Application of CBX7 in prognosis prediction of renal cell carcinoma

Abstract

The invention discloses application of CBX7 in prognosis prediction and treatment of renal cell carcinoma. The reagent for detecting the expression level of CBX7 is applied to preparation of auxiliary diagnosis or auxiliary prediction products for metastasis and prognosis of renal cell carcinoma. The relationship between expression of CBX7 in renal cell carcinoma tissues and patient metastasis and prognosis is researched for the first time, and it is found that the overall lifetime and progression-free lifetime of CBX7 low-expression renal cell carcinoma patients are short. Therefore, the CBX7 can be used as a marker for predicting metastasis and prognosis of the renal cell carcinoma, can assist in prognosis evaluation of patients with the renal cell carcinoma by detecting the expression level of the CBX7 in renal cell carcinoma tissues, and also has important guiding significance for postoperative monitoring and treatment of the patients with the renal cell carcinoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of CBX7 in the prognosis prediction of renal cell carcinoma. Background Art

[0002] Renal cell carcinoma is one of the most common malignancies of the urinary system. Despite advances in systemic treatment of renal cell carcinoma in recent years, some patients with renal cancer still have regional or distant metastases, among which distant metastases most often occur in the lungs, lymph nodes, liver, bones, and brain. The prognosis of patients with metastatic renal cell carcinoma is very poor, with only 12% of patients surviving for more than 5 years. Therefore, studying biomarkers closely related to renal cell carcinoma metastasis and prognosis will help in the early intervention and treatment of advanced renal cancer in clinic.

[0003] The Chromobox (CBX) family consists of eight members, namely CBX1-8. Studies have shown that each CBX protein exhibits different functions and prognostic effects in specific tumor types. Through database and domestic multicenter cohort analysis, we found that CBX7 is closely related to renal cell carcinoma metastasis and prognosis. So far, there have been no reports on the expression level, prognosis and function of CBX7 in renal cell carcinoma. Summary of the invention

[0004] The purpose of the present invention is to provide a molecular marker CBX7, specifically the use of CBX7 as a renal cell carcinoma metastasis, prognosis prediction marker, and its use in the preparation of a renal cell carcinoma metastasis, prognosis assessment kit.

[0005] In previous studies, we found that CBX7 was lowly expressed in renal cell carcinoma tissues, and CBX7 expression was lower in metastatic lesions than in primary lesions; the survival analysis results of a domestic multicenter cohort showed that the overall survival and disease-free survival of patients with low CBX7 expression were significantly shortened; in vitro functional experiments showed that CBX7 loss can promote the proliferation and migration of renal cancer cells.

[0006] Therefore, we believe that CBX7 has the potential to serve as a prognostic marker for renal cell carcinoma.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] Application of CBX7 as a biomarker in the preparation of auxiliary diagnosis or auxiliary prediction products for renal cell carcinoma metastasis and prognosis.

[0009] The use of a reagent for detecting CBX7 expression level in the preparation of an auxiliary diagnosis or auxiliary prediction product for renal cell carcinoma metastasis and prognosis.

[0010] As a preferred embodiment of the present invention, the reagent for detecting the expression level of CBX7 is an oligonucleotide probe that specifically recognizes the human CBX7 gene, a primer that specifically amplifies the CBX7 gene, or a binding agent that specifically binds to the protein encoded by the CBX7 gene.

[0011] As a further preferred embodiment of the present invention, the reagent for detecting the expression level of CBX7 is a primer for specifically amplifying the CBX7 gene.

[0012] As a further preferred embodiment of the present invention, the primer sequences for specifically amplifying the CBX7 gene are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0013] As a preferred embodiment of the present invention, the product is a kit or a chip.

[0014] A product for auxiliary diagnosis or auxiliary prediction of renal cell carcinoma metastasis and prognosis, comprising a reagent for detecting the expression level of CBX7; the reagent for detecting the expression level of CBX7 is preferably an oligonucleotide probe that specifically recognizes the human CBX7 gene, a primer that specifically amplifies the CBX7 gene, or a binder that specifically binds to the protein encoded by the CBX7 gene; the primer sequence that specifically amplifies the CBX7 gene is further preferably as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0015] Beneficial effects of the present invention:

[0016] The present invention studies the relationship between the expression of CBX7 in renal cell carcinoma tissue and the metastasis and prognosis of patients for the first time, and finds that the overall survival and progression-free survival of renal cell carcinoma patients with low CBX7 expression are shorter. Therefore, the CBX7 of the present invention can be used as a marker for predicting metastasis and prognosis of renal cell carcinoma. By detecting the expression level of CBX7 in renal cell carcinoma tissue, it can assist in the assessment of the prognosis of renal cell carcinoma patients, and it also has important guiding significance for the postoperative monitoring and treatment of renal cell carcinoma patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the CBX7 RNA expression level in renal cell carcinoma relative to adjacent adjacent tissues;

[0018] Figure 2 The expression of CBX7 in primary and metastatic lesions of renal cell carcinoma in the GEO database;

[0019] Figure 3 The results of immunohistochemical staining of CBX7 protein in primary and metastatic lesions of renal cell carcinoma;

[0020] Figure 4This is the overall survival curve of renal cell carcinoma patients grouped according to the expression of CBX7 in the TCGA database and the EGA database;

[0021] Figure 5 The overall survival curve and disease-free survival curve of the renal cell carcinoma cohort from three centers in China were grouped according to the expression of CBX7;

[0022] Figure 6 This is the ROC curve of CBX7 in renal cancer cohorts from three centers in the country;

[0023] Figure 7 These are the experimental results of proliferation, migration and invasion of CBX7 knockout cells in vitro. DETAILED DESCRIPTION

[0024] The present invention will now be described in detail in conjunction with the embodiments and drawings, but the implementation of the present invention is not limited thereto.

[0025] Example 1: CBX7 RNA expression levels in paired renal cell carcinoma and adjacent adjacent tissues

[0026] Sample acquisition: 104 pairs of surgically resected renal cell carcinoma and adjacent adjacent tissue specimens were collected from Jinling Hospital affiliated to Nanjing University and quickly frozen at -80°C.

[0027] mRNA extraction and RT-qPCR: First, the total RNA of the sample was extracted using TRIzol reagent, and then the RNA was reverse transcribed using PrimeScript RT Master Mix to obtain cDNA. Finally, SYBR Green was used for qPCR amplification, and the specific primer sequences were as follows: CBX7: forward primer is 5'-AGGAAGAGAGGTCCGAAACCCA-3' (SEQ ID NO.1); reverse primer is 5'-AGAAGCAGAGCTTCTCCTTGCC-3' (SEQ ID NO.2). β-actin: forward primer is 5'-AATCGTGCGTG ACATTAAGGAG-3' (SEQ ID NO.3); reverse primer is 5'-ACTGTGTTGGCGTACAGGTCTT-3' (SEQ ID NO.4).

[0028] The results are as follows Figure 1 As shown, CBX7 expression was decreased in 78 / 104 (75%) renal cancer tissues compared with paired adjacent adjacent tissues.

[0029] Example 2: Expression of CBX7 in primary and metastatic renal cell carcinoma in the GEO database

[0030] Relevant renal cancer expression profile chips were downloaded from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), including 2 data sets: GSE19949 and GSE22541. GSE19949 is an expression profile chip including 79 renal clear cell carcinoma primary lesions and 14 renal cancer lung metastases, and GSE22541 is an expression profile chip including 24 primary lesions and 20 renal cancer lung metastases. The expression level of CBX7 in each renal cell carcinoma sample was obtained, and the Graphpad Prism software was used to analyze and plot.

[0031] The results are as follows Figure 2 As shown, the expression of CBX7 in metastatic lesions was significantly lower than that in primary lesions in both datasets.

[0032] Example 3: Immunohistochemical staining results of CBX7 protein in primary and metastatic renal cell carcinoma

[0033] Sample acquisition: Thirteen pairs of renal cell carcinoma and metastatic specimens were collected from Jinling Hospital affiliated to Nanjing University by surgical resection or puncture biopsy.

[0034] Immunohistochemistry reagents include: xylene, anhydrous ethanol, 1% BSA blocking solution, EDTA antigen retrieval solution, 3% H2O2 methanol solution, ankG rabbit anti-human monoclonal antibody, HRP-labeled goat anti-rabbit secondary antibody, hematoxylin staining solution, hematoxylin differentiation solution and DAB color developer.

[0035] Immunohistochemistry detection method and scoring criteria, the specific steps are as follows:

[0036] Step 1: Use immunohistochemical reagents to perform immunohistochemical staining on renal cell carcinoma tissue and lung metastasis tissue sections. Specifically:

[0037] (1) Dewaxing the sections to water: xylene 10 min → xylene 10 min → xylene 10 min → anhydrous ethanol 5 min → 95% ethanol 5 min → 85% ethanol 5 min → 75% ethanol 5 min → double distilled water 5 min;

[0038] (2) Inactivation of endogenous peroxidase: 3% H2O2 methanol solution, place at room temperature for 20 min → wash three times with double distilled water, 5 min each time;

[0039] (3) Antigen repair: Place the slices in sodium citrate antigen repair solution (pH 6.0), boil under high pressure for 5 min, stop boiling for 10 min, boil again for 5 min → cool naturally to room temperature, and wash three times with double distilled water, 5 min each time;

[0040] (4) Serum blocking: 1% BSA blocking for 30 min, 37°C;

[0041] (5) Adding primary antibody: Gently shake off the blocking solution, add CBX7 primary antibody (1:500) on the slices, and incubate the slices flat in a humidified box at 4°C overnight. The primary antibody is a rabbit CBX7 polyclonal antibody (which can recognize CBX7 proteins in mammals such as humans and mice), purchased from Abcam, with a dilution ratio of 1:2000;

[0042] (6) Adding secondary antibody: Wash with PBS four times, 5 min each time → After the slices are slightly dried, add secondary antibody, which is HRP-labeled goat anti-rabbit IgG, and incubate at room temperature for 45 min;

[0043] (7) DAB color development: Wash with PBS four times, 5 min each time → DAB color development for 2-10 min. Observe under a microscope. If positive, it will be brown-yellow. Rinse the sections with tap water to stop color development.

[0044] (8) Counterstaining of cell nuclei: Counterstain with hematoxylin for about 2 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, and rinse with tap water to return to blue;

[0045] (9) Dehydration and sealing: Dehydrate the sections in 75% alcohol for 5 min → 85% alcohol for 5 min → 95% alcohol for 10 min → anhydrous ethanol for 10 min → anhydrous ethanol for 10 min → xylene for 15 min to make them transparent, let them dry slightly, and seal the sections with neutral gum.

[0046] Step 2: Use a microscope to score the immunohistochemical staining. The score is mainly based on the intensity of tumor cell staining and the percentage of positive cells. Staining intensity: no staining is defined as 0 points, light yellow is defined as 1 point, brown is defined as 2 points, and tan is defined as 3 points. Positive cell percentage: 1 point (0-25%), 2 points (26%-50%), 3 points (51%-75%), 4 points (76%-100%). The semi-quantitative analysis results are obtained by multiplying the two scores.

[0047] The results are as follows Figure 3 As shown in Figure 3, the expression of CBX7 protein in metastatic lesions was significantly lower than that in primary lesions.

[0048] Example 4: Prognostic analysis in TCGA database and EGA database

[0049] This study used renal clear cell carcinoma data from TCGA and EGA to complete survival validation, and used R (version 4.3.0) tools for statistical analysis and visualization.

[0050] 1. Data download and quality control: Use TCGAbiolinks and EGADownloadR packages to download the expression matrix and clinical survival information of TCGA and EGA renal clear cell carcinoma, perform quality control on the downloaded data, remove outliers, fill in missing values, and perform standardization; extract and process clinical survival information, including survival time and survival status.

[0051] 2. Grouping processing: Use the Tidyverse package to divide the samples into high expression group and low expression group according to the median expression of the CBX7 gene: samples above the median value are classified as the high expression group, and patients below or equal to the median value are classified as the low expression group; the number of people in each group is equal to ensure the balance of statistical analysis.

[0052] 3. Survival analysis: The surv function of the survival package was used to convert the survival time and event status data into survival objects. The survfit function was used to fit the Kaplan-Meier survival curve according to the survival objects and grouping information. The log-rank test was used to compare the survival differences between the two groups. The statistical significance level was set at P < 0.05. The main outcome variable was overall survival, that is, the time from the first diagnosis to death or the last follow-up (in months, the maximum follow-up time for TCGA and EGA was 151 and 145 months, respectively). The final results were visualized using the ggsurvplot function of the survminer package.

[0053] The results are as follows Figure 4 As shown, patients with low CBX7 expression in both databases had shorter overall survival and worse prognosis.

[0054] Example 5: Domestic multi-center prognostic analysis

[0055] We collected renal cancer postoperative specimens from three centers in China, including 1,072 cases from Changhai Hospital, 317 cases from Changzheng Hospital, and 207 cases from Jinling Hospital Affiliated to Nanjing University, followed up the prognosis information, made tissue chips, and performed CBX7 immunohistochemical staining. Digital photos were taken using a microscope and imaging device, and the positive signal intensity of the tumor tissue was analyzed using biological image processing software to give a score. The specific steps are as follows:

[0056] The tissue chip was scanned using Image Scope (Aperio) software, and the "positive pixel" of each sample was calculated using the Algorithms (Positive Pixel Count) program of the software. Calculation method: Positive 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), which is the immunohistochemical score of the tissue.

[0057] According to the median CBX7 histochemical score, the patients in each center were divided into high expression group and low expression group, and the overall survival curve and progression-free survival curve were drawn. Figure 5 AC), the results showed that patients with low expression of CBX7 in tumors had shorter overall survival and progression-free survival after surgery. Further, the diagnostic efficacy of CBX7 was analyzed by receiver operating characteristic curve (ROC). For overall survival, the area under the ROC curve (AUC) of CBX7 in the three cohorts was 0.772, 0.714, and 0.686 ( Figure 6 A); for progression-free survival, the areas under the ROC curve (AUC) of CBX7 in the three cohorts were 0.702, 0.666, and 0.664, respectively ( Figure 6 B). The above results indicate that CBX7 can be used to guide the prognosis of patients with renal cell carcinoma.

[0058] Example 6: Experimental results of proliferation, migration and invasion of CBX7 knockout cells in vitro

[0059] We used CRISPR / Cas9 dual vector lentiviral infection to knock out the CBX7 gene. The steps are as follows:

[0060] (1) Cas9 lentiviral packaging: pMD2.G, psPAX2, and lentiCas9-Blast plasmids were transfected into 293T cells, and the medium was changed 12 hours after transfection. 48 hours after transfection, the cell culture medium was aspirated with a 20 ml syringe, and the filtrate was filtered into a virus concentration column with a 0.45 μm cell filter, and centrifuged at 2500 rpm for 20 minutes to obtain the Cas9 lentiviral supernatant.

[0061] (2) Obtaining Cas9-expressing cells: Add Cas9 lentiviral supernatant to ACHN cells and replace with fresh culture medium 24 hours after infection. Add Blasticidin S 48 hours after infection. Collect cells after 7 days of screening and detect the expression of Cas9 protein.

[0062] (3) sgRNA lentiviral packaging: design and synthesis of sgRNA sequences targeting CBX7, F:

[0063] caccgAGGAGAGAGACCGAGCATCG, R: aaacCGATGCTCGGTCTCTCTCCTc. The oligo single-stranded DNA was annealed into double strands, and the double-stranded gRNA oligo was inserted into the lentiCRISPR v2 vector. The sgRNA lentivirus packaging steps targeting CBX7 were the same as above.

[0064] (4) Infect cells stably expressing Cas9 protein with sgRNA lentivirus, following the same steps as above. Add puromycin for selection for 7-10 days.

[0065] (5) Cultivation of monoclonal cell lines: Digest and count the cells after 7-10 days of puromycin selection, dilute the cell suspension to 10 cells / ml, aspirate 100 μl and seed it in a 96-well plate, add 100 μl of complete culture medium to each well, and culture it in a cell culture incubator overnight. Observe the cells in each well under a microscope, select the wells with only a single cell, and mark them. After the cells are fully grown, subculture them, and multiple monoclonal cell lines can be obtained.

[0066] (6) Identification of CBX7 knockout cells: Extract cell genomic DNA or protein and identify the CBX7 knockout effect, and ultimately obtain a monoclonal cell line with CBX7 gene knockout.

[0067] Using CBX7 knockout (KO) cells ( Figure 7 A) CCK8, scratch, and transwell invasion experiments were performed and it was found that knocking out CBX7 could promote the proliferation of renal cancer cells ( Figure 7 B) Migration Figure 7 C) and invasion ( Figure 7 D). The results of in vitro cell experiments prove that CBX7 plays a tumor suppressor role in renal cell carcinoma, which is consistent with the results of prognostic analysis. Therefore, CBX7 can be used as a marker for predicting metastasis and prognosis of renal cell carcinoma. By detecting the expression level of CBX7 in renal cell carcinoma tissue, it can assist in the assessment of the prognosis of renal cell carcinoma patients.

[0068] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of CBX7 as a biomarker in the preparation of auxiliary diagnosis or auxiliary prediction products for renal cell carcinoma metastasis and prognosis.

2. Use of reagents for detecting CBX7 expression levels in the preparation of auxiliary diagnosis or auxiliary prediction products for renal cell carcinoma metastasis and prognosis.

3. The use according to claim 2, characterized in that: The reagent for detecting the expression level of CBX7 is an oligonucleotide probe that specifically recognizes the human CBX7 gene, a primer that specifically amplifies the CBX7 gene, or a binding agent that specifically binds to the protein encoded by the CBX7 gene.

4. The use according to claim 3, characterized in that: The reagent for detecting the expression level of CBX7 is a primer for specifically amplifying the CBX7 gene.

5. The use according to claim 4, characterized in that: The primer sequences for specifically amplifying the CBX7 gene are shown in SEQ ID NO.1 and SEQ ID NO.

2.

6. The use according to any one of claims 2 to 5, characterized in that: The product is a test kit or a chip.

7. A product for auxiliary diagnosis or auxiliary prediction of renal cell carcinoma metastasis and prognosis, characterized in that: It contains reagents for detecting the expression level of CBX7; the reagents for detecting the expression level of CBX7 are preferably oligonucleotide probes that specifically recognize the human CBX7 gene, primers that specifically amplify the CBX7 gene, or binding agents that specifically bind to the protein encoded by the CBX7 gene; the primer sequences for specifically amplifying the CBX7 gene are further preferably as shown in SEQ ID NO.1 and SEQ ID NO.

2.

8. A substance for treating renal cell carcinoma, characterized in that The drug comprises CBX7 or an agonist thereof.

9. The substance according to claim 8, characterized in that The agonist is selected from CBX7 nucleic acid agonist and CBX7 protein agonist.

10. Use of the substance according to claim 8 in the preparation of a drug for inhibiting the proliferation, migration and invasion of renal cell carcinoma.

Images