CN115128271A - Application of interferon gamma-induced lysosomal thiol reductase in breast cancer diagnosis and prognosis evaluation - Google Patents
Application of interferon gamma-induced lysosomal thiol reductase in breast cancer diagnosis and prognosis evaluation Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and medicine, and relates to application of interferon gamma-induced lysosomal thiol reductase in breast cancer diagnosis and prognosis evaluation. Specifically, the invention discloses application of interferon gamma-induced lysosomal thiol reductase as a biomarker for breast cancer diagnosis and prognosis evaluation. The serum concentration of the marker of the invention in breast cancer patients is obviously higher than that of normal people, and the protein expression level of the marker in breast cancer patients is obviously higher than that of normal tissues. The marker of the invention is closely related to the survival of breast cancer patients, namely, the survival rate of breast cancer patients with high expression of interferon gamma-induced lysosomal thiol reductase is low, and the survival rate of breast cancer patients with low expression of interferon gamma-induced lysosomal thiol reductase is high. Therefore, the invention provides a novel marker for the diagnosis and the prognosis evaluation of the breast cancer.
Description
Technical Field
The invention belongs to the field of biotechnology and medicine, and particularly relates to a tumor biomarker and application thereof in preparation of breast cancer diagnosis and prognosis judgment.
Background
According to the latest cancer data published by the world health organization international cancer research institution in 2020 worldwide, the number of female breast cancer diseases exceeds that of lung cancer for the first time in 2020, and the breast cancer becomes the most common cancer in the world, which accounts for about 11.7% of new cancer cases. In recent years, the worldwide morbidity and mortality of breast cancer is continuously increased, the breast cancer is one of the diseases with the highest worldwide incidence of female cancer, and is also one of the main causes of cancer-related deaths of women at home and abroad, and the incidence of breast cancer gradually shows a low-age trend in recent years. Currently, the immunohistochemical method is clinically used, and breast cancers are mainly classified into luminal breast cancers, human epidermal growth factor receptor 2 positive breast cancers and triple negative breast cancers according to detection results of estrogen receptors, progestogen receptors and human epidermal growth factor receptor-2 of breast cancer patients. The patient with triple negative breast cancer lacks the expression of the three receptors, has the worst prognosis, is easy to generate metastasis, has a five-year survival rate lower than 5 percent, and is the most troublesome breast cancer subtype. At present, the breast cancer is mainly diagnosed through clinical manifestations of patients, breast molybdenum target X-ray films and breast color ultrasonography, or fine needle puncture cytology, breast biopsy and the like, the compliance of the patients is poor, and the patients are mostly found to be in a late stage, so that the patients lose good treatment opportunities. Prevention and treatment of breast cancer still has a large number of problems to be solved urgently, including how to discover earlier, intervene in time, how to realize curative effect evaluation and monitoring, how to achieve real-time accurate relapse monitoring to postoperative patient, is the problem to be researched urgently. Therefore, the development of a breast cancer diagnosis product with high sensitivity, strong specificity and convenience is one of the keys for improving the early detection rate of breast cancer and improving the prognosis of breast cancer patients.
Interferon gamma-induced lysosomal thiol reductase (gamma-interferon-lyso-thiol reductase) is the only known thiol reductase localized in lysosomes and phagocytes at present, has the optimum pH of 4.5-5.5, and is the only enzyme in the family which acts in an acidic environment. Interferon gamma induces lysosomal thiol reductase constitutively expressed in professional antigen presenting cells, and can be induced to express or up-regulated by some inflammatory factors (such as interleukin 1 beta, tumor necrosis factor alpha, interferon gamma, etc.) in other cells. The primary function of interferon gamma-induced lysosomal thiol reductases is to catalyze the reduction of protein disulfide bonds. On the one hand, interferon gamma-induced lysosomal thiol reductase can promote the presentation of MHC-class II molecule-restricted antigens, thereby affecting the activation of CD4+ T cells; on the other hand, the CD8+ T cells are promoted to recognize MHC-I molecule restricted antigens and participate in the anti-virus and tumor immune response reaction of the organism. There is increasing evidence that interferon gamma induces lysosomal thiol reductases to participate in the tumor immune response process, but its role and mechanism in breast cancer is unclear.
To date, there is no report of high expression of interferon gamma-induced lysosomal thiol reductase in breast cancer, and there is no report of interferon gamma-induced lysosomal thiol reductase as a marker for diagnosis and prognostic evaluation of breast cancer.
Disclosure of Invention
One of the purposes of the invention is to prove the application of interferon gamma-induced lysosomal thiol reductase in high expression of breast cancer and the application of interferon gamma-induced lysosomal thiol reductase as a breast cancer biomarker, and the application improves a new idea for diagnosis of breast cancer.
The second purpose of the invention is to prove that the expression of the interferon gamma-induced lysosomal thiol reductase is closely related to the prognosis of breast cancer patients, and the interferon gamma-induced lysosomal thiol reductase can be used as a biomarker for prognosis judgment of breast cancer patients.
The technical scheme of the invention provides application of interferon gamma-induced lysosome thiol reductase (gamma-interferon-induced lysosome thiol reductase, abbreviated as interferon gamma-induced lysosome thiol reductase) as a breast cancer diagnosis marker or breast cancer prognosis judgment marker.
The serum concentration of the interferon gamma-induced lysosomal thiol reductase in breast cancer patients is obviously higher than that of normal people.
The tissue chip immunohistochemical result shows that the protein expression of the interferon gamma-induced lysosome thiol reductase in the tissues of breast cancer patients is obviously higher than that of paracancerous and normal tissues.
The expression of the interferon gamma induced lysosome thiol reductase in the breast cancer tissues is closely related to the survival of the breast cancer. The survival rate of patients with high interferon gamma-induced lysosomal thiol reductase expression is low, and the survival rate of patients with low interferon gamma-induced lysosomal thiol reductase expression is high.
Interferon gamma-induced lysosomal thiol-reductase is a biomarker for the diagnosis and prognosis of breast cancer.
The interferon gamma-induced lysosome thiol reductase is used as a biomarker in the preparation of a breast cancer diagnosis and prognosis evaluation kit.
The invention has the beneficial technical effects that the invention provides a new biomarker for the diagnosis and prognosis evaluation of breast cancer.
Drawings
FIG. 1 is a standard curve equation and correlation coefficient R for the detection of interferon gamma-induced lysosomal thiol reductase in breast cancer patients and normal controls.
Figure 2 is a graph of the concentration of interferon gamma-induced lysosomal thiol reductase in 34 patients of triple negative breast cancer, 21 patients of non-triple negative breast cancer and 18 normal controls in the Beijing cooperative Hospital.
FIG. 3 shows the expression of human interferon gamma-induced lysosomal thiol reductase in breast cancer tissue and paracancerous tissue in breast cancer tissue chips.
FIG. 4 is a graph of the relationship between survival of breast cancer patients and interferon gamma-induced lysosomal thiol reductase expression.
The specific implementation mode is as follows:
example 1 analysis of the content of interferon gamma-induced lysosomal thiol reductase in patients with breast cancer and in normal control sera by Using the interferon gamma-induced lysosomal thiol reductase kit
1.1 name of interferon gamma-induced lysosomal thiol reductase kit: human interferon gamma induction lysosome sulfhydryl reductase enzyme-linked immunosorbent assay kit. Shopping deviceFrom
Company (wuxi, Jiangsu, China), cargo number: DL-IFI30-Hu 96T
1.2 interferon gamma induced lysosome thiol reductase kit experimental principle: the kit adopts a double-antibody sandwich ELISA method. The antibody of anti-human interferon gamma induced lysosome sulfhydryl reductase is coated on the enzyme label plate, the human interferon gamma induced lysosome sulfhydryl reductase in the sample or the standard substance can be combined with the coated antibody during the experiment, and the free component is washed away. Biotinylated anti-human interferon gamma induced lysosome thiol reductase antibody and horseradish peroxidase labeled avidin are added in sequence. The anti-human interferon gamma induced lysosome thiol reductase antibody is combined with the human interferon gamma induced lysosome thiol reductase antibody combined on the coating antibody, biotin is combined with avidin specifically to form immune complex, and free components are washed away. Adding chromogenic substrate (TMB), wherein the TMB is blue under the catalysis of horseradish peroxidase, and becomes yellow after adding stop solution. Measuring OD value at 450nm wavelength by using an enzyme labeling instrument, wherein the concentration of interferon gamma induced lysosome thiol reductase is in direct proportion to the OD450 value, and calculating the concentration of interferon gamma induced lysosome thiol reductase in the sample by drawing a standard curve.
1.3 kit composition storage and other reagents and instruments required for test
The kit comprises the following components in percentage by weight: ELISA Plate (Detachable) Micro ELISA Plate (removable) 8 wells × 12 strips, lyophilized Standard Reference Standard 2-20 ℃, can be stored for 6 months, the Biotinylated antibody (100X) Concentrated Biotinylated Detection Ab 1 is 120. mu.L, the HRP enzyme Conjugate (100X) Concentrated HRP Conjugate 1 is 120. mu.L-20 deg.C (protected from light), can be stored for 6 months, 20mL of a 1 bottle of Standard & Sample Diluent, 4 ℃, the medium can be stored for 6 months, and is 14mL in a Biotinylated antibody Diluent Biotinylated Detection Ab Diluent 1 bottle, 14mL in an enzyme Conjugate Diluent HRP Conjugate Diluent 1 bottle, 30mL in a Concentrated washing Solution (25X) centralized Wash Buffer (25X) 1 bottle, 10mL 4 ℃ in a Substrate Solution (TMB) Substrate Reagent 1 bottle (protected from light), 10mL 4 ℃ in a reaction terminator Solution Stop Solution 1 bottle, and 5 plates covered with a Plate Sealer.
Other reagents and instruments required for the assay: microplate reader (450nm wavelength filter), high precision pipettor, EP tube and disposable tip: 0.5-10 μ L,2-20 μ L,20-200 μ L, 200-.
1.4 sample Collection method
The samples used were serum: the whole blood sample is placed at room temperature for 2 hours or at 4 ℃ overnight and then centrifuged at 1000 Xg for 20 minutes, and the supernatant is taken for detection, and the test tube for collecting blood is a disposable endotoxin-free test tube.
1.5 preparation work before detection:
1.5.1 please remove the kit from the refrigerator 20 minutes earlier and equilibrate to room temperature. The microplate reader is turned on for preheating 15 minutes before reading.
1.5.2 washing solution: the concentrated washings were diluted with double distilled water (1: 24).
And (4) prompting: the concentrated washing liquid taken out from the refrigerator may have crystallization, which is a normal phenomenon, and the washing liquid can be prepared after the crystallization is completely dissolved by micro-heating in water bath at 40 ℃. It is used on the same day.
1.5.3 working solution of standard substance the standard substance is centrifuged at 10000 Xg for 1 minute, 1.0mL of the diluent of the standard substance and the sample is added into the freeze-dried standard substance, a tube cover is screwed, the freeze-dried standard substance is kept stand for 10 minutes, the upper part and the lower part are reversed for a plurality of times, after the diluent is fully dissolved, the mixture is gently mixed, and the working solution of the standard substance with the concentration of 600ng/mL is prepared. Then, the dilution is carried out in multiple ratios as required. The following concentrations are suggested to be formulated: 600,300,150,75,37.5,18.75,9.38,0 ng/mL. The multiple ratio dilution method comprises the following steps: and taking 7 EP tubes, adding 500 mu L of standard substance and sample diluent into each tube, sucking 500 mu L of standard substance working solution from 600ng/mL into one of the EP tubes, uniformly mixing to prepare 300ng/mL of standard substance working solution, and sequentially sucking and uniformly mixing according to the steps. As follows. And (4) prompting: the last tube acts directly as a blank well, eliminating the need to aspirate liquid from the penultimate tube.
1.5.4 biotinylated antibody working solution: the amount required for the experiment (calculated as 100. mu.L/well) is calculated before the experiment, and 100-200. mu.L should be prepared in actual preparation. The 100 x concentrated biotinylated antibody was diluted to 1x working concentration with biotinylated antibody dilution 15 minutes prior to use. It is used on the same day.
1.5.5 enzyme conjugate working solution: calculating the required dosage (calculated as 100. mu.L/well) of the experiment, 100. mu.L and 200. mu.L should be prepared in actual preparation. The 100 × concentrated HRP enzyme conjugate was diluted to 1 × working concentration with enzyme conjugate diluent 15 minutes prior to use. It is used on the same day.
1.6 operating procedures
1.6.1. The standard working solution was added to the first two rows of wells in sequence, and two wells were added in parallel for each concentration of working solution, 100. mu.L per well. The sample to be tested is added to the other wells at a volume of 100. mu.L per well (if the sample concentration is higher than the detection range, the sample is diluted with the standard & sample diluent and then sampled). The microplate was coated and incubated at 37 ℃ for 90 minutes.
And (4) prompting: and adding a sample to the bottom of the ELISA plate during sample adding, keeping the sample from touching the hole wall as much as possible, slightly shaking and uniformly mixing the sample and the ELISA plate, and avoiding generating bubbles. The sample loading time is controlled within 10 minutes.
1.6.2. The liquid was discarded and spin-dried without washing. 100 mu L of biotinylated antibody working solution is added into each well, mixed evenly, coated on an enzyme label plate, and incubated for 1 hour at 37 ℃.
1.6.3. And throwing off liquid in the holes, adding 350 mu L of washing liquid into each hole, soaking for 1-2 minutes, sucking or throwing off the liquid in the ELISA plate, and patting dry on thick absorbent paper. This plate washing step was repeated 3 times.
And (4) prompting: the plate washer can be used here in combination with other plate washing steps.
1.6.4. Add 100. mu.L of the enzyme conjugate working solution to each well, add the membrane, and incubate at 37 ℃ for 30 minutes.
1.6.5. And (4) discarding liquid in the holes, spin-drying, and washing the plate for 5 times, wherein the method is the same as the step 3.
1.6.6. mu.L of substrate solution (TMB) was added to each well, and the microplate was covered with a film and incubated at 37 ℃ for about 15 minutes in the dark.
And (4) prompting: the color development can be shortened or prolonged as appropriate according to the actual color development condition, but can not exceed 30 minutes. The standard wells can be terminated when a significant gradient occurs.
1.6.7. The reaction was stopped by adding 50. mu.L of stop buffer to each well.
And (4) prompting: the order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution.
1.6.8. The optical density (OD value) of each well was immediately measured at a wavelength of 450nm with a microplate reader.
1.7 analysis of results
1.7.1 calculate the average OD value of each group of multiple wells. The mean OD value of each standard minus the OD value of the blank wells was used as the correction value. And (3) drawing a standard curve of a four-parameter logic function (removing blank group values in drawing) on the log-log graph paper by taking the concentration as an abscissa and the OD value as an ordinate.
1.7.2 if the OD of the sample is higher than the upper limit of the standard curve, it should be re-measured after appropriate dilution.
1.8. Results of the experiment
1.8.1 obtaining the standard curve equation y of interferon gamma induced lysosome sulfhydryl reductase 0.988x 2 0.3731x +0.4108 and correlation coefficient R0.9939 (concentration on ordinate and OD on abscissa), see fig. 1. The concentrations of interferon gamma-induced lysosomal thiol reductase in breast cancer patients and normal control serum were calculated by substituting the respective measured OD values into an equation, as shown in fig. 2.
Example 2 immunohistochemical techniques detection of expression of interferon gamma-induced lysosomal thiol reductase on the breast cancer tissue chip
2.1 tissue chips were purchased from Shanghai core Biotechnology, Inc. (Shanghai, China).
The breast cancer tissue chips used have the product numbers of HBred020PG01 (point 20) and HBred090PG01 (point 90). The chip is a breast cancer and marginal tissue combined chip, and contains TNM, clinical staging and pathological grading information.
2.2 immunohistochemistry interferon gamma-inducible lysosomal thiol reductase antibodies were purchased from interferon gamma-inducible lysosomal thiol reductase polyclonal antibody of Invitrogen, USA (Cat # PA 5-21533).
2.3 additional reagents required for immunohistochemistry
Xylene, 30% H 2 O 2 DAB developer (DAKO Co.), PBS, TBST, absolute alcohol, tableting agent, envision (DAKO).
2.4 immunohistochemical Experimental procedure:
2.4.1 Breast cancer chips were routinely dewaxed to water.
2.4.23% H2O2 deionized water (colorless liquid) was incubated for 10-30 minutes to inactivate endogenous peroxidase activity.
2.4.3 washing with distilled water, soaking in PBS for 5 min
2.4.4 repair with antigen: microwave (4 medium fires recommended within 30 minutes), high pressure, enzymatic remediation methods. Naturally cooled, and then 3 minutes × 3 times.
2.4.5 serum blocking: the temperature is 15-30 minutes at room temperature, and the source of the secondary antibody is consistent as much as possible. Pour out, don't wash.
2.4.6 drop-wise addition of primary antibody diluted in a suitable ratio, and incubation at 37 ℃ for 2-3 hours or overnight at 4 ℃ (preferably rewarming). PBS wash, 3 min X5 times.
2.4.7 Biotin-labeled secondary antibodies were added dropwise and incubated at room temperature or 37 ℃ for 30 minutes to 1 hour.
2.4.8 washed with PBS 3 min. times.5 times.
2.4.9 SP (streptavidin-peroxidase) was added dropwise and incubated for 30 min-1 h at room temperature or 37 ℃.
2.4.10 PBS wash, 3 minutes X5 times.
2.4.11 color developing agent develops color (DAB, etc.).
2.4.12 fully washed with tap water.
2.4.13 can be used for counter dyeing, dewatering and transparentizing.
2.4.14 select the appropriate mounting agent.
2.5 results of the experiment.
2.5.1 chips immunohistochemistry for breast cancer tissue results showed that interferon gamma-induced lysosomal thiol reductase expression was higher in breast cancer tissues than in normal breast tissues, and interferon gamma-induced lysosomal thiol reductase expression was higher in patients with higher pathological grade (see FIG. 3).
2.5.2 according to the expression information of interferon gamma-induced lysosomal thiol reductase of TCGA breast cancer patients and the clinical information analysis of patients, the survival rate of interferon gamma-induced lysosomal thiol reductase of breast cancer patients is high, and the survival rate of interferon gamma-induced lysosomal thiol reductase of breast cancer patients is high (see figure 4).
Claims (7)
1. The interferon gamma-induced lysosome thiol reductase is applied to being used as a biomarker for breast cancer diagnosis and prognosis evaluation.
2. The use according to claim 1, wherein said interferon gamma-induced lysosomal thiol reductase is present in a patient with breast cancer at a significantly higher serum level than in a normal human.
3. The use according to claim 1, wherein said interferon gamma induces the expression of a protein of a lysosomal thiol reductase in tissues of a breast cancer patient significantly higher than in paracancerous and normal breast tissues.
4. The use according to claim 1, wherein the expression of interferon gamma-induced lysosomal thiol reductase in breast cancer tissue is closely associated with survival of breast cancer patients.
5. The use according to claim 4, wherein the survival rate of breast cancer patients with high interferon gamma-induced lysosomal thiol reductase expression is low and the survival rate of breast cancer patients with low interferon gamma-induced lysosomal thiol reductase expression is high.
6. The use according to claim 1, characterized in that interferon gamma-induced lysosomal thiol-reductase is a biomarker for the diagnosis of breast cancer and for the prognostic evaluation of breast cancer.
7. The interferon gamma-induced lysosome thiol reductase is used as a biomarker in the preparation of a breast cancer diagnosis and prognosis evaluation kit.
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