CN112626210A

CN112626210A - Application of diagnosis marker GATA4 in pancreatic inflammation-cancer transformation

Info

Publication number: CN112626210A
Application number: CN202011531377.1A
Authority: CN
Inventors: 杨丽娟; 蒋魏亮; 陈聪颖; 黄丽; 沈杰
Original assignee: Shanghai First Peoples Hospital
Current assignee: Shanghai First Peoples Hospital
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2021-04-09
Also published as: CN115851942A

Abstract

The invention relates to application of a diagnostic marker GATA4 in pancreatic 'inflammatory cancer transformation'. According to the invention, the level of GATA4 is found to be positively correlated with the level of CD68 through immunofluorescence detection, which indicates that GATA4 may participate in inflammation regulation in the pancreatic cancer development process. Experiment: the results of treating pancreatic cancer cells by adding Lipopolysaccharide (LPS) with different concentrations into a macrophage culture medium LMCM inducing THP-1 differentiation show that the increase of GATA4 level in the pancreatic cancer cells can be promoted, the proliferation, migration and invasion of the pancreatic cancer cells can be promoted, and meanwhile, the over-expression of GATA4 can obviously promote the proliferation, migration and invasion capacity of the pancreatic cancer cells under the induction of LMCM with different LPS concentrations. This suggests that GATA4 may be a key factor in the pancreatitis-cancer transformation process. The invention provides a method for diagnosing 'inflammatory cancer transformation' of pancreas by molecular detection, which has the advantages of high diagnosis speed, low cost and accurate and reliable result.

Description

Application of diagnosis marker GATA4 in pancreatic inflammation-cancer transformation

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 the prediction to rise to 2 by 2030. The early diagnosis of pancreatic cancer and/or the prevention of pancreatic cancer by taking measures have important scientific significance and clinical value.

Numerous studies have shown that the occurrence of pancreatic cancer is closely related to pancreatitis. Epidemiological investigation shows that the pancreatic cancer incidence rate of patients with acute or chronic pancreatitis is increased by 5-6 times compared with that of normal people, and the pancreatic cancer incidence rate of patients with hereditary chronic pancreatitis is increased by 70 times. 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-duct metaplasiaADM (acinar-ductal metaplasiaADM), the pathological change is formed transiently during acute pancreatitis, persists during chronic pancreatitis and pancreatic cancer, the heavier and longer the pancreatic inflammation, the more similar the pathological outcome to pancreatic cancer, and the suggestion 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 by Toste P. et al in 2016 shows 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 therapeutic efficacy of anti-inflammatory and anti-oxidative stress drugs on pancreatic cancer, there are a number of studies that suggest that their use may play a prophylactic and/or therapeutic role 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 curcumin 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 is not reported at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of a diagnostic 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 as a diagnosis marker of pancreatic inflammatory cancer transformation in preparation of a reagent or a kit for distinguishing pancreatitis from pancreatic cancer.

The invention also provides application of GATA4 as a diagnosis marker of pancreatic 'inflammatory cancer conversion' in preparing a reagent or a kit for distinguishing pancreatitis and the conversion from pancreatitis to pancreatic cancer.

The invention also provides application of the inhibitor of GATA4 in preparing a medicament for preventing and treating 'inflammatory cancer transformation' of pancreas.

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 GATA4 expressed.

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 GATA4 protein or transcript thereof as a target sequence and can inhibit GATA4 protein expression or 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 inhibitor of GATA4 in preparing medicines for preventing and treating pancreatic cancer cell proliferation, migration and invasion.

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 judging pancreatic cancer prognosis risk.

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 pancreatic cancer transformation, finds that GATA4 can be used as a marker of pancreatic cancer transformation for the first time, and deeply researches the initiation link of pancreatic cancer pathogenesis, so that pancreatic cancer is diagnosed at early stage and/or measures are taken to prevent 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 the total number of the blood sample and the tissue sample library is 200 at present; and animal models of acute and chronic pancreatitis and orthotopic pancreatic cancer are well established.

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, a 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 functional research is carried out, and verification is carried out 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: the organic integration clinical oncology, molecular biology and bioinformatics method carries out multi-dimensional research on the malignant transformation mechanism of the pancreatic inflammation from a molecular level to an integral level, animal models and clinical samples, 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: it shows that GATA4 is highly expressed in pancreatic cancer tissues and cells, and 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: immunohistochemistry results showed that GATA4 is up-regulated 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 pancreatic cancer cells were induced and cultured using macrophage medium LMCM inducing THP-1 differentiation with different LPS concentrations (0, 50, 100ng/ml), show that macrophage medium promotes proliferation, migration and invasion of pancreatic cancer cells, and A: CCK-8 experiment confirms 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 shows that the promotion effect of LMCM on pancreatic cancer cell proliferation activity can be obviously reduced, 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 clone formation experiments prove that interference GATA4 can obviously reduce the clone formation capability of LMCM on pancreatic cancer cells BxPC-3 and PANC-1, and is concentration-dependent.

FIG. 6 shows the results of example 3, including FIGS. 6(A-B) and 6 (C-D): wherein, fig. 6(A-B) and fig. 6(C-D) 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(A-B) shows that Transwell cell migration and invasion capacity experiments prove 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. 6(C-D) shows that Transwell cell migration and invasion capacity experiments prove that interference GATA4 can obviously reduce the promotion effect of LMCM on the migration and invasion capacity of pancreatic cancer cells PANC-1, and is concentration-dependent.

FIG. 7 shows the results of example 3: shows that the overexpression of 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 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 promotion effect of LMCM on the migration and invasion capacity of pancreatic cancer cells ASPC-1, 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

iTRAQ mass spectrometry was used to screen for differentially expressed genes in pancreatitis and pancreatic cancer tissue samples, suggesting that GATA4 is highly expressed in pancreatic cancer tissue.

The isotope labeling relative and absolute quantitative iTRAQ technique is a polypeptide in vitro labeling technique 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 masses of the counter groups relative to the molecule are 31, 30, 29 and 28 respectively, the peptide reactive group connects the iTRAQ reagent with Lysine (K) and the N-terminal amino acid residue on the peptide fragment, and 4 reporter groups thereon are connected with the reactive group through the counter groups, thus forming 4 equal amounts of ectopic tags with the relative molecular masses of 145. After the peptide fragment sample is marked by the iTRAQ reagent, the equilibrium molecular weights of the reporter group and the balancing 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 spectrometry detection are all completely the same. In the tandem mass spectrometry (MSMS), the connecting bonds among the reporter group, the balance group and the peptide reaction group of the peptide segment marked by the same heavy element are broken, the balance group is lost in neutrality, and meanwhile, 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 the quantitative information of the peptide fragment and the protein can be obtained through software processing.

EXAMPLE 2 clinical tissue specimen experiments

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: further testing GATA4 expression condition by RT-PCR, Western-blot and immunohistochemistry in clinical pancreatic cancer samples, and performing immunofluorescence detection on the level of GATA4 and the level of CD 68;

2 results

When 7 pancreatic cancer samples and 39 pancreatic cancer samples are analyzed by using RT-PCR technology, the GATA4 RNA level expression is up-regulated (P <0.05) relative to normal pancreatic tissues, and the results of Western-blot and immunohistochemistry show that the GATA4 expression is up-regulated in the pancreatic cancer tissues in 7 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 the level of GATA4 with the level of CD68 (macrophage marker): 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 process of pancreatic cancer development;

(2) promotion of pancreatic cancer cells by LMCM: lipopolysaccharide LPS (0, 50, 100ng/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: RT-PCR and WB were used to detect the expression of GATA4 in various pancreatic cancer cell lines, and the results were observed and recorded.

(4) Interference GATA4 observation of the effect of different LPS concentrations of LMCM 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, 100ng/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 was also highly expressed in different pancreatic cancer cells, and induction of pancreatic cancer cells in culture 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 clonogenic function 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).

(4) The overexpression of GATA4 can obviously promote the proliferation, migration and invasion capacity of pancreatic cancer cells under the induction of LMCM with different LPS concentrations (figure 7).

Example 4 animal model experiments

This example is directed to the study of the effects of GATA4 on the development of pancreatic carcinogenesis and the process of "inflammatory cancer transformation" 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 above-mentioned GATA4 interfering group, overexpression group and control group were injected in situ into the SCID mouse pancreas to establish an in situ model of pancreatic cancer, with 6 cells per 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 time, tumor tissue and venous blood are taken, the blood is used for detecting the CA199 concentration, the tumor weight and size are measured, RNA and protein are extracted, partial tissue is fixed by formalin and then is subjected to paraffin embedding and flaking, and the result is observed and recorded.

2 results

The interference group and the overexpression group of GATA4 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

Application of GATA4 as a diagnosis marker of pancreatic cancer transformation in preparation of a reagent or a kit for identifying pancreatitis and pancreatic cancer.
Application of GATA4 as a diagnosis marker of pancreatic cancer transformation in preparation of a reagent or a kit for distinguishing pancreatitis and the transformation of pancreatitis into pancreatic cancer.
3. The use of any one of claims 1-2, wherein said pancreatitis comprises chronic pancreatitis and acute pancreatitis.
Use of an inhibitor of GATA4 in the manufacture of a medicament for the prevention or treatment of "inflammatory cancer metastasis" of the pancreas.
5. The use of any one of claims 1-2 and 4, wherein said "inflammatory cancer transformation" is unidirectional transformation of pancreatitis into pancreatic cancer.
6. The use of claim 4, wherein the inhibitor of GATA4 is an agent that reduces the expression of GATA 4.
7. The use according to claim 4, wherein the inhibitor of GATA4 is selected from the group consisting of a small molecule compound and a biological macromolecule.
8. The use of claim 7, wherein the biological macromolecule is a small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid targeting GATA4 protein or its transcript and capable of inhibiting GATA4 protein expression or gene transcription; or a construct capable of expressing or forming said small interfering RNA, dsRNA, microRNA, antisense nucleic acid.
Application of the inhibitor of GATA4 in preparing medicines for preventing and treating pancreatic cancer cell proliferation, migration and invasion.
The GATA4 is used as a diagnosis marker of pancreatic cancer transformation in the preparation of a reagent or a kit for pancreatic cancer prognosis risk judgment.