CN111172285A

CN111172285A - miRNA group for early diagnosis and/or prognosis monitoring of pancreatic cancer and application thereof

Info

Publication number: CN111172285A
Application number: CN202010120765.4A
Authority: CN
Inventors: 薛育政
Original assignee: Affiliated Hospital of Jiangnan University
Current assignee: Affiliated Hospital of Jiangsu University; Affiliated Hospital of Jiangnan University
Priority date: 2020-02-26
Filing date: 2020-02-26
Publication date: 2020-05-19

Abstract

The invention discloses a miRNA group related to pancreatic cancer, which comprises the following miRNAs: miR-143, miR-218, miR-217, miR-216a, miR-205, miR-145, miR-195, miR-193a and miR-127. The miRNA group can be used as an early detection marker and a prognosis biomarker of pancreatic cancer for early diagnosis and/or prognosis monitoring of pancreatic cancer, and early diagnosis of pancreatic cancer is carried out to grasp early diagnosis and treatment opportunity of pancreatic cancer; and/or evaluating whether the pancreatic cancer patient has the risks of early metastasis and drug resistance so as to adjust the medication scheme in time, prevent the occurrence of early metastasis and improve the survival time of the patient.

Description

miRNA group for early diagnosis and/or prognosis monitoring of pancreatic cancer and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a miRNA group for pancreatic cancer early diagnosis and/or prognosis monitoring and application thereof.

Background

Pancreatic cancer, one of the most aggressive malignant tumors, is a malignant tumor of the digestive tract that is highly malignant and difficult to diagnose and treat, and about 90% of the malignant tumors are ductal adenocarcinomas originating from the epithelium of the glandular duct. The incidence and mortality of pancreatic cancer has increased dramatically in recent years, with a 5-year survival rate of < 1%, one of the worst-case malignancies. The main difficulties of the existing treatment of pancreatic cancer patients are that effective early molecular markers are lacked, early metastasis easily occurs, early onset and development of pancreatic cancer cannot be diagnosed timely, the optimal diagnosis and treatment time is delayed, and the survival rate of patients is low. The early diagnosis rate of pancreatic cancer is low, the operative mortality rate is high, the cure rate is low, in clinical chemotherapy, the main problem of pancreatic cancer is early metastasis, and no effective means is available for diagnosis in the early stage, so the development of an effective means for helping pancreatic cancer patients to diagnose is the key point for inhibiting early metastasis. Failure of current diagnostic and therapeutic management is one of the major causes of low survival in pancreatic cancer patients, and therefore, new diagnostic and prognostic biomarkers are needed for early diagnosis and targeted therapy.

Micro RNA (microRNA, miRNA) is a small RNA composed of 18-25 nucleotides, does not encode protein, and plays an important role in regulating and controlling the expression of genes. The sequence of miRNA is highly species-conserved, its expression is unique temporal and spatial specificity. The existing research proves that miRNA plays a great role in regulation and control in physiological processes such as embryonic development, tissue differentiation, cell metabolism, signal channel control and the like, and especially, part of miRNA is found to have the function of tumor suppressor or protooncogene and plays an important role in diagnosis and treatment of cancer and other diseases. Thus, miRNA is one of the popular approaches in molecular biology and epigenetic research today as a representative of non-coding RNA.

Disclosure of Invention

The invention aims to provide a miRNA group related to pancreatic cancer, which is used as an early detection marker and a prognosis biomarker of the pancreatic cancer, is used for early diagnosis and/or prognosis monitoring of the pancreatic cancer, and carries out early diagnosis on the pancreatic cancer so as to grasp the early diagnosis and treatment time of the pancreatic cancer; and/or assessing whether a pancreatic cancer patient is at risk of early metastasis and drug resistance, and adjusting the dosage regimen in time to prevent early metastasis and improve the survival time of the patient.

According to a first aspect of the present invention, there is provided a miRNA set for early diagnosis and/or prognosis monitoring of pancreatic cancer, the miRNA set comprising the following mirnas (hereinafter referred to as mirnas in the miRNA set as risk mirnas): miR-143, miR-218, miR-217, miR-216a, miR-205, miR-145, miR-195, miR-193a and miR-127.

The inventor finds that partial miRNA has a phenomenon of low expression in the pancreatic cancer patient compared with the normal human body through long-term monitoring and analysis of miRNA content in the clinical pancreatic cancer patient and the normal human body, and finds that: by carrying out grading and qualitative determination on the expression level of the 9 specific low-expression risk miRNAs in the pancreatic cancer patient and the expression level of the risk miRNAs in a normal human body, the early diagnosis of pancreatic cancer and the monitoring of early metastasis and drug resistance risk of pancreatic cancer can be carried out, so that the survival rate of the pancreatic cancer patient can be improved. The present invention has been completed based on the above findings.

In some embodiments, when used for early diagnosis of pancreatic cancer, the at-risk miRNA is derived from peripheral blood, i.e., pancreatic cancer can be diagnosed early by detecting the expression level of the at-risk miRNA in a peripheral blood sample of the subject; when used for pancreatic cancer prognostic monitoring, the miRNA is derived from peripheral blood and/or tumor tissue, i.e., the prognostic risk of pancreatic cancer can be monitored by detecting the expression level of a risk miRNA in a sample of peripheral blood and/or tumor tissue of a pancreatic cancer patient.

According to a second aspect of the present invention, there is provided a use of a product for detecting the expression level of an at-risk miRNA for the preparation of a product for early diagnosis and/or prognostic monitoring of pancreatic cancer.

In some embodiments, the product for detecting the expression level of an at-risk miRNA may be a reagent, kit, chip, and/or apparatus for detecting the expression level of a miRNA in a sample by at least one of: Northern-Blotting hybridization technique, reverse transcription PCR technique, reverse transcription-real-time fluorescence quantitative PCR technique, in-situ hybridization technique and miRNA chip technique.

According to a third aspect of the present invention, there is provided a kit for early diagnosis and/or prognostic monitoring of pancreatic cancer, comprising a reagent and/or a kit for detecting the expression level of an at-risk miRNA by reverse transcription-real-time fluorescence quantitative PCR technology.

In some embodiments, the kit for early diagnosis and/or prognosis monitoring of pancreatic cancer may include the following real-time fluorescent quantitative PCR primers:

real-time fluorescent quantitative PCR primers for miR-143: the sequence of the upstream primer is shown as SEQ ID NO. 1, and the sequence of the downstream primer is shown as SEQ ID NO. 2;

real-time fluorescent quantitative PCR primers for miR-218: the sequence of the upstream primer is shown as SEQ ID NO. 3, and the sequence of the downstream primer is shown as SEQ ID NO. 4;

real-time fluorescent quantitative PCR primers for miR-217: the sequence of the upstream primer is shown as SEQ ID NO. 5, and the sequence of the downstream primer is shown as SEQ ID NO. 6;

real-time fluorescent quantitative PCR primers for miR-216 a: the sequence of the upstream primer is shown as SEQ ID NO. 7, and the sequence of the downstream primer is shown as SEQ ID NO. 8;

real-time fluorescent quantitative PCR primers for miR-145: the sequence of the upstream primer is shown as SEQ ID NO. 9, and the sequence of the downstream primer is shown as SEQ ID NO. 10;

real-time fluorescent quantitative PCR primers for miR-205: the sequence of the upstream primer is shown as SEQ ID NO. 11, and the sequence of the downstream primer is shown as SEQ ID NO. 12;

real-time fluorescent quantitative PCR primers for miR-195: the sequence of the upstream primer is shown as SEQ ID NO. 13, and the sequence of the downstream primer is shown as SEQ ID NO. 14;

real-time fluorescent quantitative PCR primers for miR-193 a: the sequence of the upstream primer is shown as SEQ ID NO. 15, and the sequence of the downstream primer is shown as SEQ ID NO. 16;

real-time fluorescent quantitative PCR primers for miR-127: the sequence of the upstream primer is shown as SEQ ID NO. 17, and the sequence of the downstream primer is shown as SEQ ID NO. 18.

In some embodiments, the kit for early diagnosis and/or prognostic monitoring of pancreatic cancer may further include reagents/kits for extracting RNA, performing reverse transcription, and real-time fluorescent quantitative PCR; wherein the reagent comprises Trizol reagent, reverse transcriptase, buffer solution, dNTPs and MgCl₂DEPC water, DNA polymerase, etc.; the kit for reverse transcription can be any kit for reverse transcription disclosed in the prior art, such as miRcute enhanced miRNAcDNA first strand synthesis kit (TIANGEN, KP 211); the kit for performing real-time fluorescent quantitative PCR can be any kit for performing real-time fluorescent quantitative PCR disclosed in the prior art, such as miRcute enhanced miRNA fluorescent quantitative detectionTest kit (SYBRGreen) (TIANGEN, FP 411).

According to a fourth aspect of the present invention, there is provided a system for early diagnosis and/or prognostic monitoring of pancreatic cancer, comprising a configuration module, a storage module, a quantitative detection module, an input module, a risk analysis module, and an output module; wherein the content of the first and second substances,

the configuration module is used for configuring an evaluation item and storing the risk analysis rule in the storage module, wherein the evaluation item can be selected from at least one of early diagnosis, drug resistance risk and transfer risk;

the storage module is configured to store the expression level of risk miRNA of the normal person;

the quantitative detection module is used for detecting the expression level of the risk miRNA of the detected person; detecting the expression level of risk miRNA in peripheral blood of the subject when the evaluation item is early diagnosis or risk transfer, and detecting the expression level of risk miRNA in tumor tissue of the subject when the evaluation item is drug-resistant risk;

the input module is used for inputting the expression level of the risk miRNA of the examinee detected by the quantitative detection module and outputting the expression level to the risk analysis module;

the risk analysis module is used for responding to the received expression level of the risk miRNA of the detected person, acquiring the evaluation items, the risk analysis rules and the expression level of the risk miRNA of the normal person for analysis and processing, generating a risk score and outputting the risk score to the output module.

The system provided by the invention can be used for grading and qualifying the expression level of the risk miRNA in the body of the examined person and obtaining the risk scoring result, so that the risk of the corresponding assessment item of the examined person can be intuitively obtained according to the risk scoring result, and early diagnosis of pancreatic cancer and early warning of pancreatic cancer metastasis risk and drug resistance risk are facilitated.

In some embodiments, the risk analysis module may include a calculation unit and an analysis unit, wherein,

the calculation unit is used for calculating the ratio of the expression level of the risk miRNA of the detected person to the expression level of the risk miRNA corresponding to the normal person and outputting the ratio to the analysis unit;

the analysis unit is used for counting and outputting the risk score to the output module according to the ratio, the evaluation items and the risk analysis rule.

In some embodiments, when the assessment item is an early diagnosis, the risk analysis rule is configured to: when the ratio of the expression level of any miRNA of the subject to the expression level of the miRNA corresponding to the normal person is greater than or equal to 0.8, the risk score is 0, wherein the expression levels of 9 miRNAs of the subject are miR-143, miR-218, miR-217, miR-216a, miR-205, miR-145, miR-195, miR-193a and miR-127; when the number of the miRNA with the ratio less than 0.8 is 1-3, the risk score is 3; when the number of the miRNAs with the ratio of less than 0.8 is 4-6, the risk score is 6; when the number of the miRNAs with the ratio less than 0.8 is more than or equal to 7, the risk score is 9;

when the assessment item is a drug resistance risk, the risk analysis rule is configured to: when the number of miRNA with the ratio of the expression level of miRNA to the expression level of miRNA corresponding to normal persons being less than 0.8 in the expression levels of 9 miRNA of the examinee miR-143, miR-218, miR-217, miR-216a, miR-205, miR-145, miR-195, miR-193a and miR-127 is more than 6, the risk score is 9; when the number of the miRNAs with the ratio of less than 0.8 is less than or equal to 6 and the ratios of miR-143, miR-218 and miR-217 are less than 0.8, the risk score is 7; when the number of the miRNAs with the ratio of less than 0.8 is less than or equal to 6, the ratio of any one of the miRNAs in miR-143, miR-218 and miR-217 is greater than or equal to 0.8 and the ratios of miR-216a, miR-205 and miR-145 are all less than 0.8, the risk score is 5; when the number of the miRNAs with the ratio of less than 0.8 is less than or equal to 6, the ratio of any one of the miRNAs in miR-143, miR-218 and miR-217 is greater than or equal to 0.8, the ratio of any one of the miRNAs in miR-216a, miR-205 and miR-145 is greater than or equal to 0.8 and the ratios of the miR-195, the miR-193a and the miR-127 are all less than 0.8, the risk score is 3, otherwise, the risk score is 0;

when the assessment item is a transfer risk, the risk analysis rule is configured to: in the expression levels of 9 miRNAs of a subject miR-143, miR-218, miR-217, miR-216a, miR-205, miR-145, miR-195, miR-193a and miR-127, when the ratios of miR-143, miR-218 and miR-217 are all less than 0.8, the risk score is 9; when the ratio of any one miRNA in miR-143, miR-218 and miR-217 is greater than or equal to 0.8 and the ratios of miR-216a, miR-205 and miR-145 are all less than 0.8, the risk score is 6; when the ratio of any one miRNA of miR-143, miR-218 and miR-217 is greater than or equal to 0.8, the ratio of any one miRNA of miR-216a, miR-205 and miR-145 is greater than or equal to 0.8 and the ratios of miR-195, miR-193a and miR-127 are all less than 0.8, the risk score is 3, otherwise, the risk score is 0.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a group of specific low-expression risk miRNAs in pancreatic cancer patients, prepares a kit and establishes a detection analysis evaluation system according to the group of risk miRNAs, is used for early diagnosis and/or prognosis monitoring of pancreatic cancer, and provides an effective detection means for early diagnosis and prognosis monitoring of pancreatic cancer patients. By taking peripheral blood or tumor tissue of a detected person, detecting the expression level of risk miRNA, and carrying out grading and qualitative determination on the expression level of risk miRNA in the detected person and the expression level of risk miRNA in a normal human body, the early diagnosis of pancreatic cancer can be carried out on the detected person, the drug resistance risk and the early transfer risk of a pancreatic cancer patient can be analyzed, the state of illness of the pancreatic cancer patient can be diagnosed and the course of disease can be monitored, the most effective medication method can be conveniently and timely made, meanwhile, long-term monitoring is provided for the patient without further disease progression after medication, the occurrence of early transfer is prevented, medication is timely carried out, the occurrence of adverse reaction is prevented, the ineffective chemotherapy rate is reduced or the low-efficiency chemotherapy is prevented in advance, and the survival rate of the pancreatic cancer patient is.

Drawings

FIG. 1 is a block diagram of a system for early diagnosis and/or prognostic monitoring of pancreatic cancer according to one embodiment of the present invention;

FIG. 2 shows risk analysis rules for early diagnosis of pancreatic cancer according to the present invention;

FIG. 3 shows risk analysis rules corresponding to the risk of drug resistance in pancreatic cancer according to the present invention;

FIG. 4 shows risk analysis rules corresponding to the risk of pancreatic cancer metastasis in accordance with the present invention;

FIG. 5 shows the document retrieval screening process in a retrospective clinical study as described in example 1.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings. The experimental procedures in the following examples are, unless otherwise specified, conventional procedures or conditions recommended by the manufacturer; materials, reagents, instruments and the like used in examples are commercially available unless otherwise specified.

Example 1

Fig. 1 schematically shows a system for early diagnosis and/or prognostic monitoring of pancreatic cancer according to an embodiment of the present invention, including a configuration module 100, a storage module 200, a quantitative detection module 300, an input module 400, a risk analysis module 500, and an output module 600.

The quantitative detection module 300 is used for detecting the expression level of at-risk miRNA of the subject. Wherein the at-risk mirnas comprise: miR-143, miR-218, miR-217, miR-216a, miR-205, miR-145, miR-195, miR-193a and miR-127. The quantitative detection module 300 may be embodied as a product for detecting the expression level of the at-risk miRNA, for example, a reagent, a kit, a chip and/or an apparatus for detecting the expression level of the at-risk miRNA in the sample by at least one of the following methods: Northern-Blotting hybridization technique, reverse transcription PCR technique, reverse transcription-real-time fluorescence quantitative PCR technique, in-situ hybridization technique and miRNA chip technique.

In this embodiment, the quantitative detection module 300 is preferably embodied as a kit for detecting the expression level of at-risk miRNA in a sample by a reverse transcription-real-time fluorescence quantitative PCR technique. The kit mainly comprises real-time fluorescent quantitative PCR primers of the risk miRNA and real-time fluorescent quantitative PCR primers of internal reference has-U6 (see Table 1). In the process of detecting the expression level of the risk miRNA in the sample by the reverse transcription-real-time fluorescence quantitative PCR technology, other components needed to be used, such as a reagent for extracting RNA, a reagent for reverse transcription, a reagent for real-time fluorescence quantitative PCR and the like, can be provided by the kit provided by the invention, and can also be obtained by a user of the kit from other commercial ways. Wherein, the extraction method of RNA can be Trizol method, and the reagent mainly comprises Trizol reagent, chloroform, isopropanol, ethanol, etc.; the specific components of the reagent for performing reverse transcription can refer to any reagent in a kit for performing reverse transcription disclosed in the prior art; the components of the reagent for performing real-time fluorescent quantitative PCR may refer to any reagent disclosed in the prior art in the kit for performing real-time fluorescent quantitative PCR.

In this embodiment, the method for detecting the expression level of the risk miRNA in the sample by the quantitative determination module 300 preferably includes the following steps:

(1) extraction of RNA from samples:

when the sample is peripheral blood, the blood sample is,

a. taking 50-250 mu L of peripheral blood whole blood, adding 10 times of volume of Trizol reagent (Invitrogen, UK; the main component of the Trizol reagent is guanidine isocyanate), fully and uniformly blowing, standing at room temperature for 5min, and completely separating nucleic acid protein compounds;

b. adding 200 mu L of chloroform into 1mL of Trizol reagent according to the using amount of the Trizol reagent, standing for 3min, and then centrifuging for 15min at 12000g and 4 ℃;

c. transferring the upper aqueous phase to an EP (eppendorf) tube, adding 500 μ L isopropanol, turning upside down, mixing for 10s, standing at room temperature for 10min, centrifuging at 12000g at 4 deg.C for 10min, and removing the supernatant;

d. adding 1mL of 75% ethanol, reversing the upside down, mixing uniformly for 10s, centrifuging at 12000g and 4 ℃ for 10min, and removing the supernatant;

e. drying at room temperature for 5-10min (not fully dried, not fully dried and not dissolved), adding 50 μ L of RNase-free-water to fully dissolve RNA;

f. OD values were measured spectrophotometrically, and total RNA concentrations were measured.

When the sample is a tumor tissue, the tumor tissue,

A. weighing the tissues, adding 1mL of Trizol reagent, uniformly mixing, and placing on ice for cracking for 5 min;

B. adding 200 mu L of chloroform into 1mL of Trizol reagent according to the using amount of the Trizol reagent, violently shaking up to enable the Trizol reagent to be emulsion, and standing for 5 min;

C. centrifuging at 12000g and 4 deg.C for 5 min;

D. carefully sucking the supernatant into another EP tube, adding isopropanol precooled to 4 ℃ in the same volume, and uniformly mixing;

E. centrifuging at 12000g at 4 deg.C for 15 min;

F. discarding the supernatant, adding 1mL of 75% ethanol, washing the precipitate, and centrifuging at 8000g and 4 ℃ for 8 min;

G. sucking supernatant, drying ethanol, adding RNase-free-water to dissolve RNA fully;

H. OD values were measured spectrophotometrically, and total RNA concentrations were measured.

(2) Reverse transcription to synthesize cDNA: taking 1 mu gRNA, and carrying out reverse transcription by using a miRcute enhanced miRNA cDNA first strand synthesis kit (TIANGEN, KP211) to synthesize cDNA;

(3) fluorescent quantitative PCR (qPCR) amplified cDNA: performing fluorescent quantitative PCR on the cDNA synthesized by reverse transcription by using a miRcute enhanced miRNA fluorescent quantitative detection kit (SYBR Green) (TIANGEN, FP411), taking has-U6 as a detected internal reference, and taking 10 mu L as a reaction system for qPCR amplification. The fluorescent quantitative PCR results were compared with the cycle number of the internal reference, and 2 was calculated^-△△CtThen, statistical analysis was performed. The primer sequences are shown in table 1 below.

TABLE 1 fluorescent quantitative PCR primers

The storage module 200 is configured to store expression levels of risk mirnas of normal persons. The expression level of the normal risk miRNA is mainly obtained by taking normal peripheral blood as a sample, extracting RNA, performing reverse transcription to synthesize cDNA, and then quantifying the risk miRNA by using the primers in table 1 and has-U6 as an internal reference, as shown in table 2.

TABLE 2 expression levels of at-risk miRNAs in normal humans

Risk miRNA	Relative expression amount
		miR-143	6.2-6.5
miR-218	6.5-8
		miR-217	6.2-6.8
miR-216a	1.9-2.4
		miR-145	1.8-1.6
miR-205	1.5-1.8
		miR-195	0.8-1.2
miR-193a	0.75-1.2
		miR-127	0.8-1.2

In the practical application process, regarding the specific expression level of the risk miRNA of the normal person, a user can extract RNA and synthesize cDNA by reverse transcription by taking the peripheral blood of the normal person as a sample, and then the risk miRNA is obtained quantitatively by using the primers shown in the table 1 and has-U6 as an internal reference; in the process of obtaining the specific expression level of the risk miRNA of the normal person, if the measured specific numerical value is smaller than the lower limit of the range shown in the table 2, taking the lower limit of the range shown in the table 2 as the specific expression level of the risk miRNA, and if the measured specific numerical value is higher than the upper limit of the range shown, rejecting the sample.

The input module 400 is used for inputting the expression level of the risk miRNA of the subject detected by the quantitative detection module 300 and outputting the expression level to the risk analysis module 500. The input module 400 may be implemented as a keyboard, a scanner, a writing pad, or other input devices in the prior art.

The configuration module 100 is used for configuring the evaluation items and storing the risk analysis rules corresponding to the evaluation items in the storage module 200. The evaluation item can be early diagnosis, drug resistance risk or transfer risk.

According to the prospective clinical research results of the inventor (see example 2 for details), and the results of the retrospective clinical research by using the clinical data collected by literature search with the keywords of "pancreatericcancer, pancreatic product adenocarinoma, miRNA, microRNA, miR" and the like (the specific process of literature search screening in the retrospective clinical research is shown in figure 5), the relation between the expression level of each risk miRNA and the clinical pathological characteristics and prognosis of a pancreatic cancer patient is analyzed, and finally, the risk analysis rule for early diagnosis, drug resistance risk assessment and metastasis risk assessment of pancreatic cancer by using the risk miRNA is established. Meanwhile, in order to avoid the occurrence of false positives and false negatives, the risk level of the risk miRNA is graded according to the data of prospective clinical research and retrospective clinical research, and according to the False Discovery Rate (FDR) and the P value in the statistical process, the FDR is an index mainly used for measuring the false positive rate of the primary test, the P value is mainly used for measuring the occurrence of the primary error, and the grading standard and the result are shown in table 3. Wherein the risk level of the risk miRNA is reduced from A to C in sequence.

TABLE 3 grading Standard of Risk miRNA

Rank of	miRNA	Grading standards
			A	miR-218、miR-143、miR-217	FDR<25％、P<0.001
B	miR-216a、miR-205、miR-145	FDR<30％、P<0.01
			C	miR-127、miR-195、miR-193a	P<0.05

In the invention, the risk analysis rule corresponding to the evaluation project is specifically as follows:

as shown in fig. 2, when the evaluation item is an early diagnosis, the corresponding risk analysis rule is:

of the expression levels of the 9 at-risk mirnas of the subject,

when the ratio of the expression level of any risk miRNA of the detected person to the expression level of the risk miRNA corresponding to the normal person is more than or equal to 0.8, the risk score is 0;

when the number of the risk miRNA with the ratio less than 0.8 is 1-3, the risk score is 3;

when the number of the risk miRNA with the ratio less than 0.8 is 4-6, the risk score is 6;

when the number of at-risk mirnas with a ratio of less than 0.8 is greater than or equal to 7, the risk score is 9.

As shown in fig. 3, when the evaluation item is a drug resistance risk, the risk analysis rule of the correspondence is:

of the expression levels of the 9 at-risk mirnas of the subject,

when the number of the risk miRNAs with the expression level ratio of the risk miRNAs corresponding to the normal person being less than 0.8 is more than 6, the risk score is 9;

when the number of the risk miRNAs with the ratio less than 0.8 is less than or equal to 6 and the ratios of the group A risk miRNAs are all less than 0.8, the risk score is 7;

when the number of the risk miRNAs with the ratio of less than 0.8 is less than or equal to 6, the ratio of any risk miRNA in the group A risk miRNAs is greater than or equal to 0.8 and the ratios of the group B risk miRNAs are all less than 0.8, the risk score is 5;

when the number of the risk miRNAs with the ratio of less than 0.8 is less than or equal to 6, the ratio of any risk miRNA in the group A risk miRNAs is greater than or equal to 0.8, the ratio of any risk miRNA in the group B risk miRNAs is greater than or equal to 0.8 and the ratios of the group C risk miRNAs are less than 0.8, the risk score is 3;

when the number of the risk miRNAs with the ratio of less than 0.8 is less than or equal to 6 and the ratio of the expression level of any one or two risk miRNAs in the group A, the group B and the group C to the expression level of the risk miRNA corresponding to the normal person is more than or equal to 0.8, the risk score is 0.

As shown in fig. 4, when the evaluation item is a transfer risk, the risk analysis rule corresponding to the evaluation item is:

of the expression levels of the 9 at-risk mirnas of the subject,

when the ratios of the group A risk miRNAs are less than 0.8, the risk score is 9;

when the ratio of any one risk miRNA in the group A risk miRNA is greater than or equal to 0.8 and the ratios of the group B risk miRNA are less than 0.8, the risk score is 6;

when the ratio of any one of the group A risk miRNAs is greater than or equal to 0.8, the ratio of any one of the group B risk miRNAs is greater than or equal to 0.8 and the ratios of the group C risk miRNAs are less than 0.8, the risk score is 3; otherwise, the risk score is 0.

The risk analysis module 500 is configured to obtain the evaluation items, the risk analysis rules, and the expression levels of the risk mirnas of the normal persons for analysis processing in response to the received expression levels of the risk mirnas of the subjects, generate a risk score, and output the risk score to the output module 600. In this embodiment, the risk analysis module 500 preferably includes a calculation unit 501 and an analysis unit 502.

The calculation unit 501 is configured to calculate a ratio of an expression level of a risk miRNA of a subject to an expression level of a risk miRNA corresponding to a normal person and output the ratio to the analysis unit 502.

The analysis unit 502 is configured to statistically output the risk score to the output module 600 according to the ratio, the evaluation item, and the risk analysis rule.

The workflow of the system for early diagnosis and/or prognosis monitoring of pancreatic cancer of the present embodiment comprises the following steps:

s1, configuring the evaluation items and storing the risk analysis rules corresponding to the evaluation items in the storage module 200; wherein, the assessment item can be selected from one or more of early diagnosis, drug resistance risk or metastasis risk, and the risk analysis rule corresponding to the assessment item is referred to the above.

S2, detecting the expression level of the risk miRNA of the peripheral blood and/or the tumor tissue sample of the detected object by the quantitative detection module 300;

s3, the expression level of the risk miRNA detected by the quantitative detection module 300 is acquired through the input module 400 and output to the risk analysis module 500;

s4, the risk analysis module 500 responds to the expression level of the risk miRNA of the subject, obtains the evaluation items, risk analysis rules and the expression level of the risk miRNA of the normal person in the storage module 200, analyzes and processes the evaluation items, risk analysis rules and the expression level of the risk miRNA, generates a risk score, and outputs the risk score to the output module 600.

Wherein, in the above work flow, the following steps can be further included: and detecting and configuring the risk miRNA of the normal person and storing the risk miRNA into a storage module.

In the invention, the higher the risk score of early diagnosis is, the higher the risk of pancreatic cancer of the detected person is, thus prompting that early medication can be adopted; a higher drug resistance risk score indicates a higher drug resistance risk for the pancreatic cancer patient, suggesting that attention should be paid to monitoring and improving the treatment regimen; the higher the metastasis risk score is, the higher the probability of occurrence of metastasis of a pancreatic cancer patient is, long-term monitoring is required, the treatment opportunity before metastasis is grasped, the medication method is adjusted in time, the occurrence of early metastasis is prevented, and the survival time of the patient is prolonged.

The system is beneficial to early diagnosis of pancreatic cancer, analyzes the drug resistance risk and the early pancreatic cancer metastasis risk of a detected person, is convenient for a doctor to make the most effective drug administration method in time, provides long-term monitoring for a patient without further disease progression after drug administration, prevents the occurrence of early metastasis, administers the drug in time, effectively treats the patient, and improves the survival rate of the pancreatic cancer patient.

In a preferred embodiment, in addition to the quantitative detection module 300 and the input module 400, other modules of the system for early diagnosis and/or prognosis monitoring of pancreatic cancer of the present invention can be implemented by a program module, which is deployed in a server or stored in an electronic device, and includes program instructions for implementing the above functions, so that the program instructions can be executed by a processor of the server or a processor in the electronic device, thereby implementing the risk analysis monitoring by an automated system.

Example 2

22 pancreatic cancer patients were treated with blood and cancer tissues and corresponding paracancerous tissues from radical resection of pancreatic cancer in general surgery in third people's hospital, Wuxi city, from 2016 (7 months) to 2018 (7 months), with informed consent and written notes. Wherein the female is 10 cases, the male is 12 cases, the age is 40-73 years, and the median age is 55 years. After the specimen is washed with DEPC water and surface dirt and blood stain in half an hour in vitro, 5 pieces of pancreatic cancer and tissues beside the cancer (more than 3cm away from the cancer tissues) are cut rapidly by a sharp blade, and blood vessels, nerves and connective blocks which can be attached are removed. All cases were not treated with chemotherapy, radiotherapy or immunotherapy before surgery, post-operative physical examination was diagnosed by 2 pathologists together, and the paraffin pathology was pancreatic cancer. Immediately placing the tissue specimen into a liquid nitrogen refrigerator at minus 80 ℃ for storage for later use after the tissue specimen is separated from the body. Follow-up visit is carried out on the patient after the operation, the follow-up visit is carried out on the patient mainly in a telephone consultation mode, the starting point of the follow-up visit is the operation date, and the ending date is 7 months and 31 days in 2019. The follow-up content is the result of the patient's periodic review, whether the patient has relapsed or has distant metastasis, and the last follow-up time or death time of the patient is recorded.

The system for early diagnosis and/or prognosis monitoring of pancreatic cancer is utilized to detect the expression levels of miR-143, miR-218, miR-217, miR-216a, miR-205, miR-145, miR-195, miR-193a and miR-127 in blood and cancer tissue samples of 22 pancreatic cancer patients, and the risk scores of early diagnosis, drug resistance risk and metastasis risk of pancreatic cancer of the patients are analyzed and obtained.

The clinical effectiveness of the evaluation results of the system of the present invention was evaluated by comparing the number of patients with a risk score of 6 points or more for early diagnosis measured by the system of the present invention with the number of patients actually diagnosed as pancreatic cancer, comparing the number of patients with a risk score of 6 points or more for metastatic diagnosis measured by the system of the present invention with the number of patients with actually developed metastasis, and comparing the number of patients with a risk score of 7 points or more for drug resistance diagnosis measured by the system of the present invention with the number of patients with actually developed drug resistance in clinic, and the results are shown in table 4.

TABLE 4 clinical effectiveness of the inventive System

In the early diagnosis evaluation, the number of clinically actually diagnosed patients is 22, and the risk score measured by the system of the invention is not less than 18 patients with 6 scores, wherein the risk score is 12 patients with 6 scores, and the risk score is 6 patients with 9 scores;

in the evaluation of the risk of metastasis, the number of patients actually clinically metastasizing in 22 pancreatic cancer patients is 14, and among 14 patients, 9 patients with early metastasis risk scores not less than 6 are measured by the system of the invention, wherein the risk score is 4 of 6 and 5 of 9;

in the drug resistance risk assessment, the number of patients actually clinically developing metastasis in 22 pancreatic cancer patients is 15, and 11 patients with early metastasis risk scores not less than 7 are selected from 15 patients, wherein the risk score is 8 of 7 and 3 of 9.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.

Sequence listing

<110> affiliated hospital of south Jiangnan university

<120> miRNA group for early diagnosis and/or prognosis monitoring of pancreatic cancer and application thereof

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Claims

1. A miRNA panel for early diagnosis and/or prognostic monitoring of pancreatic cancer, comprising mirnas as follows: miR-143, miR-218, miR-217, miR-216a, miR-205, miR-145, miR-195, miR-193a and miR-127.

2. The miRNA panel according to claim 1, wherein, when used for early diagnosis of pancreatic cancer, the miRNA is derived from peripheral blood; when used for pancreatic cancer prognostic monitoring, the miRNA is derived from peripheral blood and/or tumor tissue.

3. Use of a product for detecting the expression level of mirnas in the set of mirnas of claim 1 or 2 for the preparation of a product for early diagnosis and/or prognostic monitoring of pancreatic cancer.

4. The use according to claim 3, wherein the product for detecting the expression level of miRNA in the miRNA group of claim 1 or 2 is a reagent, kit, chip and/or apparatus for detecting the expression level of miRNA in the sample by at least one of: Northern-Blotting hybridization technique, reverse transcription PCR technique, reverse transcription-real-time fluorescence quantitative PCR technique, in-situ hybridization technique and miRNA chip technique.

5. Kit for early diagnosis and/or prognostic monitoring of pancreatic cancer, comprising reagents and/or kits for detecting the expression level of mirnas in the miRNA group of claim 1 or 2 by reverse transcription-real-time fluorescent quantitative PCR technology.

6. The kit of claim 5, comprising the following real-time fluorescent quantitative PCR primers:

7. The system for early diagnosis and/or prognosis monitoring of pancreatic cancer comprises a configuration module, a storage module, a quantitative detection module, an input module, a risk analysis module and an output module; wherein the content of the first and second substances,

the configuration module is used for configuring an evaluation item and storing the risk analysis rule into the storage module, wherein the evaluation item is selected from at least one of early diagnosis, drug resistance risk and transfer risk;

the storage module is configured to store the expression levels of the miRNAs in the miRNA group of claim 1 of the normal human;

a quantitative detection module for detecting the expression level of the mirnas in the miRNA panel of claim 1 in a subject; detecting the expression level of miRNA in the peripheral blood of the detected object when the evaluation item is early diagnosis or risk of metastasis; detecting the expression level of miRNA in the tumor tissue of the detected object when the evaluation item is the drug resistance risk;

the input module is used for inputting the expression level of the miRNA of the detected person to be detected by the quantitative detection module and outputting the expression level to the risk analysis module;

the risk analysis module is used for responding to the received expression level of the miRNA of the detected person, obtaining the evaluation items, the risk analysis rules and the expression level of the miRNA in the miRNA group of the normal person claim 1 for analysis and processing, generating a risk score and outputting the risk score to the output module.

8. The system of claim 7, wherein the risk analysis module comprises a calculation unit and an analysis unit, wherein,

the calculating unit is used for calculating the ratio of the expression level of the miRNA of the detected person to the expression level of the miRNA corresponding to the normal person and outputting the ratio to the analyzing unit;

and the analysis unit is used for counting and outputting the risk score to an output module according to the ratio, the evaluation item and the risk analysis rule.

9. The system of claim 7 or 8,

when the assessment item is an early diagnosis, the risk analysis rule is configured to: when the ratio of the expression level of any miRNA of the subject to the expression level of the miRNA corresponding to the normal person is greater than or equal to 0.8, the risk score is 0, wherein the expression levels of 9 miRNAs of the subject are miR-143, miR-218, miR-217, miR-216a, miR-205, miR-145, miR-195, miR-193a and miR-127; when the number of the miRNA with the ratio less than 0.8 is 1-3, the risk score is 3; when the number of the miRNAs with the ratio of less than 0.8 is 4-6, the risk score is 6; when the number of the miRNAs with the ratio less than 0.8 is more than or equal to 7, the risk score is 9;