CN112553335A

CN112553335A - Renal cell carcinoma biomarkers and uses thereof

Info

Publication number: CN112553335A
Application number: CN202011493984.3A
Authority: CN
Inventors: 张永胜; 曹志飞; 涂健; 杨天宇; 钮慧; 沈珊珊
Original assignee: Nuclear Industry General Hospital
Current assignee: Nuclear Industry General Hospital
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2021-03-26

Abstract

The invention discloses a renal cell carcinoma biomarker which is used as a metastasis marker for diagnosing and/or evaluating the prognosis of renal cell carcinoma and the risk of metastasis; or, as a specific molecular target for renal cell carcinoma, for use in the treatment of renal cell carcinoma; the biomarkers include one or more of THBS2, SCGB1a1, NKX2-1, COL11a1, DCN, COL1a1, and SFTPB. By detecting the expression levels of THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and SFTPB in the tissues of the renal cell carcinoma patient, the method can judge whether the renal cell carcinoma patient has metastasis or not and the prognosis, and the accuracy rate is more than 93%. Therefore, the detection of molecular biomarkers such as THBS2 and the like in the tissues of the renal cell carcinoma patients can evaluate the disease progression, metastasis and prognosis of the renal cell carcinoma patients, can timely perform relevant treatment on the symptoms, improves the survival time and quality of the patients, and has great economic value and social benefit.

Description

Renal cell carcinoma biomarkers and uses thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a renal cell carcinoma biomarker and application thereof.

Background

Renal Cell Carcinoma (RCC) is a common cancer worldwide, accounting for approximately 2-3% of adult malignancies. About 63990 new cases of RCC and over 14400 related deaths of renal cancer in the us were reported in 2017. In China, the incidence of RCC is on the rise, and in 2014, there are 68300 new RCC and 25600 renal cancer-related deaths. Metastatic spread is the most important factor affecting the prognosis of RCC. The 5-year survival rate for primary metastatic patients is about 10%, and the 5-year survival rate for non-metastatic and late metastatic patients is 70-90%. At the initial diagnosis, nearly 60% of patients have local cancer and 20% have distant metastasis. Metastatic spread of RCC occurs mainly in bone, lung, liver or brain. The metastasis of RCC is the cause of high incidence and poor prognosis. More than 80% of RCC patients survive less than 5 years after confirmed distant metastases.

Therefore, understanding the action mechanism of the RCC, especially the action mechanism of the RCC, the development and the metastasis, and carrying out proper intervention are of great significance for improving the clinical diagnosis and treatment level of the RCC and prolonging the survival time of patients. However, there is currently no clinically ideal molecular marker to assess prognosis and risk of metastasis in RCC patients. Therefore, the search for novel markers for RCC metastasis and prognosis is currently in prime for the hotspot problem to be solved.

Disclosure of Invention

In order to make up the defects of the prior art, the invention screens out molecular markers which are expressed differentially in renal cell carcinoma tissues and metastatic cancer tissues by a high-tech biotechnology, and judges the risk of the renal cell carcinoma patient of generating metastasis and prognosis evaluation by detecting the expression level of the molecular markers and comparing the expression level with a reference level. Meanwhile, the biomarker can be used as a specific molecular target of renal cell carcinoma and applied to the precise treatment of the renal cell carcinoma.

In order to achieve the purpose, the invention provides the following technical scheme: a renal cell carcinoma biomarker as a metastasis marker for diagnosing and/or assessing the prognosis of renal cell carcinoma and the risk of developing metastasis; or, as a specific molecular target for renal cell carcinoma, for use in the treatment of renal cell carcinoma; the biomarkers include one or more of THBS2, SCGB1a1, NKX2-1, COL11a1, DCN, COL1a1, and SFTPB. When THBS2, SCGB1a1, NKX2-1, COL11a1, DCN, COL1a1 and/or SFTPB expression is up-regulated, renal cell carcinoma patients are at potential risk of developing metastasis and poor prognosis.

A detection reagent for detecting the expression level of THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB genes in a sample by a sequencing technology, a nucleic acid hybridization technology and a nucleic acid amplification technology; the detection reagent is selected from probes specifically recognizing THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB; or the detection reagent is selected from primers for specifically amplifying THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB; or the detection reagent is selected from a chip for specifically analyzing THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB.

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 it is less stable in cells and 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), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing 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 tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). 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 embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can 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 then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is a reverse Northern blot, in which 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., ncRNA) 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 certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

Generally, PCR uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; RT-PCR Reverse Transcriptase (RT) is used to prepare complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of the DNA; TMA autocatalytically synthesizes multiple copies of a target nucleic acid sequence under substantially constant conditions of temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies, TMA optionally including the use of blocking, partial, terminating and other modifying moieties to improve the sensitivity and accuracy of the TMA process; LCR with target nucleic acid adjacent region hybridization of two sets of complementary DNA oligonucleotides. The DNA oligonucleotides are covalently linked by DNA ligase in repeated cycles of heat denaturation, hybridization, and ligation to produce a detectable double-stranded ligated oligonucleotide product; the SDA uses multiple cycles of the following steps: primer sequence pairs anneal to opposite strands of the target sequence, primer extension in the presence of dNTP α S to produce double-stranded hemiphosphorothioated (phosphorothioated) primer extension products, endonuclease-mediated nicking of the hemimodified 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, thereby causing geometric amplification of the products.

Preferably, the primer sequence for specifically amplifying THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB is shown as SEQ ID NO. 1-14.

Preferably, the related product for in vitro detection of the expression level of THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB comprises a gene chip or a kit.

Preferably, the chip comprises a solid phase carrier and a probe which is attached to the solid phase carrier and specifically recognizes THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB.

As non-limiting examples, the solid phase carrier can be made of various materials commonly used in the field of gene chips, such as but not limited to nylon membrane, glass or silicon slice modified with active groups (such as aldehyde group, amino group, etc.), unmodified glass slice, plastic slice, etc.

The chip of the present invention can be prepared by a conventional method for manufacturing a biochip known in the art. For example, if a modified glass slide or silicon wafer is used as the solid support, and the 5' end of the probe contains a poly-dT string modified with an amino group, the oligonucleotide probe can be prepared into a solution, and then spotted on the modified glass slide or silicon wafer using a spotting apparatus, 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 THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB, a probe for specifically recognizing THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB, or a chip for specifically analyzing THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB.

Preferably, the kit further comprises one or more of a container, instructions for use, a positive control, a negative control, and an excipient. The excipient comprises a buffer, an auxiliary agent or a solvent.

Pharmaceutical compositions comprising inhibitors of THBS2, SCGB1a1, NKX2-1, COL11a1, DCN, COL1a1 and/or SFTPB.

Preferably, the inhibitor is capable of reducing the expression level of THBS2, SCGB1a1, NKX2-1, COL11a1, DCN, COL1a1 and/or SFTPB; alternatively, the inhibitor is an interfering RNA. The inhibitor is any agent that can reduce the levels of THBS2, SCGB1a1, NKX2-1, COL11a1, DCN, COL1a1, and/or SFTPB. As non-limiting examples, include: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

The terms "differential molecular marker expression" and "differential expression" are used interchangeably and refer to a molecular marker whose expression 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 who responds differently to a particular treatment or has a different prognosis. The term also includes molecular markers whose expression is activated to higher or lower levels at different stages of the same disease. It is also understood that differentially expressed molecular markers may be activated or inhibited at the nucleic acid level or at the protein level, or may be subject to alternative splicing to produce a different polypeptide product. This difference can be evidenced by a variety of changes including mRNA levels, microrna levels, lncRNA levels, antisense transcript levels, or other divisions of protein surface expression, secretion, or polypeptide. Differential molecular marker expression may include a comparison of expression between two or more genes or gene products thereof; or a comparison of the ratio of expression between two or more genes or gene products thereof; or even a comparison of the products of two different processes of the same gene, which differ 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 transient or cellular expression patterns in molecular markers between normal and diseased cells or between cells undergoing different disease events or disease stages.

When a molecular marker indicates or is a marker for an abnormal process, disease or other condition in an individual, the molecular marker is generally described as being over-expressed or under-expressed compared to the expression level or value of the molecular marker that indicates or is a marker for a normal process, no disease or other condition in an individual. "upregulation," "upregulated," "overexpression," "overexpressed," 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 typically 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 the value or level (or range of values or levels) of the molecular marker that is detectable at different stages of a particular disease.

"downregulated," "under-expressed," and used interchangeably, 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 typically 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, the reagents, means and/or instructions for use described herein may be provided in a kit. For example, a kit may comprise reagents, tools, and instructions for determining an appropriate treatment for a cancer patient. The kit may include reagents for collecting a tissue sample from a patient, for example by biopsy, and reagents for processing the tissue. The kit may further comprise one or more reagents for performing an expression analysis of the molecular marker, such as reagents for performing RT-PCR, qPCR, northern blot, etc. to determine the expression level of the molecular marker in a sample of the patient. For example, primers for performing RT-PCR, probes for performing northern 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 an instruction card describing how to perform the assay for measuring expression of the molecular marker. The instruction card may also include instructions for how to determine the reference cohort, including how to determine the expression levels of the molecular markers in the reference cohort and how to aggregate the expression data to establish a reference for comparison to the test patient. The instruction card may also include instructions for determining expression of the molecular marker in a test patient and for comparing the expression level to expression in a reference cohort to determine an appropriate chemotherapy for the subject.

The informational material included in the kit may be described, instructional, marketing, or other material related to the use of the methods described herein and/or the reagents used in the methods described herein. For example, the informational material of the kit may contain contact information, such as a physical address, an email address, a website, or a telephone number, where the user of the kit may obtain a wealth of information regarding the results of performing gene expression analysis and analysis, particularly when applied to a human that may have a positive response to a particular therapeutic agent.

Use of the above-mentioned renal cell carcinoma metastasis marker, the above-mentioned detection reagent, the above-mentioned related product or the above-mentioned pharmaceutical composition for the preparation of a diagnostic and/or evaluation and/or treatment of renal cell carcinoma.

According to the invention, whether renal cell carcinoma patients have metastasis or not and prognosis can be judged by detecting the expression levels of THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and SFTPB in the tissues of the renal cell carcinoma patients, and the accuracy rate is more than 93%. Therefore, the detection of molecular biomarkers such as THBS2 and the like in the tissues of the renal cell carcinoma patients can evaluate the disease progression, metastasis and prognosis of the renal cell carcinoma patients, can timely perform relevant treatment on the symptoms, improves the survival time and quality of the patients, and has great economic value and social benefit.

Drawings

FIG. 1 shows that the expression of thrombospondin 2(THBS2) in RCC tissue is significantly higher than that in paracancerous normal tissue;

fig. 2 shows that secretin protein 1a1(SCGB1a1) is significantly more expressed in RCC tissues than in paraneoplastic normal tissues;

FIG. 3 shows that NKX homeobox-1 (NKX2-1) is significantly higher in RCC tissue than in paracancerous normal tissue;

FIG. 4 is a type XI collagen alpha 1 chain (COL11A1) with significantly higher expression in RCC tissues than in paracancerous normal tissues;

FIG. 5 shows that Decorin (DCN) expression in RCC tissue is significantly higher than that in paracancerous normal tissue;

FIG. 6 is a graph showing that type I collagen alpha 1 chain (COL1A1) is significantly more expressed in RCC tissues than in paracancerous normal tissues;

FIG. 7 shows that the expression of lung surfactant protein B (SFTPB) is significantly higher in RCC tissues than in paracancerous normal tissues;

FIG. 8 shows that the expression of thrombospondin 2(THBS2) in RCC transfer tissues was significantly higher than that in RCC tissues;

fig. 9 shows that secretin protein 1a1(SCGB1a1) is significantly more expressed in RCC metastatic tissues than in RCC tissues;

FIG. 10 shows that NKX homeobox-1 (NKX2-1) is significantly higher in expression in RCC metastatic tissues than in RCC tissues;

FIG. 11 is a type XI collagen alpha 1 chain (COL11A1) with significantly higher expression in RCC transferred tissues than in RCC tissues;

FIG. 12 shows that Decorin (DCN) expression in RCC metastatic tissue is significantly higher than in RCC tissue;

FIG. 13 shows that type I collagen alpha 1 chain (COL1A1) is significantly more expressed in RCC metastatic tissues than in RCC tissues;

FIG. 14 shows that lung surfactant protein B (SFTPB) expression in RCC metastatic tissues is significantly higher than in RCC tissues;

FIG. 15 shows that high expression of thrombospondin 2(THBS2) is closely correlated with poor prognosis in RCC patients;

FIG. 16 shows that the high expression of secretin protein 1A1(SCGB1A1) is closely related to the poor prognosis of RCC patients;

FIG. 17 shows that high expression of NKX homeobox-1 (NKX2-1) is closely related to poor prognosis in RCC patients;

FIG. 18 shows that type XI alpha-1 collagen chain (COL11A1) high expression is closely related to RCC patient poor prognosis;

FIG. 19 is a graph showing that high Decorin (DCN) expression correlates well with poor prognosis in RCC patients;

FIG. 20 is a graph showing that high expression of type I collagen alpha 1 chain (COL1A1) is closely associated with poor prognosis in RCC patients;

FIG. 21 shows that high expression of lung surfactant protein B (SFTPB) is closely related to poor prognosis of RCC patients.

Detailed Description

The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The conditions employed in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments.

Example 1 preparation of a kit for predicting metastasis and prognosis in RCC patients (50 reactions)

1.Trizol：50ml；

2. Chloroform: 20ml of the solution;

3. isopropyl alcohol: 30 ml;

4.75% ethanol; 60 ml;

DEPC water: 10 ml;

mixture of oligo (dT) primers: 100 ul;

7.200U/ul M-MLV reverse transcriptase: 50 ul;

8.SYBR qPCR Mix：500ul；

9.10uM target gene specific primers (the sequence is shown in SEQ NO: 1-14): each 100 ul;

10.10uM specific primer of reference gene beta-actin (the sequence is shown in SEQ NO: 15-16): each 100 ul.

Example 2 detection and analysis of molecular markers in tissue samples

1. And collecting the RCC tissue to be detected, the paracancer normal tissue and the RCC tissue transferred from the far end, cleaning with normal saline, and putting into liquid nitrogen for freezing storage.

2. Extracting tissue RNA: adding liquid nitrogen into a mortar, shearing the tissue, grinding and crushing the tissue in the liquid nitrogen, adding 100mg of the tissue into 1ml of Trizol, and uniformly mixing. After standing at room temperature for 5 minutes, 200 uL/tube of chloroform was added thereto, followed by vigorous shaking and mixing for 15 seconds. Centrifuge at 12000 rpm for 15 minutes. Carefully pipette the upper aqueous phase into a fresh Ep tube, add an equal volume of isopropanol, mix by inversion, stand at room temperature for 5 minutes, and centrifuge at 12000 rpm for 10 minutes. The supernatant was carefully discarded, 75% ethanol was added, and the mixture was centrifuged at 8000 rpm for 8 minutes after mixing. The supernatant was removed, dried and DEPC was added to dissolve the RNA. The concentration and purity of RNA were measured with a microplate reader. The ratio OD260/OD280 was between 1.8 and 2.0, indicating good RNA purity.

3. Reverse transcription: reverse transcription was performed using reverse transcription kit (R233) of the biotechnology company of nujing nuozokenza, as follows: the following mixtures were prepared in a centrifuge tube: 4 XgDNA wiper Mix 4ul, Oligo (dT)23VN (10. mu.M) 1ul, template RNA 1pg-1ug, DEPC water to 20 ul. After mixing, incubation was carried out at 42 ℃ for 2 minutes. Then 5 XHiScript II Select qRT SuperMix 4ul was added and mixed well and incubated at 50 ℃ for 15 minutes and then at 85 ℃ for 5 seconds. The obtained cDNA was stored at-80 ℃ by freezing or subjected to real-time quantitative PCR.

4. Real-time quantitative PCR: the PCR primer is synthesized by Suzhou Jinzhi Zhi Biotechnology GmbH, and has the primer sequence of SEQ NO 1-14 and the primer sequence of internal reference gene beta-actin of SEQ NO 15-16. The primer sequences are as follows:

THBS2：

a forward primer: 5'-CGTGGACAATGACCTTGTTG-3' (SEQ ID NO.1)

Reverse primer: 5'-GCCATCGTTGTCATCATCAG-3' (SEQ ID NO.2)

SCGB1A1：

A forward primer: 5'-TTCAGCGTGTCATCGAAACCC-3' (SEQ ID NO.3)

Reverse primer: 5'-ACAGTGAGCTTTGGGCTATTTTT-3' (SEQ ID NO.4)

NKX2-1：

A forward primer: 5'-ATGTACCGGGACGACTTGGAA-3' (SEQ ID NO.5)

Reverse primer: 5'-CAATGCCTGTCAGGGCTAGAA-3' (SEQ ID NO.6)

COL11A1：

A forward primer: 5'-TGGTGATCAGAATCAGAAGTTCG-3' (SEQ ID NO.7)

Reverse primer: 5'-AGGAGAGTTGAGAATTGGGAATC-3' (SEQ ID NO.8)

DCN：

A forward primer: 5'-ATGAAGGCCACTATCATCCTCC-3' (SEQ ID NO.9)

Reverse primer: 5'-GTCGCGGTCATCAGGAACTT-3' (SEQ ID NO.10)

COL1A1：

A forward primer: 5'-GAGGGCCAAGACGAAGACATC-3' (SEQ ID NO.11)

Reverse primer: 5'-CAGATCACGTCATCGCACAAC-3' (SEQ ID NO.12)

SFTPB：

A forward primer: 5'-TGGAGCAAGCATTGCAGTG-3' (SEQ ID NO.13)

Reverse primer: 5'-ACTCTTGGCATAGGTCATCGG-3' (SEQ ID NO.14)

β-actin:

A forward primer: 5'-CACCATTGGCAATGAGCGGTTCC-3' (SEQ ID NO.15)

Reverse primer: 5'-GTAGTTTCGTGGATGCCACAGG-3' (SEQ ID NO.16)

The other reagent of the real-time quantitative PCR is a ChamQ Universal SYBR qPCR Master Mix (Q711-02) of Biotech company of Nanjing Novowed, and the specific steps are as follows: the following mixtures were prepared in qPCR tubes: 2XChamQ Universal SYBR qPCR Master Mix 10ul, 10uM upstream primer 1ul, 10uM downstream primer 1ul, template cDNA 4ul and H2O 4 ul. After mixing, centrifuging for several seconds to make it sink at the bottom of the tube. The PCR procedure included the following steps:

a. pre-denaturation: 30 seconds at 95 ℃;

b. denaturation: 10 seconds at 95 ℃;

c. annealing/extending: 30 seconds at 60 ℃;

d. repeating steps b and c for a total of 40 cycles

e. Dissolution curve analysis: 95 ℃ for 15 seconds, 60 ℃ for 60 seconds, 95 ℃ for 15 seconds.

5. Statistical analysis: and calculating the relative expression quantity of the gene according to the real-time quantitative PCR result and the 2-delta-Ct. Analysis was performed using a nonparametric t-test. Gene expression and prognosis of RCC were analyzed using the KM PLOTTER database (https:// kmplot. com). The Kaplan-Meier method is used for drawing a survival curve, and the Log-rank method is used for comparing survival difference among groups. P <0.05 indicates that the difference is statistically significant.

6. As a result: the real-time quantitative PCR results for RCC tissues and paracancer normal tissues are shown in fig. 1-7, and compared with paracancer normal tissues, the expression of thrombospondin 2(THBS2), secretin 1a1(SCGB1a1), NKX homeobox-1 (NKX2-1), type collagen α 1 chain of xi (COL11a1), Decorin (DCN), type I collagen α 1 chain (COL1a1), and lung surfactant protein b (sftpb) was significantly up-regulated in RCC tissues by 10.864-fold, 2.804-fold, 1.834-fold, 1.919-fold, 1.891-fold, 2.672-fold, and 2.236-fold, respectively, and the differences were statistically significant (P <0.01, P < 0.05). In addition, we also examined the expression difference of the above 7 genes in the RCC tissue and the RCC transfer tissue, and the results are shown in FIGS. 8-14. Compared with RCC tissues, THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and SFTPB are all significantly up-regulated in the RCC tissues, the up-regulation times are respectively 2.199-fold, 35.682-fold, 11.973-fold, 3.788-fold, 1.383-fold, 1.025-fold and 100.610-fold, and the differences are statistically significant (P <0.01 and P < 0.05). Finally, the correlation between the expression of the THBS2 and other genes in the RCC and the prognosis is analyzed, and as shown in FIGS. 15-21, the high expressions of the THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and SFTPB of the RCC patients are closely related to the poor prognosis of the RCC patients (the P is less than 0.01). The above experimental results show that whether renal cell carcinoma patients have metastasis or not and prognosis can be judged by detecting the expression levels of THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and SFTPB in the tissues of the renal cell carcinoma patients, and the accuracy rate is more than 93%. Therefore, the detection of molecular biomarkers such as THBS2 and the like in the tissues of the renal cell carcinoma patients can evaluate the disease progression, metastasis and prognosis of the renal cell carcinoma patients, can timely perform relevant treatment on the symptoms, improves the survival time and quality of the patients, and has great economic value and social benefit.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. A renal cell carcinoma biomarker, characterized by being a metastasis marker for diagnosing and/or assessing the prognosis of renal cell carcinoma and the risk of developing metastasis; or, as a specific molecular target for renal cell carcinoma, for use in the treatment of renal cell carcinoma; the biomarkers include one or more of THBS2, SCGB1a1, NKX2-1, COL11a1, DCN, COL1a1, and SFTPB.

2. A detection reagent for detecting the expression level of THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB genes in a sample by a sequencing technology, a nucleic acid hybridization technology and a nucleic acid amplification technology; the detection reagent is selected from probes specifically recognizing THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB; or the detection reagent is selected from primers for specifically amplifying THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB; or the detection reagent is selected from a chip for specifically analyzing THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB.

3. The detection reagent of claim 2, wherein the primer sequence for specific amplification of THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB is shown in SEQ ID No. 1-14.

4. A related product for in vitro detection of THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB expression level is characterized in that the related product comprises a gene chip or a kit.

5. A related product according to claim 4, wherein said chip comprises a solid support and attached thereto probes specifically recognizing THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB.

6. A related product according to claim 4, wherein said kit comprises primers for specifically amplifying THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB, probes specifically recognizing THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB, or chips specifically analyzing THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, COL1A1 and/or SFTPB.

7. The related product of claim 6, wherein the kit further comprises one or more of a container, instructions for use, a positive control, a negative control, and an excipient.

8. Pharmaceutical composition comprising an inhibitor of THBS2, SCGB1a1, NKX2-1, COL11a1, DCN, COL1a1 and/or SFTPB.

9. The pharmaceutical composition of claim 8, wherein the inhibitor is capable of reducing the expression level of THBS2, SCGB1a1, NKX2-1, COL11a1, DCN, COL1a1, and/or SFTPB; alternatively, the inhibitor is an interfering RNA.

10. Use of a renal cell carcinoma metastasis marker according to claim 1, a detection reagent according to claims 2 and 3, a related product according to any one of claims 4 to 7 or a pharmaceutical composition according to claims 8 and 9 for the preparation of a diagnostic and/or assessment and/or treatment of renal cell carcinoma.