CN108624688B

CN108624688B - Application of hsa _ circ _0012755 as prostate cancer molecular target in preparation of medicines and kits

Info

Publication number: CN108624688B
Application number: CN201810516627.0A
Authority: CN
Inventors: 李瑶; 吴海; 孔哲; 万学超; 鲁亚莉; 张亚龙
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2018-05-26
Filing date: 2018-05-26
Publication date: 2022-01-28
Anticipated expiration: 2038-05-26
Also published as: CN108624688A

Abstract

The invention belongs to the field of biotechnology, and particularly relates to application of hsa _ circ _0012755 as a prostate cancer molecular target in preparation of medicines and diagnostic kits. The nucleotide sequence of hsa _ circ _0012755 is shown in SEQ ID NO. 1. The invention comprises the application of the prostate cancer molecular target in the preparation of screening drugs for preventing, relieving and/or treating prostate cancer; an inhibitor comprising the molecular target of the prostatic cancer and the application of the inhibitor in preparing a medicament for preventing, relieving and/or treating the prostatic cancer; comprises a prostate cancer diagnosis kit containing a prostate cancer molecular target; also includes the application of the prostate cancer molecular target and the diagnosis kit in screening, diagnosing, treating and condition monitoring, prognosis monitoring and the like of high risk group of prostate cancer. The molecular target and the diagnostic kit containing the molecular target are used for diagnosing the prostate cancer, have the characteristics of simple operation, convenient material taking, safety, no wound, higher specificity and sensitivity and easy mass screening.

Description

Application of hsa _ circ _0012755 as prostate cancer molecular target in preparation of medicines and kits

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a prostate cancer molecular target hsa _ circ _0012755 in preparation of medicines and diagnostic kits, which comprises application of the biological molecular target in the fields of screening of high risk groups of prostate cancer, diagnosis of prostate cancer, monitoring of prostate cancer treatment and conditions, monitoring of prostate cancer guiding drugs, prostate cancer prognosis detection and the like.

Background

According to the annual report of ASCO: 2015 clinical oncology progress, liquid biopsy technique is listed as one of the next decade trends in the field of tumor therapy. Some molecular markers in blood have been used for diagnosis of some diseases, such as glutamic-pyruvic transaminase for diagnosis of hepatitis and leukocyte count for diagnosis of inflammation. Circulating tumor DNA (ctDNA) is extracellular free DNA in a cell-free state, exists in body fluids such as blood, synovial fluid and cerebrospinal fluid, and body cell DNA is released into the circulatory system after shedding or apoptosis, mainly consists of short single-stranded or double-stranded DNA and a mixture of single-stranded and double-stranded DNA, and exists in the form of a DNA protein complex or free DNA. ctDNA is from somatic mutation of tumor cells, therefore, ctDNA is a characteristic novel tumor biomarker, can be qualitatively, quantitatively and tracked, can be used for early diagnosis, prognosis judgment, tracking follow-up and the like of clinical tumors, and provides a series of convenient, quick, specific, noninvasive or minimally invasive and molecular biological detection means, and can avoid complex and invasive biopsy particularly for tumors which do not have typical clinical symptoms, have no specificity in examination and are difficult to diagnose. Incorporation of next generation sequencing technology (NGS) will become a "fluid biopsy" and replace invasive tissue biopsies.

The hsa _ circuit _0012755 belongs to Circular RNA (Circular RNA), is a Circular non-coding RNA, is discovered to be a RNA with important biological functions in recent years, participates in a plurality of important regulation processes such as genomic imprinting, chromosome silencing, chromatin modification, transcriptional activation, transcriptional interference, intranuclear transportation and the like, and plays an important regulation role in the life activities such as cell differentiation and development, gene transcription and translation, heredity, epigenetics and the like. In recent years, more and more authoritative researches prove that the circRNA plays a role in inhibiting or promoting tumors in the occurrence and development of the tumors and plays an important role in regulating the proliferation, apoptosis, cell cycle, invasion and metastasis capacity and the like of tumor cells. Currently, many circrnas have been demonstrated to differentially express and perform important regulatory functions in various human tumors including breast cancer, prostate, melanoma, liver cancer, colon cancer, bladder cancer, and the like. The early detection of the occurrence and development of tumors and the improvement of the curative effect of anticancer drugs are particularly important for the treatment of cancers, and the discovery of novel tumor markers as targets for diagnosis and treatment is always a hotspot of tumor research.

Prostate cancer is one of the common malignancies of the male reproductive system. Worldwide, the incidence of prostate cancer is second among all malignancies in men, and has exceeded the incidence of lung cancer in the united states, becoming the first tumor to compromise male health. The incidence of asian prostate cancer is much lower than in europe and the united states, but has been on the rise in recent years and has grown more rapidly than in developed countries in europe and the united states. Prostate cancer patients are mainly elderly men, generally develop disease after the age of 50 years, 95% of them develop in elderly men over the age of 60 years, and the incidence rate continuously increases with age. Early stage prostate cancer is mostly asymptomatic, even if uncomfortable, it is not enough to attract attention of patients, and when tumors are enlarged and press against urethra, the prostate cancer is often confused with benign prostatic hyperplasia. Prostate cancer is found only when about 80% of patients in China find distant metastatic lesions. At this time, the lesion has reached an advanced stage, and the prognosis is poor.

The clinical diagnosis modes of the prostate cancer mainly comprise digital rectal examination, serum Prostate Specific Antigen (PSA) detection, rectal ultrasonic detection, biopsy pathological examination and the like. The digital rectal examination is the simplest, most economical and practical method, and is mainly used for finding many asymptomatic patients with prostate cancer by touching the prostate with the index finger of a doctor, so that the early diagnosis and radical treatment opportunities are possibly obtained. However, the above methods have limitations. The limitations of digital rectal examination, for example, are mainly in 4 areas: (1) when the prostate tumor of a patient is not large, the patient is easy to leak a diagnosis; (2) some patients have unobvious prostate cancer swelling, but have been in the advanced stage, and are not easy to cure radically; (3) this test cannot be used when the patient has a rectal disorder; (4) when the doctor is inexperienced, missed diagnosis or misdiagnosis may occur. Normally, the PSA in blood is not high (not higher than 4 ng/ml), and when in the diseased state of prostate cancer and other prostate diseases, the PSA is increased to become the tumor marker most sensitive to screening prostate cancer at present, but the PSA has certain limitations: (1) blood is required to be taken for detection, and certain damage is caused to a patient; (2) increased PSA is also common in non-prostate cancer diseases such as prostatitis, prostatic hypertrophy, etc., and is therefore not easily diagnosed; (3) when prostate cancer is diagnosed with increased PSA, patients often fall into the middle and late stages and do not achieve the purpose of early diagnosis. The prostate ultrasonic detection is simple, intuitive and non-invasive, and provides positioning and qualitative signs and judges the nature of pathological changes by displaying the size, number, position, density, edge, body, presence or absence of calcification, the shape, size, number and distribution of the tumor, the surrounding halo, skin changes and the like; the limitations are as follows: (1) the diagnosis of compact small cancer foci is easy to miss; (2) sometimes fail to provide a definitive qualitative diagnosis; (3) the diagnostic coincidence rate is low because it cannot show the internal structure and surrounding tissues of the tumor; (4) the misdiagnosis rate of some solid benign and malignant tumors lacking typical signs is high. Biopsy cannot be used as a primary screening means due to its invasiveness and complexity, but is the gold standard for accurate diagnosis of prostate cancer, and is generally used in combination with other methods and technologies.

Recent studies show that circRNA is closely related to prostate cancer, and the circRNA can be involved in the occurrence, development and metastasis of tumors, so that the circRNA can have corresponding effects on the pathogenesis, early diagnosis, individualized treatment, detection and prognosis of metastasis and the like of the tumors.

Disclosure of Invention

The invention aims to provide a novel prostate cancer molecular target related to prostate cancer and application thereof in preparing medicines and prostate cancer diagnostic kits.

According to the invention, the content of hsa _ circ _0012755 in the prostate cancer tissue sample and the serum is higher than that in the normal prostate tissue and the plasma sample, and the fact that hsa _ circ _0012755 has close correlation with the tumorigenesis and the development of the prostate cancer is shown. Therefore, the invention provides hsa _ circ _0012755 as a novel prostate cancer molecular target related to prostate cancer, a diagnostic kit containing the prostate cancer molecular target hsa _ circ _0012755 (as a marker), and application of the molecular target or the diagnostic kit in screening, diagnosis, treatment, condition monitoring and prognosis monitoring of high-risk groups of prostate cancer.

The prostate cancer molecular target hsa _ circ _0012755 provided by the invention is a circRNA with the total length of 516bp, and the nucleotide sequence of the circular RNA is shown in SEQ ID NO. 1.

The prostate cancer molecule target hsa _ circ _0012755 can be used for screening drugs for preventing, relieving and/or treating prostate cancer.

The prostate cancer molecule target hsa _ circ _0012755 can be used for preparing a medicament for preventing, relieving and/or treating prostate cancer.

The present invention also provides a diagnostic kit for diagnosing prostate cancer, which contains the molecular target hsa _ circ _0012755 for prostate cancer.

The invention also provides a diagnostic kit for diagnosing prostate cancer, which specifically comprises:

(1) extracting reagent for total RNA in tissue sample, blood and urine;

(2) a reverse transcription reagent;

(3) and (4) quantifying PCR reagents.

The control used was β -ACTIN, whose primer sequences were as follows:

beta-ACTIN upstream primer sequence: 5 '-CCTCTCCCAAGTCCACACAG-3' (SEQ ID NO. 2)

beta-ACTIN downstream primer sequence: 5 '-GGGCACGAAGGCTCATCATT-3' (SEQ ID NO. 3).

The primer sequences of hsa _ circ _0012755 detected by the detection kit are as follows:

hsa _ circ _0012755 upstream primer sequence: 5 '-GGTTTCAGCAGCCCCTCC-3' (SEQ ID NO. 4)

hsa _ circ _0012755 downstream primer sequence: 5 '-CCTTAATGTCAGCGTCACTTGG-3' (SEQ ID NO. 5).

The diagnosis kit provided by the invention can be used for screening and diagnosing high risk group of prostatic cancer, monitoring treatment condition, monitoring prognosis and the like.

The invention also provides application of the prostate cancer molecular target hsa _ circ _0012755 as a marker in preparation of a kit for screening, diagnosing, monitoring treatment conditions and monitoring prognosis of high risk group of prostate cancer, which detects the content of hsa _ circ _0012755 in tissue samples, blood and urine of a tested person and compares the content with the normal level of hsa _ circ _0012755 so as to screen, diagnose, monitor treatment conditions and monitor prognosis of the high risk group of prostate cancer.

The application of the prostate cancer molecular target hsa _ circ _0012755 as a marker in preparing a kit for screening, diagnosing, designing medicines, monitoring treatment and conditions and monitoring prognosis of high risk group of prostate cancer can detect the content of hsa _ circ _0012755 in serum, urine and tissue samples of a tested person through total RNA extraction, reverse transcription and quantitative PCR.

Specifically, the diagnostic kit for diagnosing prostate cancer described above comprises:

(1) extracting reagent for total RNA in tissue sample, blood and urine;

every 50mg tissue or 200ul blood or 200ul urine

(2) A reverse transcription reagent;

(3) quantitative PCR reagent;

the primers include two pairs of hsa _ circ _0012755 and control β -ACTIN primers;

(4) normal prostate tissue cDNA

As a negative control, the PCR assay was quantified together with the cDNA of the test sample, and the same amount of cDNA as the test sample was used for each reaction system.

The result analysis detection sample of the diagnosis kit for diagnosing the prostatic cancer and the negative control are compared by adopting t test, the difference is obvious when P is less than 0.05, and the result is judged to be positive in sample detection.

The control used by the detection kit is beta-ACTIN, and the primer sequences are as follows:

beta-ACTIN upstream primer sequence: 5 '-CCTCTCCCAAGTCCACACAG-3' (SEQ ID NO. 2)

beta-ACTIN downstream primer sequence: 5 '-GGGCACGAAGGCTCATCATT-3' (SEQ ID NO. 3)

The invention provides a novel molecular target hsa _ circ _0012755 for diagnosis and treatment of prostate cancer. The inhibitor of the molecular target can be used for preparing a medicament for preventing, relieving and/or treating prostatic cancer. The molecular target and the diagnostic kit containing the molecular target are used for diagnosing the prostate cancer, have the characteristics of simple operation, convenient material taking, safety, no wound, higher specificity and sensitivity and convenience for large-scale screening. The molecular target is suitable for being applied to the fields of screening of high risk groups of prostatic cancer, identification of prostatic cancer, monitoring of prostatic cancer treatment and condition, monitoring of prostatic cancer guiding drugs, monitoring of prostatic cancer prognosis and the like.

Drawings

FIG. 1 shows the measurement of hsa _ circ _0012755 content in prostate cancer tissue samples and normal tissue samples.

FIG. 2 shows the measurement of hsa _ circ _0012755 content in prostate cancer serum samples and normal serum samples.

FIG. 3 is a graph of the inhibition efficiency of hsa _ circ _0012755siRNA on hsa _ circ _0012755 in prostate cancer PC-3 cell line.

FIG. 4 shows that siRNA of hsa _ circ _0012755 significantly inhibited the cycle of prostate cancer cells.

FIG. 5 is a graph showing that siRNA to hsa _ circ _0012755 significantly promoted apoptosis in prostate cancer cells.

FIG. 6 shows that siRNA of hsa _ circ _0012755 significantly inhibited prostate cancer cell proliferation.

Detailed Description

In order to make the present invention more understandable, the present invention is further illustrated below with reference to specific examples, which are only for illustrating the present invention and do not limit the scope of the present invention, and specific experimental methods not mentioned in the following examples are performed according to conventional experimental methods.

Example 1: hsa _ circ _0012755 was assayed for its content in prostate cancer tissue samples and in normal tissue samples.

The hsa _ circ _0012755 detection primer sequence used in this example was:

hsa _ circ _0012755 upstream primer sequence: 5 '-CCTTAATGTCAGCGTCACTTGG-3' (SEQ ID NO. 5).

The main steps of this example are as follows:

1. extraction of total RNA from tissue samples

Tissue samples were obtained after surgery in patients with radical prostate cancer, while excised tissue samples from patients with lymph node dissection were obtained as controls. Extracting the total RNA of the tissue sample in a 1.5ml centrifuge tube without DNA and RNase pollution;

the kit for extracting total RNA from tissue samples is purchased from Beijing kang, a century Biotechnology Co., Ltd. Concentration determination of extracted total RNA by determination of the ratio of 260/280nm ultraviolet wavelength using a thermo NanoDrop2000c spectrophotometer;

2. quantitative detection of circRNA in tissue samples

(1) Reverse transcription of RNA to obtain cDNA single strand

The reverse transcription system was prepared according to table 1 below, the preparation being carried out on ice. The prepared system is subjected to reverse transcription on a PCR instrument. The reaction conditions are 38 ℃ for 15 minutes and 85 ℃ for 5 seconds;

TABLE 1

In table 1, X indicates that the volume of RNA added is determined by the RNA concentration, which in this experiment is 500 ng/RNA concentration. The aforementioned reverse transcription kit was purchased from Dalibao Biotechnology Ltd (Takara);

(2) quantitative qPCR detection

cDNA diluent is used as a real-time quantitative PCR template, the final concentration of the primer is 200nM, and the total reaction volume is 10 mul. Real-time quantitative PCR instrument was performed using Roche LightCycler480 ii, in 384 well plates. Each sample was repeated two to three times. Under the condition that the dissolution curve basically meets the requirement and no non-specific amplification exists, the relative expression quantity is obtained by using a threshold cycle (Ct) method with reference to an operation manual, and beta-ACTIN is used as an internal reference. See table 2 for specific procedures;

TABLE 2

3. Data analysis was performed using Array Tools 4.1.0

The Ct value levels of the target circRNA and the reference circRNA in the sample can be measured by the method, and the relative content of the target circRNA in the tissue can be obtained. Correction of the amount of circRNA Using beta-ACTIN as a reference, classical 2 in QPCR assays^-ΔΔCtThe method of (1) represents the level of circRNA in the tissue sample (- Δ Ct is the difference between Ct values for target circRNA and control);

4. diagnosis of prostate cancer by hsa _ circ _0012755 levels in tissue samples

As shown in FIG. 1, the tissue samples from prostate cancer patients had a relatively high level of hsa _ circ _0012755 compared to the low level of hsa _ circ _0012755 in normal control tissue samples, and was generally higher than the average of normal tissue samples, with statistical differences.

Example 2: hsa _ circ _0012755 was assayed for its content in prostate cancer plasma samples and in normal plasma samples.

The hsa _ circ _0012755 detection primer sequence used in this example was:

The main steps of this example are as follows:

1. extraction of total RNA from plasma samples

Plasma samples were obtained from 26 prostate cancer patients, while 19 normal human plasma samples were obtained as controls. Extracting the total RNA of the tissue sample in a 1.5ml centrifuge tube without DNA and RNase pollution;

kits for total RNA extraction from plasma samples were purchased from QIAGEN. Concentration determination of extracted total RNA by determination of the ratio of 260/280nm ultraviolet wavelength using a thermo NanoDrop2000c spectrophotometer;

2. quantitative detection of circRNA in tissue samples

(1) Reverse transcription of RNA to obtain cDNA single strand

The reverse transcription system was prepared according to Table 3 below, and the preparation was carried out on ice. The prepared system is subjected to reverse transcription on a PCR instrument. The reaction conditions are 38 ℃ for 15 minutes and 85 ℃ for 5 seconds;

TABLE 3

In table 3, X indicates that the volume of RNA added is determined by the RNA concentration, which in this experiment is 500 ng/RNA concentration. The aforementioned reverse transcription kit was purchased from Dalibao Biotechnology Ltd (Takara);

(2) quantitative qPCR detection

cDNA diluent is used as a real-time quantitative PCR template, the final concentration of the primer is 200nM, and the total reaction volume is 10 mul. Real-time quantitative PCR instrument was performed using Roche LightCycler480 ii, in 384 well plates. Each sample was repeated two to three times. Under the condition that the dissolution curve basically meets the requirement and no non-specific amplification exists, the relative expression quantity is obtained by using a threshold cycle (Ct) method with reference to an operation manual, and beta-ACTIN is used as an internal reference. See table 4 for specific procedures;

TABLE 4

；

3. Data analysis was performed using Array Tools 4.1.0

The Ct value levels of the target circRNA and the reference circRNA in the sample can be measured by the method, and the relative content of the target circRNA in the tissue can be obtained. Correction of the amount of circRNA Using beta-ACTIN as reference, classical 2 in qPCR assay^-ΔΔCtThe method of (1) represents the level of circRNA in the plasma sample (- Δ Ct is the difference between Ct values for target circRNA and control);

4. diagnosis of prostate cancer by hsa _ circ _0012755 levels in plasma samples

As shown in FIG. 2, the plasma samples from prostate cancer patients had a relatively high hsa _ circ _0012755 content compared to the low hsa _ circ _0012755 content of the normal control plasma samples, and was generally higher than the mean of the normal plasma samples, with statistical differences.

Example 3: the silencing efficiency of hsa _ circ _0012755siRNA was tested against hsa _ circ _0012755 in prostate cancer cell lines.

The siRNA sequence of the prostate cancer molecule target hsa _ circ _0012755 used in this example is:

hsa _ circ _0012755siRNA sense strand sequence: 5 '-CCCAGAGAUUCAAGUCAAU-3' (SEQ ID NO. 6)

hsa _ circ _0012755siRNA antisense strand sequence: 5 '-AUUGACUUGAAUCUCUGGG-3' (SEQ ID NO. 7)

The siRNA sequence of the Negative Control (NC) used was

NC siRNA sense strand sequence: 5 '-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO. 8)

NC siRNA antisense strand sequence: 5 '-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO. 9).

The main steps of this example are as follows:

1. siRNA transfection of cells (six well plates as an example)

The prostate cancer PC-3 cell line was seeded in a six-well plate to reach a cell density of about 60-70% after two days of plating, and 5. mu.L of Lipofectamine 2000 and 200. mu.L of Opti-MEM were mixed and incubated, while 5. mu.L of 20. mu.M siRNA and 200. mu.L of Opti-MEM were mixed and incubated. Standing for 5min, and mixing the two solutions. Cells were washed once with PBS. Standing the transfection mixture for 15min, adding 1mL of serum-free and double-antibody-free culture medium, uniformly mixing, and adding into a six-hole plate, wherein the final concentration of siRNA is 50 uM/mL; replacing the culture medium with a complete culture medium after 4-6 hours for continuous culture;

2. extraction of Total RNA from cells

After the cells are transfected and cultured for 48 hours, the cells are collected and total RNA of the cells is extracted, and the extraction method is the same as that of the embodiment 1;

3. quantitative detection of circRNA in cells and data analysis

The quantitative detection and data analysis methods were the same as in example 1;

4. silencing efficiency of hsa _ circ _0012755siRNA to hsa _ circ _0012755

As shown in FIG. 3, the expression level of hsa _ circ _0012755 was reduced by more than 80% after PC-3 was transfected with hsa _ circ _0012755 siRNA. It was demonstrated that hsa _ circ _0012755siRNA could effectively knock down the expression of hsa _ circ _0012755 in prostate cancer PC-3 cell line.

Example 4: the hsa _ circ _0012755siRNA significantly inhibited the cycle of prostate cancer cells.

The main steps of this example are as follows:

1. siRNA transfection of cells (six well plates as an example)

The transfection method was the same as in example 3;

2. cell cycle experiments

After cell digestion, resuspending, inoculating in six holes, culturing for 48h, collecting cells, and detecting the cell cycle by a flow cytometer;

3. hsa _ circ _0012755siRNA influences the cycle status of prostate cancer cells

As shown in figure 4, hsa _ circ _0012755siRNA significantly inhibited the cycle of prostate cancer cells.

Example 5: the application of the prostate cancer diagnostic kit.

The main steps of this example are as follows:

1. extraction of total RNA from tissue sample, blood and urine

Treating tissue samples, blood and urine according to the amount of 1mL Trizol reagent used for every 50mg of tissue, 200ul of blood or 200ul of urine, uniformly blowing and then transferring the tissue samples, the blood and the urine into a 1.5mL Ep tube without RNase; adding 1/5 volume of chloroform into the homogenate, oscillating the centrifuge tube until the solution emulsifies into milky white without phase separation, and centrifuging at 12000 rpm and 4 ℃ for 15 min; sucking the supernatant to a new tube, adding isopropanol with the same volume, gently mixing uniformly, and precipitating at-20 ℃ for 2 h; centrifuging at 12000 rpm and 4 ℃ for 10 min; discarding the supernatant, gently adding 1ml of 75% ethanol along the tube wall, gently washing the EP tube, and centrifuging at 12000 rpm and 4 ℃ for 2 min; discarding the supernatant, centrifuging for a short time, sucking the rest supernatant with a gun, and air drying in a fume hood; adding appropriate amount of DEPC water, and blowing with a gun to dissolve the precipitate; quantification: detecting extraction quality by electrophoresis, and detecting OD260/OD280 and sample concentration by an ultraviolet spectrophotometer; obtaining total RNA in tissue samples, blood and urine, and storing at-80 ℃;

2. detecting the expression change of hsa _ circ _0012755 in tissue sample, blood and urine relative to normal prostate tissue

Using the reagent in the kit, according to the reaction system and conditions in the example 1, using the prostate normal tissue cDNA in the kit as the control cDNA in QPCR quantitative detection, detecting the expression change of hsa _ circ _0012755 in tissue samples, blood and urine relative to the prostate normal tissue, analyzing the detection result, comparing the samples and the control by adopting t test, wherein P <0.05 shows significant difference, and the detection sample is judged to be positive.

The invention is shown and described above without being limited to the above-described embodiments, the advantages of the invention and the embodiments. The present invention is not limited to the above-described embodiments, which are described in the specification and illustrated only for illustrating the principle of the present invention, but various changes and modifications may be made within the scope of the present invention as claimed without departing from the spirit and scope of the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Claims

The application of the molecular target hsa _ circ _0012755 prostate cancer as a marker in the preparation of a kit for screening and diagnosing high risk group of prostate cancer is to detect the content of hsa _ circ _0012755 in serum, urine and tissue samples of a subject and compare the content with the content of hsa _ circ _0012755 in normal samples to screen and diagnose the high risk group of prostate cancer; the nucleotide sequence of the hsa _ circ _0012755 is shown as SEQ ID NO. 1.
2. The use of claim 1, wherein the hsa _ circ _0012755 content in serum, urine and tissue samples of said subject is determined by total RNA extraction, reverse transcription, and quantitative PCR.
3. The use according to claim 1, in a kit comprising:

(1) extracting reagent for total RNA in tissue sample, blood and urine;

(2) a reverse transcription reagent;

(3) quantitative PCR reagent;

the control used was β -ACTIN, whose primer sequences were as follows:

beta-ACTIN upstream primer sequence: 5' -CCTCTCCCAAGTCCACACAG-3

beta-ACTIN downstream primer sequence: 5 '-GGGCACGAAGGCTCATCATT-3'.