Gemcitabine drug sensitive marker, detection kit and application of gemcitabine drug sensitive marker

Abstract

The invention discloses a gemcitabine drug sensitive marker, a detection kit and application of the gemcitabine drug sensitive marker, the gemcitabine drug sensitive marker is a gemcitabine drug-resistant marker or / and a gemcitabine sensitive marker, and the gemcitabine drug-resistant marker is one or more of ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL and ENSG00000286264; the sensitive marker is one or more of FAM163A, KREMEN2, VPS37D, GGCT (Growth Growth Computed Tomography), ADSL (Asymmetrical Digital Subscriber Loop), FOXL2 (Focal OXL2), MAST1 (MAST1), DNAAF3 (Deoxyribose Nucleic Acid Amplified The gemcitabine drug sensitive marker is used for predicting the drug effect of gemcitabine. According to the present invention, the expression of the related molecules is detected through the sequencing of the tissue transcriptome of the urothelial carcinoma patient, or through the PCR, the gene chip, the tissue chip, the NanoString technology, the immunohistochemistry and the ELISA method, and the guidance is provided for the drug use of gemcitabine and the precise treatment of bladder cancer.

Description

TECHNICAL FIELD

[0001] The application relates to an application of a molecular marker of an anticancer drug and a related reagent, in particular to an application of a molecular marker of the anticancer drug gemcitabine for bladder cancer and a related reagent, and belongs to the field of biological medicines. BACKGROUND

[0002] Bladder cancer is one of the common malignant tumors of the urinary system, and the incidence rate ranks the thirteenth, but due to the large population base in China, the number of new patients accounts for 15% of the global number of cases. Bladder cancer is divided into non-muscular and muscular invasive bladder cancer according to whether it invades the muscle layer. Non-muscular invasive bladder cancer can be treated by intravesical chemotherapy drugs, intravesical injection of bacillus Calmette-Guerin and tumor transurethral resection. However, a part of non-muscular invasive bladder cancer can progress to muscular invasive bladder cancer. The 5-year survival rate of patients with muscular invasive bladder cancer is less than 50%, and the patients are mainly manifested as muscle layer invasion of the tumor and metastasis of lymph nodes and other parts, and usually need to be treated by total bladder resection, which seriously affects the quality of life of the patients; for a long time, due to the few drug treatment schemes and the low clinical effective rate, the prognosis of the patients with bladder cancer in the middle and late stages is poor, and the survival rate is low.

[0003] Gemcitabine is one of the cornerstone chemotherapy drugs for treating bladder cancer, and its core anticancer mechanism lies in that as a pyrimidine nucleoside analogue, it belongs to a cell cycle-specific antimetabolite anticancer drug, can inhibit nucleotide reductase and induce chain termination by being incorporated into the DNA of cancer cells, interfere with the DNA synthesis function, and finally induce cancer cell apoptosis.

[0004] At present, as a first-line treatment drug for bladder cancer, the clinical effective rate of gemcitabine is only about 50%, and most of the population is not suitable for it, and if gemcitabine is blindly used, the tumor control of the patients who are not effective may be delayed, leading to tumor progression.

[0005] By performing transcriptome sequencing on the tumor tissues of the patients to perfect the molecular typing, the patients can be stratified for precise treatment, and after the stratification, it can be known that which potential sensitive drugs and which drug-resistant drugs the patients have. Therefore, it can be avoided that all the patients adopt the first-line treatment drug, and the patients who are originally resistant to the first-line drug lose the best treatment opportunity and treatment selection.

[0006] Therefore, in order to improve the curative effect of gemcitabine chemotherapy or combined target therapy for bladder cancer and reduce the risk of drug resistance, it is urgent to perfect the drug sensitivity molecular typing scheme of gemcitabine, find the patients suitable for the treatment of gemcitabine and the patients not suitable for the treatment of gemcitabine, and adopt different treatment modes for different patients, and moreover, an alternative treatment scheme can be provided for the patients not resistant to gemcitabine, and the curative effect of the treatment for bladder cancer is improved. SUMMARY

[0007] The present application aims at the technical problems of low sensitivity to gemcitabine, high drug resistance to gemcitabine, poor clinical treatment effect, poor prognosis, low survival rate and loss of optimal treatment opportunity and treatment options for patients with primary drug resistance to gemcitabine in the existing treatment process of bladder cancer by using gemcitabine as a therapeutic drug, and provides a gemcitabine drug sensitivity marker, related reagents, drugs and applications thereof. The present application provides different treatment methods for different patients by molecular typing of the gemcitabine drug sensitivity marker, and provides precise treatment for patients suitable for gemcitabine treatment, other alternative treatment options for patients intolerant to gemcitabine, clues for gemcitabine medication and precise treatment of bladder cancer, improved efficacy of gemcitabine chemotherapy or combined targeted drug treatment of bladder cancer, and reduced drug resistance risk.

[0008] To achieve the object of the present application, the present application provides a gemcitabine drug sensitivity marker in one aspect, which is a gemcitabine drug resistance marker and a gemcitabine sensitivity marker, wherein the drug resistance marker is one or more of ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264; and the sensitivity marker is one or more of FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3.

[0009] The high expression of the gene drug resistance markers ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264 is related to gemcitabine resistance; the high expression of the gene sensitivity markers FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3 is related to the sensitivity of cells to gemcitabine, and the high expression is sensitive to the effect of gemcitabine.

[0010] The second aspect of the present application provides the use of the above-mentioned gemcitabine drug sensitivity marker in screening patients suitable for gemcitabine drug treatment of bladder cancer, and guiding the use of gemcitabine.

[0011] The application discloses a bladder cancer treatment drug resistance or sensitivity monitoring method, and relates to the field of bladder cancer treatment.

[0012] The third aspect of the application provides a use of a gemcitabine drug sensitivity marker in non-treatment purpose gemcitabine drug use research.

[0013] The non-treatment purpose drug use research is basic research on gemcitabine pharmacodynamics and research on gemcitabine improvement research.

[0014] In particular, the effect of gemcitabine drugs in different batches is analyzed, and the killing detection of organoids on different batches of gemcitabine is carried out, and the effect of gemcitabine drugs in different batches is analyzed through the killing effect.

[0015] The fourth aspect of the application provides a quantitative reagent for the expression amount of a gemcitabine drug sensitivity marker, wherein the gemcitabine drug sensitivity marker is a gemcitabine drug resistance marker or a gemcitabine sensitivity marker, the gemcitabine drug resistance marker is one or more of ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264, and the gemcitabine sensitivity marker is one or more of FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3 and RAC3.

[0016] In particular, the quantitative reagent for the expression amount of the gemcitabine drug sensitivity marker is any one of a transcriptome sequencing reagent, a PCR reagent, a gene chip, a tissue chip, a NanoString technology, an immunohistochemical reagent or an ELISA reagent.

[0017] In particular, the quantitative reagent comprises an RNA extraction reagent, a reverse transcription reagent and a qPCR reagent.

[0018] The fifth aspect of the application provides a use of the quantitative reagent for the expression amount of the gemcitabine drug sensitivity marker in preparation of a gemcitabine drug sensitivity detection reagent, wherein the drug sensitivity detection is gemcitabine drug resistance detection or gemcitabine drug sensitivity detection.

[0019] In particular, the gemcitabine drug resistance detection refers to detecting the expression level of gemcitabine drug resistance markers ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264, and the increased expression level indicates that the patient is gemcitabine drug resistance, and the decreased expression level indicates that the patient is gemcitabine sensitive; the gemcitabine drug sensitivity detection refers to detecting the expression level of gemcitabine sensitive markers FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3, and the increased expression level indicates that the patient is gemcitabine sensitive, and the decreased expression level indicates that the patient is gemcitabine drug resistance.

[0020] The sixth aspect of the present application provides a kit for detecting the drug resistance of gemcitabine in the treatment of bladder cancer, comprising: PCR detection primers of gemcitabine drug sensitivity markers, an RNA extraction kit, and a reverse transcription kit.

[0021] In particular, the gemcitabine drug sensitivity markers are gemcitabine drug resistance markers or / and gemcitabine sensitive markers; the gemcitabine drug resistance markers are one or more of ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264; and the gemcitabine sensitive markers are one or more of FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3.

[0022] In particular, the PCR detection primers of the gemcitabine drug resistance markers ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264 are respectively SEQ ID NO. 1 to SEQ ID NO. 26.

[0023] In particular, the gemcitabine drug resistance marker detection primers are preferably:

[0024] In particular, the PCR detection primers of the gemcitabine sensitive markers FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, and RAC3 are SEQ ID NO. 27-SEQ ID NO. 44.

[0025] In particular, the gemcitabine sensitive marker detection primers are preferably:

[0026] The seventh aspect of the present application provides a gemcitabine drug sensitivity evaluation kit for bladder cancer treatment, comprising a quantitative reagent for detecting the expression amount of a gemcitabine drug sensitivity marker, wherein the drug sensitivity evaluation is drug resistance evaluation or drug sensitivity evaluation; and the gemcitabine drug sensitivity marker is a gemcitabine drug resistance marker or a gemcitabine sensitivity marker.

[0027] In particular, the quantitative reagent for detecting the expression amount of the gemcitabine drug sensitivity marker is any one of a transcriptome sequencing reagent, or a PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry, or ELISA reagent.

[0028] The eighth aspect of the present application provides a gemcitabine drug sensitivity analysis system for treating bladder cancer, comprising: A target expression amount detection device for detecting the expression amount of the gemcitabine drug sensitivity marker in a sample; the sample is a bladder cancer tissue of a patient; A drug sensitivity analysis device for determining the drug effect of bladder cancer based on the expression amount of the gemcitabine drug sensitivity marker;

[0029] A result output device for outputting the results obtained by the drug sensitivity analysis device; the drug sensitivity analysis is drug resistance analysis or sensitivity analysis.

[0030] In particular, the target expression amount detection device is a qPCR detection instrument for detecting and determining the expression amount of the drug resistance or sensitivity gene.

[0031] In particular, the drug sensitivity analysis device is a qPCR detection instrument for analyzing the increase or decrease of the expression amount of the drug resistance or sensitivity gene.

[0032] In particular, the result output device outputs the Ct value of the related drug resistance or sensitivity gene.

[0033] The ninth aspect of the present application provides a pharmaceutical composition for treating bladder cancer, comprising gemcitabine and an inhibitor or promoter of a gemcitabine drug sensitivity marker, wherein the gemcitabine drug sensitivity marker is a gemcitabine drug resistance marker or a gemcitabine sensitivity marker.

[0034] In particular, the pharmaceutical composition for treating bladder cancer comprises gemcitabine, an inhibitor of a gemcitabine drug resistance marker. The pharmaceutical composition improves the efficacy of gemcitabine on patients with high expression of drug resistance genes by inhibiting the expression of gemcitabine drug resistance genes.

[0035] In particular, the pharmaceutical composition for treating bladder cancer comprises gemcitabine, an inhibitor of a gemcitabine drug resistance marker. The pharmaceutical composition improves the efficacy of gemcitabine on patients with high expression of drug resistance genes by inhibiting the expression of gemcitabine drug resistance genes.

[0036] In particular, the gemcitabine drug resistance gene is ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264; and the gemcitabine sensitivity gene is FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3.

[0037] The purpose of the present application is achieved according to the following technical solutions:

[0038] The first purpose of the present application provides a gemcitabine drug sensitivity marker, which can be used to prompt the drug resistance and sensitivity of gemcitabine in the population, and provide a basis for precise treatment of bladder cancer.

[0039] The second purpose of the present application provides the application of a gemcitabine drug sensitivity marker for treating bladder cancer in the research of non-treatment purpose gemcitabine drug use.

[0040] The third purpose of the present application provides the application of a gemcitabine drug sensitivity marker, which includes quantitative reagents for detecting its expression, inhibitors, promoters and the like. The application of these related reagents can fill the gap in the field of precise diagnosis and treatment of bladder cancer.

[0041] The above purposes of the present application are achieved by the following technical solutions: In the first aspect of the present application, it is provided that the high expression of a group of genetic drug resistance markers ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264 is related to gemcitabine resistance. It is found that the high expression of genes FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3 is related to gemcitabine sensitivity, that is, the high expression of genes is sensitive to the effect of gemcitabine. The gene expression can be used as a potential drug sensitivity biomarker for guiding the treatment of primary bladder cancer. That is, the present application provides a gemcitabine drug sensitivity marker, which is a gemcitabine resistance marker or a gemcitabine sensitivity marker; the gemcitabine resistance marker is any one or several of a group of markers ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264; the gemcitabine sensitivity marker is FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3. The gemcitabine drug sensitivity marker is RNA or reverse-transcribed cDNA or protein.

[0042] The gemcitabine drug sensitivity marker can indicate the gemcitabine resistance and sensitivity of the population, and provide a basis for precise treatment of bladder cancer.

[0043] When the expression amount of the gemcitabine resistance marker is significantly high, it is determined that the bladder cancer is gemcitabine-resistant; when the expression amount of the gemcitabine sensitivity marker is significantly high, it is determined that the bladder cancer is gemcitabine-sensitive.

[0044] In particular, the gemcitabine drug sensitivity marker is RNA or reverse-transcribed cDNA or protein.

[0045] In particular, the gemcitabine drug sensitivity marker is screened in the following manner: A large number of surgical specimens of primary bladder cancer patients are collected, and multiple samples are taken to establish a bladder cancer organoid biological library covering hundreds of sites by three-dimensional culture method.

[0046] The inventors found that the organoids derived from low-grade urothelial carcinoma patients also showed small cell atypia, consistent cells, and few mitotic figures by Hematoxylin-Eosin (HE) staining; the organoids derived from high-grade urothelial carcinoma patients with squamous epithelial metaplasia also showed common mitotic figures, large cell atypia, and also had the histological characteristics of squamous metaplasia, which confirmed that the organoids of the application retained the pathological characteristics of the patients.

[0047] By immunohistochemical staining of the organoids and the corresponding surgical specimens of the patients, it was found that the molecular markers of the bladder cancer organoids were consistent with those of the clinically bladder cancer patients.

[0048] About one hundred sites were selected for exon sequencing and transcriptome sequencing of the tissues and corresponding organoids, and the genomic characteristics of the tissues and corresponding organoids were analyzed. The molecular characteristics of the organoid library were verified from multiple dimensions such as somatic mutations, copy number variations, and transcriptome similarities, and the mutation characteristics of the original tissue were retained.

[0049] The bladder cancer data studied in the application reflect inter-tumor and intra-tumor heterogeneity of different degrees, which provides a basis for studying the drug sensitivity heterogeneity of bladder cancer.

[0050] In order to study the drug sensitivity molecular typing of gemcitabine for bladder cancer, the inventors performed transcriptome sequencing on multiple (a total of 83) organoid sites, and through Lasso model and bioinformatics analysis, it was found that the sensitive markers of gemcitabine were FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3, and the drug resistance markers were ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264. The AUC of the training set was 0.899. And through the same method, multiple bladder cancer specimens were collected for organoid culture, drug sensitivity test and transcriptome sequencing, and molecular typing research was carried out, and the verification set of gemcitabine was 0.842.

[0051] Among them, bioinformatics analysis includes differential gene analysis, KEGG pathway enrichment analysis, drug sensitivity and gene expression correlation analysis.

[0052] The second aspect of the application provides a quantitative reagent for the expression amount of the gemcitabine drug sensitivity marker.

[0053] The application of the gemcitabine drug sensitivity marker expression quantitative reagent in preparing a gemcitabine drug sensitivity detection reagent.

[0054] The application of the gemcitabine drug sensitivity marker expression quantitative reagent in preparing a gemcitabine drug sensitivity detection reagent.

[0055] The sample detected by the gemcitabine drug sensitivity detection reagent is a bladder cancer tissue sample; and the quantitative reagent is any one of a transcriptome sequencing reagent, a PCR reagent, a gene chip reagent, a tissue chip reagent, a NanoString technology reagent, an immunohistochemical reagent or an ELISA reagent.

[0056] The application further provides a gemcitabine drug sensitivity marker detection reagent for detecting the expression amount of a gemcitabine drug sensitivity marker.

[0057] The application further provides a gemcitabine drug sensitivity evaluation kit comprising a quantitative reagent for detecting the expression amount of a gemcitabine drug sensitivity marker, wherein the drug sensitivity evaluation is drug resistance evaluation or drug sensitivity evaluation.

[0058] The quantitative reagent for the expression amount of the gemcitabine drug sensitivity marker is any one of a transcriptome sequencing reagent, a PCR reagent, a gene chip reagent, a tissue chip reagent, a NanoString technology reagent, an immunohistochemical reagent or an ELISA reagent.

[0059] The basic principle of real-time fluorescence quantitative PCR (qPCR) is to utilize the characteristics of DNA polymerase in the PCR process to synthesize new DNA chains, combine a probe or dye, and measure the degree of PCR reaction by monitoring the increase of fluorescence signals in real time. Fluorescent label Since there is a linear relationship between the Ct value of the template and the initial copy number of the template in the exponential phase of PCR amplification, it becomes the basis for quantification.

[0060] The relative expression amount of the related drug resistance or sensitivity gene can be calculated by comparing the CT value with that of the paracancerous tissue. It is defined that the relative expression of the gene is greater than 2 times that of the paracancerous tissue, which indicates that the gene expression is increased, and the relative expression is less than 0.5 times that of the paracancerous tissue, which indicates that the gene expression is decreased.

[0061] The application further provides a gemcitabine drug sensitivity analysis system for bladder cancer, comprising: 1) a target expression amount detection device for detecting the expression amount of the gemcitabine drug sensitivity marker in a sample; the sample is a bladder cancer tissue of a patient; 2) a drug sensitivity analysis device for determining the drug effect of the bladder cancer based on the expression amount of the gemcitabine drug sensitivity marker; 3) result output device: for outputting the result of the drug sensitivity analysis device, the drug sensitivity analysis is drug resistance analysis or sensitivity analysis.

[0062] The fourth aspect of the present application provides a pharmaceutical composition for treating bladder cancer, gemcitabine, an inhibitor or promoter of a gemcitabine drug sensitivity marker.

[0063] The advantages and beneficial effects of the present application are: The present application screens and determines a group of genes related to gemcitabine resistance (i.e. gemcitabine resistance genes), specifically ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264;

[0064] The present application also screens and determines genes related to gemcitabine sensitivity (i.e. gemcitabine sensitivity genes), specifically FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3.

[0065] The gemcitabine resistance markers and sensitivity markers screened by the present application can be used to predict the efficacy of gemcitabine in treating bladder cancer. When the expression level of the gemcitabine resistance marker is significantly high, it is judged that the patient has high drug resistance to gemcitabine, and the treatment effect of gemcitabine is poor. Conversely, it does not have drug resistance, and the treatment effect of gemcitabine is significant. When the expression level of the gemcitabine sensitivity marker is significantly high, it is judged that the patient has high sensitivity to gemcitabine, and the treatment effect of gemcitabine is significant. Conversely, the sensitivity is low, and the treatment effect of gemcitabine is poor.

[0066] By sequencing the tissues of bladder cancer patients, or by RT-qPCR, immunohistochemical methods to detect the expression of related molecules (gemcitabine drug sensitivity markers), it can provide accurate guidance for drug use and treatment of bladder cancer. Detecting the drug sensitivity markers of gemcitabine, high expression of drug resistance genes, and high expression of sensitivity genes.

[0067] The gemcitabine drug sensitivity markers of the present application can prompt the gemcitabine resistance and sensitivity population, provide a basis for precise treatment of bladder cancer, and the application of gemcitabine drug sensitivity marker related reagents can fill the gap in the field of precise diagnosis and treatment of bladder cancer. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 The organoids of the bladder cancer (Bladder cancer, BC) patient retain the pathological characteristics and tissue markers of the patient. Figure 1A is to establish BC patient different site organoids, left side figure is to construct a library containing non-muscular layer infiltration and muscular layer infiltration bladder cancer organoids, for tumor less than 2cm in diameter, single point sampling is carried out, for tumor greater than 2cm, multi-point (superficial site and infiltration site) sampling is carried out, organoid culture is carried out; right side is the superficial site, infiltration site and metastatic lymph node tumor tissue of No. 61 source to construct organoids, light microscope is used for organoid bright field observation; Figure 1 B is the microscope state and HE staining of BC patient organoids; Figure 1 C is the immunohistochemical staining of organoids, wherein the organoids of No. 10 source are cultured and the expression of main typing molecules is detected: Figure 1 The left side of C is the gross observation, HE and immunohistochemical staining of the tumor tissue of the patient; the middle and right sides are the microscope bright field observation and HE, immunohistochemical identification of the successfully cultured organoids from different sites of the patient.

[0069] Figure 2 The exons and transcriptome characteristics of the patient are retained in the organoids, wherein, Figure 2 A is that exon sequencing reveals that the mutation characteristics of bladder cancer tissue are retained in the organoids. 2B is the consistency rate of oncogene mutations in organoids and tissues, wherein the median is 85.71% (the median of the consistency rate of gene mutations in organoids and cancer tissues is 85.71%); Figure 2 C is that the cultured organoids and the corresponding cancer tissues are subjected to transcriptome sequencing, and the Heatmap shows the correlation of tumor tissues and paired organoids; Figure 2 D is the transcriptome correlation coefficient of the cultured organoids and the corresponding cancer tissues.

[0070] Figure 3 It is organoid drug sensitivity test, that is, 4 kinds of bladder cancer chemotherapy drugs are tested for 109 organoids.

[0071] Figure 4 It is gemcitabine drug sensitivity molecular typing, wherein Figure 4 A is the drug sensitivity molecular typing of gemcitabine; Figure 4 B is the AUC of the training set and the validation set of gemcitabine.

[0072] Figure 5 It is the verification of gemcitabine molecular typing in 80 bladder cancer tissues. DETAILED DESCRIPTION

[0073] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0074] Example 1: Validation of the correlation between organoids and tissues in bladder cancer

[0075] In this embodiment, a bladder cancer organoid biobank covering 208 sites was successfully established using a 3D culture method.

[0076] 1. HE staining Taking surgical specimens from patients with bladder cancer (BC) as an example ( Figure 1 A), HE staining was performed, confirming that the organoids preserved the patient's pathological features. Figure 1 B).

[0077] 2. Immunohistochemical staining Immunohistochemical staining of organoids and corresponding patient surgical specimens revealed consistent immunohistochemical expression characteristics between bladder cancer patients and their organoids. Figure 1 C) Staining results revealed that bladder cancer epithelial cell markers (CK5 / 6, CK20, GATA3, P63) and the proliferation index Ki-67 were consistently expressed in bladder cancer tissues and organoids.

[0078] This demonstrates that the organoid system established in this embodiment preserves the patient's pathological characteristics very well.

[0079] 3. Exon and transcriptome sequencing This embodiment selected 158 loci, including 75 exons and 83 transcriptome loci, for exon and transcriptome sequencing of tissues and corresponding organoids to analyze the genomic characteristics of bladder cancer tissues and corresponding organoids. Exon sequencing results (e.g.) Figure 2 A, 2B); transcriptome sequencing results (e.g. Figure 2 C, 2D).

[0080] The organoids are subjected to drug killing treatment of different concentrations of gemcitabine, and the concentration of gemcitabine corresponding to the killing of half of the organoids is calculated, that is, the gemcitabine IC50 value; then, according to the gemcitabine IC50 value 2.47 micromole, the organoids are divided into a sensitive group (less than 2.47 micromole) and a drug-resistant group (greater than 2.47 micromole), and a machine learning model of the transcriptome characteristics of different groups is established. A drug response model is established using multi-omics data, and a bootstrapping strategy based on LASSO (Least Absolute Shrinkage and Selection Operator) regression model is used to find gemcitabine-sensitive genes and drug-resistant genes.

[0081] The specific operation method is as follows: First, based on the correlation between gene expression and gemcitabine response in the training set, candidate genes are selected, the correlation coefficient is gradually increased (increased by 0.01 each time), and the number of candidate genes identified is limited (102-103); Then, multiple candidate genes are selected to construct each specific prediction model, and a LASSO regression model is used to model the drug sensitivity (-log2 IC50 value) and the expression profile of a selected group of candidate genes. The model is realized using the glmnet software package (v4.1-3), and the optimal penalty parameter 'lambda' is selected through ten-fold cross-validation (10 fold cross validation). The regression modeling process is bootstrapped (n = 100), and candidate genes that are considered important genes in more than 50% of cases are selected as the marker genes of the best model.

[0082] By fitting the selected marker genes, the best model with a specific correlation coefficient threshold is obtained. AUROC of all models related to gemcitabine drug is compared to find the best threshold of correlation coefficient. ROC curve and AUROC are analyzed by ROCR software package (v1.0-11).

[0083] The organoid biological library is highly preserved in the molecular characteristics of the tissue sample from multiple dimensions such as somatic mutations and transcriptome similarity. Exome sequencing results show that the large-scale bladder cancer tissue and organoid queue established by the present application is consistent with previous bladder cancer research, and has a high mutation rate of bladder cancer. At the transcriptome level, the organoids and the corresponding patient tissues also have a high degree of correlation.

[0084] Therefore, the results of transcriptome, exome sequencing, immunohistochemical staining, hematoxylin-eosin staining (HE staining) tests show that the organoids of the present application have high consistency with the tissues.

[0085] Example 1A Screening of gemcitabine drug sensitivity markers

[0086] The gemcitabine drug sensitivity marker is a gemcitabine sensitive marker and a gemcitabine drug resistance marker.

[0087] 1. Drug sensitivity test

[0088] The 109 organoids were subjected to drug sensitivity test of common chemotherapy drugs for bladder cancer: gemcitabine, cisplatin, mitomycin C and epirubicin, and the specific operation method was as follows: An appropriate amount of uniformly blown and resuspended liquid containing organoids was taken; 300g was centrifuged at 4°C for 5min, and the supernatant was carefully removed; an appropriate amount of culture medium containing 5% Matrigel was added to resuspend the organoids; 40μl of organoid suspension was added to each well of a 384-well plate, and 50μl of sterile pure water was added to the peripheral wells and wells without suspension; after plating for 24 hours, drugs (10-20μl / well) were added, and the culture was continued for 6 days, and the morphological and state changes of the organoids were observed, and the cell survival rate was tested.

[0089] The obtained IC50 values of each drug were arranged, and a heat map of the above-mentioned four chemotherapy drugs was drawn, as shown in Figure 3 Different colors represent different values.

[0090] The drug sensitivity reaction of patient organoids is generally consistent with the actual drug effect of patients, so the drug sensitivity test at the level of in vitro organoids can reflect the drug sensitivity level of clinical patients.

[0091] 2. Transcriptome sequencing

[0092] In order to study the drug sensitivity molecular typing of gemcitabine, a chemotherapy drug for bladder cancer, 60 organoid sites with drug sensitivity effect were selected for transcriptome sequencing, and through Lasso model and bioinformatics analysis, the sensitive markers of gemcitabine were FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3 and RAC3; the drug resistance markers were ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264 Figure 4 A), and the AUC of the training set A was 0.899 Figure 4 B).

[0093] The operation method is as follows: Total RNA was isolated using DNeasy & RNeasy isolation kit (Qiagen) and purified using poly-T oligo linked magnetic beads. RNA libraries were sequenced by Illumina NovaSeq 6000 platform and generated 150bp paired-end reads. Raw reads were quality controlled by FastQC (v0.11.9), Cutadapt (v2.5) and Trimmomatic (v0.3) tools. STAR software (v2.7.3a) was used to align reads to the reference human genome hg19 with default parameters; quantify gene expression, perform differential expression analysis on transcriptomic data, perform pathway clustering analysis on differentially expressed genes (such as Gene Ontology, KEGG, Reactome, etc.), perform Gene Set Enriment Analysis (GSEA), compare with existing pathway datasets (MSigDB Gene Sets). After uniform processing, samples can be analyzed by unsupervised clustering, principal component analysis, etc., and heat maps can be made. The results can be combined with various clinical indicators, mutation information, etc. for analysis.

[0094] 3. Verification analysis The same method was used to perform transcriptome sequencing on 26 organoid sites collected. Through Lasso model and bioinformatics analysis, 26 bladder cancer specimens were collected for organoid culture, drug sensitivity test and transcriptome sequencing, and molecular typing research (i.e. transcriptome characteristics of gemcitabine resistance and sensitivity) was performed. The verification set of gemcitabine is 0.842. Figure 4 B).

[0095] Through the construction of organoids for transcriptome sequencing, the training set AUC is 0.899 and the verification set is 0.842. The test results show that the gene set analyzed by the model of the application is reliable.

[0096] The independent verification set is composed of another organoid.

[0097] For each drug (cisplatin, gemcitabine, mitomycin C, epirubicin), the same transcriptome analysis process is used to obtain the gene expression profile of the verification set, which is substituted into the best model for drug treatment to make a prediction, and compared with the drug sensitivity of the verification set organoid; the prediction model is evaluated by ROC curve. The prediction model is used for verification of patient transcriptome data and patient clinical response, which perfectly matches the treatment effect of the patient in the clinic.

[0098] The effectiveness of the gemcitabine sensitivity molecular typing was verified in the tissues of 80 bladder cancer patients in this embodiment. In the bladder cancer tissues, the AUC of the gemcitabine sensitivity molecular typing was 0.817 Figure 5

[0099] Example 2 Application of gemcitabine markers in gemcitabine drug research for non-treatment purposes

[0100] In this embodiment, the study of the activity of gemcitabine is as follows: 1. Select bladder cancer tissues, and make different batches of gemcitabine act on the selected bladder cancer tissues, and determine the quantitative expression of the gemcitabine sensitivity markers (FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3, ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264) screened in Example 1. 2. According to the expression amount, compare the expression amounts of the gemcitabine sensitivity markers of different batches, and judge the biological activity of each batch of gemcitabine according to the high and low expression amounts.

[0101] Example 3 Gemcitabine resistance evaluation kit

[0102] The present embodiment provides an RT-qPCR gemcitabine resistance evaluation kit, which comprises: quantitative reagents for detecting the expression amount of the gemcitabine resistance markers (one or more of ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264) screened in Example 1.

[0103] Kit 1 (i.e., ATOH8 resistance marker kit) comprises: upstream and downstream primers of the RNA of the resistance marker ATOH8 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0104] Kit 2 (i.e., DAPK2 resistance marker kit) comprises: upstream and downstream primers of the RNA of the resistance marker DAPK2 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0105] ​Kit 3 (i.e. marker BCL2L2 resistance kit) comprises: the upstream and downstream primers of RNA of resistance marker BCL2L2 (as shown in Table 1), RNA extraction kit, cDNA synthesis kit and qPCR kit.

[0106] Kit 4 (i.e. marker PNPLA2 resistance kit) comprises: the upstream and downstream primers of RNA of resistance marker PNPLA2 (as shown in Table 1), RNA extraction kit, cDNA synthesis kit and qPCR kit.

[0107] Kit 5 (i.e. marker SHROOM1 resistance kit) comprises: the upstream and downstream primers of RNA of resistance marker SHROOM1 (as shown in Table 1), RNA extraction kit, cDNA synthesis kit and qPCR kit.

[0108] Kit 6 (i.e. marker FGGY resistance kit) comprises: the upstream and downstream primers of RNA of resistance marker FGGY (as shown in Table 1), RNA extraction kit, cDNA synthesis kit and qPCR kit.

[0109] Kit 7 (i.e. marker CPQ resistance kit) comprises: the upstream and downstream primers of RNA of resistance marker CPQ (as shown in Table 1), RNA extraction kit, cDNA synthesis kit and qPCR kit.

[0110] Kit 8 (i.e. marker CPO resistance kit) comprises: the upstream and downstream primers of RNA of resistance marker CPO (as shown in Table 1), RNA extraction kit, cDNA synthesis kit and qPCR kit.

[0111] Kit 9 (i.e. marker SLC10A5 resistance kit) comprises: the upstream and downstream primers of RNA of resistance marker SLC10A5 (as shown in Table 1), RNA extraction kit, cDNA synthesis kit and qPCR kit.

[0112] Kit 10 (i.e. marker ADAMTS15 resistance kit) comprises: the upstream and downstream primers of RNA of resistance marker ADAMTS15 (as shown in Table 1), RNA extraction kit, cDNA synthesis kit and qPCR kit.

[0113] Kit 11 (i.e. marker COL25A1 resistance kit) comprises: the upstream and downstream primers of RNA of resistance marker COL25A1 (as shown in Table 1), RNA extraction kit, cDNA synthesis kit and qPCR kit.

[0114] The kit 12 (i.e. marker ASMTL resistance kit) comprises: the upstream and downstream primers of the RNA of the resistance marker ASMTL (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit and a qPCR kit.

[0115] The kit 13 (i.e. marker ENSG00000286264 resistance kit) comprises: the upstream and downstream primers of the RNA of the resistance marker ENSG00000286264 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit and a qPCR kit.

[0116] Table 1 RNA primers of gemcitabine resistance markers

[0117] wherein, The cDNA synthesis kit (i.e. reverse transcription kit) comprises dNTPmix: containing dATP, dCTP, dGTP, dTTP each 2mM; Taq DNA polymerase.

[0118] The qPCR reagent comprises: Taq hot start enzyme, reaction buffer, SYBR Green fluorescent dye, dNTPs.

[0119] The qPCR reagent further comprises PCR enhancer, PCR stabilizer and Universal Passive Reference Dye.

[0120] The existing known qPCR kits are all suitable for the present application, and are used according to the operation instructions of the kits, for example: Fullengen Bio Co., Ltd.: PerfectStart® Universal Green qPCR SuperMix; Universal Dye Method Fluorescent Quantitative PCR Premix (Item No.: AQ631); Tian Gen Biological Technology Co., Ltd.: SuperReal Fluorescent Quantitative Premix Reagent Enhanced Edition (SYBR Green) (Item No.: FP205).

[0121] The existing known RNA extraction kits are all suitable for the present application, for example, TransZol Up High Performance RNA Extraction Kit (Item No.: ET111-01-V2) of Fullengen Bio Co., Ltd., and Magnetic Bead Method High Efficiency Tissue / Cell Total RNA Extraction Kit (Item No.: DP771) of Tian Gen Biological Technology Co., Ltd. All existing known cDNA synthesis kits are applicable to this invention, such as the FastKing cDNA First Strand Synthesis Kit II (Genomic De-generated) (Catalog No.: KR126) from Tiangen Biotech Co., Ltd.; the TransScript One-Step GDNA Removal and First Strand cDNA Synthesis Kit from TransGen Biotech Co., Ltd. (Catalog No.: AT311); or the SuperScript Preamplification System for First Strand cDNA Synthesis Kit from GIBICOL Co., Ltd., etc.

[0122] All existing known qPCR kits are applicable to this invention. Follow the instructions on the kits. For example: TransGen Biotech Ltd.: PerfectStart® Universal Green qPCR SuperMix; Universal Dye-Based Quantitative PCR Premix (Catalog No.: AQ631); Tiangen Biotech Ltd.: SuperReal Quantitative PCR Premix Enhanced Version (SYBR Green) (Catalog No.: FP205).

[0123] Using the kit of this invention in combination with commonly used RNA extraction reagents and reverse transcription reagents, the expression levels of gemcitabine resistance markers (ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264) in samples can be specifically detected. This helps to determine the gemcitabine resistance of bladder cancer patients in advance. If the test results show an increased expression level of the resistance markers, it indicates that the patient is gemcitabine resistant; if the expression level is decreased, it indicates that the patient is gemcitabine sensitive.

[0124] Using the gemcitabine resistance detection kit for bladder cancer of this invention, gemcitabine resistance can be detected immediately after bladder cancer tissue is obtained through surgery or pathological biopsy in bladder cancer patients. This allows for the evaluation of drug efficacy before gemcitabine treatment, providing patients with personalized and precise treatment plans and ensuring the best possible treatment. Without using the resistance assessment kit of this invention to evaluate drug efficacy, patients resistant to gemcitabine may be treated with gemcitabine, delaying cancer treatment and leading to tumor progression.

[0125] Example 4: Gemcitabine Sensitivity Assessment Kit

[0126] This embodiment provides an RT-qPCR gemcitabine susceptibility assessment kit, comprising: A quantitative reagent for detecting the expression amount of the gemcitabine sensitive marker (FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3) screened in Example 1.

[0127] Kit 1 (i.e., FAM163A sensitive marker kit) comprises: upper and lower primers for RNA of the sensitive marker FAM163A (as shown in Table 2); an RNA extraction kit; a reverse transcription kit (cDNA synthesis kit); and a qPCR kit.

[0128] Kit 2 (i.e., KREMEN2 sensitive marker kit) comprises: upper and lower primers for RNA of the sensitive marker KREMEN2 (as shown in Table 2); an RNA extraction kit; a reverse transcription kit; and a qPCR kit.

[0129] Kit 3 (i.e., VPS37D sensitive marker kit) comprises: upper and lower primers for RNA of the sensitive marker VPS37D (as shown in Table 2); an RNA extraction kit; a reverse transcription kit; and a qPCR kit.

[0130] Kit 4 (i.e., GGCT sensitive marker kit) comprises: upper and lower primers for RNA of the sensitive marker GGCT (as shown in Table 2); an RNA extraction kit; a reverse transcription kit; and a qPCR kit.

[0131] Kit 5 (i.e., ADSL sensitive marker kit) comprises: upper and lower primers for RNA of the sensitive marker ADSL (as shown in Table 2); an RNA extraction kit; a reverse transcription kit; and a qPCR kit.

[0132] Kit 6 (i.e., FOXL2 sensitive marker kit) comprises: upper and lower primers for RNA of the sensitive marker FOXL2 (as shown in Table 2); an RNA extraction kit; a reverse transcription kit; and a qPCR kit.

[0133] Kit 7 (i.e., MAST1 sensitive marker kit) comprises: upper and lower primers for RNA of the sensitive marker MAST1 (as shown in Table 2); an RNA extraction kit; a reverse transcription kit; and a qPCR kit.

[0134] Kit 8 (i.e., DNAAF3 sensitive marker kit) comprises: upper and lower primers for RNA of the sensitive marker DNAAF3 (as shown in Table 2); an RNA extraction kit; a reverse transcription kit; and a qPCR kit.

[0135] Kit 9 (i.e. sensitive marker RAC3 kit) comprises: upstream and downstream primers of RNA of sensitive marker RAC3 (as shown in Table 2); RNA extraction kit; reverse transcription kit and qPCR kit.

[0136] Table 2 RNA primers of gemcitabine sensitive markers

[0137] By using the kit of the present application in combination with commonly used RNA extraction reagents and reverse transcription reagents, the expression amount of gemcitabine sensitive markers (FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3) in the sample can be specifically detected, which is helpful for judging the sensitivity of the bladder cancer patient to gemcitabine in advance.

[0138] By using the bladder cancer gemcitabine drug sensitivity detection kit of the present application, the detection can be performed immediately after the bladder cancer tissue is obtained by surgery or pathological puncture of the bladder cancer patient, and the drug efficacy can be evaluated before the treatment using gemcitabine, so as to provide a personalized and precise treatment plan for the patient.

[0139] Example 5 Gemcitabine resistance analysis system

[0140] The present embodiment provides an analysis system for evaluating the efficacy of gemcitabine in treating bladder cancer, comprising: 1) a gemcitabine resistance evaluation kit, comprising one or more of the kits 1-13 described in Example 3; 2) a gemcitabine sensitivity evaluation kit, comprising one or more of the kits 1-9 described in Example 4; 3) a RT-qPCR target expression detection device: for detecting the expression amount of gemcitabine drug sensitivity markers in the sample, wherein the gemcitabine drug sensitivity markers include gemcitabine resistance markers and gemcitabine sensitive markers, the gemcitabine resistance markers are one or more of (ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264); the gemcitabine sensitive markers are one or more of (FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3); The RT-qPCR target expression detection device is a device commonly used in the art, i.e., a qPCR detector, a real-time fluorescent quantitative PCR instrument, such as AriaMx Real-Time PCR System (Agilent Technologies); The sample is a bladder cancer tissue of a patient; The reagent used by the target expression detection device is an RT-qPCR quantitative reagent, including upstream and downstream primers of gemcitabine resistance and gemcitabine sensitive marker genes; and reverse transcription reagent and qPCR reagent (such as PerfectStart® Green qPCR SuperMix); It also includes a dye method fluorescent quantitative PCR premix (item number: AQ601-01-V2) 4) Drug resistance analysis device: based on the expression amount of gemcitabine resistance markers (ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264), determine the drug effect of bladder cancer; based on the expression amount of gemcitabine sensitive markers (FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3), determine the drug effect of bladder cancer.

[0141] 5) Result output device: for outputting the results analyzed by the drug resistance analysis device.

[0142] The drug efficacy can be evaluated before the patient uses gemcitabine, so as to provide a personalized and precise treatment plan for the patient.

[0143] Example 6 Drug composition for treating bladder cancer

[0144] A drug composition for treating bladder cancer, comprising: Gemcitabine, an inhibitor of gemcitabine resistance markers (i.e., any one or more of ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, ENSG00000286264).

[0145] The drug composition can be an injection preparation for bladder cancer patients. Taking the medicine during treatment can improve the treatment effect of patients with gemcitabine resistance.

[0146] Example 6A Drug composition for treating bladder cancer

[0147] A pharmaceutical composition for treating bladder cancer, comprising: Gemcitabine, a promoter of a gemcitabine sensitivity marker (i.e., any one or more of FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, RAC3).

[0148] The pharmaceutical composition can be an injection preparation for a bladder cancer patient. Taking the medicine during treatment can improve the therapeutic effect of gemcitabine on the patient.

[0149] The above embodiments of the present application are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

Claims

1. A gemcitabine susceptibility marker, characterized in that, The gemcitabine susceptibility markers are gemcitabine resistance markers and gemcitabine sensitivity markers. Specifically, the resistance markers are one or more of ATOH8, DAPK2, BCL2L2, PNPLA2, SHROOM1, FGGY, CPQ, CPO, SLC10A5, ADAMTS15, COL25A1, ASMTL, and ENSG00000286264; the sensitivity markers are one or more of FAM163A, KREMEN2, VPS37D, GGCT, ADSL, FOXL2, MAST1, DNAAF3, and RAC3.

2. The use of the gemcitabine susceptibility marker as described in claim 1 in screening patients suitable for gemcitabine treatment of bladder cancer, to guide gemcitabine use.

3. The use of the gemcitabine susceptibility markers as described in claim 1 or 2 in gemcitabine use studies for non-therapeutic purposes.

4. The quantitative reagent for the expression level of gemcitabine susceptibility marker as described in claim 1.

5. The application of the quantitative reagent for the expression level of gemcitabine susceptibility markers as described in claim 4 in the preparation of gemcitabine susceptibility detection reagents, wherein the susceptibility detection is gemcitabine resistance detection or gemcitabine drug sensitivity detection.

6. A kit for detecting gemcitabine resistance during bladder cancer treatment, characterized in that, include: Primers for PCR detection of gemcitabine susceptibility markers, RNA extraction kit, and reverse transcription kit.

7. A kit for assessing the drug sensitivity of gemcitabine, a treatment drug for bladder cancer, characterized in that, The reagent includes a quantitative reagent for detecting the expression level of gemcitabine susceptibility markers, wherein the susceptibility assessment is a drug resistance assessment or a drug sensitivity assessment; and the gemcitabine susceptibility markers are gemcitabine resistance markers or gemcitabine sensitivity markers.

8. The drug susceptibility assessment kit as described in claim 7, characterized in that, The quantitative reagent for the expression level of gemcitabine susceptibility markers is a transcriptome sequencing reagent, or any one of PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry or ELISA reagents.

9. A gemcitabine susceptibility testing system for treating bladder cancer, characterized in that, include: Target expression level detection device: used to detect the expression level of gemcitabine susceptibility markers in samples; The sample was bladder cancer tissue from a patient. Drug sensitivity analysis device: Determine the efficacy of drugs for bladder cancer based on the expression levels of gemcitabine drug sensitivity markers; Result output device: used to output the results obtained by the drug sensitivity analysis device, wherein the drug sensitivity analysis is a drug resistance analysis or a sensitivity analysis.

10. A pharmaceutical composition for treating bladder cancer, characterized in that, Including inhibitors or promoters of gemcitabine and gemcitabine susceptibility markers, wherein the gemcitabine susceptibility markers are gemcitabine resistance markers or gemcitabine sensitivity markers.

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