CN116064802A - Auxiliary method, system and kit for early screening or prognosis monitoring of bladder cancer - Google Patents

Abstract

The invention discloses an auxiliary method, a system and a kit for early screening or prognosis monitoring of bladder cancer. The bladder cancer marker of the auxiliary method is a marker group formed by combining an m6A RNA methylation modulator YTHDF2 and an m6A RNA methylation modulator RBMX with Bladder Tumor Antigen (BTA); the system comprises an information acquisition module, an analysis module and a reporting module; the kit comprises detection reagents of YTHDF2, RBMX and BTA. Through verification, the auxiliary method, the system and the kit for early screening or prognosis monitoring of bladder cancer provided by the invention can be used for early screening or prognosis recurrence monitoring of bladder cancer, and have relatively ideal sensitivity, specificity and effectiveness for auxiliary screening or diagnosis of bladder cancer.

Description

Auxiliary method, system and kit for early screening or prognosis monitoring of bladder cancer

Technical Field

The invention relates to the technical field of medical auxiliary diagnosis, in particular to an auxiliary diagnosis method, an auxiliary diagnosis system and an auxiliary diagnosis kit for early bladder cancer risk screening and prognosis monitoring based on the combination of an m6A RNA methylation modulator YTHDF2, an m6A RNA methylation modulator RBMX and a Bladder Tumor Antigen (BTA).

Background

Bladder cancer is the most common urinary malignancy in people in China. The early symptoms of bladder cancer are not obvious, the disease progresses rapidly after the disease is developed, the disease is difficult to diagnose clearly, patients are usually in middle and late stages when diagnosing clearly, and the death rate is high. In addition, bladder cancer also has the characteristics of higher recurrence rate and clinical complexity. Thus, early screening or prognostic monitoring is an important approach to improving mortality in bladder cancer patients.

Although research has considered early detection of bladder cancer to be feasible, there is currently no effective screening means for bladder cancer. For the average person, a general physical examination, most bladder tumors can be found by ultrasound examination of the urinary system. The clinical screening methods of patients suspected to be bladder cancer are mainly cystoscopy, urine abscission cytology, B-ultrasonic and CT scanning. Among them, the cystoscopy is regarded as a gold standard for diagnosing bladder cancer, but this examination method is invasive and may cause complications such as urinary tract infection, urinary tract injury, bladder injury, etc., and cause various discomforts such as pain, infection, hematuria, etc., to the patient. Urine abscission cytology examination, FISH detection and the like can assist diagnosis of BCa, but the overall sensitivity and specificity are not satisfactory, namely 50% and 85% respectively. Although no tumor marker capable of replacing cystoscopy exists at present, the tumor marker has great potential of becoming effective diagnosis and treatment of bladder cancer. The single tumor markers are difficult to specifically diagnose the bladder cancer, so that the clinical application of the single tumor markers is limited, and the combined application of multiple bladder cancer tumor markers can improve the accuracy of diagnosing the bladder cancer, but also improve the false positive rate of diagnosis.

In order to better serve the auxiliary diagnosis and prognosis of bladder cancer patients, it is highly necessary to develop a detection method, system and kit for early screening of bladder cancer or prognosis recurrence monitoring, which are ideal in auxiliary diagnosis sensitivity and specificity, and a treatment scheme selection auxiliary reference for early diagnosis or prognosis recurrence of bladder cancer patients.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a detection method, a detection system and a detection kit for bladder cancer early screening and prognosis recurrence based on the combined application of m6A RNA methylation modulator YTHDF2 and m6A RNA methylation modulator RBMX and BTA, which are relatively ideal in auxiliary diagnosis sensitivity and specificity. The invention uses the combination of the m6A RNA methylation modulator YTHDF2 and the m6A RNA methylation modulator RBMX and BTA as the marker group for the first time in the auxiliary diagnosis of early screening and prognosis recurrence monitoring of bladder cancer, thereby improving the sensitivity, specificity and effectiveness of the auxiliary diagnosis of early bladder cancer and bladder cancer recurrence monitoring.

The present invention is thus achieved.

In a first aspect, the invention provides a detection method for assisting in diagnosing early bladder cancer and prognosis recurrence by using a combination of m6A RNA methylation modulator YTHDF2, m6A RNA methylation modulator RBMX and BTA as a marker set.

N6-adenylate methylation (m 6A) modification is mainly regulated and controlled by the joint participation of methyltransferase, demethylase, methylated reading protein and the like. The m6A modification of RNA is mainly enriched near 3' -UTR and stop codon, affecting transcription, processing and translation of RNA. Studies have reported that the m6A modified state of RNA is important for the development and therapeutic study of bladder cancer, such as METTL3, YTHDF1 and YTHDF3 play an important role in the adhesion, migration and invasion of bladder cancer cells. However, it is unclear whether m6A RNA methylation modification can serve as a new diagnostic marker for bladder cancer. The inventor detects the m6A methylation modification level of RNA in urine of 200 bladder cancer patients and 400 healthy people, and finds that m6A RNA methylation modulators YTHDF2 and RBMX have potential clinical application values as bladder cancer diagnosis markers, and the sensitivity and the specificity of the m6A RNA methylation modulators YTHDF2 and RBMX are 69.2 percent and 78.6 percent, 72.7 percent and 82.8 percent respectively.

In general, BTA has a threshold of 88ng/mL for bladder cancer diagnosis, a diagnostic sensitivity of 70% and a specificity of 80.7%. However, BTA in some non-bladder cancer patients may be greater than 88ng/mL, so relying on BTA single index detection results to screen early or aid diagnosis often results in false positives. The invention creatively combines the m6A RNA methylation modulator YTHDF2 and the m6A RNA methylation modulator RBMX with BTA as a marker group for screening and auxiliary diagnosis of early bladder cancer and prognosis recurrence risk, and greatly improves the sensitivity, specificity and effectiveness of screening and auxiliary diagnosis of early bladder cancer and prognosis recurrence risk. In the invention, the detection index of the m6A RNA methylation modulator YTHDF2 and the m6A RNA methylation modulator RBMX marker is the expression level of urine, serum, plasma vesicles, tissues or tissue cells.

Alternatively, in some embodiments, when the subject has no overt urological symptoms and the BTA concentration index is less than 88 ng/ml, the model in the detection method is an early bladder cancer risk screening or recurrence risk prognosis model: k (sieve) =a×r (YTHDF 2) +b×r (RBMX) -c×r (BTA); wherein, the value of A is 0.6-0.7, the value of B is 0.5-0.6, the value of C is 0.4-0.5, K (screening) is early bladder cancer risk screening or recurrence risk pre-judging threshold.

Further, in some embodiments, the value of a may range between 0.60 and 0.51.

Further, in some embodiments, the value of a may be in a range between any one or any two of 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, and 0.69.

Further, in some embodiments, the value of a may range between 0.69 and 0.7.

Further, in some embodiments, the value of B may range between 0.50 and 0.51.

Further, in some embodiments, the value of B may be in a range between any one or any two of 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, and 0.59.

Further, in some embodiments, the value of B may be in a range between 0.59 and 0.6.

Further, in some embodiments, the value of C may be in a range between 0.40 and 0.41.

Further, in some embodiments, the value of C may be in a range between any one or any two of 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, and 0.49.

Further, in some embodiments, the value of C may be in a range between 0.49 and 0.5.

Further, the early bladder cancer risk screening or recurrence risk prognosis is determined according to the following conditions: if the concentration index of BTA is less than 88 ng/ml and-0.1 < K (sieve) < 5.1, the subject is judged to be an early stage bladder cancer patient or a recurrent patient. If the BTA concentration index is greater than or equal to 88 ng/ml, the model is not applicable.

Meanwhile, the subjects of the bladder cancer risk screening or recurrence risk prognosis model are typically regular physical examination personnel or bladder cancer healers who need recurrence monitoring.

Alternatively, in some embodiments, when the subject has significant urological symptoms, the BTA concentration indicator is greater than or equal to or less than 88 ng/ml, the model in the detection method is an early bladder cancer assisted diagnosis or bladder cancer curative recurrence assisted diagnosis model: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f×r (YTHDF 2); wherein, the D value is 0.3-0.4, the E value is 0.7-0.8, the F value is 0.8-0.9, and K (auxiliary diagnosis) is the auxiliary diagnosis threshold value for early bladder cancer or the recurrence auxiliary diagnosis threshold value for bladder cancer healer.

Further, in some embodiments, D may range between 0.40 and 0.41.

Further, in some embodiments, D may have a value in a range between any one or any two of 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, and 0.49.

Further, in some embodiments, D may range between 0.49 and 0.5.

Further, in some embodiments, E may have a value in the range between 0.70 and 0.71.

Further, in some embodiments, the value of E may be in a range between any one or any two of 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, and 0.79.

Further, in some embodiments, E may have a value in the range between 0.79 and 0.8.

Further, in some embodiments, E may have a value in the range between 0.80 and 0.81.

Further, in some embodiments, the value of E may be in a range between any one or any two of 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, and 0.89.

Further, in some embodiments, E may have a value in the range between 0.89 and 0.9.

Further, the early bladder cancer auxiliary diagnosis or bladder cancer curative recurrence auxiliary diagnosis is judged according to the following conditions: if the concentration index of BTA is greater than or equal to 88 ng/ml and K (auxiliary diagnosis) is less than-6.6 and less than-1.6, the auxiliary diagnosis is carried out on the subjects, and the subjects are early-stage bladder cancer patients or patients with bladder cancer cure recurrence. In addition, if the concentration index of BTA is less than 88 ng/ml and-6.9 < K (adjuvant diagnosis) < -2.3, the subject is diagnosed as early stage bladder cancer patient or patient with recurrent bladder cancer patient. It is clear that the subjects of this model are typically patients suspected of early stage bladder cancer or those suspected of recurrent bladder cancer healers.

In the two models, R (YTHDF 2) represents the ratio of the expression level of YTHDF2 of the subject to the normal expression level of human body, R (RBMX) represents the ratio of the expression level of RBMX of the subject to the normal expression level of human body, and R (BTA) represents the ratio of the concentration value of BTA of the subject to 88 ng/ml.

Optionally, in some embodiments, the detection reagent includes, but is not limited to, any one of the following methods suitable for effecting detection of the set of markers: chemiluminescence, flow fluorescence, single-molecule array, enzyme-linked immunity, colloidal gold, real-time fluorescent quantitative Polymerase Chain Reaction (PCR), western blotting, electrochemiluminescence and quantum dot technology.

Further, the method for detecting the YTHDF2 expression level by using the real-time fluorescence quantitative PCR method comprises the following steps: the upstream primer of YTDDF 2 was designed 5 '-GTTGGAGGATGGAGCTAGAAA-3' (seq_1) and the downstream primer was designed 5 '-CAGGCAAACCACCGCACTGTC-3' (seq_2). The PCR reaction system used was 12. Mu.l of denatured RNA solution, 2. Mu.l of dNTP mixture, 1. Mu.l of RNase inhibitor (10U/. Mu.l), 4. Mu.l of 5 XRT Buffer, 1. Mu.l of reverse Traace, and a total volume of 20. Mu.l. The PCR reaction procedure is that in the first step, the PCR reaction is performed for 2 minutes at 95 ℃ and 15 seconds at 95 ℃; second, annealing at 60 ℃ for 60 seconds; thirdly, extending for 60 seconds at 95 ℃ and 30 seconds at 55 ℃, and circulating for 40 times; fourthly, extending for 30 seconds at 95 ℃; and fifthly, storing at 4 ℃. The relative expression level of YTHDF2 mRNA was calculated by the double DeltaCt method, and the experiment was repeated 5 times. Statistical analysis was performed using SPSS 19.0 statistical software, and the relative expression levels were expressed as (x.+ -. R). The comparison between the sample and reference groups uses a t-test. The difference of P less than 0.05 is statistically significant.

Further, the Western blotting method comprises the following detection steps: in the first step, 50. Mu.l of freshly prepared protein lysate was added to the sample, the adherent cells were scraped off with a curette, transferred to a 1.5 ml EP tube, and lysed in an ice bath for 1 hour. Centrifuging at 12000rpm at 4deg.C for 30 min, carefully sucking supernatant, and preserving at-80deg.C; BCA working fluid and Cu are added according to the protein standard and the number of samples ²⁺ Preparing a proper amount according to the proportion of 50:1, fully and uniformly mixing, and diluting a standard substance: the standard substance concentrations are 2000, 1500, 1000, 750, 500, 250, 125, 25 and 0 microgram/ml respectively, diluted standard substances are sequentially added into a 96-well plate, 20 microliters of each well is provided, and two compound wells are arranged; thirdly, adding 18 microliters of PBS (phosphate buffer solution) into each well, adding 2 microliters of protein to be detected, adding 200 microliters of prepared BCA working solution into each well, oscillating for 3-5 minutes by an oscillator, incubating for 30 minutes at 37 ℃, measuring an OD value (570 nanometers) by an enzyme-labeled instrument, and calculating the concentration of the protein to be detected according to a standard curve; step four, according to the measured concentration of the protein sample, calculating the loading volume of each group of samples according to the loading amount of 20-50 micrograms of each loading hole, mixing with a 6 XBuffer loading Buffer solution, balancing with a protein lysate, carrying out instantaneous centrifugation and mixing uniformly, boiling the sample at 95 ℃ for 5 minutes, and then quickly inserting into ice for cooling for 5 minutes; fifth, DSD-PAGE (12% separation gel, 5% concentration gel, 90 volts, about 1.5-2 hours, switchable voltage 120 volts after sample reaches separation gel) separates sample proteins. Taking down the gel, cutting according to the corresponding molecular weight, spreading the PVDF film on the gel, placing the gel between two layers of filter paper, placing the gel at the cathode, placing the PVDF film in a transfer electrophoresis tank, performing imprinting electrophoresis with constant current of 190 milliamperes, and transferring the proteins in the PAGE to the PVDF film. A first part After a fixed period of time, the PVDF film was removed and placed in a 5% strength blocking solution at 37℃for 1.5 hours. The blocking solution was discarded, TTBS diluted primary antibody was added, and 4 degrees celsius was left overnight. The next day TTBS was washed for 5 min x 3, and TTBS diluted peroxidase-labeled secondary antibody was added and incubated at 37 degrees celsius for 1.5 hours. TTBS washes for 5 min×3, TBS washes for 5 min×1, enhanced Chemiluminescence (ECL) detects YTHDF2 expression, and finally Image J software is used to scan the grey scale values of the resulting film. The experiment was repeated 5 times. Statistical analysis was performed using SPSS 19.0 statistical software, and the relative expression levels were expressed as (x.+ -. R). The comparison between the sample and reference groups uses a t-test. The difference of P less than 0.05 is statistically significant.

Further, the method for detecting the RBMX expression level by using the real-time fluorescence quantitative PCR method comprises the following steps: firstly, designing an upstream primer 5 '-TAGGACGCCGAGAGGACTAG-3' (seq_3) of RBMX; the downstream primer is 5 '-GTTCAGGGTCGAAGCTGATG-3' (seq_4). The PCR reaction system used was 12. Mu.l of denatured RNA solution, 2. Mu.l of dNTP mixture, 1. Mu.l of RNase inhibitor (10U/. Mu.l), 4. Mu.l of 5 XRT Buffer, 1. Mu.l of reverse Traace, and a total volume of 20. Mu.l. The PCR reaction procedure is that in the first step, the PCR reaction is performed for 2 minutes at 95 ℃ and then for 30 seconds at 95 ℃; second, annealing at 55 ℃ for 30 seconds; thirdly, extending for 30 seconds at 75 ℃, and circulating for 45 times; fourthly, extending for 60 seconds at 70 ℃; and fifthly, storing at 4 ℃. The relative expression level of RBMX mRNA was calculated by the double DeltaCt method and the experiment was repeated 5 times. Statistical analysis was performed using SPSS 19.0 statistical software, and the relative expression levels were expressed as (x.+ -. R). The comparison between the sample and reference groups uses a t-test. The difference of P less than 0.05 is statistically significant.

Further, the Western blotting method comprises the following steps: firstly, adding 600 microliters of PIPA precooled at 4 ℃ and 6 microliters of PMSF into a sample, shaking and uniformly mixing on a shaking table at 4 ℃ for 5 minutes, then placing the mixture on an oscillator to shake for 30 seconds, enabling RIPA and cells to fully contact and react, placing the mixture on the shaking table at 4 ℃ again for 5 minutes, then taking out and shaking, and performing cyclic operation for 5 times, wherein the total reaction time is about 30 minutes; step two, transferring the lysate into a 1.5 ml Eppendorf tube, and centrifuging at 14000 rpm and 4 ℃ for 25 minutes; collecting supernatant, and preserving at-80 ℃; thirdly, taking a 96-well plate, adding 200 microliters of Bradford buffer solution into each well, adding 2 microliters of protein sample to be tested, and shaking and mixing uniformly; placing the sample into an enzyme labeling instrument, measuring absorbance at 595nm, calculating 30 micrograms of system Loading quantity and 5×loading buffer according to a standard curve, and transferring into a 200 microliter Eppendorf tube according to each quantity; heating the sample at 95 degrees celsius for 5 minutes; fourthly, cleaning and manufacturing an SDS-PAGE gel glass plate, fixing the SDS-PAGE gel glass plate on a gel manufacturing frame after airing to ensure that the bottom is not leaked, manufacturing gel according to the instruction of an SDS-PAGE gel preparation kit, concentrating gel at an upper layer, and separating a layer at a lower layer; the electrophoresis buffer solution comprises the following components: 30 g of Tris, 144 g of glycine and 10 g of SDS, adding deionized water to 10000 ml, and uniformly mixing; the transfer buffer solution comprises the following components: 2.9 g glycine, 5.8 g Tris, 0.37 g SDS, 50 ml methanol, adding deionized water to 1000 ml, mixing well; TBST solution is composed of 24.2 g Tris, 80 g NaCl and deionized water to 10000 ml, 5 ml Tween-20 is added, and the mixture is uniformly mixed; the sealing liquid comprises the following components: 2.5 g of skimmed milk powder is dissolved in 50 ml of TBST liquid and evenly mixed; fifthly, fixing the prepared gel in an electrophoresis tank, adding a proper amount of electrophoresis buffer solution, adding a protein marker into a first hole of a gel hole, adding a protein sample as required at Yu Kong, electrophoresis under a constant voltage of 50 volts until the markers are separated, and then separating the protein under a constant voltage of 80 volts; after electrophoresis is finished, taking out gel, cutting the upper concentrated gel, cutting a proper PVDF film according to the size of the gel, then tightly attaching the gel and the film, smoothing, removing middle bubbles, placing two layers of filter paper for protection outside the gel and the film, putting into a film rotating clamping plate, putting into an electric rotating groove, adding a liquid rotating buffer solution, and rotating the film for 130 minutes under the constant current of 250 milliamps; sixthly, taking out the PVDF membrane after the electric transfer is finished, and washing 3 times by using TBST liquid on a shaking table at normal temperature for 5 minutes each time; after washing, taking out the PVDF membrane, putting the PVDF membrane into a sealing liquid, and sealing the PVDF membrane on a shaking table for 1 hour at normal temperature; after the sealing is finished, taking out the PVDF film, washing 3 times by TBST liquid on a shaking table at normal temperature for 10 minutes each time; eighth, after washing, taking out PVDF membrane, putting into primary antibody, and shaking on a shaker at 4 ℃ overnight; the next day, the PVDF film is taken out, and washed 3 times with TBST liquid at normal temperature on a shaking table for 10 minutes each time; then taking out the PVDF film, adding a secondary antibody, and reacting on a shaking table for 1 hour at normal temperature; ninth, taking out PVDF membrane, washing with TBST liquid on a shaker at normal temperature for 3 times, each time for 5 minutes; preparing a developing solution according to the specification of the ECL chemiluminescence hypersensitive display kit, taking out the PVDF film, adding the developing solution, detecting the expression of RBMX by an enhanced chemiluminescence method (ECL), and finally scanning the gray value of the obtained film by adopting Image J software. The experiment was repeated 5 times. Statistical analysis was performed using SPSS 19.0 statistical software, and the relative expression levels were expressed as (x.+ -. R). The comparison between the sample and reference groups uses a t-test. The difference of P less than 0.05 is statistically significant.

In a second aspect, the present invention provides a system for bladder cancer assisted diagnosis of a patient suspected of early stage bladder cancer or a bladder cancer curative suspected of recurrent bladder cancer, for bladder cancer recurrence risk screening of a regular physical examination personnel or a bladder cancer curative in need of recurrence monitoring.

The system comprises an information acquisition module, an analysis module and a reporting module.

Further, the information acquisition module is used for executing the operation of acquiring the subject detection information and the urological symptom information; the detection information comprises the expression quantity of YTHDF2 and RBMX and the concentration value of BTA of a subject; the urological symptom information is whether obvious urological symptoms exist or not; the overt urological symptoms refer to complaints from urological outpatients, such as: frequent urination, urgent urination, painful urination, leukouria, sometimes hematuria, even macroscopic hematuria, and nonspecific symptoms such as discomfort, chills, fever, headache, nausea, emesis, inappetence, etc. in the bladder area.

Further, the analysis module is used for executing the calculation analysis operation of the correlation threshold value by substituting the detection information into a calculation model.

Alternatively, in some embodiments, when the subject has no overt urological symptoms and the BTA concentration indicator is less than 88 ng/ml, the model in the detection method is an early bladder cancer risk screening or recurrence risk prognosis model: k (sieve) =a×r (YTHDF 2) +b×r (RBMX) -c×r (BTA); wherein, the value of A is 0.6-0.7, the value of B is 0.5-0.6, the value of C is 0.4-0.5, K (screening) is early bladder cancer risk screening or recurrence risk pre-judging threshold.

Further, in some embodiments, the value of a may range between 0.60 and 0.51.

Further, in some embodiments, the value of a may be in a range between any one or any two of 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, and 0.69.

Further, in some embodiments, the value of a may range between 0.69 and 0.7.

Further, in some embodiments, the value of B may range between 0.50 and 0.51.

Further, in some embodiments, the value of B may be in a range between any one or any two of 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, and 0.59.

Further, in some embodiments, the value of B may be in a range between 0.59 and 0.6.

Further, in some embodiments, the value of C may be in a range between 0.40 and 0.41.

Further, in some embodiments, the value of C may be in a range between any one or any two of 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, and 0.49.

Further, in some embodiments, the value of C may be in a range between 0.49 and 0.5.

Meanwhile, the subjects of the bladder cancer risk screening or recurrence risk prognosis model are typically regular physical examination personnel or bladder cancer healers who need recurrence monitoring.

Alternatively, in some embodiments, when the subject has overt urological symptoms, the BTA concentration indicator is greater than or equal to or less than 88 ng/ml, the model in the detection method is an early bladder cancer assisted diagnosis or bladder cancer curative recurrence assisted diagnosis model: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f×r (YTHDF 2); wherein, the D value is 0.3-0.4, the E value is 0.7-0.8, the F value is 0.8-0.9, and K (auxiliary diagnosis) is the auxiliary diagnosis threshold value for early bladder cancer or the recurrence auxiliary diagnosis threshold value for bladder cancer healer.

Further, in some embodiments, D may range between 0.40 and 0.41.

Further, in some embodiments, D may have a value in a range between any one or any two of 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, and 0.49.

Further, in some embodiments, D may range between 0.49 and 0.5.

Further, in some embodiments, E may have a value in the range between 0.70 and 0.71.

Further, in some embodiments, the value of E may be in a range between any one or any two of 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, and 0.79.

Further, in some embodiments, E may have a value in the range between 0.79 and 0.8.

Further, in some embodiments, E may have a value in the range between 0.80 and 0.81.

Further, in some embodiments, the value of E may be in a range between any one or any two of 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, and 0.89.

Further, in some embodiments, E may have a value in the range between 0.89 and 0.9.

In the two models, R (YTHDF 2) represents the ratio of the expression level of YTHDF2 of the subject to the normal expression level of human body, R (RBMX) represents the ratio of the expression level of RBMX of the subject to the normal expression level of human body, and R (BTA) represents the ratio of the concentration value of BTA of the subject to 88 ng/ml.

Further, the reporting module is configured to perform a reporting operation of determining a health condition of the subject based on the threshold and BTA concentration information.

Further, the early bladder cancer risk screening or recurrence risk prognosis is determined as follows: if the concentration index of BTA is less than 88 ng/ml and-0.1 < K (sieve) < 5.1, the subject is judged to be an early stage bladder cancer patient or a recurrent patient. If the BTA concentration index is greater than or equal to 88 ng/ml, the model is not applicable.

Further, the early bladder cancer auxiliary diagnosis or bladder cancer curative recurrence auxiliary diagnosis is judged according to the following conditions: if the concentration index of BTA is greater than or equal to 88 ng/ml and K (auxiliary diagnosis) is less than-6.6 and less than-1.6, the auxiliary diagnosis is carried out on the subjects, and the subjects are early-stage bladder cancer patients or patients with recurrent bladder cancer patients. In addition, if the concentration index of BTA is less than 88 ng/ml and-6.9 < K (adjuvant diagnosis) < -2.3, the subject is diagnosed as early stage bladder cancer patient or patient with recurrent bladder cancer patient. It is clear that the subjects of this model are typically patients suspected of early stage bladder cancer or those suspected of recurrent bladder cancer healers.

Alternatively, in some embodiments, the reporting module may display, by means of a screen display, voice broadcast or print, the results of bladder cancer recurrence risk screening by a regular physical examination person or a bladder cancer curative who requires recurrence monitoring and/or the results of bladder cancer assisted diagnosis by a patient suspected of early stage bladder cancer or a bladder cancer curative who is suspected of recurrence.

In a third aspect, the invention provides a kit for screening and/or aiding diagnosis of bladder cancer, the kit comprising detection reagents for YTHDF2, RBMX and BTA.

Optionally, in some embodiments, the kit includes instructions for use that record a model for bladder cancer screening and/or bladder cancer assisted diagnosis.

When the subject has no obvious urological symptoms and the concentration index of BTA is less than 88 ng/ml, the model in the detection method is an early bladder cancer risk screening or recurrence risk prediction model: k (sieve) =a×r (YTHDF 2) +b×r (RBMX) -c×r (BTA); wherein, the value of A is 0.6-0.7, the value of B is 0.5-0.6, the value of C is 0.4-0.5, K (screening) is early bladder cancer risk screening or recurrence risk pre-judging threshold.

When the subject has obvious urological symptoms and the concentration index of BTA is greater than or equal to or less than 88 ng/ml, the model in the detection method is an early bladder cancer auxiliary diagnosis or bladder cancer curative recurrence auxiliary diagnosis model: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f×r (YTHDF 2); wherein, the D value is 0.3-0.4, the E value is 0.7-0.8, the F value is 0.8-0.9, and K (auxiliary diagnosis) is the auxiliary diagnosis threshold value for early bladder cancer or the recurrence auxiliary diagnosis threshold value for bladder cancer healer.

In the two models, R (YTHDF 2) represents the ratio of the expression level of YTHDF2 of the subject to the normal expression level of human body, R (RBMX) represents the ratio of the expression level of RBMX of the subject to the normal expression level of human body, and R (BTA) represents the ratio of the concentration value of BTA of the subject to 88 ng/ml.

Drawings

FIG. 1 is a flow chart of screening and/or auxiliary diagnosis of early screening and prognosis of bladder cancer recurrence in accordance with the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. FIG. 1 is a flow chart of screening and/or auxiliary diagnosis of early screening and prognosis of bladder cancer recurrence in accordance with the present invention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1 differential expression detection of YTHDF2 in patients with early stage bladder cancer and individuals not with bladder cancer

YTDDF 2 can detect the expression difference between patients with early bladder cancer and individuals without bladder cancer (200 sites each) by different detection methods;

the real-time fluorescence quantitative PCR method is used for detecting the expression quantity of YTHDF2, and the result shows that: the average value of the expression level of early bladder cancer patients is 2.81+/-0.67, the average value of benign bladder disease patients is 1.23+/-0.09, and the p value of two groups of statistical tests is less than 0.0001. The average value of healthy individuals is 1.04+/-0.06, and the p value of the statistical test of the early bladder cancer patient group and the healthy individual group is less than 0.0001;

The Western blotting method is used for detecting the expression level of YTHDF2, and the result shows that: the average value of the expression level of early bladder cancer patients is 2.98+/-0.93, the average value of benign bladder disease patients is 1.34+/-0.12, and the p value of two groups of statistical tests is less than 0.0001. The average value of healthy individuals is 1.09+/-0.07, and the p value of the statistical test of the early bladder cancer patient group and the healthy individual group is less than 0.0001;

different detection methods show that YTHDF2 has expression difference between patients with early bladder cancer and individuals without bladder cancer.

Example 2 detection of expression of YTHDF2 in different cancer patients

The present example detects YTDDF 2 expression levels in different cancer patients by a real-time fluorescent quantitative PCR method. The results show that YTHDF2 does not have significant differences in plasma of other tumor patients or benign disease patients commonly found in the urinary system; there was no significant difference in expression of YTHDF2 in renal pelvis carcinoma, ureter carcinoma, renal tumor, urinary tract carcinoma, renal cyst, renal hamartoma and corresponding healthy individuals (200 sites each), and the average values for each group were: 1.37.+ -. 0.16,1.30.+ -. 0.14,1.42.+ -. 0.17,1.33.+ -. 0.15,1.25.+ -. 0.09,1.27.+ -. 0.10,1.07.+ -. 0.08. The results indicate that YTHDF2 has specific expression in bladder cancer patients.

Example 3 differential expression detection of RBMX in patients with early stage bladder cancer and individuals not having bladder cancer

The RBMX can detect the expression difference of patients with early bladder cancer and individuals without bladder cancer (200 sites each) by different detection methods;

the RBMX expression quantity is detected by a real-time fluorescence quantitative PCR method, and the result shows that: the average value of the expression level of early bladder cancer patients is 3.74+/-0.85, the average value of benign bladder disease patients is 1.37+/-0.13, and the p value of two groups of statistical tests is less than 0.0001. The average value of healthy individuals is 1.12+/-0.08, and the p value of the statistical test of the early bladder cancer patient group and the healthy individual group is less than 0.0001;

the RBMX expression level is detected by a Western blotting method, and the result shows that: the average value of the expression level of early bladder cancer patients is 3.88+/-0.93, the average value of benign bladder disease patients is 1.46+/-0.15, and the p value of two groups of statistical tests is less than 0.0001. The average value of healthy individuals is 1.15+/-0.09, and the p value of the statistical test of the early bladder cancer patient group and the healthy individual group is less than 0.0001;

different detection methods show that RBMX has expression difference between patients with early bladder cancer and individuals without bladder cancer.

Example 4 detection of RBMX expression in patients with different cancers

The present example detects RBMX expression levels in different cancer patients by real-time fluorescent quantitative PCR. The results show that RBMX does not have significant differences in plasma of other tumor patients or benign disease patients commonly found in the urinary system; there was no significant difference in expression of YTHDF2 in renal pelvis carcinoma, ureter carcinoma, renal tumor, urinary tract carcinoma, renal cyst, renal hamartoma and corresponding healthy individuals (200 sites each), and the average values for each group were: 1.53+ -0.19,1.56 + -0.23,1.63 + -0.27,1.59 + -0.25,1.50 + -0.17,1.48 + -0.16,1.16 + -0.10. The results indicate that RBMX has specific expression in bladder cancer patients.

Example 5 RBMX, YTDDF 2 and BTA in combination for auxiliary diagnosis of early bladder cancer

200 urological outpatients were enrolled. The group-entering standard is that BTA is greater than or equal to 88 ng/ml, urinary discomfort symptoms exist in urinary system ultrasonic examination, urinary system ultrasonic examination indicates that bladder cancer occupying lesions and the like are found, but cystoscopy is not performed yet. Detecting RBMX and YTHDF2 in urine of the 200 patients by using a real-time fluorescence quantitative PCR method, and substituting the RBMX and YTHDF2 into an auxiliary diagnosis model of bladder cancer by combining BTA values: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f× (YTHDF 2); wherein, the value of D is 0.3-0.4, the value of E is 0.7-0.8, the value of F is 0.8-0.9, K (auxiliary diagnosis) is an auxiliary diagnosis threshold value of early bladder cancer; auxiliary diagnosis mode: reporting the subject as an early stage bladder cancer patient if-6.6 < K (adjuvant diagnosis) < -1.6;

Based on whether K (co-diagnosis) of the 200 model results is in the corresponding interval, the results are given whether patients meeting the group-entry criteria have early bladder cancer. According to the bladder cancer diagnosis and treatment guide, 200 patients are followed up until a clear clinical diagnosis result is achieved. Comparing the clinical diagnosis result with the bladder cancer auxiliary diagnosis model result, the result shows that: the sensitivity of the auxiliary diagnosis of the independent BTA bladder cancer is 66.7%, the specificity is 76.2%, and the comprehensive accuracy is 64.1%; the sensitivity of the single YTDDF 2 for auxiliary diagnosis of early bladder cancer is 69.2 percent, the specificity is 78.6 percent, and the comprehensive accuracy is 67.4 percent; the sensitivity of the single RBMX for auxiliary diagnosis of early bladder cancer is 72.7%, the specificity is 82.8%, and the comprehensive accuracy is 70.4%; the sensitivity of the auxiliary diagnosis of the early bladder cancer is 74.1 percent, the specificity is 77.3 percent, the comprehensive accuracy is 70.6 percent, the sensitivity of the auxiliary diagnosis of the early bladder cancer is 75.1 percent, the specificity is 80.3 percent, the comprehensive accuracy is 72.6 percent, the sensitivity of the auxiliary diagnosis of the early bladder cancer is 86.7 percent, the specificity is 84.3 percent, the comprehensive accuracy is 82.9 percent, the sensitivity of the auxiliary diagnosis of the early bladder cancer is 98.5 percent, the specificity is 92.1 percent, and the comprehensive accuracy is 90.4 percent.

Example 6 RBMX, YTDDF 2 and BTA in combination for auxiliary diagnosis of early bladder cancer

200 urological outpatients were enrolled. The group-entering standard is that BTA is less than 88 ng/ml, urinary discomfort symptoms exist in the urinary system, urinary system ultrasonic examination indicates that bladder cancer occupying lesions and the like exist, but cystoscopy is not performed yet. Detecting RBMX and YTHDF2 in urine of the 200 patients by using a real-time fluorescence quantitative PCR method and substituting BTA values into a bladder cancer auxiliary diagnosis model: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f× (YTHDF 2); wherein, the value of D is 0.3-0.4, the value of E is 0.7-0.8, the value of F is 0.8-0.9, K (auxiliary diagnosis) is an auxiliary diagnosis threshold value of early bladder cancer; auxiliary diagnosis mode: reporting the subject as an early stage bladder cancer patient if-6.9 < K (adjuvant diagnosis) < -2.3;

based on whether K (co-diagnosis) of the 200 model results is in the corresponding interval, the results are given whether patients meeting the group-entry criteria have early bladder cancer. According to the bladder cancer diagnosis and treatment guide, 200 patients are followed up until a clear clinical diagnosis result is achieved. Comparing the clinical diagnosis result with the bladder cancer auxiliary diagnosis model result, the result shows that: the sensitivity of the auxiliary diagnosis of the independent BTA bladder cancer is 0%, the specificity is 0%, and the comprehensive accuracy is 0%; the sensitivity of the single YTDDF 2 for auxiliary diagnosis of early bladder cancer is 73.5 percent, the specificity is 80.4 percent, and the comprehensive accuracy is 71.7 percent; the sensitivity of the single RBMX for auxiliary diagnosis of early bladder cancer is 78.8%, the specificity is 87.5%, and the comprehensive accuracy is 76.4%; the sensitivity of the auxiliary diagnosis of the early bladder cancer is 88.5%, the specificity is 87.3% and the comprehensive accuracy is 84.9% by combining RBMX and YTDDF 2, and the sensitivity of the auxiliary diagnosis of the early bladder cancer is 98.9%, the specificity is 95.4% and the comprehensive accuracy is 92.8% by combining RBMX, THDF2 and BTA.

EXAMPLE 7 RBMX, YTDDF 2 in combination with BTA for the recurrent auxiliary diagnosis of bladder cancer patients

200 bladder cancer healers were enrolled. The group-entering standard is that BTA is greater than or equal to 88 ng/ml, urinary discomfort symptoms exist in urinary system ultrasonic examination, urinary system ultrasonic examination indicates that bladder cancer occupying lesions and the like are found, but cystoscopy is not performed yet. Detecting RBMX and YTHDF2 in urine of the 200 patients by using a real-time fluorescence quantitative PCR method and substituting BTA values into a bladder cancer auxiliary diagnosis model: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f× (YTHDF 2); wherein, the value of D is 0.3-0.4, the value of E is 0.7-0.8, the value of F is 0.8-0.9, K (auxiliary diagnosis) is the recurrent auxiliary diagnosis threshold of the patient with bladder cancer cure; auxiliary diagnosis mode: reporting the subject as a recurrent patient for a bladder cancer curative if-6.6 < K (adjuvant diagnosis) < -1.6;

based on whether K (adjuvant diagnosis) of the 200 model results are in the corresponding interval, the results of whether the patients meeting the group-entering standard recur or not the bladder cancer healer are given. According to the bladder cancer diagnosis and treatment guide, 200 patients are followed up until a clear clinical diagnosis result is achieved. Comparing the clinical diagnosis result with the bladder cancer auxiliary diagnosis model result, the result shows that: the sensitivity of the single BTA for auxiliary diagnosis of recurrence of bladder cancer healers is 68.2%, the specificity is 75.7%, and the comprehensive accuracy is 65.4%; the sensitivity of the single YTHDF2 for auxiliary diagnosis of recurrence of bladder cancer healers is 68.1%, the specificity is 77.3%, and the comprehensive accuracy is 66.6%; the sensitivity of the single RBMX for auxiliary diagnosis of recurrence of the bladder cancer curative is 74.1%, the specificity is 83.2%, and the comprehensive accuracy is 71.7%; the sensitivity of the auxiliary diagnosis of the recurrence of the bladder cancer curative is 72.2 percent, the specificity is 76.9 percent, the comprehensive accuracy is 70.1 percent, the sensitivity of the auxiliary diagnosis of the recurrence of the bladder cancer curative is 76.8 percent, the specificity is 82.5 percent, the comprehensive accuracy is 73.5 percent, the sensitivity of the auxiliary diagnosis of the recurrence of the bladder cancer curative is 85.9 percent, the specificity is 86.7 percent, the comprehensive accuracy is 83.2 percent, the sensitivity of the auxiliary diagnosis of the recurrence of the bladder cancer curative is 98.7 percent, the specificity is 93.5 percent, and the comprehensive accuracy is 91.2 percent.

EXAMPLE 8 RBMX, YTDDF 2 in combination with BTA for the recurrent auxiliary diagnosis of bladder cancer curers

200 bladder cancer healers were enrolled. The group-entering standard is that BTA is less than 88 ng/ml, urinary discomfort symptoms exist in the urinary system, urinary system ultrasonic examination indicates that bladder cancer occupying lesions and the like exist, but cystoscopy is not performed yet. Detecting RBMX and YTHDF2 in urine of the 200 patients by using a real-time fluorescence quantitative PCR method and substituting BTA values into a bladder cancer auxiliary diagnosis model: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f× (YTHDF 2); wherein, the value of D is 0.3-0.4, the value of E is 0.7-0.8, the value of F is 0.8-0.9, K (auxiliary diagnosis) is the recurrent auxiliary diagnosis threshold of the patient with bladder cancer cure; auxiliary diagnosis mode: reporting the subject as a recurrent patient for a bladder cancer curative if-6.9 < K (adjuvant diagnosis) < -2.3;

based on whether K (adjuvant diagnosis) of the 200 model results are in the corresponding interval, the results of whether the patients meeting the group-entering standard are bladder cancer healers or not are recurrent are given. According to the bladder cancer diagnosis and treatment guide, 200 patients are followed up until a clear clinical diagnosis result is achieved. Comparing the clinical diagnosis result with the bladder cancer auxiliary diagnosis model result, the result shows that: the sensitivity of the auxiliary diagnosis of the independent BTA bladder cancer is 0%, the specificity is 0%, and the comprehensive accuracy is 0%; the sensitivity of the single YTHDF2 for auxiliary diagnosis of recurrence of the bladder cancer curative is 72.8%, the specificity is 81.1%, and the comprehensive accuracy is 70.8%; the sensitivity of the single RBMX for auxiliary diagnosis of recurrence of the bladder cancer curative is 80.2%, the specificity is 88.9%, and the comprehensive accuracy is 77.8%; the sensitivity of auxiliary diagnosis on recurrence of bladder cancer patients is 89.3%, the specificity is 88.4% and the comprehensive accuracy is 86.7% by combining RBMX and YTDDF 2, and the sensitivity of auxiliary diagnosis on recurrence of bladder cancer patients is 99.0%, the specificity is 94.7% and the comprehensive accuracy is 92.6% by combining RBMX, THDF2 and BTA.

Example 9 RBMX, YTHDF2 in combination with BTA for Risk management for screening of bladder cancer in physical examination patients

400 normal physical examination persons between 30 and 60 years old were enrolled, had no urological symptoms, and had BTA concentration examination results of less than 88 ng/ml. Detecting the expression quantity of RBMX and YTHDF2 in the urine of the 400 persons by using a real-time fluorescence quantitative PCR method and substituting the expression quantity into an early bladder cancer risk screening model by combining with a BTA value: k (sieve) =a×r (YTHDF 2) +b×r (RBMX) -c×r (BTA); wherein, the value of A is 0.6-0.7, the value of B is 0.5-0.6, the value of C is 0.4-0.5, K (screening) is the early bladder cancer risk screening threshold; auxiliary screening mode: -0.1 < K (sieve) < 5.1, determining the subject as an early stage bladder cancer patient;

according to whether the K (screening) results of the 400 models are in the corresponding intervals, the results of whether the group entering personnel without any urological symptoms are at risk of early bladder cancer are obtained. The patients who are prompted with risks are followed for 6 months until clear clinical diagnosis results are achieved, such as no urinary system diseases, benign lesions of the urinary system and early bladder cancer. Comparing the clinical diagnosis result with the model result before 6 months, the result shows that the sensitivity of single BET for auxiliary screening of early bladder cancer is 0%, the specificity is 0%, and the comprehensive accuracy is 0%; the sensitivity of single YTHDF2 for auxiliary screening of early bladder cancer is 75.7%, the specificity is 83.4%, and the comprehensive accuracy is 73.2%; the sensitivity of the single RBMX for auxiliary screening of early bladder cancer is 81.1%, the specificity is 86.0%, and the comprehensive accuracy is 78.2%; the sensitivity of the auxiliary screening of the early bladder cancer is 87.8%, the specificity is 89.6% and the comprehensive accuracy is 86.3% by combining RBMX and YTDDF 2, and the sensitivity of the auxiliary screening of the early bladder cancer is 98.6%, the specificity is 95.1% and the comprehensive accuracy is 93.4% by combining RBMX, THDF2 and BTA.

EXAMPLE 10 Risk management of RBMX, YTHDF2 in combination with BTA for asymptomatic bladder cancer curative relapse screening

200 asymptomatic bladder cancer healers in the group, without urological symptoms, and the examination result of BTA concentration is less than 88 ng/ml. Detecting the expression quantity of RBMX and YTHDF2 in urine of the person by using a real-time fluorescence quantitative PCR method, and substituting the expression quantity into a recurrence risk screening model of asymptomatic bladder cancer healers by combining with BTA values: k (sieve) =a×r (YTHDF 2) +b×r (RBMX) -c×r (BTA); wherein, the value of A is 0.6-0.7, the value of B is 0.5-0.6, the value of C is 0.4-0.5, K (screen) is the recurrence risk screening threshold of the asymptomatic bladder cancer healer; auxiliary screening mode: -0.1 < K (sieve) < 5.1, then determining that the subject is a patient with asymptomatic bladder cancer cure recurrence;

and obtaining the results of whether the group-in asymptomatic bladder cancer healers without any urological symptoms have the risk of bladder cancer recurrence according to whether K (screening) results of the 200 models are in the corresponding intervals. Persons who are prompted for risk are followed for 6 months until a clear clinical diagnosis of recurrence is achieved. Comparing the clinical diagnosis result with the model result before 6 months, wherein the result shows that the sensitivity of the single BTA for auxiliary diagnosis of early bladder cancer is 0%, the specificity is 0%, and the comprehensive accuracy is 0%; the sensitivity of the single YTHDF2 for auxiliary screening of early bladder cancer is 77.8%, the specificity is 84.1%, and the comprehensive accuracy is 75.6%; the sensitivity of the single RBMX for auxiliary screening of early bladder cancer is 82.4%, the specificity is 87.7%, and the comprehensive accuracy is 79.6%; the sensitivity of the auxiliary screening of the early bladder cancer is 88.1%, the specificity is 90.2% and the comprehensive accuracy is 86.9% by combining RBMX and YTDDF 2, and the sensitivity of the auxiliary screening of the early bladder cancer is 98.8%, the specificity is 96.2% and the comprehensive accuracy is 94.3% by combining RBMX, THDF2 and BTA.

Comparative example 1 marker for bladder cancer detection and detection kit

The prior art document 1 (marker and detection kit for bladder cancer detection, patent application No. 202110746113.6, application publication No. CN 113759115A) uses nuclear matrix protein 22 (NMP 22), carcinoembryonic antigen (CEA), bladder Tumor Antigen (BTA) and cytokeratin 19 fragment (CYFRA 21-1) as combined markers, and the detection sensitivity and specificity of bladder cancer are 98.4% and 80%, respectively, and furthermore, the model adopted in the document is constructed directly by the concentration of NMP22, CEA, BTA and CYFRA21-1, and bladder cancer risk screening for recurrence of physical examination personnel or asymptomatic bladder cancer healers is not considered.

Comparative example 2 primer probe composition, kit and system for early screening or prognosis monitoring of bladder cancer

The primer probe composition, kit and system of the prior document 2 (early screening or prognosis monitoring of bladder cancer, patent application number 202111147804.0, application publication number: CN 113930509A) is optimized in combination with the primer probe composition of RASSF1A gene, TWIST1 gene and ZNF154 gene, and the detection sensitivity and specificity of the early screening and prognosis monitoring of bladder cancer for patients for prevention of bladder cancer are 85.71% and 100% respectively, and the screening of bladder cancer risk for recurrence by physical examination personnel or asymptomatic bladder cancer healers is not considered.

Comparative example 3 diagnostic value of urine nuclear matrix protein 22 in combination with bladder tumor antigen detection for bladder cancer

The prior document 3 (urine nuclear matrix protein 22 combined with bladder tumor antigen detection has diagnostic value on bladder cancer, tumor research and clinic, 2020, 32 (11) 772-775) uses urine nuclear matrix protein 22 combined with bladder tumor antigen as detection markers, the detection sensitivity and specificity on bladder cancer are respectively 96.98% and 61.11%, and the screening of bladder cancer risk of recurrence of physical examination personnel or asymptomatic bladder cancer healers is not considered.

In summary, neither single detection nor multiple combined detection, nor combined diagnosis with the existing bladder cancer markers bladder tumor antigens, is capable of exceeding 90% simultaneously in early screening or auxiliary diagnostic sensitivity and specificity at risk for bladder cancer. In addition, the prior art schemes do not have detection methods, systems and kits for screening the risk of bladder cancer of physical examination personnel and screening the recurrent risk of a bladder cancer curative and assisting diagnosis, thereby influencing the integrity of the risk screening and or assisting diagnosis of bladder cancer.

According to the embodiment and the comparative example, RBMX, THDF2 and BTA are combined for the first time, so that early bladder cancer, bladder cancer cured person recurrence and bladder benign diseases and early bladder cancer (early stage) and healthy individual differential diagnosis are realized, and further, auxiliary diagnosis of early bladder cancer and or bladder cancer cured person recurrence and bladder cancer risk management of healthy individuals can be better realized clinically. As the sensitivity and the specificity are simultaneously over 90 percent, the auxiliary diagnosis and the early risk screening effectiveness of the recurrence of the early bladder cancer and/or bladder cancer healers are greatly improved, and the auxiliary diagnosis and the early risk screening accuracy are ideal.

The RBMX and THDF2 expression level detection in the invention can be realized by a real-time fluorescence quantitative PCR method and a Western blotting method, so that the auxiliary diagnosis result of individuals at risk of bladder cancer can be verified by a clinically optional method.

The urine sample treatment in the THDF2 expression level detection in the above embodiment can be performed with reference to the following steps:

firstly, 50-100 milliliters of morning urine cleaning midrange urine is left; -storing the sample in a refrigerator at 20 ℃; requiring that the subject does not operate in urinary tract cavities such as cystoscope, catheterization and the like within 1 week;

secondly, adding 1 ml of urine into an EP tube, centrifuging at 4 ℃ and 12 rpm for 10 minutes, and discarding the supernatant; adding Trizol, shaking with a vortex mixer to completely lyse urinary sediment cells, and standing at room temperature for 3-5 min; 200 microliters of chloroform was added thereto, vigorously shaken for 15-30 seconds, and allowed to stand at room temperature for 5 minutes. Centrifugation at 12000rpm at 4℃for 15 min, visible liquid stratification;

third, the upper clear water was gently phase shifted to a new 1.5 ml EP tube, then 500 μl isopropanol was added, mixed gently upside down, left at room temperature for 15 minutes, centrifuged at 12000rpm for 15 minutes at 4 degrees celsius, the supernatant gently discarded, 1 ml 75% ethanol was added, centrifuged at 7000rpm for 5 minutes at 4 degrees celsius, and the supernatant discarded. Standing at room temperature for several minutes until the ethanol residue of RNA at the bottom of the tube volatilizes completely;

Fourthly, adding 200 microliters of RIPA lysate into the tube in the third step, flushing the mixed solution by a pipette, and placing a centrifuge tube filled with the solution on ice for 15 minutes to extract THDF2 and RBMX respectively;

fifth, the extracted solution was centrifuged at 10000rpm at 4℃for 15 minutes to leave an upper solution. The upper layer solution can be directly used for analysis or stored in a refrigerator at-80 ℃ for a long time;

sixthly, respectively detecting the THDF2 expression level and the RBMX expression level by a real-time fluorescence quantitative PCR method and a Western blotting method; wherein, the method for detecting the THDF2 expression quantity by using the real-time fluorescence quantitative PCR method comprises the following steps: the upstream primer of YTDDF 2 was designed 5 '-GTTGGAGGATGGAGCTAGAAA-3' (seq_1) and the downstream primer was designed 5 '-CAGGCAAACCACCGCACTGTC-3' (seq_2). The PCR reaction system used was 12. Mu.l of denatured RNA solution, 2. Mu.l of dNTP mixture, 1. Mu.l of RNase inhibitor (10U/. Mu.l), 4. Mu.l of 5 XRT Buffer, 1. Mu.l of reverse Traace, and a total volume of 20. Mu.l. The PCR reaction procedure is that in the first step, the PCR reaction is performed for 2 minutes at 95 ℃ and 15 seconds at 95 ℃; second, annealing at 60 ℃ for 60 seconds; thirdly, extending for 60 seconds at 95 ℃ and 30 seconds at 55 ℃, and circulating for 40 times; fourthly, extending for 30 seconds at 95 ℃; and fifthly, storing at 4 ℃. The relative expression level of YTHDF2 mRNA was calculated by the double DeltaCt method, and the experiment was repeated 5 times. Statistical analysis was performed using SPSS 19.0 statistical software, and the relative expression levels were expressed as (x.+ -. R). The comparison between the sample and reference groups uses a t-test. The difference of P less than 0.05 is statistically significant;

However, the steps for detecting the THDF2 expression level by the real-time fluorescent quantitative PCR method are as follows: the method for detecting the RBMX expression by using the real-time fluorescence quantitative PCR method comprises the following steps: the upstream primer of RBMX was designed 5 '-TAGGACGCCGAGAGGACTAG-3' (seq_3) and the downstream primer was designed 5 '-GTTCAGGGTCGAAGCTGATG-3' (seq_4). The PCR reaction system used was 12. Mu.l of denatured RNA solution, 2. Mu.l of dNTP mixture, 1. Mu.l of RNase inhibitor (10U/. Mu.l), 4. Mu.l of 5 XRT Buffer, 1. Mu.l of reverse Traace, and a total volume of 20. Mu.l. The PCR reaction procedure is that in the first step, the PCR reaction is performed for 2 minutes at 95 ℃ and then for 30 seconds at 95 ℃; second, annealing at 55 ℃ for 30 seconds; thirdly, extending for 30 seconds at 75 ℃, and circulating for 45 times; fourthly, extending for 60 seconds at 70 ℃; and fifthly, storing at 4 ℃. The relative expression level of RBMX mRNA was calculated by the double DeltaCt method and the experiment was repeated 5 times. Statistical analysis was performed using SPSS 19.0 statistical software, and the relative expression levels were expressed as (x.+ -. R). The comparison between the sample and reference groups uses a t-test. The difference of P less than 0.05 is statistically significant.

In addition, the Western blotting method was used to detect the expression level of YTHDF2 as follows: in the first step, 50. Mu.l of freshly prepared protein lysate was added to the sample, the adherent cells were scraped off with a curette, transferred to a 1.5 ml EP tube, and lysed in an ice bath for 1 hour. Centrifuging at 12000rpm at 4deg.C for 30 min, carefully sucking supernatant, and preserving at-80deg.C; BCA working fluid and Cu are added according to the protein standard and the number of samples ²⁺ Preparing a proper amount according to the proportion of 50:1, fully and uniformly mixing, and diluting a standard substance: the standard substance concentrations are 2000, 1500, 1000, 750, 500, 250, 125, 25 and 0 microgram/ml respectively, diluted standard substances are sequentially added into a 96-well plate, 20 microliters of each well is provided, and two compound wells are arranged; thirdly, adding 18 microliters of PBS (phosphate buffer solution) into each well, adding 2 microliters of protein to be detected, adding 200 microliters of prepared BCA working solution into each well, oscillating for 3-5 minutes by an oscillator, incubating for 30 minutes at 37 ℃, measuring an OD value (570 nanometers) by an enzyme-labeled instrument, and calculating the concentration of the protein to be detected according to a standard curve; step four, according to the measured concentration of the protein sample, calculating the loading volume of each group of samples according to the loading amount of 20-50 micrograms of each loading hole, mixing with a 6 XBuffer loading Buffer solution, balancing with a protein lysate, carrying out instantaneous centrifugation and mixing uniformly, boiling the sample at 95 ℃ for 5 minutes, and then quickly inserting into ice for cooling for 5 minutes; fifth, DSD-PAGE (12% separation gel, 5% concentration gel, 90 volts, about 1.5-2 hours, switchable voltage 120 volts after sample reaches separation gel) separates sample proteins. Taking down the gel, cutting according to the corresponding molecular weight, spreading the PVDF film on the gel, placing the gel between two layers of filter paper, placing the gel at the cathode, placing the PVDF film in a transfer electrophoresis tank, performing imprinting electrophoresis with constant current of 190 milliamperes, and transferring the proteins in the PAGE to the PVDF film. After a certain period of time, the PVDF film is taken out and put into a sealing liquid with the concentration of 5% for sealing for 1.5 hours at 37 ℃. The blocking solution was discarded, TTBS diluted primary antibody was added, and 4 degrees celsius was left overnight. The next day TTBS was used to wash the membrane for 5 min 3, and TTBS diluted peroxy was added The chemoattractant enzyme-labeled secondary antibody was incubated at 37℃for 1.5 hours. TTBS washes for 5 min×3, TBS washes for 5 min×1, enhanced Chemiluminescence (ECL) detects YTHDF2 expression, and finally Image J software is used to scan the grey scale values of the resulting film. The experiment was repeated 5 times. Statistical analysis was performed using SPSS 19.0 statistical software, and the relative expression levels were expressed as (x.+ -. R). The comparison between the sample and reference groups uses a t-test. The difference of P less than 0.05 is statistically significant.

Meanwhile, the Western blotting method is used for detecting the RBMX expression quantity as follows:

firstly, adding 600 microliters of PIPA precooled at 4 ℃ and 6 microliters of PMSF into a sample, shaking and uniformly mixing on a shaking table at 4 ℃ for 5 minutes, then placing the mixture on an oscillator to shake for 30 seconds, enabling RIPA and cells to fully contact and react, placing the mixture on the shaking table at 4 ℃ again for 5 minutes, then taking out and shaking, and performing cyclic operation for 5 times, wherein the total reaction time is about 30 minutes; step two, transferring the lysate into a 1.5 ml Eppendorf tube, and centrifuging at 14000 rpm and 4 ℃ for 25 minutes; collecting supernatant, and storing at-80deg.C; thirdly, taking a 96-well plate, adding 200 microliters of Bradford buffer solution into each well, adding 2 microliters of protein sample to be tested, and shaking and mixing uniformly; placing the sample into an enzyme labeling instrument, measuring absorbance at 595nm, calculating 30 micrograms of system Loading quantity and 5×loading buffer according to a standard curve, and transferring into a 200 microliter Eppendorf tube according to each quantity; heating the sample at 95 degrees celsius for 5 minutes; fourthly, cleaning and manufacturing an SDS-PAGE gel glass plate, fixing the SDS-PAGE gel glass plate on a gel manufacturing frame after airing to ensure that the bottom is not leaked, manufacturing gel according to the instruction of an SDS-PAGE gel preparation kit, concentrating gel at an upper layer, and separating a layer at a lower layer; the electrophoresis buffer solution comprises the following components: 30 g of Tris, 144 g of glycine and 10 g of SDS, adding deionized water to 10000 ml, and uniformly mixing; the transfer buffer solution comprises the following components: 2.9 g glycine, 5.8 g Tris, 0.37 g SDS, 50 ml methanol, adding deionized water to 1000 ml, mixing well; TBST solution is composed of 24.2 g Tris, 80 g NaCl and deionized water to 10000 ml, 5 ml Tween-20 is added, and the mixture is uniformly mixed; the sealing liquid comprises the following components: 2.5 g of skimmed milk powder is dissolved in 50 ml of TBST liquid and evenly mixed; fifthly, fixing the prepared gel in an electrophoresis tank, adding a proper amount of electrophoresis buffer solution, adding a protein marker into a first hole of a gel hole, adding a protein sample Yu Kong according to requirements, electrophoresis under a constant voltage of 50 volts until the marker is separated, and then separating the protein under a constant voltage of 80 volts; after electrophoresis is finished, taking out gel, cutting the upper concentrated gel, cutting a proper PVDF film according to the size of the gel, then tightly attaching the gel and the film, smoothing, removing middle bubbles, placing two layers of filter paper for protection outside the gel and the film, putting into a film rotating clamping plate, putting into an electric rotating groove, adding a liquid rotating buffer solution, and rotating the film for 130 minutes under the constant current of 250 milliamps; sixthly, taking out the PVDF membrane after the electric transfer is finished, and washing 3 times by using TBST liquid on a shaking table at normal temperature for 5 minutes each time; after washing, taking out the PVDF membrane, putting the PVDF membrane into a sealing liquid, and sealing the PVDF membrane on a shaking table for 1 hour at normal temperature; after the sealing is finished, taking out the PVDF film, washing 3 times by TBST liquid on a shaking table at normal temperature for 10 minutes each time; eighth, after washing, taking out PVDF membrane, putting into primary antibody, and shaking on a shaker at 4 ℃ overnight; the next day, the PVDF film is taken out, and washed 3 times with TBST liquid at normal temperature on a shaking table for 10 minutes each time; then taking out the PVDF film, adding a secondary antibody, and reacting on a shaking table for 1 hour at normal temperature; ninth, taking out PVDF film, washing with TBST liquid on a shaking table at normal temperature for 3 times, each time for 5 minutes; preparing a developing solution according to the specification of the ECL chemiluminescence hypersensitive display kit, taking out the PVDF film, adding the developing solution, detecting the expression of RBMX by an enhanced chemiluminescence method (ECL), and finally scanning the gray value of the obtained film by adopting Image J software. The experiment was repeated 5 times. Statistical analysis was performed using SPSS 19.0 statistical software, and the relative expression levels were expressed as (x.+ -. R). The comparison between the sample and reference groups uses a t-test. The difference of P less than 0.05 is statistically significant.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. An auxiliary method, an auxiliary system and an auxiliary kit for early screening or prognosis monitoring of bladder cancer are characterized in that the bladder cancer marker of the auxiliary method is a marker group formed by combining an m6A RNA methylation modulator YTHDF2 and an m6A RNA methylation modulator RBMX with Bladder Tumor Antigen (BTA); the system comprises an information acquisition module, an analysis module and a reporting module; the kit comprises detection reagents of YTHDF2, RBMX and BTA.

2. The set of markers of claim 1, wherein the computational model of the set of markers for early screening and prognosis recurrence analysis of bladder cancer is subdivided into: when the subject has no obvious urological symptoms and the concentration index of BTA is less than 88 ng/ml, the model in the detection method is an early bladder cancer risk screening or recurrence risk prediction model: k (sieve) =a×r (YTHDF 2) +b×r (RBMX) -c×r (BTA); wherein, the value of A is 0.6-0.7, the value of B is 0.5-0.6, the value of C is 0.4-0.5, K (screening) is early bladder cancer risk screening or recurrence risk pre-judging threshold; when the subject has obvious urological symptoms and the concentration index of BTA is greater than or equal to or less than 88 ng/ml, the model in the detection method is an early bladder cancer auxiliary diagnosis or bladder cancer curative recurrence auxiliary diagnosis model: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f×r (YTHDF 2); wherein, the value of D is 0.3-0.4, the value of E is 0.7-0.8, the value of F is 0.8-0.9, K (auxiliary diagnosis) is the auxiliary diagnosis threshold value of early bladder cancer or the auxiliary diagnosis threshold value of recurrence of bladder cancer healer; in the two models, R (YTHDF 2) represents the ratio of the expression level of YTHDF2 of the subject to the normal expression level of human body, R (RBMX) represents the ratio of the expression level of RBMX of the subject to the normal expression level of human body, and R (BTA) represents the ratio of the concentration value of BTA of the subject to 88 ng/ml.

3. The combination of YTHDF2, RBMX and BTA as defined in claim 1, wherein the detection index of YTHDF2 and RBMX markers is the amount of expression of urine, serum, plasma vesicles, tissue or tissue cells; the detection index of the BTA marker reflects the BTA concentration in urine of a subject, and the unit is ng/ml.

4. The bladder cancer according to any one of claims 1-3, wherein the bladder cancer is early stage bladder cancer or recurrent bladder cancer in a bladder cancer curative.

5. A method according to any one of claims 1 to 3, wherein the detection reagent of the method is adapted to any one of the following methods to effect detection of the marker set consisting of the combination of YTHDF2, RBMX and BTA: chemiluminescence, flow fluorescence, single-molecule array, enzyme-linked immunity, colloidal gold, real-time fluorescent quantitative Polymerase Chain Reaction (PCR), western blotting, electrochemiluminescence and quantum dot technology.

6. The system of claim 1, wherein the system comprises an information acquisition module, an analysis module, and a reporting module; the information acquisition module is used for executing the operation of acquiring the detection information of the subject and the information of urological symptoms; the detection information comprises the expression level of YTHDF2, the expression level of RBMX and the concentration value of BTA of a subject, and the urological symptom information is whether obvious urological symptoms exist or not; the overt urological symptoms refer to complaints from urological outpatients, such as: frequent urination, urgent urination, painful urination, leukouria, sometimes hematuria, even macroscopic hematuria, and nonspecific symptoms such as discomfort, shivering, fever, headache, nausea, emesis, inappetence, etc. in the bladder area; the analysis module is used for executing the calculation analysis operation of substituting the detection information into a calculation model to perform a relevant threshold value, and when the subject has no obvious urological department symptoms and the BTA concentration index is less than 88 ng/ml, the model in the detection method is an early bladder cancer risk screening or recurrence risk pre-judging model: k (sieve) =a×r (YTHDF 2) +b×r (RBMX) -c×r (BTA); wherein, the value of A is 0.6-0.7, the value of B is 0.5-0.6, the value of C is 0.4-0.5, K (screening) is early bladder cancer risk screening or recurrence risk pre-judging threshold; when the subject has obvious urological symptoms and the concentration index of BTA is greater than or equal to or less than 88 ng/ml, the model in the detection method is an early bladder cancer auxiliary diagnosis or bladder cancer curative recurrence auxiliary diagnosis model: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f×r (YTHDF 2); wherein, the value of D is 0.3-0.4, the value of E is 0.7-0.8, the value of F is 0.8-0.9, K (auxiliary diagnosis) is the auxiliary diagnosis threshold value of early bladder cancer or the auxiliary diagnosis threshold value of recurrence of bladder cancer healer; in the two models, R (YTHDF 2) represents the ratio of the expression level of the YTHDF2 of the subject to the normal expression level of the human body, R (RBMX) represents the ratio of the expression level of the RBMX of the subject to the normal expression level of the human body, and R (BTA) represents the ratio of the concentration value of the BTA of the subject to 88 ng/ml; the reporting module is used for executing reporting operation of judging the health condition of the subject according to the threshold value and the concentration information of BTA, and reporting the early bladder cancer risk screening or recurrence risk pre-judging according to the following conditions for conventional physical examination personnel or bladder cancer healers needing recurrence monitoring: if the concentration index of BTA is less than 88 ng/ml and-0.1 < K (sieve) < 5.1, judging that the subject is an early bladder cancer patient or a recurrent patient, and if the concentration index of BTA is greater than or equal to 88 ng/ml, not applying the model; for a bladder cancer patient suspected of having obvious urological symptoms or a bladder cancer curative suspected of having a recurrence, reporting that the subject is an early stage bladder cancer patient or a bladder cancer curative patient if the concentration index of BTA is greater than or equal to 88 ng/ml and the concentration index of-6.6 < K (co-diagnosis) < -1.6; furthermore, if the concentration index of BTA is less than 88 ng/ml and-6.9 < K (adjuvant diagnosis) < -2.3, the subject is reported to be an early stage bladder cancer patient or a patient with recurrent bladder cancer cure.

7. The system of claim 1, wherein the reporting module displays, by means of a screen display, voice broadcast or print, the results of bladder cancer recurrence risk screening by a regular physical examination person or a bladder cancer curative who requires recurrence monitoring and/or the results of bladder cancer assisted diagnosis by a patient suspected of early stage bladder cancer or a bladder cancer curative who is suspected of recurrence.

8. The kit of claim 1, wherein the kit comprises instructions for use that record a model for bladder cancer screening and/or bladder cancer assisted diagnosis; when the subject has no obvious urological symptoms and the concentration index of BTA is less than 88 ng/ml, the model in the detection method is an early bladder cancer risk screening or recurrence risk prediction model: k (sieve) =a×r (YTHDF 2) +b×r (RBMX) -c×r (BTA); wherein, the value of A is 0.6-0.7, the value of B is 0.5-0.6, the value of C is 0.4-0.5, K (screening) is early bladder cancer risk screening or recurrence risk pre-judging threshold; when the subject has obvious urological symptoms and the concentration index of BTA is greater than or equal to or less than 88 ng/ml, the model in the detection method is an early bladder cancer auxiliary diagnosis or bladder cancer curative recurrence auxiliary diagnosis model: k (co-diagnosis) =d×r (BTA) -e×r (RBMX) -f×r (YTHDF 2); wherein, the value of D is 0.3-0.4, the value of E is 0.7-0.8, the value of F is 0.8-0.9, K (auxiliary diagnosis) is the auxiliary diagnosis threshold value of early bladder cancer or the auxiliary diagnosis threshold value of recurrence of bladder cancer healer; in the two models, R (YTHDF 2) represents the ratio of the expression level of YTHDF2 of the subject to the normal expression level of human body, R (RBMX) represents the ratio of the expression level of RBMX of the subject to the normal expression level of human body, and R (BTA) represents the ratio of the concentration value of BTA of the subject to 88 ng/ml.

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