CN112646885B - Kidney cell carcinoma miRNA molecular marker and application thereof - Google Patents

Kidney cell carcinoma miRNA molecular marker and application thereof Download PDF

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CN112646885B
CN112646885B CN202011498572.9A CN202011498572A CN112646885B CN 112646885 B CN112646885 B CN 112646885B CN 202011498572 A CN202011498572 A CN 202011498572A CN 112646885 B CN112646885 B CN 112646885B
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cell carcinoma
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CN112646885A (en
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曹志飞
张永胜
涂健
杨天宇
钮慧
沈珊珊
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Nuclear Industry General Hospital
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Abstract

The invention discloses a kidney cell carcinoma miRNA molecular marker which is used as a metastasis marker for diagnosing and/or evaluating prognosis of kidney cell carcinoma and risk of metastasis; alternatively, as a specific molecular target for renal cell carcinoma, it is applied to the treatment of renal cell carcinoma; the miRNA molecular marker comprises one or more of miR-328, miR-502 and miR-504. The invention can judge whether the renal cell carcinoma patient is metastasized and prognosis by detecting the expression level of miR-328, miR-502 and/or miR-504 in the tissue of the renal cell carcinoma patient, and the accuracy rate is more than 95%. Therefore, detection of molecular biomarkers of miR-328, miR-502 and/or miR-504 in renal cell carcinoma patient tissues can evaluate the disease progress, metastasis and prognosis of renal cell carcinoma patients, can timely perform relevant treatment on symptoms, improves the survival time and the survival quality of the patients, and has great economic and social benefits.

Description

Kidney cell carcinoma miRNA molecular marker and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a kidney cell carcinoma miRNA molecular marker and application thereof.
Background
Renal Cell Carcinoma (RCC) is a common cancer worldwide, accounting for about 2-3% of adult malignancies. About 63990 new cases of RCC in the united states in 2017 were reported, more than 14400 cases of renal cancer-related death. In China, the incidence of RCC is on the rise, and there are 68300 new RCC cases and 25600 renal cancer-related deaths in 2014. Metastatic spread is the most important factor affecting the prognosis of RCC. The 5-year survival rate of primary metastatic patients is about 10%, and the 5-year survival rate of non-metastatic and late metastatic patients is 70% -90%. At the beginning of the visit, nearly 60% of patients are localized cancers and 20% are distant metastases. Metastatic spread of RCC occurs primarily in bone, lung, liver or brain. The metastasis of RCC is responsible for its high incidence and poor prognosis. More than 80% of RCC patients survive less than 5 years after distant metastasis. Therefore, understanding the mechanism of action of RCC occurrence, development, and especially metastasis, and performing appropriate intervention are of great importance to improve the clinical diagnosis level of RCC and prolong the survival time of patients. However, there is currently no clinically ideal molecular index to assess the prognosis of RCC patients and the risk of developing metastasis. Therefore, finding new RCC metastasis and prognosis markers is a hotspot problem that is currently in need of solution.
With the rapid development of genomics and transcriptomes, there is increasing evidence that non-coding RNAs play an important role in the occurrence, development and metastasis of various cancers and can be used as molecular markers for cancer diagnosis and treatment. It is well known that micrornas (miRNAs) are negatively regulated by transcriptional degradation or inhibition of expression of their target genes. Many studies have shown that miRNAs play a key regulatory role in the development of human tumors, affecting the progression of cancer by regulating the expression of genes associated with disease development and progression. However, it is still to be investigated which miRNAs can be used as RCC metastasis and prognosis markers.
Disclosure of Invention
In order to make up for the defects of the prior art, miRNA molecular markers which show differential expression in renal cell carcinoma tissues and metastatic carcinoma tissues are screened out through a high-tech biotechnology, and the risk of metastasis and prognosis evaluation of renal cell carcinoma patients are judged by detecting the expression level of the miRNAs molecular markers and comparing the expression level with a reference level. Meanwhile, the miRNA molecular marker can be used as a specific molecular target of renal cell carcinoma and applied to accurate treatment of renal cell carcinoma.
In order to achieve the above purpose, the present invention provides the following technical solutions: a kidney cell carcinoma miRNA molecular marker as a metastasis marker for diagnosing and/or assessing kidney cell carcinoma prognosis and risk of metastasis; alternatively, as a specific molecular target for renal cell carcinoma, it is applied to the treatment of renal cell carcinoma; the miRNA molecular marker comprises one or more of miR-328, miR-502 and miR-504.
A detection reagent for detecting the expression level of miR-328, miR-502 and/or miR-504 genes in a sample by using a sequencing technology, a nucleic acid hybridization technology and a nucleic acid amplification technology; the detection reagent is selected from probes specifically recognizing miR-328, miR-502 and/or miR-504; alternatively, the detection reagent is selected from primers that specifically amplify miR-328, miR-502 and/or miR-504; alternatively, the detection reagent is selected from the group consisting of chips that specifically analyze miR-328, miR-502 and/or miR-504.
Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because RNA is less stable in cells and is more susceptible to nuclease attack in experiments.
Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), a single molecular cluster-based sequencing-by-synthesis technique based on the principle of proprietary reversible termination chemistry. During sequencing, random fragments of DNA of genome are attached to the surface of optically transparent glass, hundreds of millions of clusters are formed on the surface of the glass after the DNA fragments are extended and bridge amplified, each cluster is a single molecular cluster with thousands of identical templates, and then the template DNA to be tested is sequenced by using four special deoxyribonucleotides with fluorescent groups through a reversible synthesis sequencing-by-synthesis technology.
Illustrative non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, in Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a portion or section of tissue (in situ) or in the whole tissue if the tissue is small enough (whole tissue embedded ISH) using labeled complementary DNA or RNA strands as probes. DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts within tissue sections or whole tissue implants. Sample cells and tissues are typically treated to immobilize target transcripts in situ and to increase probe entry. The probe hybridizes to the target sequence at an elevated temperature and then excess probe is washed away. The probe labeled with a base labeled with a radiation, fluorescence or antigen in the tissue is localized and quantified using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH may also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.
Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Hybridization probes bound to the filters are detected. One variation of this procedure is reverse Northern blotting, where the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments, and the probe is RNA extracted from the tissue and labeled.
The invention can amplify nucleic acids (e.g., ncrnas) prior to or simultaneously with detection. Illustrative, non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription Mediated Amplification (TMA), ligase Chain Reaction (LCR), strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that some amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while others directly amplify RNA (e.g., TMA and NASBA).
Typically, PCR increases the copy number of a target nucleic acid sequence exponentially using multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension; RT-PCR uses Reverse Transcriptase (RT) to prepare complementary DNA (cDNA) from mRNA, which is then amplified by PCR to produce multiple copies of the DNA; autocatalytically synthesizing multiple copies of a target nucleic acid sequence under substantially constant temperature, ionic strength, and pH conditions, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies, TMA optionally including the use of blocking, terminating, and other modifying moieties to improve sensitivity and accuracy of the TMA process; LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of a target nucleic acid. The DNA oligonucleotides are covalently linked by a DNA ligase in repeated multiple cycles of heat denaturation, hybridization, and ligation to produce a detectable double-stranded linked oligonucleotide product; SDA uses multiple cycles of the following steps: the primer sequence anneals to the opposite strand of the target sequence, primer extension in the presence of dNTpαS to produce a double-stranded phosphorohalidated (phosphorothioated) primer extension product, endonuclease-mediated nicking at the semi-modified restriction enzyme recognition site, and polymerase-mediated extension from the 3' end of the nick to displace the existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, resulting in geometric amplification of the product.
Preferably, the primer sequences for specifically amplifying miR-328, miR-502 and/or miR-504 are shown in SEQ ID No. 5-10.
A related product for detecting miR-328, miR-502 and/or miR-504 expression levels in vitro, the related product comprising a gene chip or a kit.
Preferably, the gene chip comprises a solid phase carrier and probes attached to the solid phase carrier, wherein the probes specifically recognize miR-328, miR-502 and/or miR-504.
As non-limiting examples, the solid support may be a variety of materials commonly used in the gene chip arts, such as, but not limited to, nylon membranes, glass or silicon wafers modified with reactive groups (e.g., aldehyde groups, amino groups, etc.), unmodified glass slides, plastic sheets, and the like.
The chips of the present invention can be prepared by conventional methods for fabricating biochips known in the art. For example, if a modified slide or a silicon wafer is used as the solid phase carrier, and the 5' -end of the probe contains an amino-modified poly dT string, the oligonucleotide probe can be prepared into a solution, then spotted on the modified slide or the silicon wafer by a spotting instrument, arranged into a predetermined sequence or array, and then fixed by standing overnight, thereby obtaining the gene chip of the present invention.
Preferably, the kit comprises a primer for specifically amplifying miR-328, miR-502 and/or miR-504, a probe for specifically recognizing miR-328, miR-502 and/or miR-504, or a chip for specifically analyzing miR-328, miR-502 and/or miR-504.
Preferably, the kit further comprises one or more of a container, instructions for use, positive control, negative control, and excipient. The excipient includes a buffer, an adjuvant, or a solvent.
A pharmaceutical composition comprising an activator of miR-328, miR-502 and/or miR-504.
Preferably, the activator is capable of increasing the expression level of miR-328, miR-502 and/or miR-504.
The application of the miRNA molecular marker of the renal cell carcinoma, the detection reagent, the related product or the pharmaceutical composition in preparing diagnosis and/or evaluation and/or treatment of the renal cell carcinoma.
The terms "differential molecular marker expression" and "differential expression" are used interchangeably to refer to the expression of a molecular marker that is activated to a higher or lower level in a subject with a particular disease relative to its expression in a normal subject, or relative to its expression in a patient that responds differently to a particular treatment or has a different prognosis. The term also includes molecular markers whose expression is activated to a higher or lower level at different stages of the same disease. It is also understood that the differentially expressed molecular markers may be activated or inhibited at the nucleic acid level or protein level, or may undergo alternative splicing to produce different polypeptide products. Such differences may be demonstrated by a variety of changes including mRNA levels, microrna levels, lncRNA levels, antisense transcript levels, or protein surface expression, secretion, or other partitioning of the polypeptide. Differential molecular marker expression may include a comparison of expression between two or more genes or between gene products thereof; or a comparison of the ratio of expression between two or more genes or between their gene products; or even a comparison of two differently processed products of the same gene, which is different between normal and diseased subjects; or different at different stages of the same disease. Differential expression includes, for example, quantitative and qualitative differences in the temporal expression pattern or cellular expression pattern in a molecular marker between normal cells and diseased cells or between cells undergoing different disease events or disease stages.
When a molecular marker indicates or is indicative of an abnormal process, disease or other condition in an individual, the molecular marker is generally described as over-expressed or under-expressed as compared to the level or value of the molecular marker in the individual that indicates or is indicative of normal process, no disease or other condition. "up-regulated", "over-expressed" are used interchangeably and refer to a value or level of a molecular marker in a biological sample that is greater than the value or level (or range of values or levels) of the molecular marker that is normally detected in a healthy or normal individual. The term may also refer to a value or level of a molecular marker in a biological sample that is greater than (or a range of values or levels of) the value or level of the molecular marker that is detectable at different stages of a particular disease.
"down-regulated", "under-expressed" are used interchangeably and refer to a value or level of a molecular marker in a biological sample that is less than the value or level (or range of values or levels) of the molecular marker that is normally detected in a healthy or normal individual. The term may also refer to a value or level of a molecular marker in a biological sample that is less than the value or level (or range of values or levels) of the molecular marker that is detectable at different stages of a particular disease.
In the present invention, reagents, tools, and/or instructions for use described herein may be provided in a kit. For example, the kit may comprise reagents, tools, and instructions for determining appropriate treatment for a cancer patient. The kit may include reagents for collecting a tissue sample from a patient, such as by biopsy, and reagents for processing the tissue. The kit may also include one or more reagents for performing molecular marker expression analysis, such as reagents for performing RT-PCR, qPCR, RNA blotting or the like to determine the expression level of the molecular marker in a sample from the patient. For example, primers for performing RT-PCR, probes for performing RNA blot analysis may be included in the kit. Suitable buffers for the assay may also be included. Detection reagents required for any of these assays may also be included.
The kits characterized herein may also include instruction cards describing how to perform assays for measuring the expression of molecular markers. The instructional card may also include instructions on how to determine the reference cohort, including how to determine the expression level of the molecular markers in the reference cohort and how to aggregate the expression data to establish a reference for comparison with the test patient. The instruction card may also include instructions for determining the expression of the molecular marker in the test patient and for comparing the expression level to the expression in the reference cohort to determine an appropriate chemotherapy for the subject.
The informational material included in the kit may be described, directed, marketed, or other material related to the methods described herein and/or the use of the reagents for the methods described herein. For example, the informational material of the kit may contain contact information, such as a physical address, email address, website, or telephone number, where the user of the kit may obtain a large amount of information about the results of performing the gene expression analysis and analysis, particularly when applied to humans that may have a positive response to a particular therapeutic agent.
The invention can judge whether the renal cell carcinoma patient is metastasized and prognosis by detecting the expression level of miR-328, miR-502 and/or miR-504 in the tissue of the renal cell carcinoma patient, and the accuracy rate is more than 95%. Therefore, detection of molecular biomarkers of miR-328, miR-502 and/or miR-504 in renal cell carcinoma patient tissues can evaluate the disease progress, metastasis and prognosis of renal cell carcinoma patients, can timely perform relevant treatment on symptoms, improves the survival time and the survival quality of the patients, and has great economic and social benefits.
Drawings
FIG. 1 is a graph showing that microRNA-328 (miR-328) is significantly less expressed in RCC tissue than in paracancerous normal tissue;
FIG. 2 is a graph showing that microRNA-502 (miR-502) is expressed significantly lower in RCC tissue than in paracancerous normal tissue;
FIG. 3 is a graph showing that microRNA-504 (miR-504) is significantly less expressed in RCC tissue than in paracancerous normal tissue;
FIG. 4 is a graph showing that microRNA-328 (miR-328) is significantly less expressed in RCC transfer tissue than in RCC tissue;
FIG. 5 is a graph showing that microRNA-502 (miR-502) is significantly less expressed in RCC transfer tissue than in RCC tissue;
FIG. 6 is a graph showing that microRNA-504 (miR-504) is significantly less expressed in RCC transfer tissue than in RCC tissue;
FIG. 7 shows that microRNA-328 (miR-328) high expression is closely related to the good prognosis of RCC patients;
FIG. 8 shows that microRNA-502 (miR-502) high expression is closely related to the good prognosis of RCC patients;
FIG. 9 shows that microRNA-504 (miR-504) high expression is closely related to good prognosis of RCC patients.
Detailed Description
The above scheme is further described below with reference to specific examples. It should be understood that these examples are illustrative of the invention and are not intended to limit the scope of the invention. The implementation conditions employed in the examples may be further adjusted according to the conditions of the particular manufacturer, and the implementation conditions not noted are typically those in routine experiments.
Example 1 preparation of a kit for predicting metastasis and prognosis of RCC patients (50 reactions)
1.Trizol：50ml；
2. Chloroform: 20ml;
3. isopropyl alcohol: 30ml;
4.75% ethanol; 60ml;
depc water: 10ml;
miRNA reverse transcription specific primers (the sequences of which are shown in SEQ NO: 1-4): 100ul;
7.200U/ul M-MLV reverse transcriptase: 50ul;
8.SYBR qPCR Mix：500ul；
9.10uM target gene specific primer (its sequence is shown in SEQ NO: 5-10): 100ul each;
10.10uM reference gene U6 specific primer (its sequence is shown in SEQ NO: 11-12): 100ul each.
Example 2 detection and analysis of MiRNA molecular markers in tissue samples
1. And collecting RCC tissues to be detected, paracancerous normal tissues and RCC tissues with far-end metastasis, cleaning with normal saline, and then putting into liquid nitrogen for freezing.
2. Tissue RNA extraction: liquid nitrogen is added into a mortar, the tissues are crushed by shearing and then are ground and crushed in the liquid nitrogen, 100mg of the tissues are added into 1ml of Trizol, and the mixture is uniformly mixed. The mixture was left at room temperature for 5 minutes, 200 uL/tube of chloroform was added thereto, and the mixture was stirred vigorously for 15 seconds. Centrifuge at 12000 rpm for 15 minutes. Carefully aspirate the upper aqueous phase into a new Ep tube, add an equal volume of isopropanol, mix upside down, leave at room temperature for 5 minutes, centrifuge for 10 minutes at 12000 rpm. Carefully discard the supernatant, add 75% ethanol, mix well and centrifuge at 8000 rpm for 8 minutes. Removing supernatant, and adding DEPC water to dissolve RNA after drying. The concentration and purity of RNA were measured by an enzyme-labeled instrument. An OD260/OD280 ratio between 1.8 and 2.0 indicates good RNA purity.
3. Reverse transcription: reverse transcription was performed using reverse transcription kit (R233) from the biotechnology company of nanking nuozan, as follows: the following mixtures were prepared in centrifuge tubes: 4 XgDNA wind Mix 4ul, 1ul of miRNA specific reverse transcription primer (10. Mu.M, the sequence of which is shown as SEQ NO: 1-4), 1pg-1ug of template RNA, and DEPC water to 20ul. After mixing, incubation was carried out at 42℃for 2 minutes. Then, 5X HiScript II Select qRT SuperMix ul of the mixture was added and incubated at 50℃for 15 minutes, followed by 5 seconds at 85 ℃. The obtained cDNA was frozen at-80℃or subjected to real-time quantitative PCR.
4. Real-time quantitative PCR: the PCR primer is synthesized by the Souzhou Jin Weizhi biotechnology company, and has the primer sequence of SEQ NO 5-10 and the primer sequence of the internal reference gene U6 of SEQ NO 11-12. The primer sequences were as follows:
miR-328 reverse transcription primer:
5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGGAA-3'(SEQ ID NO.1)
miR-502 reverse transcription primer:
5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGAATC-3'(SEQ ID NO.2)
miR-504 reverse transcription primer:
5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGATAGA-3'(SEQ ID NO.3)
u6 reverse transcription primer:
5'-CGAGCACAGAATCGCTTCACGAATTTGCGTGTCAT-3'(SEQ ID NO.4)miR-328：
forward primer: 5'-GTCGTATCCAGTGCAGGG-3' (SEQ ID NO. 5)
Reverse primer: 5'-CTAATCTGGCCCTCTCTGCC-3' (SEQ ID NO. 6)
miR-502：
Forward primer: 5'-GTCGTATCCAGTGCAGGG-3' (SEQ ID NO. 7)
Reverse primer: 5'-CGACTAATGCACCTGGGCAA-3' (SEQ ID NO. 8)
miR-504：
Forward primer: 5'-GTCGTATCCAGTGCAGGG-3' (SEQ ID NO. 9)
Reverse primer: 5'-CGACTAGACCCTGGTCTGCA-3' (SEQ ID NO. 10)
U6：
Forward primer: 5'-CGAGCACAGAATCGCTTCA-3' (SEQ ID NO. 11)
Reverse primer: 5'-CTCGCTTCGGCAGCACATAT-3' (SEQ ID NO. 12)
The other real-time quantitative PCR reagent is ChamQ Universal SYBR qPCR Master Mix (Q711-02) of Nanjinopran biotechnology company, and the specific steps are as follows: the following mixture was prepared in qPCR tube: 2XChamQ Universal SYBR qPCR Master Mix 10ul, 10uM upstream primer 1ul, 10uM downstream primer 1ul, template cDNA 4ul, H 2 And O4 ul. After the above-mentioned materials are uniformly mixed, they are centrifuged for several seconds so as to make them be settled on the bottom of the tube. PCR procedure: a. pre-denaturation:95 ℃ for 30 seconds;
b. denaturation: 95 ℃ for 10 seconds;
c. annealing/extension: 60 ℃ for 30 seconds;
d. repeating steps b and c for a total of 40 cycles
e. Dissolution profile analysis: 95℃for 15 seconds, 60℃for 60 seconds, 95℃for 15 seconds.
5. Statistical analysis: the real-time quantitative PCR result calculates the relative expression level of the gene according to the 2-delta Ct. Analysis was performed using a non-parametric t-test. Gene expression and RCC prognosis were analyzed using the KM PLOTTER database (https:// kmlot. Com). The Kaplan-Meier method draws survival curves, and the Log-rank method compares the survival differences among groups. The difference in P <0.05 is statistically significant.
6. Results: the results of real-time quantitative PCR on RCC tissues and paracancerous normal tissues are shown in figures 1-3, and compared with the paracancerous normal tissues, the expression of miR-328, miR-502 and miR-504 in the RCC tissues is significantly down-regulated, the down-regulation times are 2.725 times, 2.282 times and 2.957 times respectively, and the differences are statistically significant (P < 0.01). Furthermore, we also examined the difference in expression of the above 3 miRNAs in RCC tissue and RCC transfer tissue, and the results are shown in fig. 4-6. Compared with RCC tissues, the expression of miR-328, miR-502 and miR-504 in the RCC tissues is significantly down-regulated, the down-regulation times are 81.169 times, 178.699 times and 45.226 times respectively, and the differences are statistically significant (P is equal to 0.01). Finally, we also analyze the relevance of the expression and prognosis of miRNAs such as miR-328, miR-502 and miR-504 in RCC, and as shown in FIGS. 7-9, it is found that the high expression of miR-328, miR-502 and miR-504 in RCC patients is closely related to the good prognosis of RCC patients (P <0.01, P < 0.05). The experimental results show that by detecting the expression levels of miR-328, miR-502 and miR-504 in the tissue of a renal cell carcinoma patient, whether the renal cell carcinoma patient is metastasized and prognosis can be judged, and the accuracy rate is more than 95%. Therefore, detection of molecular biomarkers of miR-328, miR-502, miR-504 and the like in renal cell carcinoma patient tissues can evaluate the disease progress, metastasis and prognosis of renal cell carcinoma patients, can timely perform relevant treatment on symptoms, improves the survival time and the survival quality of the patients, and has great economic value and social benefit.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
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<211> 19
<212> DNA
<213> Artificial Sequence(Artificial Sequence)
<400> 11
cgagcacaga atcgcttca 19
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence(Artificial Sequence)
<400> 12
ctcgcttcgg cagcacatat 20

Claims (3)

1. Use of a product for detecting miR-328 in the preparation of a kit for diagnosing and/or assessing the prognosis of renal cell carcinoma and the risk of developing metastasis.
2. The use of claim 1, wherein the miR-328-detecting product is a primer pair that specifically amplifies miR-328.
3. The use of claim 2, wherein the primer sequence for specifically amplifying miR-328 is shown in SEQ ID No. 5-6.
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CN113862365A (en) * 2021-11-01 2021-12-31 上海市第十人民医院 Application of lncRNA LINC00113 as marker in preparation of kidney cancer detection reagent or detection kit

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