CN117106914A - ODC1 gene marker for prognosis evaluation of lung adenocarcinoma by combining pemetrexed/cisplatin with chemotherapy and application thereof - Google Patents
ODC1 gene marker for prognosis evaluation of lung adenocarcinoma by combining pemetrexed/cisplatin with chemotherapy and application thereof Download PDFInfo
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Abstract
The invention discloses an ODC1 gene marker for prognosis evaluation of lung adenocarcinoma by pemetrexed/cisplatin combined chemotherapy and application thereof, and on one hand, provides application of an ODC1 gene in preparation of a reagent for prognosis evaluation of lung adenocarcinoma by pemetrexed/cisplatin combined chemotherapy, wherein Ensembl number of the ODC1 gene is ENST00000234111.9, and simultaneously provides a kit for evaluating sensitivity of lung adenocarcinoma to pemetrexed/cisplatin combined chemotherapy, which comprises a reagent for detecting expression quantity of ODC 1. The invention discovers that the ODC1 gene has important mediating effect on iron death based on cell experiments and clinical data verification, the expression level of the ODC1 gene is related to the sensitivity of cells to an iron death inducer and combined chemotherapy treatment, and the ODC1 gene is used as a biomarker to assist in predicting the sensitivity or tolerance of lung adenocarcinoma patients to chemotherapy, thus the ODC1 gene has important clinical application value.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to an ODC1 gene marker for prognosis evaluation of lung adenocarcinoma by combining pemetrexed/cisplatin with chemotherapy and application thereof.
Background
Lung cancer is the most common, highest mortality tumor, with morbidity and mortality ranking the first of all tumors, with lung adenocarcinoma being one of the most common subtypes. Cisplatin/pemetrexed has killing or inhibiting effect on tumor cells by destroying cell DNA and blocking folic acid metabolism respectively, and cisplatin/pemetrexed combined chemotherapy is a classical first-line treatment scheme of lung adenocarcinoma, but a significant part of lung adenocarcinoma patients are relatively tolerant to combined chemotherapy and hardly benefit from the combined chemotherapy. Therefore, a series of reliable biomarkers are needed to assist clinicians in predicting the sensitivity of lung adenocarcinoma patients to chemotherapy, and the method has important significance for improving the treatment effect of the patients and reducing the social burden. At present, some possible biomarkers (such as thymidylate synthase and the like) are reported to be used for predicting the sensitivity of a lung adenocarcinoma patient to combined chemotherapy, but due to the reasons of complicated detection flow, low accuracy, verification in a cell or animal model and the like, the application of the biomarkers in clinic is still very limited, and a personalized chemotherapy scheme of the lung adenocarcinoma patient needs to be searched for with higher sensitivity, specificity and application value.
Iron death (ferrotosis) was first proposed by the university of columbia dr.brunt r.stock well in 2012 as a novel programmed death mode of iron dependence, which is distinguished from apoptosis, cell necrosis and cell scorch, and has the main mechanism that external stimulus is used for inducing imbalance of intracellular Reactive Oxygen Species (ROS), so that in Fenton reaction (Fenton reaction) catalyzed by ferrous iron, peroxidation of polyunsaturated fatty acids (PUFA) on biological membranes is caused, thereby leading to structural change of phospholipid bilayer and rupture of cell membranes, and finally leading to cell death. Iron death, which is an important cell death mode, participates in a series of pathophysiological processes, and in recent years, iron death has been widely focused by researchers, and research layers on the mechanism of iron death and application of the mechanism of iron death in disease diagnosis and treatment, especially in tumors, have been reported to cause iron death by chemotherapy, and the iron death level of tumors can be regulated by intervention of iron death-related genes, so that drug resistance of tumors to pemetrexed/cisplatin chemotherapy is improved.
Disclosure of Invention
The invention aims to provide an ODC1 gene marker for prognosis evaluation of lung adenocarcinoma by combining pemetrexed/cisplatin with chemotherapy, and the ODC1 gene (ornithine decarboxylase) has important mediating effect on iron death based on cell experiments and clinical data verification, has the expression level related to sensitivity of cells to an iron death inducer and combined chemotherapy treatment, and is used as a biomarker for assisting in predicting sensitivity or tolerance of lung adenocarcinoma patients to chemotherapy, so that the blank in the field is filled.
Another object of the present invention is to provide a kit for evaluating susceptibility of lung adenocarcinoma to pemetrexed/cisplatin combination chemotherapy, which intuitively judges whether a lung adenocarcinoma patient is susceptible or tolerant to iron death of tumor cells caused by pemetrexed/cisplatin combination chemotherapy, thereby evaluating the suitability of the patient for treating lung adenocarcinoma using combination chemotherapy.
The above object of the present invention is achieved by the following technical solutions:
the invention provides application of an ODC1 gene in preparing a reagent for prognosis evaluation of lung adenocarcinoma by combining pemetrexed/cisplatin chemotherapy, wherein the ODC1 gene is totally named as Ornithine Decarboxylase, and Ensembl number is ENST00000234111.9.
Preferably, the prognosis evaluation of lung adenocarcinoma treated with pemetrexed/cisplatin combination chemotherapy includes determining whether a lung adenocarcinoma patient is sensitive or tolerant to pemetrexed/cisplatin combination chemotherapy.
The invention also provides application of the reagent for detecting the ODC1 expression level in preparing a detection product for prognosis evaluation of lung adenocarcinoma by combining pemetrexed/cisplatin chemotherapy.
The invention also provides a kit for assessing susceptibility of lung adenocarcinoma to pemetrexed/cisplatin combination chemotherapy, comprising a reagent for detecting ODC1 expression level.
The invention also provides a method for evaluating sensitivity of lung adenocarcinoma to pemetrexed/cisplatin combined chemotherapy, which comprises the following steps:
step 1: collecting a detection sample;
step 2: and detecting the expression quantity of ODC1 in the detection sample by using the kit for evaluating the sensitivity of lung adenocarcinoma to pemetrexed/cisplatin combined chemotherapy, wherein a lung adenocarcinoma patient with high ODC1 expression is sensitive to the pemetrexed/cisplatin combined chemotherapy, and the response to the pemetrexed/cisplatin combined chemotherapy is good.
Preferably, the test sample is derived from tumor tissue of a lung adenocarcinoma patient.
Preferably, taking the median of delta CT value of ODC1 relative to the reference glyceraldehyde phosphate dehydrogenase (GAPDH) as a cut-off value, if the ODC1 expression in the detection sample is low, namely delta CT is more than 8.31, prompting the patient to tolerate the combined chemotherapy of pemetrexed/cisplatin; if the ODC1 expression in the detection sample is high, namely delta CT is less than or equal to 8.31, the patient is prompted to be sensitive to pemetrexed/cisplatin combined chemotherapy.
Compared with the prior art, the invention has the beneficial effects that:
the whole genome CRISPR/Cas9 knockout screening technology is adopted to initially find that the knockout ODC1 remarkably improves the tolerance of A549 lung adenocarcinoma cells to the iron death inducer Erastin, and the result is further proved in cell experiments. Considering that pemetrexed/cisplatin combined chemotherapy also has the effect of leading to iron death, ODC1 is knocked out or overexpressed in two lung adenocarcinoma cell lines of A549 and H358, and the corresponding dose of pemetrexed/cisplatin combined chemotherapy treatment is adopted, so that after the ODC1 is knocked out, the tolerance of cells to chemotherapy is obviously improved, and the killing capacity of chemotherapy to lung adenocarcinoma cells is obviously enhanced due to the overexpression of the ODC 1. Also, treatment of a549, H358 cells with the ODC 1-specific inhibitor DFMO significantly increased the sensitivity of lung adenocarcinoma cells to chemotherapy. ODC was thus proposed as a prognostic biomarker to determine the susceptibility of lung adenocarcinoma patients to pemetrexed/cisplatin combination chemotherapy. In addition, the qPCR method is adopted to detect the ODC1 expression condition in the surgical excision specimens of 34 patients receiving pemetrexed/cisplatin combined chemotherapy, and the prognosis results are analyzed, so that the patients with high ODC1 expression are more sensitive to patients with lower chemotherapy response, and better prognosis is indicated. Thus, the expression level of ODC1 can be used to predict whether a patient with lung adenocarcinoma is susceptible or resistant to pemetrexed/cisplatin combination chemotherapy.
Drawings
FIG. 1 is a graph showing the drug toxicity profiles of ODC1 knocked out or overexpressed and control A549, H358 lung adenocarcinoma cells, respectively, for different concentrations of Erastin or pemetrexed/cisplatin in the examples.
FIG. 2 shows the viability of A549, H358 lung adenocarcinoma cells pretreated with the ODC 1-specific inhibitor DFMO and control, and further treated with Erastin or pemetrexed/cisplatin in the examples.
FIG. 3 shows lipid peroxidation levels of A549, H358 lung adenocarcinoma cells from which ODC1 was knocked out or overexpressed, and controls, respectively, in the examples, after treatment with Erastin or pemetrexed/cisplatin.
FIG. 4 is a prognostic result of the expression level of ODC1 in lung adenocarcinoma tissue in the example.
Detailed Description
The invention is further illustrated by the following examples:
example 1
Crispr/Cas9 whole genome high throughput drug resistance Gene screening
The method comprises the following steps: firstly, a series of sgrnas are constructed according to the existing cripr/Cas 9 knockout library, transfected into lung adenocarcinoma cells a549, each cell is guaranteed to be transferred into only one sgRNA or not transferred into the sgrnas by adopting the proper virus dosage, and then the cells which are not transferred into the sgrnas are killed by puromycin treatment, so that a random certain gene is knocked out in each A549 cell. Subsequently, the knocked-out cells were treated with the iron death inducer Erastin for screening, sensitive cells were inhibited or even killed, and normal proliferation of drug-resistant cells was not affected. By high throughput sequencing analysis of sgrnas in pre-and post-treatment cells, respectively, changes in cellular activity following knock-out of each gene can be inferred.
Results: through this step, the ODC1 knockout cell subpopulation was found to be significantly more elevated than the control after the Erastin treatment, and experimental data are shown in table 1:
TABLE 1
| Gene | Log(Fc) Erastin | Log(Fc) DMSO | Log(Fc) Erastin -Log(Fc) DMSO |
| ODC1 | 2.736 | 0.253 | 2.483 |
Here, log (Fc) Erastin Log value representing the fold change in expression of a certain sgRNA after 3 days of Erastin treatment versus before drug administration, but Log (Fc) DMSO The control group, i.e. the log values of the same sgrnas after 3 days of DMSO treatment, which were changed from the expression values before dosing treatment, are shown. From this, it can be inferred that the lung adenocarcinoma cells had an enhanced resistance to Erastin after the ODC1 knockout.
2. Change of Erastin and pemetrexed/cisplatin resistance to lung adenocarcinoma cells after knockout or overexpression of ODC1
The method comprises the following steps: ODC1 and corresponding controls were knocked out or overexpressed in both A549 and H358 lung adenocarcinoma cells, respectively, followed by treatment of transfected cells with Erastin or pemetrexed/cisplatin, respectively, and detection of cell activity using CCK 8.
Results: it was found that the tolerance of ODC1 knockout cells to Erastin and pemetrexed/cisplatin was significantly higher than that of the control group, whereas over-expression of ODC1 resulted in increased sensitivity of cells to both drugs (fig. 1). Also, treatment of lung adenocarcinoma cells with the ODC1 specific inhibitor DFMO resulted in a significant increase in the ability of the cells to tolerate both Erastin and pemetrexed/cisplatin (fig. 2).
Furthermore, BODIPY lipid peroxidation experiments showed that pemetrexed/cisplatin combination treatment significantly increased lung adenocarcinoma cell lipid peroxidation levels, suggesting that combination chemotherapy resulted in iron death, while knockout of ODC1 significantly alleviated this effect (fig. 3). From this, it can be inferred that knockout of ODC1 significantly enhanced the resistance of lung adenocarcinoma cells to pemetrexed/cisplatin-induced iron death.
Expression of ODC1 in tumor tissue of lung adenocarcinoma patients receiving Pemetrexed/cisplatin combination chemotherapy and prognosis analysis of patients
The expression of ODC1 in tumor tissues of 34 lung adenocarcinoma patients receiving pemetrexed/cisplatin combined chemotherapy was detected by qRT-PCR. The patients were then followed for more than 5 years to compare the ODC1 correlation with the prognosis of the patient, as follows:
(1) Total RNA in the tissue sample is extracted.
(2) Detection of ODC1 expression using qRT-PCR: provided by assist Saint Co LtdqPCR SYBR Green Master Mix detection was performed on a quantsudio 5 fluorescent quantitative PCR instrument supplied by Thermo Fisher company, the primer sequences were:
ODC1：
Forward：GGCTGTACCGATCCTGAGACCTT；
Reverse：GCCACCGCCAATATCAAGCAGA；
reference gene GAPDH:
Forward：AGAAGGCTGGGGCTCATTTG；
Reverse：AGGGGCCATCCACAGTCTTC。
the primers are all provided by Shanghai Biotechnology Co.
(3) Patient survival was followed (> 5 years). The follow-up period is once a year, with a maximum follow-up time of 10 years.
(4) And (5) analyzing the detection result by a survival curve. Taking the median of 8.31 of the delta CT value of ODC1 relative to the internal reference GAPDH as a cut-off value, thereby distinguishing high/low expression groups and corresponding different survival conditions, as shown in FIG. 4, the result shows that for a lung adenocarcinoma patient receiving pemetrexed/cisplatin combined treatment, the high expression of ODC1 in a tumor tissue sample means better prognosis, and the patient is indicated to respond well to combined chemotherapy. Thus, the expression level of ODC1 can be used to predict the susceptibility of lung adenocarcinoma patients to pemetrexed/cisplatin treatment.
In conclusion, the invention firstly adopts a high-throughput technology to comprehensively and systematically analyze the change of the sensitivity condition of the A549 lung adenocarcinoma cells to the iron death inducer Erastin after randomly knocking out a certain gene, discovers that the lung adenocarcinoma cells are more tolerant to Erastin after ODC1 knocking out, and further verifies that the ODC1 knocking out leads to the tolerance of pemetrexed/cisplatin combined chemotherapy, thereby indicating that the ODC1 expression level is suitable for predicting the sensitivity degree of the lung adenocarcinoma patients to the pemetrexed/cisplatin combined chemotherapy. Then further confirming the expression difference of the gene in lung adenocarcinoma patients and the application value in judging the sensitivity degree to pemetrexed/cisplatin combined chemotherapy, and detecting the expression of ODC1 in tumor samples of the lung adenocarcinoma patients, wherein if the expression of ODC1 in the samples is low (namely delta CT is more than 8.31), the patients are tolerant to the pemetrexed/cisplatin combined chemotherapy; if ODC1 expression is high (i.e., ΔCT is less than or equal to 8.31), the patient is susceptible to pemetrexed/cisplatin combination chemotherapy.
The foregoing is a preferred embodiment of the present invention, but the present invention should not be limited to the disclosure of this embodiment. So that equivalents and modifications will fall within the scope of the invention, all within the spirit and scope of the invention as disclosed.
Claims (7)
- Use of the ODC1 gene, wherein the ODC1 gene is designated as Ornithine Decarboxylase and the Ensembl number is ENST00000234111.9, for the preparation of a reagent for prognosis evaluation of lung adenocarcinoma by pemetrexed/cisplatin combination chemotherapy.
- 2. The use according to claim 1, comprising determining whether a patient with lung adenocarcinoma is sensitive or tolerant to pemetrexed/cisplatin combination chemotherapy.
- 3. Application of a reagent for detecting ODC1 expression quantity in preparing a detection product for prognosis evaluation of lung adenocarcinoma by combining pemetrexed/cisplatin chemotherapy.
- 4. A kit for assessing susceptibility of lung adenocarcinoma to pemetrexed/cisplatin combination chemotherapy comprising reagents for detecting ODC1 expression.
- 5. A method for assessing susceptibility of lung adenocarcinoma to pemetrexed/cisplatin combination chemotherapy, comprising the steps of:step 1: collecting a detection sample;step 2: detecting the expression level of ODC1 in the detection sample by the kit for assessing susceptibility of lung adenocarcinoma to pemetrexed/cisplatin combination chemotherapy, wherein: patients with lung adenocarcinoma with high ODC1 expression are sensitive to the combined chemotherapy of pemetrexed/cisplatin, which suggests that the combined chemotherapy of pemetrexed/cisplatin is good.
- 6. The method of assessing susceptibility of lung adenocarcinoma to pemetrexed/cisplatin combination chemotherapy as claimed in claim 5 wherein said test sample is derived from lung adenocarcinoma tumor tissue.
- 7. The method for evaluating susceptibility of lung adenocarcinoma to pemetrexed/cisplatin combination chemotherapy as claimed in claim 5, wherein median of Δct value of ODC1 relative to reference glyceraldehyde phosphate dehydrogenase is 8.31 as cut-off:if delta CT in the detected sample is more than 8.31, ODC1 expression is low, and resistance to pemetrexed/cisplatin combined chemotherapy is indicated;if the delta CT in the detected sample is less than or equal to 8.31, the ODC1 expression is high, which indicates that the combined chemotherapy of pemetrexed/cisplatin is sensitive.
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