CN117625791B - Biomarker for colorectal cancer diagnosis and prognosis and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biological medicines, in particular to a biomarker for colorectal cancer diagnosis and prognosis judgment and application thereof. More specifically, the present invention relates to a biomarker for diagnosis and prognosis of colorectal cancer, said biomarker being ZNF823 and/or ASCL5. The inventors have found that the expression levels of ZNF823 and ASCL5 in colorectal cancer patients are significantly higher than in healthy people. More particularly, the inventors found that high levels of ZNF823 and/or ASCL5 expression have good sensitivity and specificity when used for diagnosing colorectal cancer and are correlated with prognosis of colorectal cancer patients, and thus can be used as biomarkers for colorectal cancer diagnosis and prognosis.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a biomarker for colorectal cancer diagnosis and prognosis judgment and application thereof.
Background
Recent statistics show that cancer death accounts for 23.91% of all the death causes of residents in China, and is one of the most powerful road blocking tigers on human health. At the end of month 1 in 2019, the national cancer center issues a latest completed report of the epidemic situation of the malignant tumor in China journal, and analyzes the latest cancer situation in China in 2015 (the data of the national tumor registration center generally lags 3 years, so the current report issue data is 2015 registration data). It is reported that the incidence rate of cancer is about 392.9 tens of thousands in 2015 nationally, the incidence rate is 285.83/10 tens of thousands, the death rate is 170.05/10 tens of thousands in year. This also means that on average 7.5 people per minute were diagnosed with cancer and 4.4 people die from cancer. For more than 10 years, the incidence of cancer and death rate in China continuously rise, the incidence rate of cancer is averagely increased by about 3.9% every year, and the death rate is averagely increased by 2.5% every year. Colorectal cancer is one of the most common digestive tract tumors at present. Recent epidemiological data of malignant tumors have found that colorectal cancer morbidity rates are ranked at positions 4 and 3 in males and females, respectively, mortality rates are at positions 5 and 4, and urban residents are higher. On the whole world, colorectal cancer and lung cancer incidence rate are parallel first, and human health is seriously threatened.
Colorectal cancer has a tendency to develop younger and most patients have developed to advanced or locally advanced stages at the time of diagnosis and have a poor prognosis due to the lack of early diagnosis and efficient screening methods. With the continuous development of various medical technologies and the development of chemotherapeutic drugs and targeted drugs, the clinical treatment effect of colorectal cancer patients is greatly improved, but the survival rate of most patients is not improved from the perspective of long-term survival rate. With the rapid increase in multiple sets of high throughput data, some molecular biomarkers were found to be abnormally expressed in colorectal cancer and correlated with prognosis, which makes it possible to diagnose colorectal cancer more accurately and efficiently.
For example, CDX2 is an intestinal specific transcription factor that regulates regeneration and differentiation of intestinal epithelial cells, playing a role in maintaining intestinal epithelial cell morphology and function. CDX2 was expressed in all normal mucosa, only in 35.2% of tumor tissues. CDX2 gene is rarely mutated, but the gene carrying it is often amplified and it may be shown that CDX2 acts as a mitochondrial survival oncogene, involved in the potential molecular pathways leading to cancer formation, through cell proliferation and migration by cross-talk with Wnt signaling pathway, tumor stroma proteins and inflammatory cytokines. The application of CDX2 to the detection of colorectal cancer circulating tumor cells is currently being studied, and auxiliary diagnosis is made for the clinical diagnosis and treatment of colorectal cancer patients. However, there remains a need to find other novel biomarkers for prostate cancer diagnosis and prognosis.
Circulating tumor cells are a subset of tumor cells that shed from a primary tumor or metastatic tumor and are released into the blood circulation. Recent studies have found that, on the one hand, circulating tumor cells may appear in the peripheral blood of patients very early in tumorigenesis, which aids in early diagnosis of cancer. On the other hand, these circulating tumor cells can also be used to predict prognosis in cancer patients, and the discovery of circulating tumor cells often predicts recurrence or metastasis of a tumor, which also suggests poor prognosis in patients. How to use circulating tumor cells for diagnosis or prognosis of cancer, especially specific cancers such as colorectal cancer, is also an important direction in our future in the search of circulating tumor cell lines. A great benefit of using circulating tumor cells for diagnosis or prognosis is that it can effectively replace tumor biopsies, which is a good surrogate indicator for those patients who cannot take a pathological tissue biopsy, and can help clinicians to dynamically monitor and determine the biological characteristics of cancer in real time. However, due to the scarcity of circulating tumor cells, the use thereof as a means of diagnosing cancer, particularly specific cancers such as colorectal cancer, presents challenges, and not all cancer-related markers can be detected in circulating tumor cells.
Therefore, it is of great clinical value to find new colorectal cancer diagnosis-related markers, in particular biomarkers suitable for diagnosis by means of circulating tumor cells.
Disclosure of Invention
The object of the present invention is to provide a novel diagnostic and prognostic marker for colorectal cancer. In particular, the inventors have found that the expression levels of ZNF823 and ASCL5 in colorectal cancer patients are significantly higher than in healthy people. More particularly, the inventors found that high levels of ZNF823 and/or ASCL5 expression have good sensitivity and specificity when used for diagnosing colorectal cancer and are correlated with prognosis of colorectal cancer patients, and thus can be used as biomarkers for colorectal cancer diagnosis and prognosis.
As used herein ZNF823 is an abbreviation for zinc finger protein 823 (zinc finger protein 823, ZNF 823) with NCBI Gene ID 55552.
As used herein, ASCL5 is an abbreviation for the acetate-scale family bHLH transcription factor 5 (acetate-scute family bHLH transcription factor, ASCL 5), which has an NCBI Gene ID of 647219.
In particular, the present invention provides a biomarker for colorectal cancer diagnosis, wherein the biomarker is ZNF823 and/or ASCL5.
In other aspects, the invention also provides a biomarker for prognosis of colorectal cancer, wherein the biomarker is ZNF823 and/or ASCL5.
In other aspects, the invention also provides a kit for colorectal cancer diagnosis comprising reagents for detecting ZNF823 and/or ASCL5 expression.
In other aspects, the invention also provides a kit for prognosis of colorectal cancer, the kit comprising reagents for detecting ZNF823 and/or ASCL5 expression.
In other aspects, the invention also provides the use of an agent that detects ZNF823 and/or ASCL5 expression in the manufacture of a tool for the diagnosis of colorectal cancer.
In other aspects, the invention also provides the use of an agent that detects ZNF823 and/or ASCL5 expression in the manufacture of a tool for prognosis of colorectal cancer.
Further, the diagnosis of colorectal cancer comprises the steps of:
(1) Collecting a sample of a test subject, and collecting a control sample;
(2) Detecting and comparing the expression level of ZNF823 and/or ASCL5 in the sample of the subject to be detected and the control sample;
diagnosing the test subject as suffering from or at risk of suffering from colorectal cancer if the expression level of ZNF823 in the sample of the test subject is increased compared to the expression level of ZNF823 in the control sample and/or the expression level of ASCL5 in the sample of the test subject is increased compared to the expression level of ASCL5 in the control sample.
Further, the control sample is derived from healthy tissue of a healthy population or test subject.
Further, the prognosis of colorectal cancer comprises the following steps:
(1) Collecting samples of patients with colorectal cancer in advance as a group to be tested, and taking the samples of the patients with colorectal cancer in advance as a control group;
(2) Detecting and comparing the expression level of ZNF823 and/or ASCL5 in the samples of the to-be-detected group and the control group;
and if the expression level of ZNF823 in the sample of the test group is reduced compared with the expression level of ZNF823 in the sample of the control group and/or the expression level of ASCL5 in the sample of the test group is reduced compared with the expression level of ASCL5 in the sample of the control group, judging that the prognosis of the test group is good.
As used herein, the subject includes a mammal, preferably a primate mammal, more preferably a human.
As used herein, a sample of the test subject includes a clinical biological sample of the subject, including, but not limited to, one or more of serum, plasma, whole blood, secretions, cotton swabs, pus, body fluids, tissues, organs, paraffin sections, tumor tissue, biopsy samples, circulating tumor cells, circulating tumor DNA, or urine shed cells. In a preferred embodiment, the sample of the test subject comprises intestinal tissue of the test subject, such as an intestinal biopsy sample, and the control sample is derived from intestinal tissue of a healthy subject, such as an intestinal biopsy sample, or healthy tissue of the test subject, such as a paracancerous tissue. In a preferred embodiment, the sample of the test subject is a circulating tumor cell.
As used herein, the samples of the prognostic and pre-colorectal cancer patients include clinical biological samples of the subject, including, but not limited to, one or more of serum, plasma, whole blood, secretions, cotton swabs, pus, body fluids, tissues, organs, paraffin sections, tumor tissue, biopsy samples, circulating tumor cells, circulating tumor DNA, or urine shed cells. In a preferred embodiment, the sample of the prognostic and pre-colorectal cancer patient comprises intestinal tissue of the subject to be tested, such as an intestinal biopsy sample. In a preferred embodiment, the samples of the patients with prognosis and pre-colorectal cancer are circulating tumor cells.
As used herein, the reagent for detecting the expression of ZNF823 and/or ASCL5 in a sample of a test subject is not particularly limited and is a reagent for detecting the expression of ZNF823 and/or ASCL5 at mRNA or protein level in a sample of a subject, which is well known and readily available to those skilled in the art. For example, reagents for detecting expression of ZNF823 and/or ASCL5 in a subject sample may include corresponding reagents for real-time fluorescent quantitative PCR, enzyme-linked immunosorbent assay (ELISA), protein/peptide fragment chip detection, chemiluminescence, immunoblotting, microbead immunodetection, microfluidic immunization.
The beneficial effects of the invention are that
The inventors have found that the expression levels of ZNF823 and ASCL5 in colorectal cancer patients are significantly higher than in healthy people. More particularly, the inventors found that high levels of ZNF823 and/or ASCL5 expression, especially both, when used in combination, have good sensitivity and specificity for use in diagnosing colorectal cancer and are correlated with prognosis of colorectal cancer patients and thus can be used as biomarkers for colorectal cancer diagnosis and prognosis judgment. Furthermore, the present invention also finds that diagnosis and prognosis of colorectal cancer can be performed by harvesting circulating tumor cells of a subject and detecting expression levels of ZNF823 and ASCL5 therein.
Drawings
Fig. 1 shows the expression levels of ZNF823 and ASCL5 in colorectal cancer tissue samples and paracancerous normal tissue samples.
Fig. 2 shows the detection of ZNF823 and ASCL5 expression levels in colorectal cancer tissue samples and paracancestral normal tissue samples by immunohistochemistry.
Fig. 3 shows the expression levels of ZNF823 and ASCL5 in circulating tumor cells of colorectal cancer patients.
FIG. 4 shows the expression levels of ZNF823 and ASCL5 in human colorectal cancer cells HCT116, HT29, HCT15 and human normal colon tissue cells CCD-18 Co.
Fig. 5 shows the change in the migratory and invasive capacity of human colorectal cancer cells HCT116 after interfering with ZNF823 and/or ASCL5 expression.
Fig. 6 shows ROC curve analysis of ZNF823 and ASCL5 alone and in combination in colorectal cancer patients and healthy people.
FIG. 7 shows analysis of Kaplan-Meier survival curves of ZNF823 and ASCL5, alone and in combination, in colorectal cancer patients.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1: expression profiling chip analysis of human colorectal cancer and paired normal tissues
Tumor genome map (TCGA) project, which was planned to be initiated jointly in 2006 by us National Cancer Institute (NCI) and National Human Genome Research Institute (NHGRI), used large-scale experiments with large-scale sequencing-based genomic analysis techniques for 36 cancers, TCGA genomic analysis centers (GCCs) aligned tumor and normal tissues, looking for mutations, amplifications or deletions of genes associated with each cancer or subtype. To understand the molecular mechanism of cancer, help is provided for improving the scientific understanding of the molecular basis of cancer pathogenesis.
The TCGA standard method downloads 124 whole gene expression profile data and clinical information of colorectal cancer tissues and normal tissues, statistical analysis adopts R language (version 3.1.1) software, program packages (hetmap, vennediagram, hist, etc.) to be installed and loaded, and then uses DESeq and edge program packages to analyze to find out differentially expressed genes. Judgment standard: (1) expression level of cancer/paracancerous agent < -2, (2) P <0.05, and (3) was not reported in colorectal cancer. Two genes that were significantly highly expressed in colorectal cancer, ZNF823 and ASCL5, were finally screened.
Example 2: ZNF823 and ASCL5 are highly expressed in colorectal cancer
Collecting 65 clinical colorectal cancer tissue samples and 42 near-cancer normal tissue samples, respectively extracting RNA of the colorectal cancer tissue samples and the near-cancer normal tissue samples by using a TRIzol method, and respectively detecting mRNA levels of ZNF823 and ASCL5 by using an RT-qPCR method. The results are depicted in figure 1, which demonstrates high expression of ZNF823 and ASCL5 in colorectal cancer.
Further, tissue chips were fabricated and ZNF823 and ASCL5 protein levels were detected using immunohistochemical methods. The immunohistochemical staining is carried out by adopting a kit (Zhonghua gold bridge, goods number: PV-9000), and the specific steps are as follows:
a. tissue chip xylene dewaxing and gradient ethanol hydration;
b. adopting EDTA antigen retrieval liquid to carry out antigen retrieval;
c. the peroxidase blocking agent (reagent a) was incubated for 10min to block the activity of endogenous peroxidase;
d. serum (reagent B) was blocked for 30min;
e.znf823 or ASCL5 antibodies (all purchased from Abcam,1:500 dilution) were incubated overnight at 4 ℃;
f. incubating the biotin-labeled secondary antibody (reagent C) for 30min at room temperature;
g. reacting streptomycin avidin-peroxidase (reagent D) for 20min at room temperature;
color development of DAB solution, counterstaining of mature hematoxylin for 5min, color separation of 1% (v/v) ethanol hydrochloride for 3s, and blue returning of running water for 15min;
i. sequentially dehydrating gradient ethanol, transparency to xylene for 5min, sealing with neutral resin, storing, and photographing under microscope.
As shown in FIG. 2, ZNF823 or ASCL5 immunohistochemical H-Score was significantly increased in colorectal cancer tissue compared to paracancerous normal tissue.
Example 3: detection of ZNF823 and ASCL5 expression levels in circulating tumor cells of colorectal cancer patients
1) Extracting 10mL of venous blood of a colorectal cancer patient in an ACD anticoagulation tube, and conventionally centrifuging and separating plasma for later use;
2) Enrichment and separation of CTC cells in plasma comprises the following specific steps: extracting single cell layer from blood plasma by adding sample density separating liquid (Cytelligen), and removing CD45 in the extracted single cell layer by adding immunocyte to remove magnetic bead + Immune cells are removed, and CTC in a single cell layer is concentrated and enriched through differential enrichment;
3) The enriched CTC cells were harvested by centrifugation and 1ml of RNA lysate was added to the enzyme-free EP tube; 200ul of chloroform is added into an EP tube, vigorously oscillated for 15 seconds, and kept still at room temperature for 3 minutes, and repeated for 3 times; centrifuging at 12000 Xg and 4 ℃ for 15min; adding the upper water phase into a new enzyme-free EP pipe, adding equal volume of isopropanol into the EP pipe, reversing, mixing uniformly, and standing for 10min; centrifuging at 12000 Xg and 4 ℃ for 15min; the EP tube liquid was discarded, 1ml of 75% ethanol was added, and the EP tube was shaken; centrifuging at 12000 Xg and 4 ℃ for 5min; discarding the supernatant, and standing at room temperature for drying; adding a proper amount of DEPC water to dissolve RNA; the purity and concentration of RNA were examined and expression of ZNF823 and ASCL5 in CTC cells was examined by RT-qPCR and compared with expression of ZNF823 and ASCL5 in cells harvested from normal intestinal tissue, and as shown in fig. 3, it was confirmed that ZNF823 and ASCL5 were highly expressed in CTC cells of colorectal cancer patients.
Example 4: ZNF823 and ASCL5 affect invasion and migration of colorectal cancer cells
Human colorectal cancer cells HCT116, HT29, HCT15 and human normal colon tissue cells CCD-18Co were cultured in RPMI-1640 medium (100 U.mL containing 10% fetal bovine serum) -1 Penicillin and 0.1 mg.mL -1 Streptomycin), at 37 ℃,5% co 2 Culturing in a constant temperature incubator.
After digestion and collection of the cultured cells, RNA was extracted and expression of ZNF823 and ASCL5 in normal and cancer cells was detected by RT-qPCR as described in example 3. The results are shown in FIG. 4, which shows that both ZNF823 and ASCL5 are significantly more expressed in human colorectal cancer cells HCT116, HT29, HCT15 than in human normal colon tissue cells CCD-18 Co.
The expression of ZNF823 and ASCL5 in colorectal cancer cells was interfered with by siRNA (siRNA sequence: siNC:5'-UUCUCCGAACGUGUCACGUUCAUACTT-3' (SEQ ID No. 1), siZNF823:5'-CAUCAGAGUUGACACUGGACACAAATT-3' (SEQ ID No. 2), siASCL5:5'-AGCUGCAUGCAGCUGGGCGUCAUGCTT-3' (SEQ ID No. 3)) (the interference results are shown in FIG. 4), and then Transwell cell migration and invasion experiments were performed. The results are shown in figure 5, which shows a significant decrease in the migratory and invasive capacity of human colorectal cancer cells HCT116 after interfering with the expression of ZNF823 and ASCL5 genes alone and in combination, wherein the decrease after the combined interference is more pronounced.
Example 5: colorectal cancer diagnostic value of ZNF823 and ASCL5
mRNA levels of ZNF823 and ASCL5 in colorectal cancer tissue samples and paracancerous normal tissue samples, as determined in example 2, were analyzed by subject working curve (ROC) for independent and combined diagnostic test results of ZNF823 and ASCL5. The results are shown in fig. 6, which shows that mRNA expression of ZNF823 (sensitivity 53.85%, specificity 88.1%) and ASCL5 (sensitivity 69.23%, specificity 73.81%) has independent diagnostic effects on colorectal cancer, but the combined diagnostic effect is better, the area AUC (area under the ROC curve) = 0.9221 under ROC curve can reach 83.08%, and the specificity can reach 92.86%. From this result, ZNF823 and ASCL5 alone have a certain diagnostic effect, but the diagnostic specificity and sensitivity are still insufficient, and higher sensitivity and specificity can be achieved when the two are used in combination. Thus, ZNF823 and ASCL5 may be used to diagnose colorectal cancer alone and in combination.
Example 6: relationship of ZNF823 and ASCL5 to clinical prognosis of colorectal cancer
The relationship of ZNF823 and ASCL5 overall survival to colorectal cancer patients was statistically analyzed using mRNA levels of ZNF823 and ASCL5 in colorectal cancer tissue samples and paracancerous normal tissue samples determined in example 2. As a result, as shown in fig. 7, it can be seen that the five-year overall survival rate of the colorectal cancer patient group in which ZNF823 was Low-expressed (ZNF 823 low+ascl5high) or ASCL5 was Low-expressed (ZNF 823 high+ascl5low) was significantly higher than that of the colorectal cancer patient group in which ZNF823 and ASCL5 were High-expressed (ZNF 823 high+ascl5high), and that the five-year overall survival rate of the colorectal cancer patient group in which ZNF823 was Low-expressed and ASCL5 was simultaneously Low-expressed (ZNF 823 low+ascl5low) was highest. This illustrates: high expression of ZNF823 and/or ASCL5 may lead to poor prognosis for colorectal cancer patients.
It should be noted that the description of the present invention and the accompanying drawings illustrate preferred embodiments of the present invention, but the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, which are not to be construed as additional limitations of the invention, but are provided for a more thorough understanding of the present invention. The above-described features are further combined with each other to form various embodiments not listed above, and are considered to be the scope of the present invention described in the specification; further, modifications and variations of the present invention may be apparent to those skilled in the art in light of the foregoing teachings, and all such modifications and variations are intended to be included within the scope of this invention as defined in the appended claims.
Claims (6)
1. Use of an agent for detecting ZNF823 and/or ASCL5 expression in the manufacture of a tool for the diagnosis of colorectal cancer.
2. The use according to claim 1, wherein the diagnosis of colorectal cancer comprises the steps of:
(1) Collecting a sample of a test subject, and collecting a control sample;
(2) Detecting and comparing the expression level of ZNF823 and/or ASCL5 in the sample of the subject to be detected and the control sample;
diagnosing the test subject as suffering from or at risk of suffering from colorectal cancer if the expression level of ZNF823 in the sample of the test subject is increased compared to the expression level of ZNF823 in the control sample and/or the expression level of ASCL5 in the sample of the test subject is increased compared to the expression level of ASCL5 in the control sample.
3. The use according to claim 2, wherein the control sample is derived from healthy tissue of a healthy population or a subject to be tested,
the sample of the subject to be tested is one or more of serum, plasma, whole blood, organs, tumor tissues, circulating tumor cells and circulating tumor DNA.
4. Use of an agent for detecting ZNF823 and/or ASCL5 expression in the manufacture of a tool for prognosis of colorectal cancer.
5. The use according to claim 4, wherein the prognosis of colorectal cancer comprises the steps of:
(1) Collecting samples of patients with colorectal cancer in advance as a group to be tested, and taking the samples of the patients with colorectal cancer in advance as a control group;
(2) Detecting and comparing the expression level of ZNF823 and/or ASCL5 in the samples of the to-be-detected group and the control group;
and if the expression level of ZNF823 in the sample of the test group is reduced compared with the expression level of ZNF823 in the sample of the control group and/or the expression level of ASCL5 in the sample of the test group is reduced compared with the expression level of ASCL5 in the sample of the control group, judging that the prognosis of the test group is good.
6. The use according to claim 5, wherein the sample of the patient with prognostic and pre-colorectal cancer is one or more of serum, plasma, whole blood, organs, tumor tissue, circulating tumor cells, circulating tumor DNA.
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