit (Thermo Fisher) after 48 hrAnd (3) cell lysis, adding reaction liquid, reading an absorbance value on a bioluminescence detector, counting the difference multiple of luciferase expression intensity between DOX + and DOX-according to a result, screening the selected monoclonal through a luciferase reporter gene technology, and selecting a clone which is well regulated and controlled by tetracycline as a candidate Tet-off clone. Among them, clones #4, #5, #18 and #23 had better gene on-off/DOX fold-control (see FIGS. 2 and 3), and among them, A549 Tet-off- #4 cell strain was selected for subsequent experiments.
Secondly, establishing a random gene mutation library: the gene search vector GSV based on PiggyBac was transfected into A549 Tet-off- #4 cell line according to FugeneHD instructions every 105Cotransfecting 500ng GSV and 125ng transposase (mPB), transferring the cells into a 10cm culture dish after 24h of transfection, adding G418 with the final concentration of 500 mug/ml for screening, replacing the culture solution once every 3-4 days, establishing a whole genome random gene mutation library, estimating the size of the mutation library according to G418 resistant clone, and obtaining more than 10 ten thousand mutant cell clones in total.
And thirdly, screening cisplatin-resistant mutant lung cancer cell strains: selecting a clone capable of surviving from a gene mutation library of the whole genome lung cancer cell by using cisplatin (with the concentration of 40 mu M), repeatedly carrying out multiple rounds of screening, picking a single clone, detecting the IC50 of the single clone to the chemotherapeutic drug by using a WST-1 kit, and comparing the difference of the resistance of the mutant cell strain to the chemotherapeutic drug between the wild type and the mutant cell strain to determine whether the screened lung cancer cell strain is a drug-resistant cell strain, wherein the candidate drug-resistant cell strain is more resistant to the cisplatin when the IC50 of the candidate drug-resistant cell strain to the cisplatin is higher, thereby obtaining a CIS-platin-resistant clonal cell strain CIS4, and the IC50 of the clone to the cisplatin (see figure 4).
And fourthly, verification of the drug resistance characteristics of the cisplatin-resistant mutant lung cancer cell strain: detecting whether CIS-platinum-resistant property of CIS4 mutant clone is regulated by DOX by using a WST-1 kit, respectively setting four groups of A549-DOX-, A549-DOX +, CIS4-DOX-, CIS4-DOX + and the like, adding CIS-platinum according to the final concentration of 200uM, 100uM, 50uM, 25uM, 12.5uM and 0uM of the CIS-platinum, each group of 3 multiple holes, culturing for 48 hours, adding a WST-1 reagent of 5ul, and culturing at 37 ℃ in CO2After the incubator is incubated for 4 hours, the light absorption value of each hole is measured on an enzyme linked immunosorbent assay with the wavelength of 450nm, and the light absorption value is recordedThe results are recorded, the cell growth curve is drawn by taking the dose as the abscissa and the absorbance value as the ordinate, and the IC50 is calculated, after DOX is added, the IC50 of the CIS4 to the CIS-platinum is reduced to 21.652uM, while before and after the DOX is added to the A549, the IC50 has no obvious difference, so that the A549 cells in the control group are not regulated by the DOX, the drug resistance of the CIS4 mutant clone is regulated by the DOX, and the mutant gene is directly related to the CIS-platinum drug resistance.
Fifthly, cloning candidate drug resistance genes: CIS4 genome, which is a cell line resistant to CIS-platin mutation, was extracted using a genome extraction kit (QIAGEN) according to the instructions, the genomic DNA concentration was measured using Nanodrop, 1ug of genomic DNA was digested with Sau3a1 restriction enzyme (NEB) according to the instructions, and then the genomic DNA was digested with T4DNA ligase instruction and Adaptor (5 μ l 100 μm SPL and 5 μ l 100 μm SPR, adding 90 μ l water, heating to 100 deg.C, reacting for 10min, slowly cooling to room temperature) are connected at room temperature overnight, and the specific reaction system comprises 5 μ l Sau3AI single-cut genomic DNA, 1 μ l Adaptor, 10 XT4The Buffer 1. mu.l, ATP (0.01M) 0.1. mu.l, T were ligated4Ligase 0.5. mu.l and deionized water was added to a total volume of 10. mu.l. The DNA sequences at the two ends of the GSV insertion are obtained by Splinkette PCR and sequencing, and the insertion site is selected at the common integration site TTAA of piggyBac transposon, as shown in figure 5 (the sequences of the joint and the primer are all described in the published patent CN102747096A, Gene search vector, random Gene mutation control method and application).
And (3) positioning an insertion site through Blat alignment of a UCSC genome, wherein the GSV insertion site is positioned at 30,655bp (Chr 4: 52399941) downstream of the gene TXDDC 16, reversely activating the transcription of the gene, and generating antisense RNA so as to regulate the expression level of the gene.
Analysis of the relationship between the TXNDC16 gene function and drug resistance: TXNDC16 encodes a 825 amino acid protein (thioredoxin domain 16), a luminal glycoprotein of the Endoplasmic Reticulum (ER), also known as endoplasmic reticulum protein 90(ERp90), which is localized primarily in the endoplasmic reticulum, cell membranes, and mitochondria. Thioredoxin plays an important role in maintaining cellular redox homeostasis and cell survival, and makes cells resistant to various chemical agents, including doxorubicin, prednisone, paclitaxel, tamoxifen, and the like. Functional enrichment analysis suggests that TXNDC16 is primarily involved in adenylate cyclase regulation of the G-protein coupled receptor signaling pathway and guanosine triphosphatase activity. G protein-coupled receptors (GPCRs) are involved in a large number of human pathophysiological and pharmacological activities and are the most studied drug targets. TXNDC16 may be a novel pathway involved in the drug-resistant cisplatin chemotherapy of lung cancer cells by mediating the alteration of GPCRs signaling pathways.
Nucleotide sequence listing
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Application of TXDDC 16 gene in preparation of detection kit for detecting drug resistance of lung cancer chemotherapeutic drugs
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taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct 2880
gagctaccaa ctctttgaac cgaggtaact ggcttggagg agcgcagtca ccaaaacttg 2940
tcctttcagt ttagccttaa ccggcgcatg acttcaagac taactcctct aaatcaatta 3000
ccagtggctg ctgccagtgg tgcttttgca tgtctttccg ggttggactc aagacgatag 3060
ttaccggata aggcgcagcg gtcggactga acggggggtt cgtgcataca gtccagcttg 3120
gagcgaactg cctacccgga actgagtgtc aggcgtggaa tgagacaaac gcggccataa 3180
cagcggaatg acaccggtaa accgaaaggc aggaacagga gagcgcacga gggagccgcc 3240
agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccaccact gatttgagcg 3300
tcagatttcg tgatgcttgt caggggggcg gagcctatgg aaaaacggct ttgccgcggc 3360
cctctcactt ccctgttaag tatcttcctg gcatcttcca ggaaatctcc gccccgttcg 3420
taagccattt ccgctcgccg cagtcgaacg accgagcgta gcgagtcagt gagcgaggaa 3480
gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg 3540
ccacatgaag cacttcactg acaccctcat cagtgccaac atagtaagcc agtatacact 3600
ccgctagagg atctaagctt ccaacaagct cgtcatcgct ttgcagaaga gcagagagga 3660
tatgctcatc gtctaaagaa ctacccattt tattatatat tagtcacgat atctataaca 3720
agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat 3780
cgtatgagtt aaaacttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc 3840
ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc 3900
tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga 3960
gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa 4020
Claims (2)
1. The application of a reagent for detecting the TXDDC 16 gene expression level in preparing a detection kit for detecting the drug resistance of lung cancer chemotherapeutic drugs, wherein the lung cancer chemotherapeutic drugs are cisplatin.
2. The use according to claim 1, wherein the nucleotide sequence of the TXNDC16 gene is GenBank accession No. NM _ 020784.
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