Application of methylation modification of YAP protein at arginine 124 site in individualized diagnosis and treatment of tumor

Abstract

The application relates to the technical field of medical biological detection, and provides application of YAP protein asymmetric dimethylation modification at an arginine 124 site (YAP-R124me2a) in preparation of a tumor early diagnosis, prognosis evaluation or drug selection reagent or kit, wherein the methylation modification is asymmetric dimethylation modification, and the tumor is a YAP-related malignant solid tumor; further provided are a tumor early diagnosis, prognosis evaluation and drug selection kit and an evaluation system. The modification level of YAP-R124me2a in tumor tissue is determined by using an immunohistochemical technology, microscope photographing and computer image processing software, and combined with postoperative follow-up information, the correlation between the expression amount of YAP-R124me2a and the prognosis of tumor patients after surgery is determined, YAP-R124me2a can be used as a protein molecular marker for early diagnosis of liver cancer, lung adenocarcinoma, gastric cancer and colon cancer, judgment of postoperative prognosis and drug selection, and has important guiding significance for postoperative monitoring and individualized sequential treatment of tumor patients.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical biological detection, and relates to application of YAP protein arginine 124 site methylation modification in individualized diagnosis and treatment of tumors, in particular to application of YAP protein arginine 124 site asymmetric methylation modification in early diagnosis, prognosis evaluation and drug selection of tumors. BACKGROUND

[0002] As a major disease, the morbidity and mortality of tumors have been high for a long time. The pathogenic mechanism of tumors is very complex, and is the result of multiple pathways, multiple factors and multiple steps long-term effects. Most of the tumor patients in China are in the middle and late stages when they are found, and the overall treatment effect of tumors is not optimistic. Early diagnosis and postoperative prognosis evaluation are crucial for tumor prevention and treatment. In addition, the progress of chemotherapy and targeted drugs for middle and late stage tumors is changing with each passing day, and individualized drug selection significantly affects the effect of later tumor drug treatment. For a long time, finding a tumor marker that can be applied to early diagnosis, prognosis evaluation and drug selection has become one of the focuses of tumor research.

[0003] The Hippo signaling pathway is one of the signaling pathways newly found to be closely related to the occurrence and development of tumors. The activation of the Hippo signaling pathway caused by various exogenous factors leads to the continuous enhancement of the activity of the downstream effector YAP, and participates in the occurrence and development of tumors. Abnormal activity of YAP protein is related to various tumors, such as liver cancer, gastric cancer, breast cancer, intestinal cancer and lung adenocarcinoma. Post-translational modification of amino acids is considered to be an important mechanism for regulating the activity and function of YAP. We first found that the arginine 124 site of YAP is methylated by protein mass spectrometry, synthesized a methylated polypeptide as an antigen, prepared an antibody capable of specifically recognizing YAP-R124 asymmetric dimethylation (R124me2a), and applied it to the detection of various adenocarcinoma tissues including liver cancer and drug treatment related liver cancer tissues. It has important significance for early diagnosis, prognosis analysis and individualized drug selection of related tumors. No related report has been found so far. SUMMARY

[0004] The application aims to provide a tumor marker applied to individualized diagnosis and treatment, and a new use of YAP protein arginine 124 site methylation modification, in particular, application in early diagnosis and prognosis evaluation of tumors and in tumor drug selection. In addition, a tumor early diagnosis, prognosis evaluation and drug selection evaluation system is also provided, which improves the accuracy of early diagnosis and prognosis evaluation of tumors, and in particular, improves the curative effect of individualized drug treatment of tumors.

[0005] The inventors have found that PRMT1 inhibitors significantly inhibit the activity of YAP protein through extensive and in-depth research, and found that YAP protein can be directly combined with PRMT1 protein through immunoprecipitation and Biacore technology. It is found through mass spectrometry technology that the arginine at position 124 of the YAP protein is methylated, and a related antigen polypeptide is synthesized and coupled with a carrier protein keyhole limpet hemocyanin to form an antigen and immunize a host animal. A specific affinity purification method is used to obtain a polyclonal antibody that specifically recognizes two R124 methylation modifications, namely monomethylation and asymmetric dimethylation antibodies.

[0006] Through protein immunoprecipitation, the two purified R124 methylation modification polyclonal antibodies are used to detect the modification level of the above two methylation modifications in human liver cancer tissues, and it is found that R124 in human liver cancer tissues only exists in asymmetric dimethylation modification. At the same time, experiments show that the YAP-R124 asymmetric dimethylation modification presents a specific high expression phenomenon in liver cancer, gastric cancer, lung adenocarcinoma and intestinal cancer, which is significantly higher than that in the adjacent tissues of the cancer, and the R124me2a modification is negatively correlated with the prognosis of tumor patients. Therefore, YAP-R124me2a modification can be used as a marker for tumor diagnosis and prognosis analysis.

[0007] In addition, in vitro experiments use the antibody to verify that drug treatment of various tumors including liver cancer, lung cancer, gastric cancer and colon adenocarcinoma, YAP-R124me2a modification is significantly increased, and inhibition of YAP-R124me2a modification can significantly increase the sensitivity of drug treatment of tumors; in vivo experiments use sorafenib combined with small molecule drug MS023 that inhibits YAP-R124me2a modification, which can significantly increase the sensitivity of sorafenib treatment. The antibody is used to detect 101 cases of liver cancer tissues of patients using sorafenib after surgery and 95 cases of liver cancer tissues of patients not using sorafenib after surgery, and the median of the Image Scope score of YAP-R124me2a expression is 0.0528. When higher than 0.0528, it is considered that YAP-R124me2a is highly expressed, and lower than 0.0528 is YAP-R124me2a low expression. In the high expression group, the survival period of patients using sorafenib is not significantly prolonged; in the low expression group, the survival period of patients using sorafenib is significantly prolonged. Therefore, YAP-R124me2a modification can be used as a molecular marker to guide the application of chemotherapy and targeted drugs in tumor patients.

[0008] In a first aspect of the present application, the use of YAP protein arginine 124 site methylation modification in the preparation of a tumor early diagnosis and prognosis evaluation reagent or kit is provided.

[0009] In a second aspect of the present application, the use of YAP protein arginine 124 site methylation modification in the preparation of a tumor drug selection reagent or kit is provided.

[0010] In the above two applications, the methylation modification is asymmetric dimethylation modification; the tumor is a YAP-related malignant solid tumor, including lung cancer, breast cancer, gastric cancer, intestinal cancer, liver cancer, ovarian cancer, and pancreatic cancer.

[0011] Preferably, the reagent is a reagent for detecting the level of asymmetric methylation modification of arginine 124 of YAP protein in a biological sample; the kit comprises a reagent for detecting the level of asymmetric methylation modification of arginine 124 of YAP protein in a biological sample.

[0012] Further preferably, the reagent for detecting the level of asymmetric methylation modification of arginine 124 of YAP protein in a biological sample is an immunohistochemical detection reagent comprising a specific antibody (anti-YAP-R124me2a) that recognizes the asymmetric methylation modification form of YAP-R124.

[0013] The 124th arginine modification site of YAP protein and the related sequence are highly conserved in mammals, such as in humans, mice, and rabbits, and the sequence is the same, only the position is different, and different antibodies can be used to detect the level of asymmetric methylation modification of arginine 124 of human YAP protein.

[0014] The antibody preparation method used in the application is as follows: the artificially synthesized R124 asymmetric methylation modification polypeptide is injected into an animal (such as a rabbit), and then the specific antibody anti-YAP-R124me2a is obtained by separation and extraction from the serum.

[0015] Preferably, the biological sample is selected from any one of surgical tumor tissue, puncture-obtained tumor tissue, or circulating tumor cells collected from patient blood. For early diagnosis and tumor patients who have lost the opportunity for surgery, the expression level of YAP-R124me2a can be detected by using puncture-obtained cancer tissue, or the level of YAP-R124me2a can be detected by collecting circulating tumor cells from patient blood, thereby achieving early diagnosis and prognosis evaluation.

[0016] In a third aspect of the application, a tumor early diagnosis and prognosis evaluation kit is provided, which is composed of a tumor tissue paraffin section preparation reagent system, an antigen repair reagent system, and an antibody system. The antibody system comprises a specific antibody that recognizes the asymmetric dimethylation modification form of YAP-R124, and the tumor is a YAP-related malignant solid tumor.

[0017] In a fourth aspect of the present application, a tumor drug screening kit is provided, which is composed of a tumor tissue paraffin section preparation reagent system, an antigen repair reagent system and an antibody system. The antibody system comprises a specific antibody for recognizing the asymmetric dimethylation modified form of YAP-R124, and the tumor is a YAP-related malignant solid tumor.

[0018] In a fifth aspect of the present application, a tumor early diagnosis, prognosis evaluation or drug screening evaluation system is provided, which comprises a tumor early diagnosis and prognosis evaluation kit or a tumor drug screening kit and an immunohistochemical scoring module installed on a terminal carrier.

[0019] The tumor early diagnosis and prognosis evaluation kit and the tumor drug screening kit are as described above, respectively.

[0020] The immunohistochemical scoring module is based on the different field photos randomly selected from the tumor tissue and the paracancerous tissue under a microscope, and the immunohistochemical score is calculated according to the following formula, and the calculation result is compared with the preset median:

[0021] The immunohistochemical score = Log10[255 / Iavg],

[0022] Wherein, Iavg = (Iwp + Ip + Isp) / (Nwp + Np + Nsp), that is, the average intensity = (total intensity of weak positive pixels + total intensity of positive pixels + total intensity of strong positive pixels) / (number of weak positive pixels + number of positive pixels + number of strong positive pixels).

[0023] In the tumor early diagnosis and prognosis evaluation system, the medians of different cancer tissues are different, and for the same tissue, the medians of different detection methods are also different. In the specific embodiment of the present application, the immunohistochemical method is used for detection, the median of liver cancer tissue is 0.0269, the median of gastric cancer tissue is 0.02058, the median of lung adenocarcinoma tissue is 0.1192, and the median of intestinal cancer tissue is 0.04274. Taking the median of the YAP-R124me2a expression score as the boundary, if the Image Scope score is higher than the median, it is determined that the YAP-R124me2a protein is highly expressed, and if it is lower than the median, it is determined that the YAP-R124me2a protein is lowly expressed. The expression level of YAP-R124 methylation modification is negatively correlated with the prognosis of the above four tumors.

[0024] In the drug selection evaluation system, in order to realize quantitative evaluation, taking liver cancer and sorafenib as an example, the modification level of YAP-R124me2a in tumor tissue is determined by using immunohistochemical technology, microscope photographing and computer image processing software. Taking the median (0.0528) of YAP-R124me2a expression score as the boundary, the liver cancer patients are grouped, the patients with immunohistochemical score higher than 0.0528 are grouped into the high expression group, and the patients with immunohistochemical score lower than 0.0528 are grouped into the low expression group. Combined with the medication and postoperative follow-up information, it is found that the survival of YAP-R124me2a low expression patients in the medication group is obviously better than that of the low expression patients in the non-medication group, and the survival analysis of the two groups has statistical difference (P=0.024), which indicates that the YAP-R124me2a low expression patients have better prognosis after taking sorafenib; the survival period of YAP-R124me2a high expression patients in the medication group is not statistically different from that of the high expression patients in the non-medication group, which indicates that whether the YAP-R124me2a high expression patients take sorafenib or not does not affect the prognosis of the patients.

[0025] The beneficial guarantees and effects of the present application are as follows:

[0026] In terms of technology, the detection of the modification level of YAP-R124me2a is essentially the immunohistochemical detection of biological samples, which is mature in technology and has the characteristics of simple operation, sensitive detection, good specificity, high repeatability and the like, and is now increasingly applied in clinical examination technology.

[0027] The present application is based on the fact that YAP is known to be abnormally expressed and active in various malignant solid tumors, and it is found that R124 methylation modification regulates the continuous enhancement of YAP activity, and is highly expressed in liver cancer, lung adenocarcinoma, gastric cancer and colon cancer, and shows a negative correlation with the prognosis of patients. The modification level of YAP-R124me2a in tumor tissue is determined by using immunohistochemical technology, microscope photographing and computer image processing software, and combined with postoperative follow-up information, it is determined that the expression amount of YAP-R124me2a is related to the prognosis of tumor patients after surgery, and YAP-R124me2a can be used as a protein molecular marker for early diagnosis of liver cancer, lung adenocarcinoma, gastric cancer and colon cancer, judgment of postoperative prognosis and drug selection, which has important guiding significance for postoperative monitoring and sequential individualized treatment of tumor patients.

[0028] Therefore, the present application provides a detection method with good development prospect, a tumor marker for representing tumor diagnosis and patient prognosis analysis, and a modified antibody for specific detection, which provides an important reference basis for clinical diagnosis and treatment scheme. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 YAP-R124 is found by protein spectrum technology;

[0030] Figure 2 Conservation analysis of YAP-R124 site in different species;

[0031] Figure 3 Verification of rabbit-derived antibodies specifically recognizing different methylation modifications of YAP-R124, wherein WT: unmodified polypeptide; R124K: R124K mutant polypeptide; R124A: R124A mutant polypeptide; MMA: R124 monomethylation modified polypeptide; ADMA: R124 asymmetric dimethylation modified polypeptide; SDMA: R124 symmetric dimethylation modified polypeptide;

[0032] Figure 4 Detection of YAP-R124 methylation modification present in human liver cancer tissue using the above-mentioned antibodies

[0033] Figure 5 Analysis of the correlation between YAP-R124me2a methylation modification expression in liver cancer tissue and prognosis of tumor patients;

[0034] Figure 6 Analysis of the correlation between YAP-R124me2a methylation modification expression in lung adenocarcinoma tissue and prognosis of tumor patients;

[0035] Figure 7 Analysis of the correlation between YAP-R124me2a methylation modification expression in gastric cancer tissue and prognosis of tumor patients;

[0036] Figure 8 Analysis of the correlation between YAP-R124me2a methylation modification expression in colon cancer tissue and prognosis of tumor patients.

[0037] Figure 9 Effect of chemical or targeted drugs on the modification level of YAP-R124me2a in various tumor cells;

[0038] Figure 10 Effect of YAP-R124 mutation (R124K) on the sensitivity of chemical or targeted drugs in various tumor cells;

[0039] Figure 11 Representative images of immunohistochemical staining of YAP-R124me2a in liver cancer tissues of 196 patients treated by surgery (101 patients took sorafenib after surgery, and 95 patients did not take sorafenib after surgery), showing the expression level of YAP-R124me2a in tumor tissues.

[0040] Figure 12The survival analysis chart of YAP-R124me2a high expression group patients in the sorafenib non-use group and use group shows that whether taking sorafenib or not does not affect the prognosis of patients in the high expression group. DETAILED DESCRIPTION

[0041] The embodiments of the present application will be described in detail below with reference to the embodiments of the present application. The following embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0042] Example 1: YAP protein arginine 124 site methylation modification site is highly conserved in mammals

[0043] 2x10 6 Hep3B liver cancer cells overexpressing YAP and PRMT1 were inoculated in a 90 mm culture dish, YAP protein was separated by immunoprecipitation using a rabbit monoclonal YAP antibody (Cell Signaling Technology, #14704), and protein mass spectrometry was used to identify potential arginine methylation modification sites of YAP eukaryotic protein. The results show that the arginine modification site at position 124 of the YAP protein is methylated ( Figure 1 ), and this site is very conserved in mammals ( Figure 2 ).

[0044] Example 2: Antibodies for two methylation modification forms of YAP-R124

[0045] Using the synthesized methylated polypeptide as an antigen, antibodies (rabbit-derived) that recognize two methylation modification forms of YAP-R124 were prepared: anti-YAP-R124me1 and anti-YAP-R124me2a. Dot blot experiments verified that both antibodies can specifically recognize the corresponding modified polypeptides, and do not recognize other methylated modified polypeptides, non-methylated modified polypeptides or R124 mutant polypeptides ( Figure 3 ), and the relevant sensitivity and specificity meet the experimental requirements. The results show that the two methylation antibodies can be used to detect the potential methylation modification of YAP-R124 site in human liver cancer tissues or cells.

[0046] Experimental Example 3: Only YAP-R124me2a methylation modification exists in human liver cancer tissues

[0047] Six human liver cancer specimens were randomly selected, and 50 mg of frozen tissue samples were taken from each, YAP-R124me1 and YAP-R124me2a antibodies were used to detect the methylation modification of YAP-R124 site in the samples. Tissue Protein (ThermoFisher, 78510) was used to isolate proteins, and rabbit-derived YAP monoclonal antibody (Cell Signaling Technology, #14704) was used to immunoprecipitate YAP proteins. Western blotting was used to enrich YAP-R124me1 and YAP-R124me2a as primary antibodies to identify the potential methylation modification of YAP-R124 in human liver cancer samples. The experimental results show that only YAP-R124me2a methylation modification exists in human liver cancer tissue Figure 4

[0048] Experimental Example 4: YAP-R124me2a modification expression level is negatively correlated with liver cancer prognosis

[0049] Randomly selected 90 cases of liver cancer patients treated by surgery (HCAD, all from Shanghai Xichao Biotechnology Co., Ltd.), the expression level of YAP-R124me2a in liver cancer tissue was detected by immunohistochemical method and the immunohistochemical score was calculated. The modification of this site in liver cancer tissue is significantly higher than that in paracancerous tissue, and the expression level of YAP-R124me2a modification is negatively correlated with the prognosis of liver cancer Figure 5

[0050] Experimental Example 5: YAP-R124me2a modification expression level is negatively correlated with lung adenocarcinoma prognosis

[0051] Randomly selected 98 cases of lung adenocarcinoma patients treated by surgery (LUAD, all from Shanghai Xichao Biotechnology Co., Ltd.), the expression level of YAP-R124me2a in lung adenocarcinoma tissue was detected by immunohistochemical method and the immunohistochemical score was calculated. The modification of this site in lung adenocarcinoma tissue is significantly higher than that in paracancerous tissue, and the expression level of YAP-R124me2a modification is negatively correlated with the prognosis of lung adenocarcinoma Figure 6

[0052] Experimental Example 6: YAP-R124me2a modification expression level is negatively correlated with gastric cancer prognosis

[0053] Randomly selected 90 cases of gastric cancer patients treated by surgery (STAD, all from Shanghai Xichao Biotechnology Co., Ltd.), the expression level of YAP-R124me2a in gastric cancer tissue was detected by immunohistochemical method and the immunohistochemical score was calculated. The modification of this site in gastric cancer tissue is significantly higher than that in paracancerous tissue, and the expression level of YAP-R124me2a modification is negatively correlated with the prognosis of gastric cancer Figure 7

[0054] Experimental Example 7: YAP-R124me2a modification expression level is negatively correlated with colon cancer prognosis​​​​

[0055] Randomly selected 98 cases of surgical treatment of colon cancer patients (COAD, all from Shanghai Xianchao Biotechnology Co., Ltd.), the expression level of YAP-R124me2a in colon cancer tissue was detected by immunohistochemical method and the immunohistochemical score was calculated. The site modification of colon cancer tissue is significantly higher than that of the adjacent cancer tissue, and the expression level of YAP-R124me2a modification is negatively correlated with the prognosis of colon cancer Figure 8 ).

[0056] Example 8: Detection of YAP-R124me2a modification expression level and prognosis analysis of clinical samples

[0057] The method for detecting the expression of YAP-R124me2a in tumor tissue in vitro in this embodiment comprises the following steps:

[0058] (a) Using xylene, ethanol, 3% H2O2 solution, 1% BSA blocking solution, DAB color reagent, hematoxylin and horseradish peroxidase labeled goat anti-mouse IgG in the kit to perform immunohistochemical staining on the liver cancer tissue section;

[0059] (b) Using a microscope and an imaging device to take digital photos;

[0060] (c) Using biological image processing software to analyze the positive signal intensity in the tumor and give a score.

[0061] The specific steps are as follows:

[0062] (1) Prepare liver cancer tissue paraffin sections, and bake in a 60°C oven overnight;

[0063] (2) De-wax the section to water;

[0064] (Dimethylbenzene I ① 10 min→ Dimethylbenzene II ② 10 min→ Dimethylbenzene III ③ 10 min→ 100% ethanol 5 min→

[0065] 95% ethanol 5 min→ 85% ethanol 5 min→ 75% ethanol 5 min→ double distilled water 5 min)

[0066] (3) 3% H2O2 solution, room temperature for 20 min;

[0067] (4) Wash with double distilled water for 5 min x 3;

[0068] (5) Antigen repair: put the section into 0.01M citrate buffer (pH 6.0) and boil for 30 min;

[0069] (6) Naturally cool to room temperature, wash with double distilled water for 5 min x 3;

[0070] (7) 1% BSA blocking for 30 min, 37°C;

[0071] (8) Remove the blocking solution without washing, directly add the primary antibody (rabbit YAP-R124me2a polyclonal antibody, dilution ratio 1:1000). Put into the wet box and place in the 4°C refrigerator overnight for 16 hours;

[0072] (9) Take out at 4°C, re-warm at room temperature for 15 min, then wash with 0.01M PBS for 5 min x 4;

[0073] (10) Add the secondary antibody (horseradish peroxidase labeled goat anti-mouse IgG, purchased from DAKO Company in Denmark, ready-to-use, no need to dilute) dropwise for 45 min at 37°C;

[0074] (11) Wash with 0.01M PBS for 5 min x 4, develop with DAB for 2-10 min, and observe under the microscope;

[0075] (12) Stop the color development with double distilled water, and then stain with hematoxylin for 10 seconds;

[0076] (13) After differentiation, return to blue with running water, and then soak with distilled water;

[0077] (14) Dehydrate and transparentize, and then cover with a cover glass;

[0078] (15) Observe the positive staining under the microscope, and then randomly select 3 fields in the liver cancer tissue and take photos;

[0079] (16) Scan the tissue samples by using the Image Scope (Aperio Company) software, and then calculate the "positive Pixel" of each sample by using the Algorithms (Positive Pixel Count) program of the software after scanning, and obtain the calculation data as shown in Table 1:

[0080] Table 1 "Positive Pixel" calculation content

[0081]

[0082] The calculation method of the immunohistochemical score of each tissue is Log10[255 / Iavg], wherein Iavg=(Iwp+Ip+Isp) / (Nwp+Np+Nsp), that is, the average intensity, and the calculation method is: average intensity=(total intensity of weak positive pixels+total intensity of positive pixels+total intensity of strong positive pixels) / (number of weak positive pixels+number of positive pixels+number of strong positive pixels), that is, the immunohistochemical score of the tissue, which is used for subsequent analysis.

[0083] In the prognostic assessment of liver cancer, the discovery of the correlation between YAP-R124me2a modification and liver cancer provides a novel approach to predicting postoperative survival or death in hepatocellular carcinoma (HCC) patients. This discovery plays a crucial role in assessing the prognosis of HCC patients and offers significant guidance for postoperative monitoring and sequential treatment. In the prognostic assessment, the median YAP-R124me2a expression score (0.0269) in 90 HCC tissue samples was used as the cutoff. An Image Scope score above 0.0269 indicated high YAP-R124me2a protein expression, while a score below 0.0269 indicated low YAP-R124me2a protein expression. The results showed a significant negative correlation between YAP-R124me2a modification expression levels and liver cancer prognosis.

[0084] In the prognostic assessment of lung adenocarcinoma, the YAP-R124me2a expression level was determined using the median YAP-R124me2a expression score (0.1192) from 98 lung adenocarcinoma tissue samples. An Image Scope score above 0.1192 was considered high YAP-R124me2a protein expression, while a score below 0.1192 was considered low YAP-R124me2a protein expression. The results showed a significant negative correlation between YAP-R124me2a modification expression levels and lung adenocarcinoma prognosis.

[0085] In the prognostic assessment of gastric cancer, the criteria for high and low expression of YAP-R124me2a were based on the median YAP-R124me2a expression score (0.02058) in 90 gastric cancer tissue samples. An Image Scope score above 0.02058 was considered high expression of YAP-R124me2a protein, while a score below 0.02058 was considered low expression. The results showed a significant negative correlation between YAP-R124me2a modification expression levels and gastric cancer prognosis.

[0086] In the prognostic assessment of colorectal cancer, the YAP-R124me2a expression level was determined using the median YAP-R124me2a expression score (0.04274) from 98 colorectal cancer tissue samples. An Image Scope score above 0.04274 indicated high YAP-R124me2a protein expression, while a score below 0.04274 indicated low YAP-R124me2a protein expression. The results showed a significant negative correlation between YAP-R124me2a modification expression levels and colorectal cancer prognosis.

[0087] Experiment Example 9: Effect of drugs on the modification level of YAP-R124me2a in tumor cells

[0088] 2×10 6Huh-7 hepatocellular carcinoma cells were seeded in 90 mm dishes, and 1 μM sorafenib was added to the culture medium. Proteins were collected on days 1, 3, and 5. Using Western blotting, rabbit-derived YAP monoclonal antibody (CellSignaling Technology, #14704) and anti-YAP-R124me2a were added as primary antibodies to detect the levels of YAP protein and YAP-R124me2a in hepatocellular carcinoma cells after sorafenib treatment. The results showed that after sorafenib treatment of hepatocellular carcinoma cells, the total amount of YAP protein did not change significantly, but the level of YAP-R124me2a modification increased significantly. Figure 9 A).

[0089] 3×10 6 HCT 116 colon adenocarcinoma cells were seeded in 90 mm dishes, and 2.5 μM oxaliplatin (Oxa) was added to the culture medium. Proteins were collected on days 1, 3, and 5. Using Western blotting, rabbit-derived YAP monoclonal antibody (Cell Signaling Technology, #14704) and anti-YAP-R124me2a were added as primary antibodies to detect the levels of YAP protein and YAP-R124me2a in colon adenocarcinoma cells after oxaliplatin treatment. The results showed that oxaliplatin treatment significantly increased the levels of YAP protein and YAP-R124me2a modification in colon adenocarcinoma cells. Figure 9 B).

[0090] 2×10 6 SGC-7901 gastric adenocarcinoma cells were seeded in 90 mm dishes. 5 μM of 5-fluorouracil (5-FU) was added to the culture medium. Proteins were collected on days 1, 3, and 5. Using Western blotting, rabbit-derived YAP monoclonal antibody (Cell Signaling Technology, #14704) and anti-YAP-R124me2a were added as primary antibodies to detect the levels of YAP protein and YAP-R124me2a in gastric cancer cells after 5-fluorouracil treatment. Results showed that after 5-fluorouracil treatment of gastric cancer cells, the total amount of YAP protein did not change significantly, but the level of YAP-R124me2a modification increased significantly. Figure 9 C).

[0091] 2×10 6A549 lung adenocarcinoma cells were seeded in 90 mm dishes, and 2 uM of gemcitabine (Gem) was added to the culture medium. The proteins were collected on days 1, 3, and 5, respectively. Western blotting was used to detect the levels of YAP protein and YAP-R124me2a in lung cancer cells after gemcitabine treatment, with rabbit-derived YAP monoclonal antibody (Cell Signaling Technology, #14704) and anti-YAP-R124me2a as primary antibodies. The results showed that after treatment of lung cancer cells with gemcitabine, the total amount of YAP protein did not change significantly, but the level of YAP-R124me2a modification increased significantly Figure 9 D).

[0092] Experimental Example 10: Effect of YAP-R124 mutation on drug sensitivity of tumor cells

[0093] Huh-7 cells infected with CMV-GFP, CMV-YAP, and CMV-YAP-R124K lentivirus (MOI: 100) were seeded in 96-well plates at a density of 3 x 10 3 Each experimental group was treated with 2-fold dilution of sorafenib (0.024 uM to 100 uM). After 24 hours of treatment, 1:10 dilution of CCK-8 reagent was added. After 1 h of standing, the TECAN infinite F200 enzyme marker OD450 was read. According to the relevant data, the IC50 curve was drawn, and the IC50 value was calculated. The results showed that after mutation of the YAP-R124 site, the half-inhibition rate of sorafenib on HuH-7 cell proliferation was significantly improved Figure 10 A).

[0094] HCT 116 colon adenocarcinoma cells infected with CMV-GFP, CMV-YAP, and CMV-YAP-R124K lentivirus (MOI: 100) were seeded in 96-well plates at a density of 3.5 x 10 3 Each experimental group was treated with 2-fold dilution of oxaliplatin (0.076 uM to 100 uM). After 48 hours of treatment, 1:10 dilution of CCK-8 reagent was added. After 1 h of standing, the TECAN infinite F200 enzyme marker OD450 was read. According to the relevant data, the IC50 curve was drawn, and the IC50 value was calculated. The results showed that after mutation of the YAP-R124 site, the half-inhibition rate of oxaliplatin on HCT 116 cell proliferation was significantly improved Figure 10 B).

[0095] SGC-7901 gastric adenocarcinoma cells infected with CMV-GFP, CMV-YAP, and CMV-YAP-R124K lentivirus (MOI: 200) were seeded in 96-well plates at a density of 3 x 103 Cells were seeded at a density of 0.15 μM to 1000 μM in 96-well plates. Each experimental group was treated with serially diluted 5-FU (0.15 μM to 1000 μM). After 72 hours of treatment, CCK-8 reagent was diluted 1:10. After standing for 1 hour, the OD450 values ​​were read using a TECAN Infinite F200 microplate reader. IC50 curves were plotted based on the data, and IC50 values ​​were calculated. Results showed that the YAP-R124 mutation significantly increased the half-maximal inhibitory rate (WHM) of 5-FU on the proliferation of SGC-7901 cells. Figure 10 C).

[0096] A549 lung adenocarcinoma cells infected with CMV-GFP, CMV-YAP, and CMV-YAP-R124K lentiviruses (MOI:200) were divided into groups of 3 × 10⁻⁶ cells. 3 The cells were seeded at a density of 0.076 μM to 500 μM in 96-well plates. Each experimental group was treated with serially diluted gemcitabine (0.076 μM to 500 μM). After 72 hours of treatment, a 1:10 dilution of CCK-8 reagent was added. After standing for 1 hour, the OD450 values ​​were read using a TECAN Infinite F200 microplate reader. IC50 curves were plotted based on the data, and IC50 values ​​were calculated. The results showed that the YAP-R124 mutation significantly increased the half-maximal inhibitory rate (WHM) of gemcitabine against the proliferation of A549 lung adenocarcinoma cells. Figure 10 D).

[0097] Example 11: Analysis of liver cancer tissue specimens treated with sorafenib postoperatively

[0098] 196 patients with liver cancer who underwent surgery were randomly selected (101 patients received sorafenib postoperatively, and 95 patients did not receive sorafenib postoperatively; all patients were from the Eastern Hepatobiliary Surgery Hospital). The expression level of YAP-R124me2a in liver cancer tissue was detected by immunohistochemistry, and the immunohistochemical score was calculated. The specific steps are as follows:

[0099] (1) Prepare paraffin sections of liver cancer tissue and bake overnight at 60°C;

[0100] (2) Slice and dewax until wet;

[0101] (Xylene I ① 10 min → Xylene II ② 10 min → Xylene III ③ 10 min → 100% ethanol 5 min → 95% ethanol 5 min → 85% ethanol 5 min → 75% ethanol 5 min → Double distilled water 5 min)

[0102] (3) 3% H2O2 solution, left at room temperature for 20 minutes;

[0103] (4) Wash with double-distilled water for 5 min × 3;

[0104] (5) Antigen retrieval: The sections were placed in 0.01 M citrate buffer (pH 6.0) and boiled for 30 min;

[0105] (6) Natural cooling to room temperature, double distilled water wash 5 min x 3;

[0106] (7) 1% BSA blocking for 30 min, 37°C;

[0107] (8) Shake off the blocking solution, do not wash, directly add the primary antibody (1:200). Place in a wet box, 4°C refrigerator overnight for 16 hours;

[0108] (9) 4°C, remove, room temperature for 15 min, then 0.01 M PBS wash 5 min x 4;

[0109] (10) Drop the secondary antibody, 45 min, 37°C;

[0110] (11) 0.01 M PBS wash 5 min x 4, DAB color development 2-10 min, observe under the microscope;

[0111] (12) Double distilled water to stop color development, hematoxylin re-staining for 10 seconds;

[0112] (13) After differentiation, tap water returns blue, distilled water immersion;

[0113] (14) Dehydration, transparency, cover glass cover;

[0114] (15) Observe positive staining under the microscope, randomly select 3 fields in each of the hepatocellular carcinoma tissue and the adjacent tissue, and take pictures;

[0115] (16) Use the biological image processing software Image-Pro plus (Media Cybernetics, Inc.) to analyze the positive signal intensity of the tumor and the tumor-adjacent tissue, and give a score; the specific method is as follows: after scanning the tissue sample by using the Image Scope (Aperio company) software, the Algorithms (Positive Pixel Count) program of the software is used to calculate the "positive Pixel" of each sample,

[0116] Region

[0117] Length (μm)

[0118] Area (μm2)

[0119] Number of Weak Positive (Nwp)

[0120] Number of Positive Pixels (Np)

[0121] Number of Strong Positive Pixels (Nsp)

[0122] Total Intensity of Weak Positive (Iwp)

[0123] Total Intensity of Positive (Ip)

[0124] Total Intensity of Strong Positive (Isp)

[0125] Average Intensity Value of Positive (Iavg)

[0126] Average Intensity Value of Strong Positive (Nsr)

[0127] Average Intensity Value of Weak Positive (Iwavg)

[0128] Number of Negative Pixels (Nn)

[0129] Total Intensity of Negative (In)

[0130] Total Number of Color Development (NTotal)

[0131] Total Area of Color Development (ATotal)

[0132] Positivity (NPositive / NTotal)

[0133] The calculation method of the histochemical score of each tissue sample is Log10[255 / Iavg], wherein the calculation method of the histochemical score of each tissue is Log10[255 / Iavg], wherein Iavg=(Iwp+Ip+Isp) / (Nwp+Np+Nsp), i.e. the average intensity, the calculation method is: average intensity=(total intensity of weak positive pixels+total intensity of positive pixels+total intensity of strong positive pixels) / (number of weak positive pixels+number of positive pixels+number of strong positive pixels), i.e. the immunohistochemical score of the tissue, which is used for subsequent analysis. The median of the YAP-R124me2a expression score in 196 liver cancer tissues (0.0528) is used as the boundary to group liver cancer patients. Patients with ImageScope scores higher than 0.0528 are included in the high expression group; patients with Image Scope scores lower than 0.0528 are included in the low expression group. Among them, there are 99 cases in the low expression group and 97 cases in the high expression group.

[0134] Example 12: Analysis of liver cancer patients with low expression of YAP-R124me2a

[0135] Combined with the prognosis information, the survival analysis of 99 liver cancer patients with low expression of YAP-R124me2a (49 cases in the drug group and 50 cases in the non-drug group) was performed. Data analysis was performed using SPSS software 18.0, survival curve analysis was performed using Kaplan-Meier method, and comparison between the two groups was performed using log-rank test. The results showed that the survival of patients with low expression of YAP-R124me2a in the drug group was significantly better than that of patients with low expression in the non-drug group, and the survival analysis of the two groups had statistical difference (P=0.024) (see Figure 11 ). The results suggest that liver cancer patients with low expression of YAP-R124me2a have better prognosis after taking sorafenib.

[0136] Example 13: Analysis of liver cancer patients with high expression of YAP-R124me2a

[0137] Combined with the prognosis information, the survival analysis of 97 liver cancer patients with high expression of YAP-R124me2a in the patients of Example 6 (52 cases in the drug group and 45 cases in the non-drug group) was performed. Data analysis was performed using SPSS software 18.0, survival curve analysis was performed using Kaplan-Meier method, and comparison between the two groups was performed using log-rank test. The results showed that the survival of patients with high expression of YAP-R124me2a in the drug group was not statistically different from that of patients with high expression in the non-drug group (P=0.633) (see Figure 12 ). The results suggest that whether liver cancer patients with high expression of YAP-R124me2a take sorafenib or not does not affect the prognosis of the patients.

[0138] The application determines the modification level of YAP-R124me2a in tumor tissues by using immunohistochemical technology, microscope photographing and computer image processing software, and determines the correlation between the expression level of YAP-R124me2a and the prognosis of tumor patients after surgery in combination with postoperative follow-up information, and more importantly, the correlation between the expression level and the survival period of drug use after surgery of tumor patients. YAP-R124me2a can be used for preparing a protein molecular marker for early diagnosis of liver cancer, judgment of postoperative prognosis and drug selection, and has important guiding significance for postoperative monitoring and individualized sequential treatment of tumor patients.

[0139] The application provides a detection method with good development prospect. YAP is known to be abnormally expressed and active in various tumors, the application finds that R124 methylation modification regulates the sustained enhancement of YAP activity, and is highly expressed in liver cancer, lung adenocarcinoma, gastric cancer and colon cancer tissues, and presents a negative correlation with the prognosis of patients; importantly, the expression level is negatively correlated with the drug efficacy of tumor patients after surgery. The application provides a tumor marker for simultaneously representing tumor diagnosis, patient prognosis analysis and drug selection, and obtains a modified antibody for specific detection, and provides an important reference basis for formulating an individualized diagnosis and treatment scheme for clinical tumor patients.

[0140] The above has specifically described the preferred embodiments of the application, but the application is not limited to the embodiments, and those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the application, and these equivalent modifications or replacements are all included in the scope defined by the claims of the application.

Claims

1. Use of methylation modification of YAP protein at arginine 124 in preparation of a reagent or kit for early diagnosis and prognosis evaluation of tumor or drug selection for tumor, characterized in that, The methylation modification is asymmetric dimethylation modification, and the tumor is liver cancer, lung adenocarcinoma, gastric cancer or colon cancer.

2. The use according to claim 1, wherein: wherein The reagent is a reagent for detecting the level of asymmetric methylation modification of YAP protein at arginine 124 in a biological sample, The kit comprises a reagent for detecting the level of asymmetric methylation modification of YAP protein at arginine 124 in a biological sample.

3. The use according to claim 2, wherein: wherein The reagent for detecting the level of asymmetric methylation modification of YAP protein at arginine 124 in a biological sample is an immunohistochemical detection reagent, comprising a specific antibody recognizing the asymmetric dimethylation modification form of YAP-R124.

4. The use according to claim 3, wherein: wherein The specific antibody is a specific antibody recognizing the asymmetric dimethylation modification form of YAP-R124: anti-YAP-R124me2a.

5. The use according to claim 2, wherein: wherein The biological sample is selected from any one of surgical tumor tissue, puncture obtained tumor tissue or circulating tumor cells collected in patient blood.

6. Use according to claim 1, characterized in that, The kit is used for early diagnosis and prognosis evaluation of tumor, and is characterized by comprising a tumor tissue paraffin section preparation reagent system, an antigen repair reagent system and an antibody system, wherein the antibody system comprises a specific antibody recognizing the asymmetric dimethylation modification form of YAP-R124.

7. Use according to claim 1, characterized in that, The kit is used for tumor drug selection, and comprises a tumor tissue paraffin section preparation reagent system, an antigen repair reagent system and an antibody system, wherein the antibody system comprises a specific antibody recognizing the asymmetric dimethylation modification form of YAP-R124.

8. A system for early diagnosis of a tumor, assessment of prognosis, or assessment of drug selection, characterized by, The tumor is liver cancer, lung adenocarcinoma, gastric cancer or colon cancer, and the system comprises a tumor early diagnosis and prognosis evaluation kit or a tumor drug selection kit and an immunohistochemical scoring module installed on a terminal carrier, The kit comprises a tumor tissue paraffin section preparation reagent system, an antigen repair reagent system and an antibody system, wherein the antibody system comprises a specific antibody recognizing the asymmetric dimethylation modification form of YAP-R124, The immunohistochemical scoring module is based on different field photos randomly selected from tumor tissue and paracancerous tissue under a microscope, and the immunohistochemical score is calculated according to the following formula, and the calculation result is compared with a preset median value: Immunohistochemical score = Log10[255 / Iavg], wherein Iavg=(Iwp+Ip+Isp) / (Nwp+Np+Nsp), i.e., average intensity=(total intensity of weak positive pixels+total intensity of positive pixels+total intensity of strong positive pixels) / (number of weak positive pixels+number of positive pixels+number of strong positive pixels).

Images