CN110317870B - miRNA marker for diagnosing prostate cancer, kit, system, application and detection method - Google Patents

miRNA marker for diagnosing prostate cancer, kit, system, application and detection method Download PDF

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CN110317870B
CN110317870B CN201910611368.4A CN201910611368A CN110317870B CN 110317870 B CN110317870 B CN 110317870B CN 201910611368 A CN201910611368 A CN 201910611368A CN 110317870 B CN110317870 B CN 110317870B
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龙丹
周彦妮
丁微
李胜富
冯莉
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West China Hospital of Sichuan University
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Abstract

The invention discloses a miRNA marker, a kit, a system, an application and a detection method for diagnosing prostate cancer; the marker is a composition of miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5 p; the marker, the kit and the system can be used for auxiliary diagnosis of the prostate cancer, and are easy to detect, accurate in quantification, and good in sensitivity and specificity.

Description

miRNA marker for diagnosing prostate cancer, kit, system, application and detection method
Technical Field
The invention relates to the technical field of biology, in particular to a miRNA marker for diagnosing prostate cancer, a kit, an application and a detection method.
Background
Prostate cancer refers to an epithelial malignancy that occurs in the prostate gland and is one of the major cancers that present a health hazard to men.
Currently, there are 4 main clinical diagnosis modalities for prostate cancer: digital rectal examination, serum Prostate Specific Antigen (PSA) examination, rectal ultrasonography, and tissue biopsy. The above methods all have certain limitations. Among the limitations of (1) digital rectal examination are: the digital rectal examination has high missed diagnosis rate, most of the missed diagnosis is in middle and advanced stages, and the clinical treatment is not easy to realize; (2) limitations of the digital rectal examination of serum Prostate Specific Antigen (PSA) are: detection of PSA has poor detection specificity, and the increase of PSA can be caused by prostatitis, prostatic hyperplasia and the like, and most of the increase of PSA also occurs in the middle and late stages of cancer, so that the PSA is not easy to be clinically treated. (3) The limitations of rectal ultrasound detection are: the misdiagnosis rate of ultrasonic detection on some benign tumor masses is high, and the diagnosis coincidence rate is low because the internal structure and the surrounding tissues of the tumor cannot be displayed; (4) limitations of tissue biopsy are: tissue biopsy is the current gold standard for prostate cancer diagnosis, but is not frequently used due to its invasiveness and complexity.
Disclosure of Invention
In view of the above, the present application provides a miRNA marker, a kit, a system, an application, and a detection method for diagnosing prostate cancer, where the marker, the kit, and the system can be used for auxiliary diagnosis of prostate cancer, and are easy to detect, accurate in quantification, and good in sensitivity and specificity.
In order to solve the technical problems, the technical scheme provided by the application is the miRNA marker for diagnosing the prostate cancer, and is characterized in that the marker is a composition of miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5 p.
Preferably, the reverse transcription primer and the detection primer of the miRNA marker are characterized in that,
the reverse transcription primer is as follows:
the reverse transcription primer sequence of miR-21-5p is shown in SEQ ID NO 1;
the reverse transcription primer sequence of miR-141-3p is shown in SEQ ID NO 2;
the reverse transcription primer sequence of miR-221-3p is shown in SEQ ID NO. 3;
the reverse transcription primer sequence of miR-206 is shown in SEQ ID NO. 4;
the reverse transcription primer sequence of miR-148a-3p is shown in SEQ ID NO. 5;
the reverse transcription primer sequence of miR-210-5p is shown in SEQ ID NO 6;
the reverse transcription primer sequence of miR-205-5p is shown in SEQ ID NO. 7;
the reverse transcription primer sequence of miR-145-5p is shown in SEQ ID NO. 8;
the detection primer is as follows:
the sequence of a forward primer of the miR-21-5p is shown as SEQ ID NO. 9;
the reverse primer sequence of miR-21-5p is shown in SEQ ID NO. 10;
the sequence of a forward primer of the miR-141-3p is shown as SEQ ID NO. 11;
the reverse primer sequence of miR-141-3p is shown in SEQ ID NO. 12;
the sequence of a forward primer of the miR-221-3p is shown as SEQ ID NO. 13;
the reverse primer sequence of miR-221-3p is shown in SEQ ID NO. 14;
the sequence of the forward primer of miR-206 is shown as SEQ ID NO. 15;
the reverse primer sequence of miR-206 is shown in SEQ ID NO. 16;
the sequence of a forward primer of miR-148a-3p is shown as SEQ ID NO: 17;
the reverse primer sequence of miR-148a-3p is shown in SEQ ID NO 18;
the sequence of a forward primer of miR-210-5p is shown as SEQ ID NO. 19;
the reverse primer sequence of miR-210-5p is shown in SEQ ID NO. 20;
the sequence of a forward primer of miR-205-5p is shown as SEQ ID NO: 21;
the reverse primer sequence of miR-205-5p is shown in SEQ ID NO. 22;
the sequence of a forward primer of the miR-145-5p is shown as SEQ ID NO. 23;
the reverse primer sequence of miR-145-5p is shown in SEQ ID NO. 24.
The invention also provides application of the miRNA marker in preparation of a diagnostic reagent for prostate cancer.
Preferably, the reagent determines the risk degree of the prostate cancer of the subject by detecting the content of the miRNA marker in a biological sample of the subject.
The invention also provides application of the reverse transcription primer and the detection primer of the miRNA marker in preparation of a diagnostic reagent for prostate cancer.
The invention also provides a diagnostic kit for the prostate cancer, which is used for detecting the content of the miRNA marker in the blood plasma; the marker is a composition of miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5 p.
Preferably, the kit comprises: the reverse transcription primer and the detection primer of the miRNA marker;
the reverse transcription primer is as follows:
the reverse transcription primer sequence of miR-21-5p is shown in SEQ ID NO 1;
the reverse transcription primer sequence of miR-141-3p is shown in SEQ ID NO 2;
the reverse transcription primer sequence of miR-221-3p is shown in SEQ ID NO. 3;
the reverse transcription primer sequence of miR-206 is shown in SEQ ID NO. 4;
the reverse transcription primer sequence of miR-148a-3p is shown in SEQ ID NO. 5;
the reverse transcription primer sequence of miR-210-5p is shown in SEQ ID NO 6;
the reverse transcription primer sequence of miR-205-5p is shown in SEQ ID NO. 7;
the reverse transcription primer sequence of miR-145-5p is shown in SEQ ID NO. 8;
the detection primer is as follows:
the sequence of a forward primer of the miR-21-5p is shown as SEQ ID NO. 9;
the reverse primer sequence of miR-21-5p is shown in SEQ ID NO. 10;
the sequence of a forward primer of the miR-141-3p is shown as SEQ ID NO. 11;
the reverse primer sequence of miR-141-3p is shown in SEQ ID NO. 12;
the sequence of a forward primer of the miR-221-3p is shown as SEQ ID NO. 13;
the reverse primer sequence of miR-221-3p is shown in SEQ ID NO. 14;
the sequence of the forward primer of miR-206 is shown as SEQ ID NO. 15;
the reverse primer sequence of miR-206 is shown in SEQ ID NO. 16;
the sequence of a forward primer of miR-148a-3p is shown as SEQ ID NO: 17;
the reverse primer sequence of miR-148a-3p is shown in SEQ ID NO 18;
the sequence of a forward primer of miR-210-5p is shown as SEQ ID NO. 19;
the reverse primer sequence of miR-210-5p is shown in SEQ ID NO. 20;
the sequence of a forward primer of miR-205-5p is shown as SEQ ID NO: 21;
the reverse primer sequence of miR-205-5p is shown in SEQ ID NO. 22;
the sequence of a forward primer of the miR-145-5p is shown as SEQ ID NO. 23;
the reverse primer sequence of miR-145-5p is shown in SEQ ID NO. 24.
Preferably, the kit is a kit based on a digital PCR platform.
The present invention also provides a prostate cancer diagnosis system, including:
an information acquisition module: the information acquisition module is used for acquiring detection information of a subject, and the detection information comprises content information of the marker;
a calculation module: the system is used for establishing a diagnosis model and calculating a risk value P according to the content information of the marker and the diagnosis model;
a judging module: for determining that the subject is at low risk for prostate cancer if the P-value is less than a first predetermined value, determining that the subject is at moderate risk for prostate cancer if the P-value of the subject is within a range from a first predetermined value to a second predetermined value, and determining that the subject is at high risk for prostate cancer if the P-value of the subject is greater than a second predetermined value;
the calculation formula of the diagnostic model is as follows:
P＝es/(1+es) (I)；
S＝0.574+0.043×m1+0.028×m2-0.004×m3+0.022×m4-0.032×m5-0.001×m6+0.017×m7-0.014×m8(II)；
wherein the content of the first and second substances,
esis the power of S of e, and e is a natural constant e which is 2.71828182845904;
m1 is the content of miR-21-5p in a biological sample of a subject;
m2 is the content of miR-141-3p in a biological sample of a subject;
m3 is the content of miR-221-3p in a biological sample of a subject;
m4 is the content of miR-206 in a biological sample of a subject;
m5 is the content of miR-148a-3p in a biological sample of a subject;
m6 is the content of miR-210-5p in a biological sample of a subject;
m7 is the content of miR-205-5p in a biological sample of a subject;
m8 is the content of miR-145-5p in a biological sample of a subject.
Preferably, the biological sample is plasma.
Preferably, the first predetermined value is 0.441, and the second predetermined value is 0.623.
Preferably, the method for detecting the content of the miRNA marker of claim 1 in the plasma comprises:
(1) collecting blood plasma, and extracting total free RNA;
(2) configuring a reverse transcription reaction system by using the obtained total free RNA, and carrying out reverse transcription reaction to obtain a reverse transcription sample;
(3) and (3) configuring a digital PCR detection reaction system by using the obtained reverse transcription sample, preparing a digital PCR reaction microdroplet, and carrying out digital PCR reaction.
Compared with the prior art, the detailed description of the application is as follows:
(1) the marker miRNA provided by the invention is easy to detect and accurate in quantification, and the sensitivity and specificity of prostate cancer diagnosis are improved. The successful development of the miRNA biomarker is helpful for the auxiliary diagnosis of the prostate cancer and provides reference for the development of other biomarkers. The diagnosis kit of the prostate cancer is used for detecting the content of miRNA markers in blood plasma; the marker is a composition of miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5p, and 8 miRNAs have good tissue specificity, a proper amount of detected targets, simplicity and strong operability.
(2) The technical scheme provided by the invention aims at detecting the miRNA marker content in the blood plasma, is minimally invasive and can be widely accepted by the public.
(3) The invention judges the degree of the prostate cancer by detecting the content of the marker miRNA in the plasma. By adopting the marker, the kit and the system provided by the invention, the risk degree of the prostate cancer of the subject can be judged by detecting the content of the miRNA marker in the biological sample of the subject and adopting a formula, so that the kit can be used for assisting diagnosis of the prostate cancer, and provides support for a clinician to quickly and accurately master the condition of the patient and timely adopt a more personalized prevention and treatment scheme.
(4) The kit provided by the invention is based on a digital PCR platform and has the characteristics of higher accuracy and sensitivity.
Drawings
FIG. 1 is a schematic diagram of a system for diagnosing prostate cancer according to the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example 1
A diagnostic kit for prostate cancer, which is used for detecting the content of miRNA markers in plasma; the kit comprises: the reverse transcription primer and the detection primer of the miRNA marker;
the marker is a composition of miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5 p;
the reverse transcription primer is as follows:
the reverse transcription primer sequence (5'-3') of miR-21-5p is shown in SEQ ID NO: 1;
the reverse transcription primer sequence (5'-3') of miR-141-3p is shown in SEQ ID NO: 2;
the reverse transcription primer sequence (5'-3') of miR-221-3p is shown in SEQ ID NO: 3;
the reverse transcription primer sequence (5'-3') of miR-206 is shown in SEQ ID NO. 4;
the reverse transcription primer sequence (5'-3') of miR-148a-3p is shown as SEQ ID NO: 5;
the reverse transcription primer sequence (5'-3') of miR-210-5p is shown in SEQ ID NO: 6;
the reverse transcription primer sequence (5'-3') of miR-205-5p is shown in SEQ ID NO: 7;
the reverse transcription primer sequence (5'-3') of miR-145-5p is shown in SEQ ID NO: 8;
the detection primer is as follows:
the forward primer sequence (5'-3') of miR-21-5p is shown in SEQ ID NO. 9;
the reverse primer sequence (5'-3') of miR-21-5p is shown in SEQ ID NO: 10;
the forward primer sequence (5'-3') of miR-141-3p is shown in SEQ ID NO: 11;
the reverse primer sequence (5'-3') of miR-141-3p is shown in SEQ ID NO: 12;
the forward primer sequence (5'-3') of miR-221-3p is shown in SEQ ID NO: 13;
the reverse primer sequence (5'-3') of miR-221-3p is shown in SEQ ID NO: 14;
the sequence (5'-3') of the forward primer of miR-206 is shown as SEQ ID NO. 15;
the reverse primer sequence (5'-3') of miR-206 is shown as SEQ ID NO: 16;
the sequence (5'-3') of the forward primer of miR-148a-3p is shown as SEQ ID NO: 17;
the reverse primer sequence (5'-3') of miR-148a-3p is shown in SEQ ID NO: 18;
the sequence (5'-3') of the forward primer of miR-210-5p is shown as SEQ ID NO: 19;
the reverse primer sequence (5'-3') of miR-210-5p is shown in SEQ ID NO: 20;
the forward primer sequence (5'-3') of miR-205-5p is shown in SEQ ID NO: 21;
the reverse primer sequence (5'-3') of miR-205-5p is shown in SEQ ID NO: 22;
the sequence (5'-3') of the forward primer of miR-145-5p is shown as SEQ ID NO: 23;
the reverse primer sequence (5'-3') of miR-145-5p is shown in SEQ ID NO: 24.
Example 2
Test example 1
(1) Collecting blood plasma, and extracting total free RNA;
collecting blood of a subject intravenously at 3ml, centrifuging at 4 deg.C for 10min at 2000g, separating upper layer plasma part, centrifuging at 4 deg.C at 12000g for 20min, and collecting upper layer plasma sample. 200ul of Plasma samples were aspirated, total free RNA (including total miRNA) was extracted using the MIRNeasy Serum/Plasma Kit from QIAGEN, and 10ul of quality control was added after shaking by adding QIAzolllysine Reagent and standing for 5 min. And measuring the concentration after extraction, and adjusting the concentration to be 20-40 ng/ul of total free RNA.
(2) Configuring a reverse transcription reaction system by using the obtained total free RNA and a reverse transcription primer, and carrying out reverse transcription reaction to obtain a miRNA reverse transcription sample; the miRNA reverse transcription reaction system is shown in Table 1(miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5p are respectively detected and independently configured into a tube)
The reverse transcription primers of the invention in table 1 are:
the reverse transcription primer is as follows:
the reverse transcription primer sequence (5'-3') of miR-21-5p is shown in SEQ ID NO: 1;
the reverse transcription primer sequence (5'-3') of miR-141-3p is shown in SEQ ID NO: 2;
the reverse transcription primer sequence (5'-3') of miR-221-3p is shown in SEQ ID NO: 3;
the reverse transcription primer sequence (5'-3') of miR-206 is shown in SEQ ID NO. 4;
the reverse transcription primer sequence (5'-3') of miR-148a-3p is shown as SEQ ID NO: 5;
the reverse transcription primer sequence (5'-3') of miR-210-5p is shown in SEQ ID NO: 6;
the reverse transcription primer sequence (5'-3') of miR-205-5p is shown in SEQ ID NO: 7;
the reverse transcription primer sequence (5'-3') of miR-145-5p is shown in SEQ ID NO: 8;
the quality Control product in Table 1 is Spike-in Control, which is a commercially available product, and the actual synthetic single-stranded miRNA is 5X 104The sequence (5'-3') of the cel-miRNA-39 of copies/ul is shown as SEQ ID NO. 25;
carrying out reverse transcription reaction by using a quality control product reverse transcription primer to obtain a quality control product reverse transcription sample; the reverse transcription primer sequence (5'-3') of the quality control product is shown as SEQ ID NO: 26;
the reverse transcription reaction was carried out according to the conditions of Table 2, the completion of the reaction was indicated at 4 ℃;
TABLE 1 reverse transcription reaction system for miRNA and quality control products
Composition (I) Volume of
100mM dNTPs(with dTTP) 0.15ul
MultiScribeTM Reverse Transcriptase,50U/μL 1ul
10X RT Buffer 1.5ul
RNase Inhibitor 0.188ul
The reverse transcription primer or the quality control product reverse transcription primer of the invention 3ul
Total free RNA samples 3ul
Ultrapure water 6.162ul
In total 15ul
TABLE 2 reverse transcription reaction conditions of miRNA and quality control
Step (ii) of Time of day Temperature of
1 30min 16℃
2 30min 42℃
3 5min 85℃
4 Forever 4℃
(3) Configuring a miRNA digital PCR detection reaction system by using the obtained miRNA reverse transcription sample and a detection primer, preparing a digital PCR reaction microdrop, and carrying out digital PCR reaction;
the miRNA digital PCR detection reaction system is shown in Table 3(miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5p are respectively detected and independently configured with a tube);
in table 3, the detection primers of the present invention are:
the forward primer sequence (5'-3') of miR-21-5p is shown in SEQ ID NO. 9;
the reverse primer sequence (5'-3') of miR-21-5p is shown in SEQ ID NO: 10;
the forward primer sequence (5'-3') of miR-141-3p is shown in SEQ ID NO: 11;
the reverse primer sequence (5'-3') of miR-141-3p is shown in SEQ ID NO: 12;
the forward primer sequence (5'-3') of miR-221-3p is shown in SEQ ID NO: 13;
the reverse primer sequence (5'-3') of miR-221-3p is shown in SEQ ID NO: 14;
the sequence (5'-3') of the forward primer of miR-206 is shown as SEQ ID NO. 15;
the reverse primer sequence (5'-3') of miR-206 is shown as SEQ ID NO: 16;
the sequence (5'-3') of the forward primer of miR-148a-3p is shown as SEQ ID NO: 17;
the reverse primer sequence (5'-3') of miR-148a-3p is shown in SEQ ID NO: 18;
the sequence (5'-3') of the forward primer of miR-210-5p is shown as SEQ ID NO: 19;
the reverse primer sequence (5'-3') of miR-210-5p is shown in SEQ ID NO: 20;
the forward primer sequence (5'-3') of miR-205-5p is shown in SEQ ID NO: 21;
the reverse primer sequence (5'-3') of miR-205-5p is shown in SEQ ID NO: 22;
the sequence (5'-3') of the forward primer of miR-145-5p is shown as SEQ ID NO: 23;
the reverse primer sequence (5'-3') of miR-145-5p is shown in SEQ ID NO: 24.
TABLE 3 miRNA digital PCR reaction system
Composition (I) Volume of
ddPCRTM Probe Supermix 10ul
Taqman primer + probe 0.5ul
miRNA reverse transcription sample 3ul
Ultrapure water 6ul
In total 20ul
In table 4, the quality control detection primers are:
the sequence of the forward primer (5'-3') is shown as SEQ ID NO: 27;
the reverse primer sequence (5'-3') is shown as SEQ ID NO. 28;
configuring a digital PCR detection reaction system of the quality control product by using the obtained quality control product reverse transcription sample and the quality control product detection primer, preparing a digital PCR reaction microdrop, and carrying out digital PCR reaction;
the quality control product digital PCR detection reaction system is shown in Table 4;
TABLE 4 quality control digital PCR reaction system
Composition (I) Volume of
ddPCRTM EvaGreen Supermix 10ul
Quality control quality inspectionDetection primer 0.5ul
Quality control product reverse transcription sample 3ul
Ultrapure water 6ul
In total 20ul
Wherein the content of the first and second substances,
preparing a digital PCR reaction microdroplet, and performing a digital PCR reaction process by using the obtained reverse transcription samples (miRNA reverse transcription samples and quality control product reverse transcription samples) specifically comprises the following steps: adding the prepared digital PCR detection reaction system (miRNA digital PCR detection reaction system, quality control product digital PCR detection reaction system) into the microdroplet generation card DG8TMCarridges sample tank, in the same time in the Oil generating tank add 70ul of Droplet Generation Oil for Probes, finally with sealing film DG8TMGaskets are sealed and placed on a microdroplet generator to prepare microdroplets for digital PCR reaction;
the prepared droplets of the digital PCR reaction were slowly transferred to a 96-well plate, and then heat-sealed with an aluminum sealing film in cooperation with a sealing film machine, followed by digital PCR reaction under the conditions shown in Table 5.
TABLE 5 digital PCR reaction conditions
Figure BDA0002122482800000111
The resulting 96-well plate after the digital PCR reaction was transferred to QX200TMAnd reading the result in a digital PCR reader.
(5) And (4) calculating a result:
1) assuming that the total free RNA sample dissolved volume after extraction is A μ L and the Bio-rad QX200 digital PCR detection result is B copies/μ L, the concentration of the quality control substance (Xcopies/μ L) is calculated according to the following formula:
the formula: x copy number/. mu.l ═ B × 20/3 × 15/3 × a)/10 ═ 10/3 × a × B
2) The quality control quality detection result should satisfy: 3X 104～4×104And (4) copies/ul, if the miRNA (miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5p) detection result of the sample is effective, otherwise, the experiment is invalid, and the sample needs to be extracted again for detection.
3) If the quality control result is qualified, the concentration of the target miRNA in the plasma sample (Ycopies/muL) is calculated according to the following formula:
the formula: y copy number/. mu.l ═ B × 20/3 × 15/3 × a)/200 ═ 1/6 × a × B
(6) The experimental results are as follows:
the number of the droplets finally detected by each group of reactions is between 10000 and 20000, and the droplets are all larger than 10000, so that the result is effective and reliable. The results are shown in Table 6.
TABLE 6
Figure BDA0002122482800000121
(7)
Grouping samples: 230 clinical diagnosis conclusions the established samples consisted of 52 normal persons, 35 benign prostatic hyperplasia, 42 prostatitis, and 101 prostate cancer patients.
The prostate cancer diagnostic kit of example 1 is used to detect the miRNA marker content of example 1 in the group-input sample by the method described above.
The experimental data are collated, analyzed and modeled to obtain a prostate cancer diagnosis model, and the prostate cancer degree of a subject is judged by detecting the content of the miRNA marker in a biological sample of the subject and calculating a risk value P through the diagnosis model;
the concrete formula is as follows:
P＝es/(1+es) (I)；
S＝0.574+0.043×m1+0.028×m2-0.004×m3+0.022×m4-0.032×m5-0.001×m6+0.017×m7-0.014×m8 (II)；
wherein the content of the first and second substances,
esis the power of S of e, and e is a natural constant e which is 2.71828182845904;
m1 is the content of miR-21-5p in a biological sample of a subject;
m2 is the content of miR-141-3p in a biological sample of a subject;
m3 is the content of miR-221-3p in a biological sample of a subject;
m4 is the content of miR-206 in a biological sample of a subject;
m5 is the content of miR-148a-3p in a biological sample of a subject;
m6 is the content of miR-210-5p in a biological sample of a subject;
m7 is the content of miR-205-5p in a biological sample of a subject;
m8 is the content of miR-145-5p in a biological sample of a subject;
the biological sample is plasma.
And (3) analyzing a result by the model:
when P <0.441, it indicates a low risk of the subject to suffer from prostate cancer;
when 0.441< P <0.623, it indicates that the subject is at moderate risk for prostate cancer;
when P >0.623, it indicates a high risk of the subject to suffer from prostate cancer.
The accuracy of the test with the kit of example 1 was determined by obtaining ROC curves for the test controls using MedCalc software, and the results are shown in table 7.
TABLE 7
Reagent kit Sensitivity of the device(％) Specificity (%) AUC 95% confidence interval
Example 1 90.3 87.4 0.891 0.83-0.94
As can be seen from Table 7, the prostate cancer marker, the kit and the system provided by the invention have high accuracy, sensitivity and specificity, and can be applied to the preparation of a diagnostic reagent for prostate cancer, the judgment of the risk degree of prostate cancer of a subject and the auxiliary evaluation of the status of prostate cancer.
Compared with normal people, prostatic hyperplasia and prostatitis, the overall expression level of miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5p in the plasma of the prostate cancer patient is greatly different.
At the same time, the user can select the desired position,
the overall expression level of miR-21-5P, miR-141-3P, miR-221-3P, miR-148a-3P, miR-210-5P and miR-148a-3P in the plasma of the prostate cancer patient group is higher than that of the overall expression level of miR-21-5P, miR-141-3P, miR-221-3P, miR-148a-3P, miR-210-5P in the plasma of a normal human control group (P is less than 0.05);
the overall expression levels of miR-206, miR-205-5P and miR-145-5P in the plasma of the prostate cancer patient group are lower than the overall expression levels of miR-206, miR-205-5P and miR-145-5P in the plasma of the prostate hyperplasia control group (P < 0.05).
Example 3
Based on the diagnosis model described in example 2, as shown in fig. 1, this embodiment provides a diagnosis system for prostate cancer, the system including:
the information acquisition module 100: the information acquisition module 100 is used for acquiring the detection information of the subject, wherein the detection information comprises the content information of the marker of embodiment 2;
the calculation module 200: the system is used for establishing a diagnosis model and calculating a risk value P according to the content information of the marker and the diagnosis model;
the judging module 300: for determining that the subject is at low risk for prostate cancer if the P-value is less than a first predetermined value, determining that the subject is at moderate risk for prostate cancer if the P-value of the subject is within a range from a first predetermined value to a second predetermined value, and determining that the subject is at high risk for prostate cancer if the P-value of the subject is greater than a second predetermined value;
the calculation formula of the diagnostic model 200 is as follows:
P＝es/(1+es) (I)；
S＝0.574+0.043×m1+0.028×m2-0.004×m3+0.022×m4-0.032×m5-0.001×m6+0.017×m7-0.014×m8 (II)；
wherein the content of the first and second substances,
esis the power of S of e, and e is a natural constant e which is 2.71828182845904;
m1 is the content of miR-21-5p in a biological sample of a subject;
m2 is the content of miR-141-3p in a biological sample of a subject;
m3 is the content of miR-221-3p in a biological sample of a subject;
m4 is the content of miR-206 in a biological sample of a subject;
m5 is the content of miR-148a-3p in a biological sample of a subject;
m6 is the content of miR-210-5p in a biological sample of a subject;
m7 is the content of miR-205-5p in a biological sample of a subject;
m8 is the content of miR-145-5p in a biological sample of a subject;
the biological sample is plasma;
the first predetermined value is 0.441 and the second predetermined value is 0.623.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Sichuan university Hospital in western China
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Claims (5)

1. A miRNA marker based on a digital PCR platform for diagnosing prostate cancer, wherein the marker is a composition of miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5 p; the diagnosis of the prostate cancer is diagnosis of the risk degree of the prostate cancer, and the risk degree of the prostate cancer is as follows: low risk of prostate cancer, moderate risk of prostate cancer, and high risk of prostate cancer.
2. The reverse transcription primer and the detection primer of the miRNA marker of claim 1,
the reverse transcription primer is as follows:
the reverse transcription primer sequence of miR-21-5p is shown in SEQ ID NO 1;
the reverse transcription primer sequence of miR-141-3p is shown in SEQ ID NO 2;
the reverse transcription primer sequence of miR-221-3p is shown in SEQ ID NO. 3;
the reverse transcription primer sequence of miR-206 is shown in SEQ ID NO. 4;
the reverse transcription primer sequence of miR-148a-3p is shown in SEQ ID NO. 5;
the reverse transcription primer sequence of miR-210-5p is shown in SEQ ID NO 6;
the reverse transcription primer sequence of miR-205-5p is shown in SEQ ID NO. 7;
the reverse transcription primer sequence of miR-145-5p is shown in SEQ ID NO. 8;
the detection primer is as follows:
the sequence of a forward primer of the miR-21-5p is shown as SEQ ID NO. 9;
the reverse primer sequence of miR-21-5p is shown in SEQ ID NO. 10;
the sequence of a forward primer of the miR-141-3p is shown as SEQ ID NO. 11;
the reverse primer sequence of miR-141-3p is shown in SEQ ID NO. 12;
the sequence of a forward primer of the miR-221-3p is shown as SEQ ID NO. 13;
the reverse primer sequence of miR-221-3p is shown in SEQ ID NO. 14;
the sequence of the forward primer of miR-206 is shown as SEQ ID NO. 15;
the reverse primer sequence of miR-206 is shown in SEQ ID NO. 16;
the sequence of a forward primer of miR-148a-3p is shown as SEQ ID NO: 17;
the reverse primer sequence of miR-148a-3p is shown in SEQ ID NO 18;
the sequence of a forward primer of miR-210-5p is shown as SEQ ID NO. 19;
the reverse primer sequence of miR-210-5p is shown in SEQ ID NO. 20;
the sequence of a forward primer of miR-205-5p is shown as SEQ ID NO: 21;
the reverse primer sequence of miR-205-5p is shown in SEQ ID NO. 22;
the sequence of a forward primer of the miR-145-5p is shown as SEQ ID NO. 23;
the reverse primer sequence of miR-145-5p is shown in SEQ ID NO. 24.
3. The use of the miRNA marker of claim 1 in the preparation of a diagnostic reagent for prostate cancer, wherein the reagent is used for determining the risk degree of prostate cancer in a subject by detecting the content of the miRNA marker in a biological sample of the subject, and the risk degree of prostate cancer is as follows: low risk of prostate cancer, moderate risk of prostate cancer, and high risk of prostate cancer.
4. The application of a reverse transcription primer and a detection primer of the miRNA marker in the claim 2 in preparing a diagnostic reagent for the prostate cancer, wherein the reagent is used for judging the risk degree of the prostate cancer of a subject by detecting the content of the miRNA marker in a biological sample of the subject, and the risk degree of the prostate cancer is as follows: low risk of prostate cancer, moderate risk of prostate cancer, and high risk of prostate cancer.
5. A kit for diagnosing prostate cancer based on a digital PCR platform, which is used for detecting the content of miRNA markers in plasma; the marker is a composition of miR-21-5p, miR-141-3p, miR-221-3p, miR-206, miR-148a-3p, miR-210-5p, miR-205-5p and miR-145-5 p; the diagnosis of the prostate cancer is diagnosis of the risk degree of the prostate cancer, and the risk degree of the prostate cancer is as follows: low risk of prostate cancer, moderate risk of prostate cancer, and high risk of prostate cancer; the kit comprises: the reverse transcription primer and the detection primer of the miRNA marker;
the reverse transcription primer is as follows:
the reverse transcription primer sequence of miR-21-5p is shown in SEQ ID NO 1;
the reverse transcription primer sequence of miR-141-3p is shown in SEQ ID NO 2;
the reverse transcription primer sequence of miR-221-3p is shown in SEQ ID NO. 3;
the reverse transcription primer sequence of miR-206 is shown in SEQ ID NO. 4;
the reverse transcription primer sequence of miR-148a-3p is shown in SEQ ID NO. 5;
the reverse transcription primer sequence of miR-210-5p is shown in SEQ ID NO 6;
the reverse transcription primer sequence of miR-205-5p is shown in SEQ ID NO. 7;
the reverse transcription primer sequence of miR-145-5p is shown in SEQ ID NO. 8;
the detection primer is as follows:
the sequence of a forward primer of the miR-21-5p is shown as SEQ ID NO. 9;
the reverse primer sequence of miR-21-5p is shown in SEQ ID NO. 10;
the sequence of a forward primer of the miR-141-3p is shown as SEQ ID NO. 11;
the reverse primer sequence of miR-141-3p is shown in SEQ ID NO. 12;
the sequence of a forward primer of the miR-221-3p is shown as SEQ ID NO. 13;
the reverse primer sequence of miR-221-3p is shown in SEQ ID NO. 14;
the sequence of the forward primer of miR-206 is shown as SEQ ID NO. 15;
the reverse primer sequence of miR-206 is shown in SEQ ID NO. 16;
the sequence of a forward primer of miR-148a-3p is shown as SEQ ID NO: 17;
the reverse primer sequence of miR-148a-3p is shown in SEQ ID NO 18;
the sequence of a forward primer of miR-210-5p is shown as SEQ ID NO. 19;
the reverse primer sequence of miR-210-5p is shown in SEQ ID NO. 20;
the sequence of a forward primer of miR-205-5p is shown as SEQ ID NO: 21;
the reverse primer sequence of miR-205-5p is shown in SEQ ID NO. 22;
the sequence of a forward primer of the miR-145-5p is shown as SEQ ID NO. 23;
the reverse primer sequence of miR-145-5p is shown in SEQ ID NO. 24.
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