CN119144728A - Primer, kit and application of MicroRNA marker of primary liver cancer - Google Patents
Primer, kit and application of MicroRNA marker of primary liver cancer Download PDFInfo
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- 201000007270 liver cancer Diseases 0.000 title claims abstract description 55
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- 108091026068 miR-2276 stem-loop Proteins 0.000 claims abstract description 33
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- 108091070501 miRNA Proteins 0.000 description 20
- 239000000523 sample Substances 0.000 description 16
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Abstract
The invention relates to the technical field of medical inspection, in particular to a primer, a kit and application of a microRNA marker of primary liver cancer, wherein the marker is miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921. The invention assists in stage diagnosis of primary liver cancer by detecting the content of the specific combined MicroRNA markers, and has the characteristics of no wound, accurate quantification, good sensitivity and specificity and the like.
Description
Technical Field
The invention relates to the technical field of medical inspection, in particular to a primer, a kit and application of a microRNA marker for primary liver cancer.
Background
Primary liver cancer is a serious public health problem. Clinically, liver cancer is generally divided into four stages (stage I-stage IV), which correspond to extremely early, progressive and terminal stages, respectively. If the patient can be actively treated in very early stages and early stages, such as surgical excision or local ablation treatment, the patient has a good prognosis, and the survival rate of five years can reach 80 to 90% in general. This is because small liver cancer does not usually invade blood vessels nor distant metastasis occurs, and thus the therapeutic effect is good. However, for mid-and late-stage liver cancer, especially when the cancer has spread to sites other than the liver or multiple tumors exist, the possibility of healing is greatly reduced, and the five-year survival rate of late-stage liver cancer is usually lower than 10% or even lower. Therefore, accurate early diagnosis of liver cancer is particularly important.
In China, many patients are already in the middle and late stages when diagnosing liver cancer. It is estimated that about 20% to 30% of liver cancer patients can be found early in the disease. This is due to the fact that liver cancer often lacks significant symptoms in early stages, and patients have only minor discomfort, such as non-specific symptoms of fatigue, loss of appetite, etc., which are easily overlooked or mistaken for other minor health problems. On the other hand, there are still major technical drawbacks due to the current major means for screening liver cancer, such as ultrasonography and Alpha Fetoprotein (AFP) detection in blood. AFP is a classical liver cancer tumor marker, but about 30% of liver cancer patients have normal AFP level, and 18% of liver cancer patients have increased AFP low concentration, so that the specificity and the sensitivity are not high. The rate of liver cancer detection by ultrasound is affected by operator experience and equipment quality, and these examinations typically require that a sufficiently large tumor be found in the liver. Therefore, there is a need for an innovative method to completely replace or assist the above traditional methods for screening and staged diagnosis of liver cancer.
Disclosure of Invention
The invention aims to solve the defects in the prior art, provides a primer, a kit and application of a microRNA marker for primary liver cancer, and assists in stage diagnosis of primary liver cancer by detecting the content of specific combined microRNA markers (miR-323 a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921), and has the characteristics of noninvasiveness, accurate quantification, good sensitivity and specificity and the like.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides an application of a microRNA marker of primary liver cancer in preparation of a reagent or a detection tool for detecting primary liver cancer, wherein the marker is miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921.
Preferably, the reagent or detection means determines the degree of liver cancer in the subject by detecting the amount of the marker in the biological sample of the subject.
Preferably, the marker is derived from exosomes.
Preferably, the exosomes are derived from peripheral blood serum or plasma.
In a second aspect, the invention provides an application of a primer for detecting the MicroRNA marker in preparation of a reagent or a detection tool for detecting primary liver cancer.
In a third aspect, the invention provides a reverse transcription primer and a detection primer for detecting a microRNA marker of primary liver cancer, wherein the marker is miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921;
The reverse transcription primer is as follows:
The sequence (5 '-3') of the reverse transcription primer of miR-323a-5p is shown in SEQ ID NO.1, and the sequence is GTCGTATCCAGTGCCACACGTCCTCGAGGCTTAAGCACTGGATACGACGCGAACGC;
the sequence (5 '-3') of the reverse transcription primer of miR-2276-5p is shown in SEQ ID NO.2, and is GTCGTATCCAGTGCGAGCCGTTGAGACCGCAAGTGCACTGGATACGACCGTCTGCAA;
The sequence (5 '-3') of the reverse transcription primer of miR-4295 is shown as SEQ ID NO.3, and the sequence is GTCGTATCCAGTGCGAGCCGTTGAGACCGCAAGTGCACTGGATACGACAAGGAAAA;
The sequence (5 '-3') of the reverse transcription primer of miR-2467-3p is shown in SEQ ID NO.4, and the sequence is GTCGTATCCAGTGCCACACGTCCTCGAGGCTTAAGCACTGGATACGACCCTGAGCC;
The sequence (5 '-3') of the reverse transcription primer of miR-921 is shown in SEQ ID NO.5, and the sequence is GTCGTATCCAGTGCAGTGTCAGAGGTAGTCGCACTGGATACGACGAATCCTG;
The detection primer is as follows:
the forward primer sequence (5 '-3') of miR-323a-5p is shown in SEQ ID NO.6, and the sequence is AGACAGGTGGTCCGTGGC;
The sequence (5 '-3') of the reverse primer of miR-323a-5p is shown in SEQ ID NO.7, and the sequence is GTATCCAGTGCCACACGTCCTC;
The forward primer sequence (5 '-3') of miR-2276-5p is shown in SEQ ID NO.8, and the sequence is AATGCGCCCTCTGTCACC;
the sequence (5 '-3') of the reverse primer of miR-2276-5p is shown in SEQ ID NO.9, and the sequence is ATCCAGTGCGAGCCGTTG;
the forward primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO.10, and the sequence is AAGCCAGTGCAATGT;
The sequence (5 '-3') of the reverse primer of miR-4295 is shown as SEQ ID NO.11, and the sequence is ATCCAGTGCGAGCCGTTG;
The forward primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO.12, and the sequence is TCTATAGCAGAGGCAGAGA;
The sequence (5 '-3') of the reverse primer of miR-2467-3p is shown in SEQ ID NO.13, and the sequence is GTATCCAGTGCCACACGTCCTC;
the forward primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO.14, and the sequence is ATTGCCTAGTGAGGGACAGAAC;
The sequence of the reverse primer (5 '-3') of miR-921 is shown in SEQ ID NO.15, and the sequence is CAGTGCAGTGTCAGAGGTAGTCG.
In a fourth aspect, the invention provides an application of the reverse transcription primer and the detection primer in preparation of a reagent or a detection tool for detecting primary liver cancer.
In a fifth aspect, the invention provides a diagnostic kit for primary liver cancer, which comprises reagents for detecting the expression levels of markers miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921.
Preferably, the diagnostic kit comprises a reverse transcription primer and a detection primer of the marker, wherein the reverse transcription primer is as follows:
The sequence (5 '-3') of the reverse transcription primer of miR-323a-5p is shown as SEQ ID NO. 1;
the sequence (5 '-3') of the reverse transcription primer of miR-2276-5p is shown as SEQ ID NO. 2;
the sequence (5 '-3') of the reverse transcription primer of miR-4295 is shown as SEQ ID NO. 3;
The sequence (5 '-3') of the reverse transcription primer of miR-2467-3p is shown as SEQ ID NO. 4;
the sequence (5 '-3') of the reverse transcription primer of miR-921 is shown in SEQ ID NO. 5;
The detection primer is as follows:
The forward primer sequence (5 '-3') of miR-323a-5p is shown in SEQ ID NO. 6;
The sequence (5 '-3') of the reverse primer of miR-323a-5p is shown as SEQ ID NO. 7;
the forward primer sequence (5 '-3') of miR-2276-5p is shown in SEQ ID NO. 8;
the sequence (5 '-3') of the reverse primer of miR-2276-5p is shown in SEQ ID NO. 9;
The forward primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 10;
The reverse primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 11;
The forward primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 12;
The reverse primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 13;
the forward primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 14;
the reverse primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 15.
Preferably, the diagnostic kit further comprises a PCR reaction reagent.
The beneficial effects of the invention are as follows:
The invention adopts MicroRNA markers of miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921 to carry out stage diagnosis on the primary liver cancer, has noninvasive property, is easy to detect and accurate in quantification, and improves the sensitivity and specificity of primary liver cancer diagnosis.
The marker is derived from exosomes of peripheral blood serum or plasma, and miRNA in the exosomes has a phospholipid bilayer membrane structure, so that the miRNA is not influenced by external environment and has better stability in detection, and therefore, the marker has good application prospect in liver cancer diagnosis.
The invention utilizes the reverse transcription primer and the detection primer of miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921 marker to detect the content of the microRNA marker of the primary liver cancer, and adopts a formula to evaluate the stage grade of the primary liver cancer of a tested person, so that the accuracy is high and the specificity is strong.
The kit based on the digital PCR platform can assist in diagnosing liver cancer and stage liver cancer timely, accurately and specifically.
Drawings
FIG. 1 is a ROC curve for diagnosing healthy subjects and liver cancer patients of stage I-IV using markers;
FIG. 2 is a ROC curve for diagnosing stage I-II and stage III-IV liver cancer patients using markers.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments thereof in order to enable those skilled in the art to better understand the technical aspects of the invention.
Example 1
The embodiment provides a diagnostic kit for primary liver cancer, which is used for detecting the content of MicroRNA markers in exosomes of peripheral blood serum/plasma, wherein the markers are miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921, and the kit comprises reverse transcription primers and detection primers of the markers;
The reverse transcription primer is as follows:
The sequence (5 '-3') of the reverse transcription primer of miR-323a-5p is shown as SEQ ID NO. 1;
the sequence (5 '-3') of the reverse transcription primer of miR-2276-5p is shown as SEQ ID NO. 2;
the sequence (5 '-3') of the reverse transcription primer of miR-4295 is shown as SEQ ID NO. 3;
The sequence (5 '-3') of the reverse transcription primer of miR-2467-3p is shown as SEQ ID NO. 4;
the sequence (5 '-3') of the reverse transcription primer of miR-921 is shown in SEQ ID NO. 5;
The detection primer is as follows:
The forward primer sequence (5 '-3') of miR-323a-5p is shown in SEQ ID NO. 6;
The sequence (5 '-3') of the reverse primer of miR-323a-5p is shown as SEQ ID NO. 7;
the forward primer sequence (5 '-3') of miR-2276-5p is shown in SEQ ID NO. 8;
the sequence (5 '-3') of the reverse primer of miR-2276-5p is shown in SEQ ID NO. 9;
The forward primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 10;
The reverse primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 11;
The forward primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 12;
The reverse primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 13;
the forward primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 14;
the reverse primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 15.
Example 2
(1) Extracting total miRNA by separating and purifying exosomes from blood plasma
Collecting blood by vein, centrifuging at 4deg.C for 10min at 2000g, separating upper layer plasma part, centrifuging at 12000g for 20min at 4deg.C, and collecting upper layer plasma sample;
1ml of plasma was taken and subjected to exosome extraction and purification using Exosome Isolation and Purification Kit (from PLASMA AND serum) from MCE;
Extracting miRNA by using exoRNeasy Kits of QIAGEN company by sucking about 200ul exosome sample, adding 10ul of quality control substance before extraction, measuring concentration after extraction, and adjusting total miRNA concentration to 20-40 ng/ul.
(2) And configuring a reverse transcription reaction system by using the obtained miRNA and the reverse transcription primer, and carrying out reverse transcription reaction to obtain a miRNA reverse transcription sample.
The reverse transcription reaction system of miRNA is shown in Table 1 (miR-323 a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921 are each separately configured in a tube).
TABLE 1 reverse transcription reaction System of miRNA and quality control substances
In table 1, the 5 miRNA reverse transcription primers are:
The sequence (5 '-3') of the reverse transcription primer of miR-323a-5p is shown as SEQ ID NO. 1;
the sequence (5 '-3') of the reverse transcription primer of miR-2276-5p is shown as SEQ ID NO. 2;
the sequence (5 '-3') of the reverse transcription primer of miR-4295 is shown as SEQ ID NO. 3;
The sequence (5 '-3') of the reverse transcription primer of miR-2467-3p is shown as SEQ ID NO. 4;
The sequence (5 '-3') of the reverse transcription primer of miR-921 is shown in SEQ ID NO. 5.
The quality control product in Table 1 is synthesized single-stranded miRNA of 5×10 4 copies/ul cel-miRNA-39, the sequence (5 '-3') is shown as SEQ ID NO.16 and UCACCGGGUGUAAAUCAGCUUG, and the quality control product reverse transcription primer sequence (5 '-3') is shown as SEQ ID NO.17 and GTCGTATCCAGTGCAGTGTCAGAGGTAGTCGCACTGGATACGACCAAGCTGA.
The reverse transcription reaction was performed under the conditions of Table 2, and 4℃indicates that the reaction was completed.
TABLE 2 reverse transcription reaction conditions for miRNAs and quality control substances
(3) And preparing a digital PCR reaction microdroplet by using the obtained miRNA reverse transcription sample and a detection primer to prepare a miRNA digital PCR detection reaction system, and performing digital PCR reaction.
The digital PCR detection reaction system of miRNA is shown in Table 3 (miR-323 a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921 are each separately configured in one tube).
Table 3.MiRNA digital PCR detection reaction system
The detection primers for the 5 mirnas in table 3 are:
The forward primer sequence (5 '-3') of miR-323a-5p is shown in SEQ ID NO. 6;
The sequence (5 '-3') of the reverse primer of miR-323a-5p is shown as SEQ ID NO. 7;
the forward primer sequence (5 '-3') of miR-2276-5p is shown in SEQ ID NO. 8;
the sequence (5 '-3') of the reverse primer of miR-2276-5p is shown in SEQ ID NO. 9;
The forward primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 10;
The reverse primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 11;
The forward primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 12;
The reverse primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 13;
the forward primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 14;
the reverse primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 15.
The detection primers for the quality control products in table 3 are:
the forward primer sequence (5 '-3') is shown as SEQ ID NO.18 and AGGCTCACCGGGTGTAAAT;
the reverse primer sequence (5 '-3') is shown as SEQ ID NO.19 and GCAGTGCAGTGTCAGAGGTAGTC.
The prepared reaction system is subjected to preparation of digital PCR reaction microdroplets, and then transferred into a 96-well plate for PCR reaction, and the reaction conditions are shown in Table 4.
TABLE 4 digital PCR reaction conditions
The 96-well plate after the reaction was transferred to a QX200TM digital PCR reader for reading the results.
(4) Result calculation
1) Quality control calculation assuming that the total RNA sample dissolution volume after extraction is A [ mu ] L and the Bio-rad QX200 digital PCR detection result is B copies/[ mu ] L, the concentration of the quality control (X copies/[ mu ] L) is calculated according to the following formula (1):
X copy number/μl= (b×20/3×15/3×a)/10=10/3×a×b (1)
2) The quality control product detection result is 4 multiplied by 10 4~5×104 copies/ul, if the quality control product detection result is satisfied, the miRNA detection result of the sample is effective, otherwise, the experiment is ineffective, and the detection is carried out by re-extraction.
3) If the quality control product results are acceptable, the concentration (Y copies/. Mu.L) of the target miRNA in the plasma sample is calculated according to the following formula (2):
Y copy number/μl= (b×20/3×15/3×a)/200=1/6×a×b (2)
(5) Risk assessment
The P1 value is first calculated as follows formulas (3) and (4):
P1=es1/（1+es1)(3)
S1=5.21+0.025×m1-0.016×m2+0.017×m3-0.033×m4+0.054×m5(4)
Wherein m1 is the content of miR-323a-5p in the sample, m2 is the content of miR-2276-5p in the sample, m3 is the content of miR-4295 in the sample, m4 is the content of miR-2467-3p in the sample, and m5 is the content of miR-921 in the sample.
When P1 is less than 0.419, the risk of the tested person to suffer from liver cancer is lower, and when P1 is more than 0.419, the tested person is at certain risk of the tested person to suffer from liver cancer.
Patients assessed as having a certain risk of liver cancer may be further assessed for their risk of extremely early and early (stage I-II) or advanced and late (stage III-IV) stages of development, and P2 values are calculated according to formulas (5) and (6):
P2=es2/（1+es2)(5)
S2=1.627+0.071×m1-0.037×m2+0.052×m3-0.042×m4+0.084×m5(6)
Wherein m1 is the content of miR-323a-5p in the sample, m2 is the content of miR-2276-5p in the sample, m3 is the content of miR-4295 in the sample, m4 is the content of miR-2467-3p in the sample, and m5 is the content of miR-921 in the sample.
When P2<0.402, the risk of the tested person for extremely early stage and early stage (stage I-II) is high, and when P2>0.402, the risk of the tested person for the progressive stage and the final stage (stage III-IV) is high.
Example 3
104 Healthy people without liver cancer, 10 liver cancer patients with stage I, 35 liver cancer patients with stage II, 41 liver cancer patients with stage III and 19 liver cancer patients with stage IV are taken in, blood plasma of the healthy people is collected according to the method of the embodiment 2, exosomes are separated and purified, total miRNA in the exosomes is extracted, digital PCR reaction is carried out, after the reaction is finished, calculation of P1 or P2 value is carried out according to a risk assessment step, and then data analysis is carried out.
Effect data-analysis was performed using GRAPHPAD PRISM software to obtain ROC curves for the detection controls, see fig. 1 and 2. Fig. 1 distinguishes auc=0.89, sensitivity=96.2%, specificity=62.5% for healthy and stage I-IV liver cancer patients, and fig. 2 distinguishes auc=0.84, sensitivity=81.7%, specificity=71.1% for stage I-II and stage III-IV patients.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended 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 such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. The application of a microRNA marker for primary liver cancer in preparation of a reagent or a detection tool for detecting primary liver cancer is characterized in that the marker is miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921.
2. The use according to claim 1, wherein the marker is derived from exosomes.
3. The use according to claim 2, wherein the exosomes are derived from peripheral blood serum or plasma.
4. The use according to any one of claims 1 to 3, wherein the reagent or the detection means determines the liver cancer level of the subject by detecting the content of the marker in the biological sample of the subject.
5. Use of a primer for detecting the MicroRNA marker of claim 1 in preparation of a reagent or a detection tool for detecting primary liver cancer.
6. The reverse transcription primer and the detection primer for detecting the MicroRNA marker of the primary liver cancer are characterized in that the marker is miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921;
The reverse transcription primer is as follows:
The sequence (5 '-3') of the reverse transcription primer of miR-323a-5p is shown as SEQ ID NO. 1;
the sequence (5 '-3') of the reverse transcription primer of miR-2276-5p is shown as SEQ ID NO. 2;
the sequence (5 '-3') of the reverse transcription primer of miR-4295 is shown as SEQ ID NO. 3;
The sequence (5 '-3') of the reverse transcription primer of miR-2467-3p is shown as SEQ ID NO. 4;
the sequence (5 '-3') of the reverse transcription primer of miR-921 is shown in SEQ ID NO. 5;
The detection primer is as follows:
The forward primer sequence (5 '-3') of miR-323a-5p is shown in SEQ ID NO. 6;
The sequence (5 '-3') of the reverse primer of miR-323a-5p is shown as SEQ ID NO. 7;
the forward primer sequence (5 '-3') of miR-2276-5p is shown in SEQ ID NO. 8;
the sequence (5 '-3') of the reverse primer of miR-2276-5p is shown in SEQ ID NO. 9;
The forward primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 10;
The reverse primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 11;
The forward primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 12;
The reverse primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 13;
the forward primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 14;
the reverse primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 15.
7. Use of the reverse transcription primer and the detection primer according to claim 6 in preparation of a reagent or a detection tool for detecting primary liver cancer.
8. The diagnostic kit for the primary liver cancer is characterized by comprising a reagent for detecting the expression levels of markers miR-323a-5p, miR-2276-5p, miR-4295, miR-2467-3p and miR-921.
9. The diagnostic kit of claim 8, wherein the kit comprises reverse transcription primers and detection primers for the markers, the reverse transcription primers being:
The sequence (5 '-3') of the reverse transcription primer of miR-323a-5p is shown as SEQ ID NO. 1;
the sequence (5 '-3') of the reverse transcription primer of miR-2276-5p is shown as SEQ ID NO. 2;
the sequence (5 '-3') of the reverse transcription primer of miR-4295 is shown as SEQ ID NO. 3;
The sequence (5 '-3') of the reverse transcription primer of miR-2467-3p is shown as SEQ ID NO. 4;
the sequence (5 '-3') of the reverse transcription primer of miR-921 is shown in SEQ ID NO. 5;
The detection primer is as follows:
The forward primer sequence (5 '-3') of miR-323a-5p is shown in SEQ ID NO. 6;
The sequence (5 '-3') of the reverse primer of miR-323a-5p is shown as SEQ ID NO. 7;
the forward primer sequence (5 '-3') of miR-2276-5p is shown in SEQ ID NO. 8;
the sequence (5 '-3') of the reverse primer of miR-2276-5p is shown in SEQ ID NO. 9;
The forward primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 10;
The reverse primer sequence (5 '-3') of miR-4295 is shown as SEQ ID NO. 11;
The forward primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 12;
The reverse primer sequence (5 '-3') of miR-2467-3p is shown in SEQ ID NO. 13;
the forward primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 14;
the reverse primer sequence (5 '-3') of miR-921 is shown in SEQ ID NO. 15.
10. The diagnostic kit of claim 8, wherein the kit further comprises PCR reaction reagents.
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