CN115851942A - Application of diagnosis marker GATA4 in pancreatic inflammatory cancer transformation - Google Patents
Application of diagnosis marker GATA4 in pancreatic inflammatory cancer transformation Download PDFInfo
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Abstract
The present application is a divisional application of 202011531377.1. The invention relates to an application of a diagnosis marker GATA4 in preparing a pancreatitis or pancreatic cancer inflammation identification regulating reagent or a kit, belonging to the technical field of molecular diagnosis. According to the invention, the relation between GATA4 and inflammatory cancer transformation is researched in cells, the correlation between the level of GATA4 and the level of CD68 (macrophage marker) is analyzed, and immunofluorescence detection in pancreatic cancer tissues finds that the two can be co-expressed, so that the GATA4 is possibly involved in inflammatory regulation in the pancreatic cancer development process; the culture of pancreatic cancer cells is induced by adding different concentrations of LPS in the THP-1 differentiation induction macrophage culture medium LMCM, and the result shows that inflammation can promote proliferation, migration and invasion of pancreatic cancer cells.
Description
The application is a divisional application of a patent application named 'application of a diagnostic marker GATA4 in' inflammatory cancer transformation 'of pancreas', the application date of the original application is 22 days 12 months after 2020, and the application number is 202011531377.1.
Technical Field
The invention relates to the technical field of molecular diagnosis, in particular to application of a diagnosis marker GATA4 in 'inflammatory cancer transformation' of pancreas.
Background
Pancreatic cancer (PDAC) has high malignancy, difficult early diagnosis and poor curative effect of the existing treatment method, so the mortality rate is high, the prognosis is very poor, and the five-year survival rate is only 5 percent. While the introduction and application of new diagnostic and therapeutic approaches have led to the control of morbidity and mortality in other cancers, the incidence and mortality in pancreatic cancer continues to rise, with PDAC ranking 4 of the U.S. mortality in 2012 and prediction going up to 2 by 2030. The early diagnosis of pancreatic cancer and/or the prevention of pancreatic cancer by taking measures has important scientific significance and clinical value.
Numerous studies have shown that the occurrence of pancreatic cancer is closely related to pancreatitis. Epidemiological investigation showed that patients with acute or chronic pancreatitis had a 5-6 fold increase in pancreatic cancer incidence compared to normal populations, while patients with hereditary chronic pancreatitis had a 70 fold increase. In clinical practice, the subject group found that two people in a high-incidence family of acute pancreatitis had been diagnosed with pancreatic cancer. Pathological studies show that pancreatitis and pancreatic cancer both originate from acinar cells, wherein the pancreatic cancer originates from acinar-produced metaplasiaADM (acinar-ductal metaplasiaADM), the pathological change is formed transiently in acute pancreatitis, persists in chronic pancreatitis and pancreatic cancer, the heavier and longer the pancreatic inflammation, the more similar the pathological outcome to pancreatic cancer, suggesting that pancreatic inflammation may promote the occurrence and development of pancreatic cancer. In addition, chronic inflammatory signals are closely associated with low grade pancreatic intraepithelial neoplasia (pancreatic intraepithelial neoplasma PanIN) to high grade PanIN and ultimately to pancreatic cancer. A publication by Mol Cancer Research in 2016, tosteP et al, indicates that KRAS gene mutation and inflammatory processes are required to be simultaneously involved in the onset of pancreatic Cancer in adult mice. In addition, despite the inconsistent efficacy of anti-inflammatory and anti-oxidative stress drugs in pancreatic cancer, a number of studies have shown that their use can be prophylactic and/or therapeutic in pancreatic cancer. As studies have shown, the ingestion of high doses of vitamin C, E and selenium can reduce the risk of pancreatic cancer in participants. Cell and animal experiments indicate that non-steroidal anti-inflammatory drugs (non-steroidal anti-inflammatory drugs-drugs NSAIDs) such as aspirin and the anti-inflammatory drug zingiberin play a role in the prevention and/or treatment of pancreatic cancer. In addition to inflammation closely associated with the occurrence of PDACs, inflammatory signaling pathways are also closely associated with the growth and metastasis of PDACs. Inflammatory factors such as IL6 and IL8 promote migration, invasion and angiogenesis of pancreatic tumor cells.
The epidemiological research, the animal model and the patient data indicate that the pancreatitis and the pancreatic cancer are closely related. The research on the molecular mechanism of pancreatic cancer transformation and further the prevention and treatment of pancreatic cancer have important theoretical significance and clinical application value.
The application of the diagnostic marker GATA4 in the pancreatic transformation of inflammatory cancer has not been reported at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of a diagnosis marker GATA4 in pancreatic 'inflammatory cancer transformation'.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention firstly provides application of GATA4 serving as a diagnosis marker of pancreatic inflammatory cancer transformation in preparation of a reagent or a kit for identifying pancreatitis and pancreatic cancer.
The invention also provides application of GATA4 serving as a diagnosis marker of pancreatic cancer transformation in preparation of a reagent or a kit for distinguishing pancreatitis and pancreatitis transformation from pancreatitis to pancreatic cancer.
The invention also provides application of the GATA4 inhibitor in preparing a medicament for preventing and treating pancreatic inflammatory transformation.
Preferably, the "inflammatory cancer transformation" refers to unidirectional transformation from pancreatitis to pancreatic cancer.
Preferably, the inhibitor of GATA4 is an agent that reduces the amount of expression of GATA 4.
Preferably, the inhibitor of GATA4 is selected from a small molecule compound or a biological macromolecule.
Preferably, the biological macromolecule is small interfering RNA, dsRNA, shRNA, micro RNA or antisense nucleic acid which takes the GATA4 protein or the transcript thereof as a target sequence and can inhibit the expression of the GATA4 protein or the gene transcription; or a construct capable of expressing or forming said small interfering RNA, dsRNA, microRNA, antisense nucleic acid.
The invention also provides application of the GATA4 inhibitor in preparing a medicament for preventing and treating proliferation, migration and invasion of pancreatic cancer cells.
The invention also provides application of the GATA4 serving as a diagnosis marker of the pancreatic cancer transformation in preparing a reagent or a kit for pancreatic cancer prognosis risk judgment.
The invention has the advantages that:
1. the research of the invention proves the critical function of the expression level of GATA4 in the process of 'inflammatory cancer transformation' of pancreas, finds that GATA4 can be used as a marker of 'inflammatory cancer transformation' of pancreas for the first time, and deeply studies the initiation link of pancreatic cancer pathogenesis, so that the pancreatic cancer is diagnosed at an early stage and/or measures are taken to prevent the pancreatic cancer, and the invention has excellent accuracy, specificity and sensitivity, and has important scientific significance and clinical value.
2. Unique resource advantages: the kit has complete pancreatitis and pancreatic cancer follow-up data, and preliminarily establishes a blood sample and tissue sample library of patients with pancreatic inflammation and pancreatic cancer, wherein 200 cases are counted at present; and animal models of acute and chronic pancreatitis and pancreatic cancer in situ have been constructed skillfully.
3. Multidisciplinary cross-study model: the research characteristics of clinical oncology, bioinformatics and molecular biology are fully exerted, and organic combination is carried out, so that the method is an innovation for transforming a multidisciplinary cross research mode of medicine.
4. Intensive study of the system: on the basis of the previous research, the transcription factor GATA4 is used as an entry point, an inflammation dynamic gene regulation and control network of pancreatic inflammation cancer transformation is analyzed, new key nodes are deeply excavated and subjected to functional research, and the research is verified in an established model animal and clinical sample library, so that the systematicness, continuity and depth of research are reflected.
5. Multidimensional nature of the study approach: the multi-dimensional research on the pancreatitis malignant transformation mechanism from a molecular level to an integral level, an animal model and a clinical sample is carried out by organically integrating clinical oncology, molecular biology and bioinformatics methods, and the obtained conclusion is more reliable.
Drawings
FIG. 1 is a flow chart of a clinical specimen tissue experiment.
FIG. 2 shows the results of example 2: indicating that GATA4 is highly expressed in pancreatic cancer tissues and cells, A: RT-PC showed increased expression of GATA4 in pancreatic cancer tissues; b: western-blot results show that GATA4 is up-regulated in pancreatic cancer tissues; c: the immunohistochemical results showed that GATA4 expression was upregulated in pancreatic cancer tissues; d: immunofluorescence assay found that the levels of GATA4 were positively correlated with CD68 levels.
FIG. 3 results of example 3, in which different concentrations of LPS (0, 50, 100 ng/ml) were added to macrophage medium LMCM inducing THP-1 differentiation to induce pancreatic cancer cells, indicate that macrophage medium promotes proliferation, migration and invasion of pancreatic cancer cells, and A: CCK-8 experiments confirm that macrophage medium promotes pancreatic cancer cell proliferation; B. a clone formation experiment proves that the macrophage culture medium promotes the clone formation of pancreatic cancer cells; and Transwell cell migration and invasion capacity experiments prove that the macrophage culture medium promotes pancreatic cancer cell migration and invasion capacity is enhanced.
FIG. 4 shows the results of example 3: it was shown that GATA4 is highly expressed in pancreatic cancer cells and is affected by tumor-associated macrophages.
FIG. 5 shows the results of example 3: the interference GATA4 is shown to be capable of obviously reducing the promotion effect of LMCM on the pancreatic cancer cell proliferation activity, and A, B: CCK-8 experiments prove that the interference GATA4 can obviously reduce the proliferation activity of LMCM on pancreatic cancer cells BxPC-3 and PANC-1, and is concentration-dependent; C-D cloning formation experiment proves that interference GATA4 can obviously reduce the cloning formation ability of LMCM on pancreatic cancer cells BxPC-3 and PANC-1, and is concentration dependent.
FIGS. 6 and 7 show the results of example 3: wherein, fig. 6 and fig. 7 both show that the interference GATA4 can obviously reduce the promotion effect of LMCM on the migration and invasion of pancreatic cancer cells, and fig. 6 is a Transwell cell migration and invasion capacity experiment that the interference GATA4 can obviously reduce the promotion effect of LMCM on the migration and invasion capacity of pancreatic cancer cells BxPC-3, and is concentration-dependent; FIG. 7 is a Transwell cell migration and invasion capacity experiment which proves that interference GATA4 can obviously reduce the promotion effect of LMCM on the migration and invasion capacity of pancreatic cancer cell PANC-1, and the concentration dependence is presented.
FIG. 8 shows the results of example 3: shows that the overexpression GATA4 can promote the proliferation, migration and invasion capacity of LMCM to pancreatic cancer cells, A: CCK-8 experiments prove that the overexpression GATA4 can obviously promote the proliferation activity of pancreatic cancer cells ASPC-1 and is time-dependent; B. a clonogenic experiment proves that the over-expression GATA4 can promote the clonogenic capacity promotion effect of the LMCM on pancreatic cancer cells ASPC-1, and is concentration-dependent; transwell cell migration and invasion capacity experiments prove that the over-expression GATA4 can obviously promote the migration and invasion capacity of the pancreatic cancer cells ASPC-1 by LMCM, and is concentration-dependent.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
Example 1
Preliminary experiments
And the iTRAQ mass spectrometry is used for screening the differentially expressed genes of pancreatitis and pancreatic cancer tissue samples, and the GATA4 is suggested to be highly expressed in the pancreatic cancer tissue.
The isotope labeling relative and absolute quantification iTRAQ technology is a polypeptide in vitro labeling technology developed by Applied Biosystems Incorporation (ABI) in the united states. The technology adopts 4 or 8 isotope labels, and can flexibly compare the relative content and the absolute content of protein in various different samples by marking Peptide (Peptide) specific amino acid sites and then performing tandem mass spectrometry. in the iTRAQ experiment, total protein in tissue cells is obtained by protein extraction, the total protein is subjected to enzymolysis (Trypsin digest) to form peptide fragments under the action of pancreatin, and the iTRAQ reagent marks all the obtained peptide fragments. iTRAQ reagent consists essentially of 3 parts: a reporter group (Reportgroup), a balancing group (Balancegroup), and a peptide reactive group (Amine-specific reactive group). Taking a standard 4 reagent as an example: the relative molecular masses of the reporter groups are 114, 115, 116 and 117 respectively; the relative molecular masses of the balancing groups are 31, 30, 29 and 28 respectively, the peptide reaction group connects the iTRAQ reagent with Lysine (K) and N-terminal amino acid residues on the peptide segment, and 4 kinds of report groups are connected with the reaction group through the balancing groups, so that 4 equivalent ectopic labels with the relative molecular masses of 145 are formed. After the peptide fragment sample is marked by the iTRAQ reagent, the equilibrium molecular weights of the reporter group and the equilibrium group are both 145, so that any iTRAQ reagent is changed, and the molecular weights of the same polypeptide marked by different isotopes in the first-stage mass spectrometric detection are all completely the same. In the tandem mass spectrometry (MSMS), the connecting bonds among the reporter group, the balancing group and the peptide reaction group of the peptide segment marked by the same heavy element are broken, the neutrality of the balancing group is lost, and simultaneously the same peptide segment of different isotope labels generates peaks with different mass-to-charge ratios (114-117). Therefore, according to the height and the area of the peak, the quantitative information of the same peptide fragment among the samples can be obtained, and then the quantitative information of the peptide fragment and the protein can be obtained through software processing.
Example 2
Clinical tissue specimen experiment
This example studies the relationship of GATA4 to "inflammatory cancer transformation" in clinical tissue specimens.
1 method
Samples of 7 parapancreatic cancers, 39 pancreatic cancers from the first human hospital in Shanghai were taken and analyzed for GATA4 expression in this clinical sample: performing RT-PCR, western-blot and immunohistochemistry on a clinical pancreatic cancer sample to further verify the GATA4 expression condition, and performing immunofluorescence to detect the level of GATA4 and the level of CD 68;
2 results
When 7 pancreatic cancer parapancreatic cancer samples and 39 pancreatic cancer samples are analyzed by using the RT-PCR technology, the expression of GATA4 RNA level is up-regulated (P < 0.05) relative to a normal pancreatic cancer tissue, and the Western-blot and immunohistochemistry results also show that the expression of GATA4 is up-regulated in the pancreatic cancer tissue in 7 pairs of paired pancreatic cancer samples. Immunofluorescence assay found that the level of GATA4 was positively correlated with the level of CD68 (tumor-associated macrophage marker), suggesting that GATA4 may be involved in inflammatory regulation during pancreatic cancer development (fig. 2).
Example 3
Cell experiments
This example investigated the relationship of GATA4 to "inflammatory cancer transformation" in cells.
(1) Correlation of GATA4 levels with CD68 (macrophage marker) levels: immunofluorescence detection in pancreatic cancer tissues shows that the two can be co-expressed, so that the GATA4 is suggested to be possibly involved in inflammation regulation in the pancreatic cancer development process;
(2) Promotion of pancreatic cancer cells by LMCM: lipopolysaccharide LPS (0, 50, 100 ng/ml) was added at various concentrations to the macrophage medium inducing THP-1 differentiation, and the results were observed and recorded.
(3) GATA4 expression in pancreatic cancer cells: and (3) detecting the GATA4 expression condition in various pancreatic cancer cell strains by using RT-PCR and WB, and observing and recording the result.
(4) Interference with GATA4 observation of the effect of LMCM at different LPS concentrations on pancreatic cancer cell proliferation, migration and invasiveness.
(5) Overexpression of GATA4 the effect of LMCM induction at different LPS concentrations on the proliferative, migratory and invasive capacity of pancreatic cancer cells was observed.
2 results
(1) Pancreatic cancer cells were induced by the culture of macrophage medium LMCM inducing THP-1 differentiation with the addition of LPS (0, 50, 100 ng/ml) at various concentrations, and the results showed that inflammation could promote the proliferation, migration and invasion of pancreatic cancer cells (FIG. 3).
(2) RT-PCR and WB detection found that GATA4 is also highly expressed in different pancreatic cancer cells, and induction of pancreatic cancer cells with LMCM added with LPS at different concentrations promoted upregulation of GATA4 levels in a concentration-dependent manner (FIG. 4).
(3) The interference of GATA4 can obviously weaken the promotion of LMCM with different LPS concentrations on the proliferation activity and the clone formation of pancreatic cancer cells, and is concentration-dependent (figure 5), and similarly, the interference of GATA4 can also obviously inhibit the migration and invasion capacity of pancreatic cancer cells induced by LMCM with different LPS concentrations (figure 6 and figure 7).
(4) The overexpression of GATA4 can obviously promote the proliferation, migration and invasion capacities of pancreatic cancer cells induced by LMCM with different LPS concentrations (figure 8).
Example 4
Animal model experiment
This example aims to study the effect of GATA4 on the development of pancreatic cancer and the "inflammatory cancer transformation" process in animal models.
1 method
Establishment of SCID mouse in situ pancreatic cancer model to study the effect of GATA4 on pancreatic neoplasia:
1) Constructing an SCID small pancreas in situ cancer model: pancreatic cancer cells (cells labeled with luciferase gene) from the GATA4 interfering group, the overexpression group and the control group were injected in situ into the pancreas of SCID mice to establish an in situ model of pancreatic cancer, and 6 cells were injected into each group. Detecting the expression of luciferase by using a Caliper IVIS imaging system every two weeks to observe the condition of each component tumor;
2) Preparation of a specimen: the SCID mice are killed within a certain period of time, tumor tissue and venous blood are taken, the blood is used for detecting the concentration of CA199, the weight and the size of the tumor are measured, RNA and protein are extracted, a part of tissue is fixed by formalin and then is subjected to paraffin embedding and flaking, and the result is observed and recorded.
2 results of
The pancreatic inflammation of the GATA4 interference group and the overexpression group can obviously promote the proliferation, migration and invasion of pancreatic cancer cells. The control group did not show this phenomenon.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.
Claims (2)
- Application of GATA4 as a diagnostic marker in preparation of a pancreatitis or pancreatic cancer identification inflammation regulating reagent or kit.
- 2. The use of claim 1, wherein said pancreatitis comprises chronic pancreatitis and acute pancreatitis.
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| CN202011531377.1A CN112626210A (en) | 2020-12-22 | 2020-12-22 | Application of diagnosis marker GATA4 in pancreatic inflammation-cancer transformation |
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