CN114214405B - Reagent, kit and method for liver cancer metastasis prediction - Google Patents
Reagent, kit and method for liver cancer metastasis prediction Download PDFInfo
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Abstract
The invention relates to a reagent for liver cancer metastasis prediction, and a kit and a method for liver cancer metastasis prediction. The reagent, the kit and the prediction method can be used for efficiently and accurately predicting liver cancer metastasis, and have important significance in guiding clinical application and improving survival rate of patients.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a reagent, a kit and a method for transfer prediction.
Background
The latest cancer statistics data show that the primary liver cancer is the fourth most common six malignant tumors worldwide, wherein the liver cell liver cancer accounts for 75% -85% of the primary liver cancer. The Chinese is the country with the highest liver cancer incidence, more than 40 thousands of new cases are increased annually, and the new cases become one of the most important public health and social problems. The malignant degree of liver cancer is high, the development is rapid, more than 50% of patients have undetectable micrometastases before treatment, and the micrometastases are direct causes of recurrence and metastasis after liver cancer treatment, and are one of the most important factors causing high death rate of the liver cancer. Therefore, the biomarker with high specificity and sensitivity is searched, and the method can be used for screening out patients which are more likely to be transferred after treatment earlier, and has important significance for guiding clinical establishment of closer follow-up, more positive treatment schemes and finally prolonging the survival time of the patients.
The current clinical method for judging liver cancer metastasis of patients mainly comprises imaging examination, including: ultrasonic examination, CT examination, MRI examination, PET-CT examination, etc., the detection effect of which is closely related to the anatomical size of the metastasis, is unfavorable for finding the micro-metastasis.
COMMD (copper metabolism related gene) has been reported to be related to tumorigenesis and development, regulating various biological processes such as NF- κb activity, copper ion balance, sodium ion transport, hypoxia, etc. COMMD10 is one of the members of the COMMD family of proteins, but so far, no study report has been made on its application in liver cancer metastasis prediction.
Disclosure of Invention
The invention aims to provide a reagent for predicting liver cancer metastasis, and also provides a kit for predicting liver cancer metastasis and a prediction method.
A reagent for liver cancer metastasis prediction, which is used for detecting the level of COMMD10 gene expression and/or the level of COMMD10 gene expressed protein in liver cancer cells and/or liver cancer tissues.
In a further preferred embodiment of the present invention, the reagent comprises an amplification primer, and the sequence of the amplification primer is:
forward primer: 5'-CTCCTCAAGCTGTGTTACAACTC-3';
reverse primer: 5'-GGAATCCAGCTGTGCTTGTATAG-3'.
In the present invention, it is further preferable that the reagent further comprises housekeeping gene primers.
In the invention, a further preferable scheme is that the housekeeping gene primer is a PCR primer of internal reference GAPDH, and the sequence is as follows:
forward primer: 5'-CCATCAATGACCCCTTCATTGACC-3' the number of the individual pieces of the plastic,
reverse primer: 5'-GAAGGCCATGCCAGTGAGCTTCC-3'.
The invention also provides a kit for liver cancer metastasis prediction, which comprises the reagent of any one of claims 1-4.
In the present invention, it is further preferable that the kit further comprises one or a combination of two or more of a standard substance, a transcriptase, a fluorescent dye, a buffer solution and an antibody of a protein expressed by the COMMD10 gene.
The invention also provides a method for predicting liver cancer metastasis, which comprises the following steps:
A. constructing a standard curve equation of the expression level of the COMMD10 gene or the expression level of the protein expressed by the COMMD10 gene and the number of cancer cell metastasis; wherein the reagent is used for detecting the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene;
B. testing the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene in the liver cancer cells and/or liver cancer tissues to be tested; wherein the reagent is used for detecting the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene;
C. substituting the data measured in the step B into the standard curve equation obtained in the step A to obtain the data.
In the invention, a further preferable scheme is that the expression level of the COMMD10 gene or the expression level of the protein expressed by the COMMD10 gene in the step A is obtained by detecting a liver in-situ tumor sample; the number of cancer metastasis in step a is detected by lung metastasis samples.
In the invention, a further preferable scheme is that the liver in-situ tumor sample is obtained by constructing a liver cancer cell strain for over-expressing the COMMD10 gene and/or knocking down the COMMD10 gene expression.
The invention also provides another method for predicting liver cancer metastasis, which comprises the following steps:
A. constructing a standard curve equation of the expression level of the COMMD10 gene or the expression level of the protein expressed by the COMMD10 gene and the number of cancer cell metastasis; wherein, the kit is adopted to detect the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene;
B. testing the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene in the liver cancer cells and/or liver cancer tissues to be tested; wherein, the kit is adopted to detect the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene;
C. substituting the data measured in the step B into the standard curve equation obtained in the step A to obtain the data.
Compared with the prior art, the invention has the following advantages: the invention discovers that the protein level expressed by the COMMD10 gene and/or the COMMD10 gene is inversely related to liver cancer metastasis (namely, the protein expressed by the COMMD10 gene and/or the COMMD10 gene is low in expression, the number of metastasis sites of cancer cells is high, the protein expressed by the COMMD10 gene and/or the COMMD10 gene is over-expressed, and the number of metastasis sites of the cancer cells is small), so that liver cancer metastasis can be well predicted by detecting the protein level expressed by the COMMD10 gene and/or the COMMD10 gene, the accuracy is high, the detection is convenient, and detection reagents, kits and prediction methods are determined based on the detection results; personalized treatment can be formulated for liver cancer patients who are easy to metastasize better, and liver cancer metastasis can be found/found in advance, so that interventional treatment is performed in advance, and the method has important significance for improving survival rate of patients.
The invention is described in detail below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a graph showing the relative expression level of COMMD10 in 10 liver cancer samples with and without metastasis detected by a real-time quantitative PCR test in example 1;
FIG. 2 shows the results of immunohistochemical detection in example 1 of the expression level of COMMD10 in 496 cases of liver cancer samples with and without metastasis;
FIG. 3 is a graph showing the correlation between COMMD10 expression and prognosis of liver cancer patients analyzed by Kaplan-Meier method in example 1;
FIG. 4 is an electrophoresis chart and a data chart of relative expression amounts of COMMD10 in Huh7 hepatoma cells infected with COMMD10 low expression (shCOMMD 10) and control (shNC) lentivirus detected by a real-time quantitative PCR experiment and a Western blotting experiment in example 1;
FIG. 5 is an electrophoresis chart and a data chart of relative expression amounts of COMMD10 in Huh7 hepatoma cells infected with COMMD10 overexpression (COMMD 10) and control (Mock) lentivirus detected by a real-time quantitative PCR experiment and a Western immunoblotting experiment in example 1;
FIG. 6 is a sample graph of in-situ model experiment of liver cancer in nude mice of COMMD10 stable knockdown Huh7 liver cancer cells in example 1,
FIG. 7 is an electron microscopic image and data diagram of lung metastasis of nude mice with COMMD10 stably knocked down Huh7 liver cancer cells in example 1;
FIG. 8 is a sample diagram of a nude mouse liver cancer in situ model experiment of a COMMD10 stable over-expressed Huh7 liver cancer cell;
FIG. 9 is an electron micrograph and data of lung metastases of nude mice of COMMD10 stably overexpressed Huh7 hepatoma cells.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined into new embodiments. Examples and experimental examples of the present invention, except as specifically illustrated, where equipment and reagent materials were obtained from commercial sources, are given by way of illustration only and are not to be construed as limiting the scope of the present application.
A reagent for liver cancer metastasis prediction, which is used for detecting the level of COMMD10 gene expression and/or the level of COMMD10 gene expressed protein in liver cancer cells and/or liver cancer tissues.
The invention discovers that the protein level expressed by the COMMD10 gene and/or the COMMD10 gene is inversely related to liver cancer metastasis (namely, the protein expressed by the COMMD10 gene and/or the COMMD10 gene is low in expression, the number of metastasis sites of cancer cells is high, the protein expressed by the COMMD10 gene and/or the COMMD10 gene is over-expressed, and the number of metastasis sites of the cancer cells is small), so that liver cancer metastasis can be well predicted by detecting the protein level expressed by the COMMD10 gene and/or the COMMD10 gene, the accuracy is high, the detection is convenient, and detection reagents, kits and prediction methods are determined based on the detection results; personalized treatment can be formulated for liver cancer patients who are easy to metastasize better, and liver cancer metastasis can be found/found in advance, so that interventional treatment is performed in advance, and the method has important significance for improving survival rate of patients.
The reagent may include an amplification primer, and the sequence of the amplification primer is:
forward primer (sequence 1): 5'-CTCCTCAAGCTGTGTTACAACTC-3';
reverse primer (sequence 2): 5'-GGAATCCAGCTGTGCTTGTATAG-3'.
The reagent can also comprise housekeeping gene primers; specifically, the housekeeping gene primer may be a PCR primer of reference GAPDH, and the sequence thereof is:
forward primer (sequence 3): 5'-CCATCAATGACCCCTTCATTGACC-3' the number of the individual pieces of the plastic,
reverse primer (sequence 4): 5'-GAAGGCCATGCCAGTGAGCTTCC-3'.
The invention also provides a kit for liver cancer metastasis prediction, which comprises the reagent; the corresponding kit can also comprise one or more than two of standard substances, transcriptases, fluorescent dyes, buffers and antibodies of proteins expressed by the COMMD10 genes.
The invention also provides a method for predicting liver cancer metastasis, which comprises the following steps:
A. constructing a standard curve equation of the expression level of the COMMD10 gene or the expression level of the protein expressed by the COMMD10 gene and the number of cancer cell metastasis; wherein the reagent is used for detecting the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene;
B. testing the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene in the liver cancer cells and/or liver cancer tissues to be tested; wherein the reagent is used for detecting the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene;
C. substituting the data measured in the step B into the standard curve equation obtained in the step A to obtain the data.
In the invention, a further preferable scheme is that the expression level of the COMMD10 gene or the expression level of the protein expressed by the COMMD10 gene in the step A is obtained by detecting a liver in-situ tumor sample; the number of cancer metastasis in step a is detected by lung metastasis samples.
In the invention, a further preferable scheme is that the liver in-situ tumor sample is obtained by constructing a liver cancer cell strain for over-expressing the COMMD10 gene and/or knocking down the COMMD10 gene expression.
The invention also provides another method for predicting liver cancer metastasis, which comprises the following steps:
A. constructing a standard curve equation of the expression level of the COMMD10 gene or the expression level of the protein expressed by the COMMD10 gene and the number of cancer cell metastasis; wherein, the kit is adopted to detect the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene;
B. testing the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene in the liver cancer cells and/or liver cancer tissues to be tested; wherein, the kit is adopted to detect the expression level of the COMMD10 gene and/or the expression protein level of the COMMD10 gene;
C. substituting the data measured in the step B into the standard curve equation obtained in the step A to obtain the data.
Experimental example 1
1. The expression level of COMMD10 in clinical liver cancer samples (from 496 southern hospital cases at the university of south medical science) was examined.
1) Real-time quantitative PCR: fresh liver cancer specimens after surgical excision were ground into powder with a mortar and liquid nitrogen, and then RNAiso Plus (Takara, japan) reagent was added to extract total RNA. Total cDNA was obtained by reverse transcription using a reverse transcription kit RT Master Mix (RR 036A, takara, japan). According toPreparing PCR reaction liquid by using a Premix Ex Taq II (Takara) instruction book, and performing real-time quantitative PCR detection under the following reaction conditions: the pre-denaturation at 95℃for 10 min, denaturation at 95℃for 8 sec, annealing at 60℃for 20 sec and extension at 72℃for 34 sec were performed for a total of 40 cycles. Data per 2 -ΔΔCt The formula calculates the relative expression level of the gene. Primers for COMMD10 and reference GAPDH were purchased from Rui Boxing Corp.
2) Immunohistochemistry: paraffin sections of human liver cancer tissue specimens were baked in an oven at 60℃for 1h, followed by xylene dewaxing, gradient alcohol hydration, autoclaving of antigen, peroxidase inhibitor treatment for 15 min, incubation of COMMD10 antibody (ab 127691, abcam, UK) overnight at 4℃and incubation of HRP-labeled immunohistochemical secondary antibody, DAB development, hematoxylin staining, dehydration, transparency and sealing. Results were scored by two pathologists according to the product of staining intensity and percentage of staining positive cells.
Referring to FIGS. 1 and 2, the results show that the COMMD10 protein is mainly localized to the cytoplasm; compared with the liver cancer patients without metastasis, the mRNA and protein expression of COMMD10 in tumor tissues of the liver cancer patients with metastasis are lower.
2. The Kaplan-Meier method analyzed COMMD10 expression for prognostic correlation with hepatoma patients (from 496 southern hospital cases at the university of medical science).
Based on the immunohistochemical results, negative expression "-" was defined as COMMD10 low expression group, positive expressions "+ ++" to "++" were defined as COMMD10 high expression groups. Referring to fig. 3, the median survival time of 240 COMMD10 low-expressing patients was 64 months, while 256 COMMD10 high-expressing patients still did not reach median survival time at the end of follow-up, indicating that the total survival time (OS) of the COMMD10 high-expressing group was significantly better than that of the COMMD10 low-expressing group. Meanwhile, the survival time of the low expression group of COMMD10 without disease is 10 months, and the survival time of the high expression group of COMMD10 without disease is 28 months, which shows that the survival time (DFS) of the high expression group of COMMD10 without disease is obviously longer than that of the low expression group of COMMD 10.
3. And (3) constructing a Huh7 liver cancer cell strain with stable over-expression and knocked down COMMD 10.
Taking 293T cells in logarithmic growth phase, transfecting target plasmid and lentivirus packaging plasmid pMD2.G plasmid and pSPAX2 plasmid, and collecting virus supernatant; spreading a logarithmic growth phase Huh7 liver cancer cell strain (purchased from Shanghai cell bank of China academy of sciences) until the cell density reaches 30% -50%, and adding 1mL of virus supernatant to transfect cells; screening was performed by adding puromycin after transfection was completed. All plasmids were purchased from guangzhou pluripotency gene limited.
Referring to fig. 4 and 5, the results show that the expression level of the COMMD10 gene and the corresponding protein in the COMMD10 knockdown group (shrom md 10) is obviously lower than that in the control group (shNC), while the expression level of the COMMD10 gene and the corresponding protein in the COMMD10 over-expression group (COMMD 10) is obviously higher than that in the control group (Mock), and the results prove that the stable over-expression and knockdown Huh7 liver cancer cell strain of the COMMD10 is successfully constructed.
4. The ability of COMMD10 to inhibit liver cancer lung metastasis was examined.
1) Constructing an in-situ liver cancer model: taking COMMD10 knockdown/over-expressed Huh7 cells in logarithmic growth phaseAfter cell counting, the cells were counted according to 3X 10 5 The required cell amount was calculated for each cell/25 μl/cell, using 1:1 and matrigel. After 1% pentobarbital sodium was intraperitoneally injected to anesthetize nude mice, the mice were opened by traversing under the xiphoid process under aseptic conditions, 25. Mu.L of the cell suspension was aspirated with an insulin needle, and the mice were horizontally injected into the left lobe of the liver, and the wounds were sutured. Killing nude mice by cervical dislocation after the observation period is over, obtaining liver in-situ tumor and lung metastasis, soaking in 10% formaldehyde solution for 24 hr, and dehydrating tissue, embedding, slicing and subsequent H&E staining.
2) H & E staining: placing paraffin sections in a 60 ℃ oven for 1 hour, respectively dewaxing and hydrating, hematoxylin staining, eosin staining, dehydrating, transparency and sealing; the number of lung metastases was observed and counted under a normal microscope.
Referring to FIGS. 6-9, compared with the control group, the number of lung metastases of mice is obviously increased by knocking down COMMD10 expression in Huh7 tumor cells; and after the COMMD10 is over-expressed, the number of lung metastasis is obviously reduced.
Through the experiment, the fact that the protein level expressed by the COMMD10 gene and/or the COMMD10 gene is inversely related to liver cancer metastasis (namely, the low expression of the protein expressed by the COMMD10 gene and/or the COMMD10 gene and the high number of cancer cell metastasis sites, the over expression of the protein expressed by the COMMD10 gene and/or the COMMD10 gene and the small number of cancer cell metastasis sites) is proved, so that the liver cancer metastasis can be well predicted by detecting the protein level expressed by the COMMD10 gene and/or the COMMD10 gene, the accuracy is high, the detection is convenient, and a detection reagent, a kit and a prediction method for predicting the liver cancer metastasis can be determined based on the detection; personalized treatment can be formulated for liver cancer patients who are easy to metastasize better, and liver cancer metastasis can be found/found in advance, so that interventional treatment is performed in advance, and the method has important significance for improving survival rate of patients.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
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Claims (5)
1. The application of a reagent for detecting the expression level of the COMMD10 gene in liver cancer cells and/or liver cancer tissues in preparing a reagent for predicting liver cancer lung metastasis is characterized in that the reagent for detecting the expression level of the COMMD10 gene in liver cancer cells and/or liver cancer tissues comprises an amplification primer, wherein the sequence of the amplification primer is as follows:
forward primer: 5'-CTCCTCAAGCTGTGTTACAACTC-3';
reverse primer: 5'-GGAATCCAGCTGTGCTTGTATAG-3'.
2. The use according to claim 1, wherein the reagent for detecting the level of COMMD10 gene expression in liver cancer cells and/or liver cancer tissues further comprises housekeeping gene primers.
3. The use according to claim 2, wherein the housekeeping gene primer is a PCR primer for reference GAPDH, the sequence of which is:
forward primer: 5'-CCATCAATGACCCCTTCATTGACC-3' the number of the individual pieces of the plastic,
reverse primer: 5'-GAAGGCCATGCCAGTGAGCTTCC-3'.
4. Use of a kit for preparing a reagent for predicting liver cancer lung metastasis, wherein the reagent for detecting the level of COMMD10 gene expression in liver cancer cells and/or liver cancer tissues according to any one of claims 1 to 3 is applied.
5. The use of claim 4, wherein the kit further comprises one or a combination of two or more of a standard, a transcriptase, a fluorescent dye, and a buffer.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320385A (en) * | 2013-05-06 | 2013-09-25 | 南方医科大学南方医院 | Human well-differentiated liver cancer cell line HL1017 and construction method thereof |
| CN113633654A (en) * | 2021-07-12 | 2021-11-12 | 官键 | Targeted drug and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320385A (en) * | 2013-05-06 | 2013-09-25 | 南方医科大学南方医院 | Human well-differentiated liver cancer cell line HL1017 and construction method thereof |
| CN113633654A (en) * | 2021-07-12 | 2021-11-12 | 官键 | Targeted drug and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| COMMD10基因在小鼠和人体组织中的表达与分布及生物信息挖掘;孙雅玲;中国博士学位论文全文数据库 基础科学辑(第1期);第5页2.4 荧光评论PCR第8页1.2试剂 * |
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