CN112725451A - Melanoma diagnosis marker and application thereof - Google Patents

Melanoma diagnosis marker and application thereof Download PDF

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CN112725451A
CN112725451A CN202110142808.3A CN202110142808A CN112725451A CN 112725451 A CN112725451 A CN 112725451A CN 202110142808 A CN202110142808 A CN 202110142808A CN 112725451 A CN112725451 A CN 112725451A
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cap2
melanoma
expression level
protein
sample
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崔健
姜薇
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Hebei Renbo Technology Co ltd
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Abstract

The invention discloses a molecular marker for diagnosing melanoma, which is CAP2 gene or protein. The invention proves that CAP2 is highly expressed in melanoma cells of mice and humans, the expression level of the CAP2 is increased along with the increase of the metastasis degree of the melanoma, the CAP2 is in negative correlation with the survival rate, and the CAP2 can be used as a diagnosis or prognosis marker of the melanoma. CAP2 expression levels were further analyzed by 451 melanoma samples in the TCGA database to have a statistical correlation with melanoma disease. The invention proves the relevance of CAP2 and melanoma for the first time, and is a very valuable potential drug screening or treatment target in the field of melanoma treatment.

Description

Melanoma diagnosis marker and application thereof
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to a melanoma diagnosis marker and application thereof.
Background
Melanoma, also known as Malignant Melanoma (MM), accounts for approximately 1% to 3% of all malignant tumors. Cutaneous melanoma (CMM) is an invasive tumour maliciously transformed from epidermal melanocytes, the most advanced and the worst prognosis of all cutaneous tumours, with later appearance of blood transport or lymph node metastases. The disease is hidden, the early stage metastasis is easy, patients who see the doctor are mostly in the middle and late stages, and the mortality rate is high within 5 years after metastasis occurs. In recent years, the incidence and mortality of MM have been on the rise, and the American Cancer Society (ACS)2017 reports that, in addition to the incidence and mortality of MM and thyroid cancer, other common malignancies have been on the fall. Epidemiological research in China shows that about 20000 new cases occur every year, Chinese instruction for melanoma diagnosis and treatment (2015 edition) shows that compared with European and American countries, the mortality of MM in China rises with the rise of morbidity, which reflects that China has a gap with the European and American countries in MM diagnosis and treatment and needs early prevention, early discovery and early treatment in clinical work.
The current research on the basic and clinical research of melanoma is increasing, but the pathogenesis of melanoma is still unknown, and many patients are diagnosed at the middle and advanced stage. There is no significant progress in clinical treatment and the current situation is still not optimistic. Therefore, further exploring the relevant mechanisms of melanoma occurrence and development, searching for new tumor markers and treating targets to prolong the life cycle of patients become problems to be solved urgently.
Certain genes have been shown to be associated with melanoma. Such as the effect of CXCR4 gene silencing on melanoma invasion and metastasis, the expression of SPINK5 in clinical tissue samples of skin malignant melanoma. The method finds more new targets of the melanoma and has important significance for biological diagnosis and treatment of the melanoma.
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims to provide a molecular marker related to melanoma occurrence, which is applied to clinic to realize early diagnosis of melanoma and improve the survival rate and the survival quality of patients.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
in a first aspect, the present invention provides a molecular marker for melanoma diagnosis, wherein the molecular marker is CAP2 gene or protein.
Further, the invention provides primers for detecting the molecular marker, which comprise an upstream primer and a downstream primer capable of detecting the CAP2 gene expression level at the mRNA level, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1 or SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.2 or SEQ ID NO. 4.
In a second aspect, the invention provides a reagent for quantitatively detecting the expression level of a CAP2 gene or protein, wherein the reagent for detecting the expression level of the CAP2 gene comprises the primer; the reagent for detecting the expression level of the CAP2 protein comprises a specific antibody of the CAP2 protein.
Further, the invention provides application of the reagent in preparation of a kit for diagnosis or prognosis analysis of melanoma.
The invention proves that CAP2 is highly expressed in melanoma cells, and the expression level of the CAP2 is increased along with the increase of the metastasis degree of the melanoma.
Further, it was demonstrated that the expression level of CAP2 in melanoma patients was negatively correlated with survival.
In some embodiments, the kit further comprises sample treatment reagents comprising at least one of sample lysis reagents, sample purification reagents, and sample nucleic acid extraction reagents.
In some embodiments, the sample is selected from at least one of a blood, serum, plasma, tissue or cell, and saliva sample of the subject.
In a third aspect, the invention provides the use of a CAP2 gene or protein as a marker in the preparation of a medicament for the prevention or treatment of melanoma.
Preferably, the CAP2 is highly expressed in melanoma cells, and the expression level is increased along with the increase of the metastasis degree of the melanoma.
In a fourth aspect, the present invention provides a pharmaceutical composition for treating melanoma comprising an inhibitor of functional expression of CAP2, and a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutically acceptable carrier includes buffers, emulsifiers, suspending agents, stabilizers, preservatives, salts, excipients, fillers, coagulants and conditioners, surfactants, dispersants, and anti-foaming agents.
Based on the technical scheme, the invention has the following beneficial effects:
the invention proves that CAP2 is highly expressed in melanoma cells of mice and humans, the expression level of the CAP2 is increased along with the increase of the metastasis degree of the melanoma, the CAP2 is in negative correlation with the survival rate, and the CAP2 can be used as a diagnosis or prognosis marker of the melanoma. CAP2 expression levels were further analyzed by 451 melanoma samples in the TCGA database to have a statistical correlation with melanoma disease. The invention proves the relevance of CAP2 and melanoma for the first time, and is a very valuable potential drug screening or treatment target in the field of melanoma treatment.
Drawings
FIG. 1 shows the measurement of the expression level of CAP2 mRNA in mouse melanoma cells;
FIG. 2 shows the detection of the expression level of CAP2 protein in mouse melanoma cells;
FIG. 3 shows the measurement of the expression level of CAP2 mRNA in human melanoma cells;
FIG. 4 shows the detection of the expression level of CAP2 protein in human melanoma cells;
FIG. 5CAP2 expression correlates with survival of melanoma patients.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
The mRNA level content of CAP2 in mouse normal epidermal cell JB6, mouse melanoma high-metastasis cell B16-F10 and mouse melanoma low-metastasis cell B16-F1 were compared.
Mouse melanoma cells B16-F10 were inoculated into DMEM medium containing 10% fetal bovine serum (with penicillin and streptomycin added at 100U/mL) and placed at 37 deg.C with 5% CO2Cultured in an incubator and passaged by digestion with 0.25% trypsin.
The operation steps are as follows:
RNA extraction and fluorescent quantitative PCR detection: extracting RNA of cells by using a total RNA rapid extraction kit (Feijie organism), carrying out reverse transcription according to conventional steps to form cDNA, and detecting the expression effect of CAP2 by real-time fluorescence quantitative PCR, wherein the sequences of cDNA amplification primer pairs are respectively:
qPCR primer F: 5'-CAGGGTTAGACGGACCTCC-3' (SEQ ID NO: 1);
qPCR primer R: 5'-CTCGGCCACCATACTGTTTATC-3' (SEQ ID NO: 2).
The results are shown in FIG. 1, that the CAP2 expression level is higher in tumor cells than in normal epidermal cells, and the expression level is higher as the degree of metastasis of melanoma cells is higher.
Example 2
Comparing the protein level content of CAP2 in the normal epidermal cells of the mice and the high-metastatic cells B16-F10 of the mice melanoma and the low-metastatic cells B16-F1 of the mice melanoma.
The operation steps are as follows:
cells with 80% confluency were collected, centrifuged, and the supernatant discarded, rinsed twice with PBS, and discarded. Adding RIPA lysis solution, and performing ice lysis for 20 min. The supernatant was collected by centrifugation at 12000g for 10 min. Adding 1xSDS loading buffer, beating, mixing, boiling and denaturing for 5 min. Total protein was separated on a 10% SDS-PAGE gel and then transferred to PVDF membrane. 5% BSA was blocked for 2h at RT, incubated with CAP2 antibody (Origene, TA305626(1:1000 dilution)) overnight at 4 ℃ and washed 3 times with TBST. The secondary antibody was incubated at room temperature for 1h and washed 3 times with TBST. ECL hypersensitive chemiluminescence liquid development is carried out, and imaging is carried out through a Tannon imaging system. The expression level of CAP2 protein in different cells was compared using Tubulin (abcam, ab7291(1:2000 dilution)) as an internal control.
As shown in FIG. 2, the expression level of CAP2 protein was significantly increased in tumor cells compared to normal epidermal cells, and increased as the degree of metastasis of melanoma cells was higher.
Example 3
Comparing the expression level of CAP2 in human normal epidermal cells HaCat and human melanoma cells SK-MEL-24, SK-MEL-28.
See the experimental procedure described in example 1 for specific procedures.
The sequences of the cDNA amplification primer pairs are respectively as follows:
qPCR primer F: 5'-caggtcctgtagcatccaca-3' (SEQ ID NO: 3);
qPCR primer R: 5'-gggcaaataaagctgagcgt-3' (SEQ ID NO: 4).
As a result, as shown in FIG. 3, it was found that the expression level of CAP2 was higher in human melanoma cells than in normal epidermal cells.
Example 4
Comparing the protein level content of CAP2 in human normal epidermal cells HaCat and human melanoma cells SK-MEL-24, SK-MEL-28.
See the experimental procedure described in example 2 for specific procedures.
The results are shown in FIG. 4, in which the expression of CAP2 protein was significantly increased in human melanoma cells compared to human normal epidermal cells.
Example 5
The gene expression data of melanoma patients in a TCGA database are analyzed, the melanoma patients are divided into two types of patients according to the CAP2 expression level, and the survival periods of the two types of patients are compared.
mRNA data for melanoma patients and clinical information for each patient were downloaded from the TCGA database (https:// cancer tumor. nih. gov /), and melanoma patient data were selected for subsequent analysis, for a total of 451 melanoma samples.
Gene expression analysis was performed using edgeR package (https:// bioconductor. org/packages/release/bioc/html/edgeR. html) in the R package, and 451 melanoma patients were classified into a low-expression group and a high-expression group according to the expression level of CAP 2. And then Kaplan-Meier survival analysis is carried out, a P value is calculated according to two-sized long-rank test, the P value is less than 0.05, the survival curve between the two groups has statistical difference (figure 5), and the survival time of the patient with low CAP2 expression level is found to be obviously better than that of the patient with high CAP2 expression level.
Further, CAP2 can be used as a melanoma diagnosis or prognosis marker.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> Hebei Renbao Macke Tech Co Ltd
<120> melanoma diagnosis marker and application thereof
<130> P210002
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
cagggttaga cggacctcc 19
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
ctcggccacc atactgttta tc 22
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
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caggtcctgt agcatccaca 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
gggcaaataa agctgagcgt 20

Claims (10)

1. A molecular marker for melanoma diagnosis, characterized in that the molecular marker is CAP2 gene or protein.
2. The primer for detecting the molecular marker of claim 1, which comprises an upstream primer and a downstream primer capable of detecting the expression level of CAP2 gene at mRNA level, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID No.1 or SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No.2 or SEQ ID No. 4.
3. A reagent for quantitatively detecting an expression level of a CAP2 gene or protein, wherein the reagent for detecting an expression level of a CAP2 gene comprises the primer of claim 2; the reagent for detecting the expression level of the CAP2 protein comprises a specific antibody of the CAP2 protein.
4. Use of the reagent according to claim 3 for the preparation of a kit for the diagnostic or prognostic analysis of melanoma.
5. The use according to claim 4, wherein the CAP2 is highly expressed in melanoma cells and its expression level increases with the degree of metastasis of melanoma.
6. The use of claim 5, wherein the CAP2 is expressed in a melanoma patient in an amount that is inversely correlated with survival.
7. The use of claim 6, wherein the kit further comprises sample treatment reagents comprising at least one of sample lysis reagents, sample purification reagents, and sample nucleic acid extraction reagents.
8. The use of claim 7, wherein the sample is selected from at least one of a blood, serum, plasma, tissue or cell and a saliva sample of the subject.
9. A pharmaceutical composition for treating melanoma, comprising an inhibitor of functional expression of CAP2, and a pharmaceutically acceptable carrier.
10. The use according to claim 9, wherein the pharmaceutically acceptable carrier comprises buffers, emulsifiers, suspending agents, stabilizers, preservatives, salts, excipients, fillers, coagulants and conditioners, surfactants, dispersants, anti-foaming agents.
CN202110142808.3A 2021-02-02 2021-02-02 Melanoma diagnosis marker and application thereof Pending CN112725451A (en)

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