CN116179704A - Application of TRIM21 as nasopharyngeal carcinoma radiotherapy efficacy prediction marker - Google Patents
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Abstract
The invention discloses an application of TRIM21 as a nasopharyngeal carcinoma radiotherapy efficacy prediction marker. In the invention, the effectiveness of TRIM21 as a nasopharyngeal carcinoma radiotherapy efficacy prediction marker is discovered for the first time, and experiments prove that the TRIM21 can be effectively used for indicating the nasopharyngeal carcinoma radiotherapy efficacy of a nasopharyngeal carcinoma patient, so that nasopharyngeal carcinoma patients unsuitable for radiotherapy treatment can be screened out early, and the delay of treatment time caused by the application of wrong treatment means is avoided.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an application of TRIM21 as a nasopharyngeal carcinoma radiotherapy efficacy prediction marker.
Background
Nasopharyngeal carcinoma is a malignant tumor occurring in the nasopharynx of the human body, and its epidemic distribution is extremely unbalanced and is highly developed in east asia. Radiation therapy is the basic treatment modality for nasopharyngeal carcinoma, and all newly diagnosed patients who are non-metastatic nasopharyngeal carcinoma need to receive radical radiation therapy. Although nasopharyngeal carcinoma cells are sensitive to radiation, about 20% of human subjects still resist radiation, i.e., tumor recurrence occurs early after treatment.
Radiotherapy is the local irradiation of tumors with radiation rays, which in the traditional view are thought to kill tumor cells by destroying their genomic DNA, thereby achieving the purpose of tumor treatment. However, recent studies have shown that the efficacy of radiotherapy depends on the activation of the body's anti-tumor immune response, in addition to killing tumor cells by DNA damage. Mechanically, radiotherapy indirectly kills tumors by activating the human immune system and mobilizing immune cells to attack tumor cells. However, it has now been found that radiotherapy has limited activation of the immune system, as tumor cells limit the immune activation of radiotherapy by means of pro-cancerous factors, ultimately resulting in radiotherapy resistance. The cancer promoting factors are clear, which is beneficial to predicting the curative effect of tumor radiotherapy and developing targeted drugs and further improving the curative effect of tumor radiotherapy.
Ubiquitination is one of the most common protein modifications. E3 ubiquitin ligase is an executor of ubiquitination, whose abnormal expression in a variety of tumors leads to abnormal ubiquitination of protein molecules, has been shown to be involved in the development and progression of a variety of tumors. Tri-motif protein 21 (TRIM 21) is a ubiquitin ligase that exists in the cytoplasm of cells, and it has been found from previous studies that TRIM21 is involved in regulating the carbohydrate metabolism, amino acid metabolism, degradation of oncostatin, etc. of tumor cells, and promoting proliferation of tumor cells. However, whether TRIM21 plays an important role in the radiotherapy immunity activation and treatment resistance of nasopharyngeal carcinoma, and whether the expression of the TRIM21 can predict the radiotherapy curative effect and guide the treatment is not reported yet.
In the related art, no means is available for predicting the resistance of a patient to the radiotherapy treatment of the nasopharyngeal carcinoma, and the mechanism of how the nasopharyngeal carcinoma resists the radiotherapy is not clear, so that the development of an effective nasopharyngeal carcinoma radiotherapy curative effect prediction marker and the further elucidation of the mechanism of the radiotherapy resistance of the nasopharyngeal carcinoma have important value for guiding the radiotherapy of the nasopharyngeal carcinoma.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems in the prior art. Therefore, the invention aims to provide the application of TRIM21 as a nasopharyngeal carcinoma radiotherapy efficacy prediction marker. In the invention, the effectiveness of TRIM21 as a nasopharyngeal carcinoma radiotherapy efficacy prediction marker is discovered for the first time, and experiments prove that the TRIM21 can be effectively used for indicating the nasopharyngeal carcinoma radiotherapy efficacy of a nasopharyngeal carcinoma patient, so that nasopharyngeal carcinoma patients unsuitable for radiotherapy treatment can be screened out early, and the delay of treatment time caused by the application of wrong treatment means is avoided.
In a first aspect, the invention provides the use of an agent for detecting TRIM21 in the manufacture of a predictive product for the efficacy of radiation therapy for nasopharyngeal carcinoma.
In some embodiments of the invention, the reagent for detecting TRIM21 includes a reagent for detecting TRIM21 at a gene level and a protein level.
In some embodiments of the invention, detection techniques at the gene level include, but are not limited to, sequencing methods, nucleic acid probe methods, PCR amplification.
In some embodiments of the invention, protein level detection techniques include, but are not limited to, western Blot, ELISA, immunofluorescence, immunohistochemistry.
In some embodiments of the invention, the reagent for detecting TRIM21 is a quantitative detection reagent. Of course, the technical scheme of the invention can be realized by a non-quantitative comparison method by a person skilled in the art, and the technical core of the method is only that the comparison of TRIM21 expression conditions of a reference substance and a sample can be realized, so that whether the quantization is needed or not is a better option.
In some embodiments of the invention, the reagent for quantifying TRIM21 is used to prepare a reagent for predicting the efficacy of radiation therapy for nasopharyngeal carcinoma using techniques that are a combination of immunohistochemical reagents or microscopic evaluation systems.
In some embodiments of the invention, the method for using the nasopharyngeal carcinoma radiotherapy efficacy prediction product comprises the following steps:
taking a sample of a subject, quantitatively detecting TRIM21 in the sample by using a nasopharyngeal carcinoma radiotherapy efficacy prediction product, and if the expression level is obviously higher than that of a reference substance, the subject is insensitive to the nasopharyngeal carcinoma radiotherapy, and the nasopharyngeal carcinoma radiotherapy has no curative effect or has no obvious curative effect on the treatment; if the expression level is not significantly higher than the control, the subject is sensitive to the nasopharyngeal carcinoma radiotherapy, and the nasopharyngeal carcinoma radiotherapy has an effect on the treatment.
In some embodiments of the invention, the detection method is selected to be combined with immunohistochemical observation by a microscope, TRIM21 expression level is obtained through immunohistochemistry, staining intensity is determined under the microscope, the staining intensity is multiplied by positive cell proportion to obtain IRS score, and when IRS=0-6, the detection method is insensitive to nasopharyngeal carcinoma radiotherapy, and the treatment effect of the nasopharyngeal carcinoma radiotherapy is not obvious; irs=7-12, is sensitive to and has therapeutic effects on nasopharyngeal carcinoma radiotherapy.
In the invention, the inventor discovers that the E3 ubiquitin ligase molecule is related to the anti-tumor immune reaction hypofunction in the nasopharyngeal carcinoma through analyzing the general tumor sequencing data and the single cell sequencing data of the nasopharyngeal carcinoma, and meanwhile, the TRIM21 high expression prompts that the radiotherapy of the nasopharyngeal carcinoma is insensitive, the immune activation of the radiotherapy is insufficient, and the poor prognosis of a patient is related. Through in vitro "tumor cell-immune cell" co-culture experiments, and mouse tumor radiotherapy models, the inventors found that: the TRIM21 can obviously enhance tumor cell antigen presentation induced by radiotherapy, increase the opportunity of immune cells to recognize tumors, and the TRIM21 can help the activation of immune effector cells such as dendritic cells, CD8+ T cells and the like, play a role in killing tumors, promote the sensitivity of the tumor radiotherapy and enhance the curative effect of the radiotherapy. The above results suggest: TRIM21 may be a marker for predicting the efficacy of radiation therapy for nasopharyngeal carcinoma.
In addition, the inventor also selects a biopsy nasopharyngeal carcinoma tissue paraffin specimen of 355 cases of initial nasopharyngeal carcinoma patients, adopts an immunohistochemical method to evaluate the expression level of TRIM21, then determines the staining intensity under a microscope, and multiplies the staining intensity by the positive cell proportion to obtain a IRS (immunoreactive score) score. 355 nasopharyngeal cancer patients were classified into TRIM21 low-expression groups (irs=0-6) and high-expression groups (irs=7-12) according to the median of IRS scores of patients. Combining with prognosis data, it was found that TRIM21 was significantly highly expressed in tumors in patients with recurrent nasopharyngeal carcinoma after radiotherapy. Kaplan-Meier survival showed that TRIM21 high expression was associated with recurrence, distant metastasis and death in nasopharyngeal carcinoma patients. Multi-factor analysis shows that: TRIM21 is a poor prognostic factor for disease progression-free survival, distant metastasis-free survival, and overall survival in nasopharyngeal cancer patients.
In some embodiments of the invention, the control is a healthy person or a sample of a nasopharyngeal carcinoma patient without recurrence, distant metastasis, and death of nasopharyngeal carcinoma.
In some embodiments of the invention, the subject is a patient with nasopharyngeal carcinoma. The applicable crowd in the invention is limited to nasopharyngeal carcinoma patients, namely the core is to screen or distinguish nasopharyngeal carcinoma patients which are not applicable to radiotherapy from nasopharyngeal carcinoma patients.
In some embodiments of the invention, the sample comprises a body fluid or tissue sample.
In some embodiments of the invention, the tissue sample is a tumor tissue sample.
In a second aspect of the invention, a nasopharyngeal carcinoma radiotherapy efficacy prediction system is provided, and the nasopharyngeal carcinoma radiotherapy efficacy prediction system includes a sampling module, a TRIM21 detection module, and an analysis module.
In some embodiments of the invention, the sampling module is used to sample samples, including body fluids or tissue samples, of the type that may be conventional in the art.
In some embodiments of the invention, the TRIM21 detection module is configured to detect TRIM21 content in a sample.
In some embodiments of the present invention, the TRIM21 detection module contains the reagent for detecting TRIM21 according to the first aspect of the present invention.
In some embodiments of the present invention, the TRIM21 detection module performs the task of detecting the TRIM21 content of the sample in an automated or non-automated manner.
In some embodiments of the invention, the TRIM21 detection module employs an autoimmune histochemical analysis device to perform TRIM21 detection. It obtains the level of TRIM21 by immune response scoring (immunoreactive score, IRS).
In some embodiments of the invention, the analysis module is configured to compare differences in TRIM21 content between different samples and output an analysis result based on the differences.
In some embodiments of the invention, the analytical method in the analytical module is determined based on a Kaplan-Meier curve or a COX proportional hazards model.
In some embodiments of the invention, the sample comparison object of the analysis module is a subject sample and a control.
In some embodiments of the invention, the control is derived from healthy people or from nasopharyngeal carcinoma patients who have no recurrence of nasopharyngeal carcinoma, distant metastasis, and death.
The beneficial effects of the invention are as follows:
1. the invention provides the TRIM21 as the marker for predicting the curative effect of the nasopharyngeal carcinoma radiotherapy for the first time, and the effect of predicting the curative effect of the nasopharyngeal carcinoma radiotherapy can be realized through experiments and verification, so that the rapid classification of patients is realized, the proportion of effective treatment is improved, and the possibility of curing and the effective survival rate of the patients are increased.
2. The prediction method is accurate and effective, can effectively and conveniently divide sensitive and insensitive people for nasopharyngeal carcinoma radiotherapy, and improves diagnosis and treatment efficiency.
Drawings
FIG. 1 shows the effect of TRIM21 knockout on tumor growth after nasopharyngeal carcinoma radiotherapy, wherein A is the effect on tumor volume and B is the effect on tumor quality.
FIG. 2 is a graph showing the effect of TRIM21 knockout on immune cell number changes following nasopharyngeal carcinoma radiotherapy.
FIG. 3 is a representative image of immunohistochemical staining of TRIM21 low and high expression groups.
FIG. 4 shows TRIM21 expression in tumors of patients with early-stage recurrent nasopharyngeal carcinoma after radiotherapy.
FIG. 5 is a graph showing the correlation between survival without local recurrence (A), survival without disease (B) and total survival (C) of TRIM 21-highly expressed nasopharyngeal carcinoma patients.
Fig. 6 is a graph of the calculated ROC curve in example 2.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The starting materials, reagents or apparatus used in the examples and comparative examples were either commercially available from conventional sources or may be obtained by prior art methods unless specifically indicated. Unless otherwise indicated, assays or testing methods are routine in the art.
Example 1 verification of TRIM21 as a marker for nasopharyngeal carcinoma in animal experiments
(1) Constructing a mouse nasopharyngeal carcinoma radiotherapy model:
normal nasopharyngeal carcinoma SUNE1 cells (human hypodifferentiated nasopharyngeal carcinoma cells) and nasopharyngeal carcinoma SUNE1 cells subjected to TRIM21 knockout treatment are inoculated into a humanized immunodeficiency mouse subcutaneously, and tumors are established. The tumors were subjected to a unified radiotherapy treatment (6 Gy, single irradiation), tumor size was monitored periodically, and tumor growth curves were drawn.
After 20 days of radiotherapy treatment, mice were sacrificed, tumor weights were measured, a portion of tumor samples were extracted, and flow cytometry was used to detect immune cell infiltration and activation inside tumors.
(2) And extracting part of tumor samples for immunohistochemical analysis:
extracting part of tumor samples, preparing paraffin sections, drying the paraffin sections in a 65 ℃ oven for 30 minutes, sequentially soaking the paraffin sections in an environment-friendly transparent agent for 5 minutes, sequentially soaking the paraffin sections in absolute ethanol for 2 minutes, 95% ethanol for 2 minutes and 75% ethanol for 2 minutes.
A3% (v/v) aqueous hydrogen peroxide solution was prepared, and the mixture was placed in an autoclave to boil, and then placed in a slide to be heated for 150 seconds. And uncovering the pot cover after heating is finished, and naturally cooling to room temperature.
Blocking was performed using immunohistochemical blocking solution (30 min incubation at room temperature) and the blocking solution was discarded. Wash, add primary antibody (12108-1-AP, proteintech) and incubate overnight at 4 ℃. Washing, adding biotin-labeled secondary antibody (Boshide, SA 1020), and incubating at 37℃for 25min. Color development was performed using freshly configured DAB. After washing with tap water, counterstaining with hematoxylin for 2-5min, washing with tap water. Sequentially placing the slices in 75% ethanol for 2min,5% ethanol for 2min, and absolute ethanol for 2min, and naturally airing. The tissues were scored for immunohistochemistry according to positive proportion and staining degree (staining intensity score multiplied by positive cell proportion score, yielding IRS (immunoreactive score) score).
The criteria for the staining intensity scores are shown in table 1.
TABLE 1 staining intensity of paraffin specimens for nasopharyngeal carcinoma tissues
| Scoring of | Dyeing intensity | Color of |
| 0 | Dyeing-free | - |
| 1 | Weak and weak | Yellowish light yellow |
| 2 | Medium and medium | Yellow brown color |
| 3 | Strong strength | Brown color |
The criteria for positive proportion scoring are shown in table 2.
TABLE 2 nasopharyngeal carcinoma tissue Paraffin sample Positive proportion
| Scoring of | Positive cell proportion |
| 1 | <25％ |
| 2 | 25-50％ |
| 3 | 50-75％ |
| 4 | >75％ |
In this example, the Kaplan-Meier method was used to plot the survival curves during data analysis, and the log-rank test was used for the difference comparison. Multivariate analysis was performed using a Cox proportional hazards model (CPH: cox proportional hazards) model, calculating the Hazard Ratio (HR) and determining independent prognostic factors to assess whether there was a difference in the effect of radiotherapy in TRIM21 high-low expression sets. Data are expressed as mean ± SEM, P-values <0.05 are considered statistically significant.
The results are shown in FIGS. 1 and 2.
It can be found that a nasopharyngeal carcinoma tumor model mouse is established by subcutaneously inoculating normal or TRIM21 knocked-out nasopharyngeal carcinoma SUNE1 cells into a humanized immunodeficiency mouse, and a nasopharyngeal carcinoma tumor radiotherapy treatment model mouse is finally obtained by carrying out radiotherapy on tumors. By comparing mice in different experimental groups, the TRIM21 is knocked out to obviously delay the growth of tumors after radiotherapy, namely, the knocking out of the TRIM21 obviously increases the sensitivity of radiotherapy, and the number and activation of CD8+ T cells in the tumors knocked out by the TRIM21 are obviously increased, so that the anti-tumor immunity is strong. The results show that TRIM21 is related to the curative effect of the nasopharyngeal carcinoma radiotherapy and can be a potential marker for predicting the curative effect of the radiotherapy.
Example 2 clinical verification of TRIM21 as a marker for nasopharyngeal carcinoma
In order to further verify the correlation of TRIM21 and the radiotherapy efficacy of the nasopharyngeal carcinoma and confirm whether the TRIM21 can be used as a potential marker for predicting the radiotherapy efficacy, the inventor selects 355 clinical patients with initial diagnosis of the nasopharyngeal carcinoma and carries out radiotherapy treatment and follow-up investigation on the patients. In follow-up, 67 (18.9%) patients were found to relapse and 108 (30.4%) patients were eliminated.
The inventor collects the paraffin specimens of the biopsied nasopharyngeal carcinoma tissues of 355 patients, evaluates the expression level of TRIM21 by adopting the immunohistochemical method, and further determines the staining intensity and the positive cell proportion under a microscope. IRS scoring criteria are the same as in the previous examples.
By statistical analysis of the patient's IRS scores, 355 nasopharyngeal cancer patients were found to be classified into TRIM21 low expression group (irs=0-6) and high expression group (irs=7-12) according to the median of IRS scores (representative pictures of immunohistochemical staining are shown in fig. 3). Prognosis data are obtained by combining tracking and collecting, and the patients with recurrent nasopharyngeal carcinoma after radiotherapy are found to have obvious high expression of TRIM21 of tumors. And Kaplan-Meier survival showed that TRIM21 high expression was associated with recurrence, distant metastasis and death in nasopharyngeal carcinoma patients (as shown in fig. 3). Further by multifactorial analysis (results are shown in table 4 and fig. 5), TRIM21 was found to be a poor prognostic factor for nasopharyngeal carcinoma patients survival without disease progression, survival without distant metastasis, and overall survival.
Table 3 Multi-factor analysis results for 355 cases of nasopharyngeal carcinoma patients
Further, ROC curve analysis was performed on the immune score IRS (0-12 minutes) of the above sample tissue in combination with the recurrence status after radiotherapy of the patient, and irs=6 was taken as a critical value, and the results are shown in fig. 6.
The sensitivity of the method for predicting the curative effect of the radiotherapy of the nasopharyngeal carcinoma is 53.7%, the specificity is 63.9%, and the method has a good prediction effect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The application of the reagent for detecting TRIM21 in preparing the nasopharyngeal carcinoma radiotherapy efficacy prediction product.
2. The use according to claim 1, wherein the reagent for detecting TRIM21 comprises a reagent for detecting TRIM21 at a gene level and a protein level.
3. The use according to claim 1, wherein the reagent for detecting TRIM21 is a quantitative detection reagent.
4. The use according to claim 1, wherein the method for using the nasopharyngeal carcinoma radiotherapy efficacy prediction product is: taking a sample of a subject, quantitatively detecting TRIM21 in the sample by using a nasopharyngeal carcinoma radiotherapy efficacy prediction product, and if the expression level is obviously higher than that of a reference substance, the subject is insensitive to the nasopharyngeal carcinoma radiotherapy, and the nasopharyngeal carcinoma radiotherapy has no curative effect or has no obvious curative effect on the treatment; if the expression level is not significantly higher than the control, the subject is sensitive to the nasopharyngeal carcinoma radiotherapy, and the nasopharyngeal carcinoma radiotherapy has an effect on the treatment.
5. The use according to claim 1, wherein the predictive product of the efficacy of radiation therapy for nasopharyngeal carcinoma is based on immunohistochemical combined microscopy, the IRS score is obtained by the obtained staining intensity and positive cell proportion, and the IRS score is 0-6, which indicates low expression, and the patient has no or insignificant efficacy of radiation therapy for nasopharyngeal carcinoma; the IRS score of 0-6 indicates high expression, and the patient has an effect on the treatment of nasopharyngeal carcinoma radiotherapy.
6. The use according to claim 4, wherein the control is a healthy person or a sample of a patient suffering from nasopharyngeal carcinoma without recurrence, distant metastasis and death of nasopharyngeal carcinoma.
7. The use of claim 6, wherein the sample comprises a body fluid or tissue sample; the tissue sample is a tumor tissue sample.
8. The nasopharyngeal carcinoma radiotherapy curative effect prediction system is characterized by comprising a sampling module, a TRIM21 detection module and an analysis module.
9. The radiation therapy efficacy prediction system for nasopharyngeal carcinoma according to claim 8, wherein said TRIM21 detection module is configured to detect TRIM21 content in the sample.
10. The system for predicting the efficacy of radiation therapy for nasopharyngeal carcinoma according to claim 8, wherein said analysis module is configured to compare the differences in TRIM21 content between different samples and output an analysis result based on the differences.
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| CN116904586A (en) * | 2023-09-12 | 2023-10-20 | 上海益诺思生物技术股份有限公司 | Application of reagent for detecting plasma-derived exosome lncRNA in preparation of diagnostic reagent for detecting kidney injury |
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| CN116904586B (en) * | 2023-09-12 | 2023-12-22 | 上海益诺思生物技术股份有限公司 | Application of reagent for detecting plasma-derived exosome lncRNA in preparation of diagnostic reagent for detecting kidney injury |
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