CN117844924A - Biomarker BUB1 for renal cell carcinoma - Google Patents
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Abstract
The invention discloses a biomarker BUB1 of renal cell carcinoma, and specifically discloses the following application of a reagent for detecting BUB1 in a sample (i) to prepare a product for predicting prognosis of renal cancer and suitability for immunotherapy; (ii) preparing a product for diagnosing renal cancer. According to the invention, through the research of integrating TCGA data, compared with normal tissues, the high expression of BUB1 in the tissues of KIRP, KIRC and KICH is found, and the analysis of specificity, sensitivity and accuracy is carried out, so that the BUB1 is suggested to be used as a molecular marker for diagnosing renal cell carcinoma. In addition, BUB1 plays a key role in regulating angiogenesis and immune infiltration in the tumor microenvironment, suggesting that it may predict the prognosis of renal cancer, with high BUB1 expression predicting a poorer prognosis for KIRP and KIRC, kirch. Thereby providing a new idea for diagnosing, prognosticating and the like of renal cell carcinoma.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a biomarker BUB1 for renal cell carcinoma and application of the biomarker BUB1 in renal cell carcinoma.
Background
Renal cancer is a tumor with high malignancy in the urinary system, one of the ten causes of cancer-related death, and one of the most common tumors. The incidence of renal cancer is rising worldwide, with various subtypes depending on the mix of histology, clinical and genetic processes. Renal cell carcinoma (renal cell carcinoma, RCC) is one of the most important types of renal cancer. Renal cell carcinoma is a malignant tumor originating in the renal parenchymal urinary tract epithelial system, accounting for 80% -90% of kidney malignant tumors. Among them, renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), and chromophobe renal cell carcinoma (KICH) account for almost 95% of all renal cell carcinomas. Progress has been made in the diagnosis, examination, surgery and medical treatment of renal cancer. However, the clinical results are still unsatisfactory.
Treatment of renal cell carcinoma remains a significant challenge in clinical practice, creating a more reliable prognostic prediction model, further elucidating the key molecular mechanisms of tumor progression is an urgent need to address. However, patients of different stages may have different prognosis due to the high heterogeneity of renal cell carcinoma. And differences in patient response to immunotherapy may also be related to their genetic and molecular background. The biological information analysis is adopted to screen out the biological markers related to the occurrence, development and prognosis of the kidney cancer, so that the method has wide prospect in the aspects of personalized treatment and prognosis prediction, and provides assistance for diagnosis of the kidney cancer, prediction of prognosis of the kidney cancer and suitability of immunotherapy.
Disclosure of Invention
In view of the above, the invention discloses a molecular marker of renal cell carcinoma, which is BUB1, provides application of the molecular marker in diagnosing renal carcinoma, predicting prognosis of renal carcinoma and suitability for immunotherapy, and provides assistance for diagnosing renal carcinoma, predicting prognosis of renal carcinoma and suitability for immunotherapy.
In order to achieve the above object, the present invention provides the following technical solutions:
the first aspect of the invention discloses the use of a reagent for detecting BUB1 in a sample, said use being any of the following:
(i) Preparing a product for predicting prognosis of renal cancer and suitability for immunotherapy;
(ii) Preparing a product for diagnosing kidney cancer.
BUB1 is a protein-encoding gene which encodes serine/threonine protein kinases and plays an important role in mitosis. The encoded protein fraction functions by phosphorylating members of the mitotic checkpoint complex and activating spindle checkpoints. This protein also plays a role in the late-stage inhibition of complex/cell cycle body activation. Such proteins may also play a role in DNA damage reactions. Mutations in this gene are associated with aneuploidy and cancer.
From the theory, the invention verifies the action mechanism of BUB1 in renal cell carcinoma by integrating TCGA database to check BUB1 expression difference in multiple renal cell carcinoma tumor tissues and normal tissues. The research of the invention finds that compared with normal tissues, BUB1 in KIRP, KIRC and KICH tissues is over-expressed, and analysis of specificity, sensitivity and accuracy is carried out, so that the BUB1 can be used as a molecular marker for diagnosing renal cell carcinoma. In addition, BUB1 plays a key role in regulating angiogenesis and immune infiltration in the tumor microenvironment, suggesting that it may predict the prognosis of renal cancer, with high BUB1 expression predicting a poorer prognosis for KIRP and KIRC, kirch.
In a second aspect, the invention discloses a product for predicting prognosis of renal cancer and suitability for immunotherapy, said product comprising an agent for detecting the level of BUB1 expression in a sample. It will be appreciated that the product employs detection methods conventional in the art, including but not limited to nucleic acid sequencing techniques, polymerase chain reaction, real-time fluorescent quantitative polymerase chain reaction or Western blotting.
Further, the reagent comprises a reagent for detecting the protein expression level of BUB1 or the mRNA expression level of BUB1 in the sample. In some specific embodiments of the invention, the agent is a primer capable of specifically amplifying gene BUB1 or a probe specifically binding gene BUBU 1; in other specific embodiments of the invention, the agent is a primer capable of reverse transcribing mRNA of gene NEDD 4. It will be appreciated that, in addition to the above-mentioned reagents, the product may further include other auxiliary reagents according to the specific detection mode, which are not described in detail herein.
Further, the sample is a body fluid or tissue of the subject.
Further, the body fluid is blood, urine, puncture fluid or cerebrospinal fluid.
Further, the tissue is kidney cancer tissue or a tissue beside the cancer, and the tissue beside the cancer refers to a tissue which is less than or equal to 2cm away from a focus.
Further, the kidney cancer is renal cell carcinoma.
Further, the renal cell carcinoma is clear renal cell carcinoma, papillary renal cell carcinoma or chromophobe renal cell carcinoma.
Further, the product is an in vitro test product, and in some embodiments of the invention, the product is a chip or a kit.
Further, the product performs the following method:
obtaining a sample of the subject and detecting the level of BUB1 expression in the sample;
based on BUB1 expression levels, prognosis prediction of renal cancer is performed.
Specifically, the prognosis prediction of the kidney cancer is performed according to the cut off value according to the BUB1 expression level, if the value is higher than the cut off value, the expression level is judged to be high, the subject belongs to a high risk group, poor prognosis is indicated, and the survival period is short; if the expression level is lower than the cut off value, the expression level is judged to be low, the subject belongs to a low risk group, and the prognosis is indicated to be good and the survival period is long.
The cut off value as described herein is the average BUB1 expression in normal kidney tissue of a given healthy population, which is obtained using the R-pack surviviner package.
In a third aspect, the invention discloses a product for diagnosing renal cancer, said product comprising an agent for detecting the level of BUB1 expression in a sample.
Further, the reagent comprises a reagent for detecting the protein expression level of BUB1 or the mRNA expression level of BUB1 in the sample.
Further, the renal cancer is renal cell carcinoma, which is clear cell carcinoma of the kidney, papillary cell carcinoma of the kidney, or chromophobe renal cell carcinoma.
Further, the product is an in vitro test product, and in some embodiments of the invention, the product is a kit or chip. It will be appreciated that the product employs detection methods conventional in the art, including but not limited to nucleic acid sequencing techniques, polymerase chain reaction, real-time fluorescent quantitative polymerase chain reaction or Western blotting.
Further, the reagent comprises a reagent for detecting the protein expression level of BUB1 or the mRNA expression level of BUB1 in the sample. In some specific embodiments of the invention, the agent is a primer capable of specifically amplifying gene BUB1 or a probe specifically binding gene BUBU 1; in other specific embodiments of the invention, the agent is a primer capable of reverse transcribing mRNA of gene NEDD 4. It will be appreciated that, in addition to the above-mentioned reagents, the product may further include other auxiliary reagents according to the specific detection mode, which are not described in detail herein.
Further, the sample is kidney cancer tissue of the subject.
Further, the product performs the following method:
obtaining a sample of the subject and detecting the level of BUB1 expression in the sample;
judging the expression level of BUB1 according to the BUB1 expression level, and diagnosing whether the patient is renal cancer.
Further, the determination is made based on a threshold value of the BUB1 expression level, and if the determination is higher than the threshold value, the patient is diagnosed with renal cancer. The threshold value described herein is the average expression level of BUB1 in its normal kidney tissue in a given healthy population.
The invention has the beneficial effects that:
the invention finds out the BUB1 related to the progress of the renal cell carcinoma by the bioinformatics technology, predicts the prognosis of the renal cell carcinoma and the suitability of immunotherapy according to the gene expression level, and predicts the survival condition of 1,3 and 5 years in KICH and KIRP. The biological informatics index provided by the invention relates to a renal cancer detection method which has the advantages of high flux, high sensitivity, low cost, simple detection method, suitability for popularization and use and the like.
The invention provides a gene as a biomarker for predicting the prognosis of kidney cancer and diagnosing kidney cancer, and provides a reliable method for diagnosing kidney cancer, analyzing the prognosis of a patient with kidney cancer and predicting the prognosis of immunotherapy.
Drawings
Fig. 1: the box plot of the mRNA Sequencing results shows: the mRNA amount of the gene related to the invention in kidney cancer is obviously increased compared with normal kidney tissue, and the expression amount of the gene in tumors is obviously different from that of the gene in kidney cancer, wherein P is less than 0.05.
Fig. 2: the expression level of the gene of the present invention in kidney cancer is significantly higher in tumors than in normal kidney tissue.
Fig. 3: violin plots show the amount of gene expression in different stages of kidney cancer.
Fig. 4: volcanic plot of gene results shows: compared with normal kidney tissues, the mRNA quantity of the gene related by the invention has obviously up-regulated expression quantity in KIRC, and P is less than 0.05, thus having statistical significance.
Fig. 5: volcanic plot of gene results shows: compared with normal kidney tissues, the mRNA quantity of the gene related by the invention has obviously up-regulated expression quantity in KIRP, and P is less than 0.05, thus having statistical significance.
Fig. 6: volcanic plot of gene results shows: compared with normal kidney tissues, the mRNA quantity of the gene related by the invention has obviously up-regulated expression quantity in KICH, and P is less than 0.05, thus having statistical significance.
Fig. 7: shows a gene one-way cox forest map in KIRC for clinical information.
Fig. 8: shows a gene one-way cox forest map in KIRP for clinical information.
Fig. 9: shows a gene one-way cox forest map in KICH for clinical information.
Fig. 10: survival analysis graph of disease-free survival rate of patients in different risk groups of KIRC.
Fig. 11: survival analysis graph of KIRC patients in different risk groups.
Fig. 12: survival analysis graph of disease-free survival rate of patients in different risk groups of KIRP.
Fig. 13: survival analysis graph of KIRP patients in different risk groups.
Fig. 14: survival analysis graph of disease-free survival rate of patients in different risk groups of KICH.
Fig. 15: survival analysis graph of KICH patients in different risk groups.
Fig. 16: the cut off value visualizes the graph.
Fig. 17: time dependent ROC analysis plots (auc=85.7) showing KIRP patient 1,3,5 year survival based on age, stage and risk scores.
Fig. 18: time dependent ROC analysis plots (auc=96.6) showing the age, stage and risk scores based on KICH patients 1,3,5 year survival.
Fig. 19: GO enrichment analysis classification histogram of genes in KIRC.
Fig. 20: KEGG enrichment of genes in KIRC analysis of bubble figures.
Fig. 21: GO enrichment analysis classification histogram of genes in KIRP.
Fig. 22: KEGG enrichment of genes in KIRP analysis bubble figures.
Fig. 23: GO enrichment analysis classification histogram of genes in KICH.
Fig. 24: KEGG enrichment of genes in KICH analysis bubble figures.
Fig. 25: GSEA enrichment analysis graph of genes in KIRC.
Fig. 26: GSEA enrichment analysis graph of genes in KIRP.
Fig. 27: graph of GSEA enrichment analysis of genes in KICH.
Fig. 28: GSEA enrichment analysis of different immune cell pathways of genes in KIRC.
Fig. 29: GSEA enrichment analysis of different immune cell pathways of genes in KIRP.
Fig. 30: GSEA enrichment analysis of different immune cell pathways of genes in KICH.
Fig. 31: using the Cibersort algorithm, the infiltration of the genes into different immune cells in the KIRC was estimated and analyzed.
Fig. 32: using the Cibersort algorithm, the infiltration of the genes into different immune cells in the KIRP was estimated and analyzed.
Fig. 33: using the Cibersort algorithm, the infiltration of the genes into different immune cells in the KICH was estimated and analyzed.
Detailed Description
The following detailed description of embodiments of the invention is exemplary and is provided merely to illustrate the invention and is not to be construed as limiting the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Unless otherwise specified, the conditions and steps are not specifically described, and the method is a conventional method.
The affy, input R, limma, tidyverse, rms, TCGAbiolinks, parallel, e1071, preprocessCore, org.Hs.eg.db, clusterifier, ggpubr, ggtheses, forest, ROCR, survivinal, etc. packages referred to in the examples below are all prior art and are sourced from https:// cran. R-project. Org or http:// www.bioconductor.org/, and run in R software after loading.
Example 1 Gene data acquisition
In the present invention, transcriptome sequencing (RNAseq) data and clinical data TCGA-KIRC, TCGA-KIRP and TCGA-KICH are downloaded from the cancer genomic map The Cancer Genome Atlas (TCGA) bioinformatics database (https:// www.cancer.gov/ccg/research/genome-sequencing/TCGA).
Example 2 expression differential analysis
1. The transcriptome data of the TCGA database is subjected to differential gene expression analysis by using an R software package of 'limma', genes related to the survival time and prognosis are screened, and 22 genes related to renal cell carcinoma are screened out according to p < 0.01 and logFC > 2.
2. And (3) respectively analyzing and screening the expression quantity of 22 genes in renal cell carcinoma through online analysis website GEPIA. The results showed that BUB1 was differentially expressed in normal and kidney cancer tissues, and that the mRNA expression level thereof in kidney cancer tissues was significantly increased compared with that in normal kidney tissues, with significant differences (P < 0.05) (FIG. 1). Expression of BUB1 in pan-cancer, the expression level of BUB1 in kidney cancer was significantly increased in tumor compared to normal kidney tissue (fig. 2). BUB1 expression levels in different phases of renal cancer, wherein BUB1 expression levels in advanced renal cancer were high (FIG. 3).
The results show that BUB1 is up-regulated in all three renal cell carcinomas, which suggests that BUB1 can be used as a tumor marker for detecting renal cell carcinomas.
3. BUB1 gene is subjected to differential analysis and verification in normal kidney tissues and kidney cancer tissues by using a tidyverse package and a Deseq-2 package in R language. As a result, it was found that the expression level of BUB1 mRNA was significantly up-regulated in KIRC, KIRP, KICH, and that P <0.05 was statistically significant as compared with normal kidney tissue (FIGS. 4 to 6). This suggests that BUB1 can be used as a marker and has sensitivity to the identification of renal cancer.
Example 3 verification of BUB1 Effect amount
Based on the sample data in the The Cancer Genome Atlas (TCGA) bioinformatics database downloaded in example 1, annotation was performed using a gene length file, the R packet applied was a tidyverse packet, and a Cox binary-classification variable forest map was obtained using a single factor regression analysis method to verify the magnitude of the BUB1 effect.
The results showed that in the KIRC, KIRP and KICH gene one-factor cox forest plots for displaying clinical information, 95% ci of the effector amount of BUB1 was greater than 1, and none of them intersected the null line, with statistical significance (fig. 7-9). The verification result proves that the HR of the BUB1 gene is more than 1, and the relative risk is high, so that the BUB1 gene is proved to be a promoting factor for the development of the kidney cancer.
EXAMPLE 4 relationship between BUB1 mRNA expression level and prognosis of renal cancer patient and renal cancer diagnosis
Gene survival analysis expression profile data was obtained based on the The Cancer Genome Atlas (TCGA) bioinformatics database. And dividing the BUB1 mRNA average expression level into a high expression group and a low expression group, carrying out survival analysis, and comparing survival rates among different groups by adopting a log-rank test. Specifically, the relationship between BUB1 gene and prognosis of renal cancer patients was studied using R software and survivin package to draw Kaplan-Meier survival curves, including total survival and disease-free incidence. ROCR and rms package are used for drawing ROC curve, area under curve (AUC value) is calculated, statistical analysis is carried out, and sensitivity, accuracy and specificity of BUB1 gene in kidney cell cancer prediction are verified according to AUC, so as to study diagnosis value of BUB1 gene on different types of kidney cancers.
The results showed that Kaplan-Meier survival curves showed that the overall survival rate was lower for the BUB1 mRNA high-expressing group patients than for the low-expressing group (family < 0.05) (fig. 11, 13 and 15); in addition, the patient survival rate was lower in the BUB1 mRNA high expression group than in the low expression group (user < 0.05) (fig. 10, 12 and 14). The results show that the expression level of BUB1 is related to the prognosis of a patient with renal cancer, and BUB1 can be used as a key target for predicting the prognosis of the renal cancer. Specifically, the average BUB1 mRNA expression level was used as the cut off value, and FIG. 16 is a visual chart of the cut-off value, and the cut off value was 1.33 based on the survival time and survival state of the patient using a surviviner package. If the threshold value is higher than the cut off value, the subject belongs to a high risk group, and the bad prognosis and the short survival time are prompted; if the expression level is lower than the cut off value, the expression level is judged to be low, the subject belongs to a low risk group, and the prognosis is indicated to be good and the survival period is long.
Furthermore, by ROC curve analysis of the survival of KIRP, KICH patients 1,3,5 years, the area under the curve (AUC) values for KIRP patients 1,3,5 years were 85.7 (fig. 17); the area under the curve (AUC) values for KICH patients 1,3,5 years were 96.6 (fig. 18). The results show that BUB1 has good prediction efficacy for the prognosis survival of kidney cancer.
Further, the sensitivity, accuracy and specificity of the BUB1 gene in kidney cell cancer prediction are shown in Table 1, and the results show that BUB1 is a good tumor marker for predicting kidney cell cancer and diagnosing kidney cell cancer.
TABLE 1
| KIRC | KIRP | KICH | |
| specificity | 0.9329502 | 0.971831 | 0.828125 |
| sensitivity | 0.8873239 | 0.8125 | 0.9130435 |
| accuracy | 0.9274874 | 0.9556962 | 0.8505747 |
Example 5 Gene enrichment analysis
The G0, KEGG and GSEA enrichment analysis is carried out by using tidyverse, clusteprofiler packets and the like, the relevant mechanism and immune function are explored, and the channel obtained by the enrichment analysis is found to be enriched in the relevant channels such as cell cycle, immunoglobulin production and the like, so that the support is provided for determining the immune therapeutic target.
The results showed that the pathway enriched in KIRC by BUB1 was enriched in the pathway such as immunoglobulin production (FIG. 19); the pathway enriched in KIRC by BUB1 shows significant enrichment in the cell cycle and other pathways (fig. 20). The pathway enriched in KIRP by BUB1 is enriched in the pathway such as immunoglobulin production (fig. 21); the pathway enriched in KIRP by BUB1 was significantly enriched in the cell cycle and the like (fig. 22). The pathway enriched in KICH by BUB1 is enriched in the pathway such as nuclear division (FIG. 23); the pathway enriched for BUB1 in KICH shows significant enrichment in the pathway such as the cell cycle (FIG. 24), where the color of the color block in FIGS. 19-24 represents significance and the number represents the number of genes enriched on this pathway.
Figures 25-27 show the most significant GSEA enrichment pathway among the three renal cell carcinoma types, showing BUB1 up-regulated cell cycle, antigen binding, etc. pathway enrichment. Fig. 28-30 show the top ten enrichment pathways of three renal cell carcinoma types, with broken lines in fig. 25-30 representing pathways up-or down-regulated, and different colors representing different pathways.
FIGS. 31-33 show the infiltration of different immune cells, with different color patches reflecting different degrees of immune infiltration.
The cell cycle enables the cells of the organism to complete life activities such as division and proliferation on the premise of following normal life laws. From the above results, the enrichment of BUB1 in the cell cycle suggests that it plays a role in the process of participating in cancer cell division, and the same effect is also demonstrated by the pathway of nuclear division. The enriched immunoglobulin production and antigen binding pathways indicate that the gene plays a key role in cancer cell immunity and plays a role in the curative effect and the reactivity of immunotherapy.
The correlation of BUB1 with immune cells in this tumor was clarified by immune cell scoring. According to FIGS. 31-32, BUB1 is associated with T helper cell, CD4 memory cell activation, etc., and the surface BUB1 mediates to some extent the immune response and inflammatory response.
The researches show that the BUB1 gene is remarkably increased in the expression of kidney cancer as a whole, and the increase of mRNA expression quantity and immune infiltration of the gene can be used as molecular markers for early screening, operation effect evaluation, recurrence monitoring, drug resistance monitoring, metastasis monitoring, molecular typing, assisted pathological tissue typing and the like of the kidney cancer. The gene can be detected by a molecular biological method, thereby providing a trace, minimally invasive, quantitative, low-cost, high-flux, effective and objective method for identifying renal canceration cells.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. Use of a reagent for detecting BUB1 in a sample, characterized in that it is any one of the following:
(i) Preparing a product for predicting prognosis of renal cancer and suitability for immunotherapy;
(ii) Preparing a product for diagnosing kidney cancer.
2. A product for predicting prognosis of renal cancer and suitability for immunotherapy, said product comprising an agent for detecting the expression level of BUB1 in a sample.
3. A product for predicting the prognosis of renal cancer and the suitability for immunotherapy as claimed in claim 2, wherein said reagent comprises a reagent for detecting the protein expression amount of BUB1 or the mRNA expression amount of BUB1 in the sample.
4. A product for predicting the prognosis of renal cancer and suitability for immunotherapy as claimed in claim 2, wherein the sample is a body fluid or tissue of the subject;
preferably, the body fluid is blood, urine, puncture fluid or cerebrospinal fluid;
preferably, the tissue is kidney cancer tissue or paracancerous tissue.
5. A product for predicting the prognosis of renal cancer and suitability for immunotherapy as claimed in claim 2, wherein the renal cancer is renal cell carcinoma;
preferably, the renal cell carcinoma is clear cell carcinoma of the kidney, papillary cell carcinoma of the kidney or chromophobe renal cell carcinoma.
6. A product for predicting prognosis of renal cancer and suitability for immunotherapy as claimed in claim 2, wherein the product is a chip or kit.
7. A product for predicting the prognosis of renal cancer and suitability for immunotherapy as claimed in any one of claims 2 to 6, wherein the product performs the following method:
obtaining a sample of the subject and detecting the level of BUB1 expression in the sample;
performing prognosis prediction of kidney cancer according to BUB1 expression level;
preferably, the prognosis prediction of the kidney cancer is performed according to a cut off value according to the BUB1 expression level, if the value is higher than the cut off value, the expression level is judged to be high, the subject belongs to a high risk group, the prognosis is poor, and the survival period is short; if the expression level is lower than the cut off value, judging that the expression level is low, and the subjects belong to a low risk group, so that the prognosis is good and the survival period is long;
preferably, the cut off value is the average BUB1 expression in normal kidney tissue, which is obtained using the R-pack surviviner pack.
8. A product for diagnosing renal cancer, comprising an agent for detecting the level of BUB1 expression in a sample;
preferably, the reagent comprises a reagent for detecting the protein expression level of BUB1 or the mRNA expression level of BUB1 in a sample;
preferably, the renal cancer is renal cell carcinoma, which is clear cell carcinoma of the kidney, papillary cell carcinoma of the kidney, or chromophobe renal cell carcinoma;
the product is a kit or a chip;
preferably, the sample is kidney cancer tissue of the subject.
9. A product for diagnosing renal cancer as claimed in claim 8 wherein the product performs the following method:
obtaining a sample of the subject and detecting the level of BUB1 expression in the sample;
judging the expression level of BUB1 according to the BUB1 expression level, and diagnosing whether the patient is renal cancer.
10. The product according to claim 8 or 9, characterized in that said determination is made according to a threshold value of the expression level of BUB1, above which it is diagnosed as suffering from cancer;
preferably, the threshold is the average expression level of BUB1 in its normal kidney tissue in a given healthy population.
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