CN112362872A - Pancreatic cancer tumor marker and application thereof - Google Patents
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Abstract
The invention provides a pancreatic cancer tumor marker, wherein the RNA sequence of the pancreatic cancer tumor marker is shown as SEQ ID NO. 1. Also provides the application of the pancreatic cancer tumor marker. Also provides a detection primer pair of the pancreatic cancer tumor marker and application thereof. Also provides an accelerant of the pancreatic cancer tumor marker and application thereof. Also provides an inhibitor of the pancreatic cancer tumor marker and application thereof. The pancreatic cancer tumor marker disclosed by the invention can be used for simply, conveniently, accurately and quickly diagnosing pancreatic cancer, improving the early diagnosis level of pancreatic cancer and being suitable for large-scale popularization and application.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to the technical field of tumor markers, and particularly relates to a pancreatic cancer tumor marker and application thereof.
Background
Pancreatic cancer is a highly invasive, highly malignant, difficult to diagnose and treat malignant neoplasm of the digestive tract, and about 90% of it is ductal adenocarcinoma originating from the epithelium of the glandular duct. Its morbidity and mortality has increased dramatically in recent years. The treatment of pancreatic cancer is mainly a comprehensive treatment combining surgery, chemotherapy and radiotherapy, the 5-year survival rate of the pancreatic cancer is less than 1 percent, and the pancreatic cancer is one of the worst malignant tumors. The early diagnosis rate of pancreatic cancer is low, the operative mortality rate is high, and the cure rate is low. The incidence rate of pancreatic cancer is higher for men than for women, the ratio of men to women is 1.5-2: 1, male patients are more common than for women before menopause, and the incidence rate of postmenopausal women is similar to that of men.
Therefore, early diagnosis is particularly important for the treatment and prognosis of pancreatic cancer, and peripheral blood detection is the most common and easily popularized diagnostic method. The pancreatic cancer diagnostic marker commonly used in clinic at present, such as CA19-9, plays an important role in diagnosing the occurrence of tumor and monitoring the recurrence thereof. However, the specificity and sensitivity of these markers are often too low to meet clinical needs, and a relatively sensitive method is still lacking, especially in terms of early diagnostic value. Therefore, finding a novel pancreatic cancer tumor marker with high sensitivity and specificity would be the key to increasing the level of early diagnosis of pancreatic cancer.
Therefore, it is desirable to provide a tumor marker for pancreatic cancer, which can make diagnosis of pancreatic cancer simple, accurate and rapid and can improve the level of early diagnosis of pancreatic cancer.
Disclosure of Invention
In order to overcome the defects in the prior art, an object of the present invention is to provide a pancreatic cancer tumor marker, which can make diagnosis of pancreatic cancer simple, accurate and rapid, improve early diagnosis level of pancreatic cancer, and is suitable for large-scale popularization and application.
The invention also aims to provide a pair of detection primer pairs for pancreatic cancer tumor markers, which can simply, rapidly and accurately detect the pancreatic cancer tumor markers, improve the early diagnosis level of pancreatic cancer and is suitable for large-scale popularization and application.
The other purpose of the present invention is to provide a promoter for pancreatic cancer tumor markers, which can promote the expression of the pancreatic cancer tumor markers, further inhibit the growth of pancreatic cancer tumor cells, and is suitable for large-scale popularization and application.
The invention also aims to provide an inhibitor of the pancreatic cancer tumor marker, which can inhibit the expression of the pancreatic cancer tumor marker, further promote the growth of normal pancreatic cells, and is suitable for large-scale popularization and application.
The invention also aims to provide application of the pancreatic cancer tumor marker as a pancreatic cancer diagnostic reagent, which can enable pancreatic cancer to be diagnosed simply, accurately and quickly, improve early diagnosis level of pancreatic cancer, and is suitable for large-scale popularization and application.
The invention also aims to provide application of the detection primer pair of the pancreatic cancer tumor marker as a pancreatic cancer diagnostic reagent, which can simply, conveniently, rapidly and accurately detect the pancreatic cancer tumor marker, improve the early diagnosis level of pancreatic cancer and is suitable for large-scale popularization and application.
The invention also aims to provide application of the promoter for the pancreatic cancer tumor marker in serving as a pancreatic cancer treatment drug, which can promote the expression of the pancreatic cancer tumor marker so as to inhibit the growth of pancreatic cancer tumor cells, and is suitable for large-scale popularization and application.
The invention also aims to provide application of the inhibitor of the pancreatic cancer tumor marker in serving as a pancreatic cancer treatment drug, which can inhibit the expression of the pancreatic cancer tumor marker so as to promote the growth of normal pancreatic cells, and is suitable for large-scale popularization and application.
In order to achieve the above objects, in a first aspect of the present invention, there is provided a pancreatic cancer tumor marker, characterized in that the RNA sequence of the pancreatic cancer tumor marker is represented by SEQ ID No. 1.
In a second aspect of the present invention, a pair of the above-mentioned detection primer pairs for pancreatic cancer tumor markers is provided, wherein the detection primer pairs for pancreatic cancer tumor markers have the nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4.
In a third aspect of the present invention, there is provided an accelerator for a tumor marker of pancreatic cancer as described above, wherein the inhibitor for a tumor marker of pancreatic cancer has a nucleotide sequence shown in SEQ ID NO. 5.
In a fourth aspect of the present invention, there is provided an inhibitor of the above pancreatic cancer tumor marker, which is characterized in that the inhibitor of the pancreatic cancer tumor marker has a nucleotide sequence shown in SEQ ID NO. 6.
In a fifth aspect of the present invention, there is provided a use of the above-mentioned pancreatic cancer tumor marker as a pancreatic cancer diagnostic reagent.
In a sixth aspect of the present invention, an application of the detection primer pair for pancreatic cancer tumor markers as a pancreatic cancer diagnostic reagent is provided.
In a seventh aspect of the present invention, there is provided a use of the promoter for a tumor marker of pancreatic cancer as a medicament for treating pancreatic cancer.
In the eighth aspect of the present invention, there is provided a use of the above-mentioned inhibitor for a tumor marker of pancreatic cancer as a therapeutic agent for pancreatic cancer.
The invention has the beneficial effects that:
a. the RNA sequence of the pancreatic cancer tumor marker is shown as SEQ ID NO. 1, the pancreatic cancer marker can be used for simply, conveniently, accurately and quickly diagnosing pancreatic cancer, improving the early diagnosis level of pancreatic cancer, and is suitable for large-scale popularization and application.
b. The detection primer pair of the pancreatic cancer tumor marker has the nucleotide sequence shown in SEQ ID NO. 3 and the nucleotide sequence shown in SEQ ID NO. 4, can simply, conveniently, quickly and accurately detect the pancreatic cancer tumor marker, improves the early diagnosis level of pancreatic cancer, and is suitable for large-scale popularization and application.
c. The promoter of the pancreatic cancer tumor marker has a nucleotide sequence shown in SEQ ID NO. 5, can promote the expression of the pancreatic cancer tumor marker, further inhibits the growth of pancreatic cancer tumor cells, and is suitable for large-scale popularization and application.
d. The inhibitor of the pancreatic cancer tumor marker has a nucleotide sequence shown in SEQ ID NO. 6, can inhibit the expression of the pancreatic cancer tumor marker, further promotes the growth of normal pancreatic cells, and is suitable for large-scale popularization and application.
e. The pancreatic cancer tumor marker disclosed by the invention is applied to serving as a pancreatic cancer diagnostic reagent, can be used for simply, conveniently, accurately and quickly diagnosing pancreatic cancer, improving the early diagnosis level of pancreatic cancer, and is suitable for large-scale popularization and application.
f. The application of the detection primer pair of the pancreatic cancer tumor marker in serving as a pancreatic cancer diagnostic reagent can simply, conveniently, quickly and accurately detect the pancreatic cancer tumor marker, improve the early diagnosis level of pancreatic cancer, and is suitable for large-scale popularization and application.
g. The promoter for the pancreatic cancer tumor marker can promote the expression of the pancreatic cancer tumor marker and further inhibit the growth of pancreatic cancer tumor cells when being applied to a pancreatic cancer treatment drug, and is suitable for large-scale popularization and application.
h. The inhibitor of the pancreatic cancer tumor marker can inhibit the expression of the pancreatic cancer tumor marker and further promote the growth of normal pancreatic cells when being applied to a pancreatic cancer treatment drug, and is suitable for large-scale popularization and application.
These and other objects, features and advantages of the present invention will become more fully apparent from the following detailed description, the accompanying drawings and the appended claims, wherein like reference numerals refer to like parts throughout the several views, and wherein like reference numerals refer to like parts throughout the several views.
Drawings
FIG. 1 is a schematic diagram of the quantitative RT-PCR method for detecting the expression of hsa-miR-6779-5p gene in blood samples of pancreatic cancer patients and blood samples of healthy persons.
FIG. 2 is a schematic diagram of the detection of the expression of hsa-miR-6779-5p gene in pancreatic cancer cell lines and human normal pancreatic ductal epithelial cells HPDE by quantitative RT-PCR, wherein: p < 0.0001.
FIG. 3 is a schematic diagram of the detection of the expression of hsa-miR-6779-5p gene in the pancreatic cancer cell line BxPC-3 overexpressing hsa-miR-6779-5p gene (A and B) and in normal HPDE cells inhibiting the expression of hsa-miR-6779-5p gene (C and D) by quantitative RT-PCR, in which: p < 0.0001.
FIG. 4 is a schematic diagram showing the cell proliferation potency changes of pancreatic cancer cell line BxPC-3 overexpressing hsa-miR-6779-5p gene and normal HPDE cells inhibiting the expression of hsa-miR-6779-5p gene.
Detailed Description
In order to make the diagnosis of pancreatic cancer simple, convenient, accurate and rapid and improve the early diagnosis level of pancreatic cancer, the invention provides a pancreatic cancer tumor marker, and the RNA sequence of the pancreatic cancer tumor marker is shown as SEQ ID NO. 1. The pancreatic cancer tumor marker is hsa-miR-6779-5p gene.
The invention also provides a pair of detection primer pairs of the pancreatic cancer tumor marker, and the detection primer pairs of the pancreatic cancer tumor marker have a nucleotide sequence shown as SEQ ID NO. 3 and a nucleotide sequence shown as SEQ ID NO. 4.
The invention also provides an accelerant of the pancreatic cancer tumor marker, and an inhibitor of the pancreatic cancer tumor marker has a nucleotide sequence shown as SEQ ID NO. 5.
The invention also provides an inhibitor of the pancreatic cancer tumor marker, which has a nucleotide sequence shown as SEQ ID NO. 6.
The invention also provides application of the pancreatic cancer tumor marker in serving as a pancreatic cancer diagnostic reagent.
The invention also provides application of the detection primer pair of the pancreatic cancer tumor marker in serving as a pancreatic cancer diagnostic reagent.
The invention also provides application of the promoter of the pancreatic cancer tumor marker in serving as a pancreatic cancer treatment drug.
The invention also provides application of the inhibitor of the pancreatic cancer tumor marker in serving as a pancreatic cancer treatment drug.
In order to clearly understand the technical contents of the present invention, the following examples are given in detail. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Example 1 quantitative RT-PCR detection of expression of hsa-miR-6779-5p Gene in blood samples from patients with pancreatic cancer and blood samples from healthy persons
1. Specimen source: after the consent of the patients and healthy persons was obtained, blood samples of 19 pancreatic cancer patients and 10 healthy persons were collected, the ages and sexes of the healthy persons were matched with those of the pancreatic cancer patients, and the blood sampling time was in the early morning in an empty stomach state.
2. Extraction of ribonucleic acid (RNA): total RNA was extracted using Trizol kit (Invitrogen), the extraction method referring to the Trizol kit instructions. When RNA is to be used, the following method may be used to precipitate RNA: NaAc was added to 0.3M and centrifuged at 12,000 Xg for 5 minutes.
3. Synthesis of cDNA:
(1) adding 4 muL (0.5 mug/muL) of template RNA, 2 muL of 20 pmol/muL of RT primer of hsa-miR-6779-5p gene (shown as the nucleotide sequence of SEQ ID NO: 2) and 20 pmol/muL of RT primer of U6 gene (also shown as the reverse amplification primer of U6 gene, namely the nucleotide sequence shown as SEQ ID NO: 8) into an ice-bath centrifuge tube, uniformly mixing, and centrifuging for 3-5 seconds;
(2) water bath at 70 ℃ for 5 minutes and ice bath for 30 seconds;
(3) adding 5 XM-MLV reverse transcriptase reaction solution 4 μ L, RNase inhibitor 1 μ L, dNTP 2 μ L (100mM), and mixing;
(4) water bath at 37 ℃ for 5 minutes, adding 1 mu L M-MLV reverse transcriptase (200 enzyme activity units/. mu.L), and mixing uniformly;
(5) water bath at 37 ℃ for 1 hour;
(6) the reaction was terminated at 70 ℃ for 10 minutes, the product was placed on ice for the next PCR experiment, and the remaining was stored at-70 ℃.
4. Primer design and amplification for quantitative RT-PCR
A primer pair is designed according to the RNA sequence and the RT primer sequence of the hsa-miR-6779-5p gene shown in SEQ ID NO. 1, and is respectively shown as the nucleotide sequence shown in SEQ ID NO. 3 and the nucleotide sequence shown in SEQ ID NO. 4.
In addition, U6 is used as an internal control, and the amplification primer pair is respectively shown as the nucleotide sequence shown in SEQ ID NO. 7 and the nucleotide sequence shown in SEQ ID NO. 8.
Reagent of experimental instrument: thermal Cycler dice Real Time System TP800, Takara; premix Ex TaqTM, Takara.
Reaction system of PCR: total volume 20. mu.L, SYBR Premix Ex Taq 10. mu.L, top and bottom0.4. mu.L each of the primers (10. mu. mol/L), cDNA 1. mu.L, ddH2O8.2 mu L, mixing the reagent evenly, centrifuging and putting the reagent into a PCR instrument for amplification reaction. The PCR conditions were 94 ℃ for 4 min; 30s at 94 ℃, 20s at 60 ℃ and 40 cycles. The PCR amplification products were detected by agarose gel electrophoresis, and the results are shown in FIG. 1.
5. The experimental results are as follows: as can be seen from the results in FIG. 1, the expression level of the hsa-miR-6779-5p gene in the blood sample of pancreatic cancer patients is obviously lower than that in the blood sample of healthy people, and the p value is less than 0.05 (statistically significant) through a t test.
Example 2 quantitative RT-PCR detection of expression of hsa-miR-6779-5p gene in pancreatic cancer cell lines and cell supernatant exosomes
The expression of hsa-miR-6779-5p gene in human pancreatic cancer cell strains AsPC-1, BxPC-3, CFPAC-1, HPAF-II, MIA PaCa-2, PANC-1 and human normal pancreatic duct epithelial cell HPDE and the expression in cell supernatant exosomes are detected by quantitative RT-PCR. Wherein the cell strain is derived from Shanghai cell bank of Chinese academy of sciences.
The RNA of the cell line and the exosome RNA of the cell supernatant were extracted according to the method described in example 1, and the cDNA was synthesized by reverse transcription, and PCR reaction was performed according to the method described in example 1 using U6 as an internal control and the cDNA of the exosome of the different cell lines and the cell supernatant as a template, and the PCR amplification product was detected by agarose gel electrophoresis, and the results are shown in FIG. 2.
As can be seen from the results in FIG. 2, the hsa-miR-6779-5p gene is expressed very little in AsPC-1, BxPC-3, MIA PaCa-2 and PANC-1 and is expressed less in CFPAC-1 and HPAF-II. The hsa-miR-6779-5p gene has very low expression in cell supernatant exosomes of AsPC-1, BxPC-3, MIA PaCa-2 and PANC-1, and has low expression in cell supernatant exosomes of CFPAC-1 and HPAF-II.
Example 3 overexpression of hsa-miR-6779-5p in pancreatic cancer cell line BxPC-3 and inhibition of expression of hsa-miR-6779-5p and change in proliferation capacity of cells after expression of hsa-miR-6779-5p in normal pancreatic ductal epithelial cells HPDE
Aiming at the mRNA of the hsa-miR-6779-5p gene, designing and synthesizing an hsa-miR-6779-5p mimics sequence and an hsa-miR-6779-5p inhibitor sequence, which are respectively shown as a nucleotide sequence shown in SEQ ID NO. 5 and a nucleotide sequence shown in SEQ ID NO. 6; unrelated sequences NC mimics and NC inhibitor are respectively set as negative controls, and are respectively shown as a nucleotide sequence shown in SEQ ID NO. 9 and a nucleotide sequence shown in SEQ ID NO. 10.
Using liposome (Lipofectamine 2000) transfection reagent, hsa-miR-6779-5p mimics and NC mimics were transfected into BxPC-3 cells at a final concentration of 50nmol/L and hsa-miR-6779-5p inhibitor and NC inhibitor were transfected into HPDE cells (supplied from Shanghai cell Bank of China academy of sciences) at a final concentration of 50nmol/L, respectively, and cultured with DMEM containing 10% fetal bovine serum. RNA of different cell lines was extracted according to the method described in example 1, cDNA was synthesized by reverse transcription, and the expression of hsa-miR-6779-5p gene in pancreatic cancer cell line BxPC-3 and normal HPDE cells was detected by quantitative RT-PCR assay using U6 as an internal control and cDNA of different cell lines as a template according to the method described in example 1, and the results are shown in FIG. 3.
As can be seen from the results of FIGS. 3 to 4, compared with the control group (NC mimics), the hsa-miR-6779-5p mimics can effectively promote the mRNA level of the hsa-miR-6779-5p gene in the pancreatic cancer cell line BxPC-3, and can inhibit the growth of the pancreatic cancer cell line BxPC-3 cells, the survival rate of the pancreatic cancer cells is obviously lower than that of the cells of the control group, and the results show that the expression up-regulation of the human hsa-miR-6779-5p gene can inhibit the growth of the pancreatic cancer cells to a certain extent. Compared with a control group (NC inhibit), the hsa-miR-6779-5p inhibit can effectively inhibit the mRNA level of the hsa-miR-6779-5p gene in the HPDE cell, can promote the growth of the HPDE cell, has the survival rate obviously higher than that of the cell of the control group, and shows that the expression of the human hsa-miR-6779-5p gene is reduced and the growth of normal pancreatic cells can be promoted to a certain extent.
Therefore, the hsa-miR-6779-5p gene and the expression product thereof are used as markers for diagnosing pancreatic cancer, so that the pancreatic cancer can be diagnosed more accurately and quickly, and the hsa-miR-6779-5p gene and the expression product thereof are used as target genes for preparing medicaments for treating pancreatic cancer, so that new treatment targets and treatment ways are provided for treating pancreatic cancer.
In conclusion, the pancreatic cancer tumor marker disclosed by the invention can be used for simply, conveniently, accurately and quickly diagnosing pancreatic cancer, improving the early diagnosis level of pancreatic cancer and being suitable for large-scale popularization and application.
In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.
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<223> qPCR forward primer for amplifying human hsa-miR-6779-5p gene
<400> 3
aacacaccug ggaggggcug g 21
<210> 4
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)...(19)
<223> qPCR reverse primer for amplifying human hsa-miR-6779-5p gene
<400> 4
gtcgtatcca gtgcagggt 19
<210> 5
<211> 21
<212> RNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)...(21)
<223> mimics sequence of RNA sequence of human hsa-miR-6779-5p gene
<400> 5
cugggagggg cuggguuugg c 21
<210> 6
<211> 21
<212> RNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)...(21)
<223> inhibitor sequence of RNA sequence of human hsa-miR-6779-5p gene
<400> 6
gccaaaccca gaaaauccca g 21
<210> 7
<211> 17
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)...(17)
<223> qPCR forward primer for amplifying human U6 gene
<400> 7
ctcgcttcgg cagcaca 17
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)...(20)
<223> qPCR reverse primer for amplifying human U6 gene
<400> 8
<210> 9
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)...(21)
<223> NC mimics sequence of human hsa-miR-6779-5p gene
<400> 9
uucuccgaac gugucacgut t 21
<210> 10
<211> 21
<212> RNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)...(21)
<223> NC inhibitor sequence of human hsa-miR-6779-5p gene
<400> 10
uucuccgaac gugucacgut t 21
Claims (8)
1. A pancreatic cancer tumor marker is characterized in that the RNA sequence of the pancreatic cancer tumor marker is shown as SEQ ID NO. 1.
2. The pair of primers for detecting pancreatic cancer tumor markers according to claim 1, wherein the pair of primers for detecting pancreatic cancer tumor markers has the nucleotide sequence shown in SEQ ID NO. 3 and the nucleotide sequence shown in SEQ ID NO. 4.
3. The promoter for a tumor marker of pancreatic cancer according to claim 1, wherein said promoter for a tumor marker of pancreatic cancer has a nucleotide sequence shown in SEQ ID NO. 5.
4. The inhibitor of pancreatic cancer tumor marker according to claim 1, wherein said inhibitor of pancreatic cancer tumor marker has a nucleotide sequence shown in SEQ ID NO. 6.
5. The pancreatic cancer tumor marker of claim 1 for use as a pancreatic cancer diagnostic reagent.
6. The use of the primer pair for detecting a tumor marker of pancreatic cancer according to claim 2 as a diagnostic reagent for pancreatic cancer.
7. The use of the promoter for a tumor marker of pancreatic cancer according to claim 3 as a therapeutic agent for pancreatic cancer.
8. The use of the inhibitor of a tumor marker for pancreatic cancer according to claim 4 as a therapeutic agent for pancreatic cancer.
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Cited By (1)
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CN113151464A (en) * | 2021-03-29 | 2021-07-23 | 武汉博越致和生物科技有限公司 | Method for diagnosing pancreatic cancer by miRNA detection and application thereof |
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CA2951008A1 (en) * | 2014-06-11 | 2015-12-17 | Toray Industries, Inc. | Biliary tract cancer detection kit or device, and detection method |
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CA2951008A1 (en) * | 2014-06-11 | 2015-12-17 | Toray Industries, Inc. | Biliary tract cancer detection kit or device, and detection method |
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CN113151464A (en) * | 2021-03-29 | 2021-07-23 | 武汉博越致和生物科技有限公司 | Method for diagnosing pancreatic cancer by miRNA detection and application thereof |
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