CN117625790B - Biomarker for diagnosis and prognosis of prostate cancer 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 diagnosing and prognosis judging prostate cancer and application thereof. More specifically, the present invention relates to a biomarker for diagnosis and prognosis of prostate cancer, which is DHFRL and/or TMEM87A. The inventors have found that the expression levels of DHFRL a and TMEM87A in prostate cancer patients are significantly higher than in healthy humans. More particularly, the present inventors have found that high levels of DHFRL a and/or TMEM87A are expressed with good sensitivity and specificity when used for diagnosing prostate cancer and are correlated with prognosis of prostate cancer patients and thus can be used as biomarkers for prostate cancer diagnosis and prognosis.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a biomarker for diagnosing and prognosis judging prostate cancer and application thereof.
Background
Prostate cancer is a malignant tumor that occurs in the prostate epithelium, and the number of patients has a rapidly growing trend. The early stage of the prostate cancer patient has no obvious symptoms, the diagnosis is late, the survival period is not ideal, and the early stage or the local advanced stage of the prostate cancer patient is more than 2/3 of the patients at the initial diagnosis. Thus, there is a need for early screening, early diagnosis, early treatment to improve survival and quality of life for prostate cancer patients.
Due to limitations of current methods for prostate cancer diagnosis, prostate cancer, which is progressive in nature, may have metastasized prior to detection, while the survival rate of individuals with metastatic prostate cancer is very low. Surgical removal of the prostate is generally effective in patients with prostate cancer that metastasizes but not yet metastasized. Thus, determining tumor extent is important in selecting optimal treatment and improving patient survival.
Currently, diagnosis of prostate cancer is generally based on elevated Prostate Specific Antigen (PSA) blood tests or, less commonly, on abnormal digital rectal tests (DRE). PSA is a glycoprotein produced by prostate epithelial cells and the PSA test measures the amount of PSA in blood samples. Although elevated PSA levels do not necessarily indicate the presence of prostate cancer, most men with prostate cancer have elevated PSA concentrations (e.g., above 4 ng/mL) and there are no PSA levels at risk of 0 with prostate cancer. In fact, the most common cause of elevated PSA is Benign Prostatic Hyperplasia (BPH), a noncancerous increase in the prostate.
Several biomarkers, including prostate specific membrane antigen (PSMA, prostate-specific membrane antigen), have also been found to be a transmembrane glycoprotein expressed on the cell membrane, potentially useful for diagnosis and prognosis of prostate cancer. PSMA exhibits a specific high expression pattern in prostate cancer compared to normal prostate tissue and other parts of the body, and its expression level is highly correlated with the aggressiveness of prostate cancer. AR-V7 is an AR-shearing variant (AR-V), which is one of the most frequently detected shearing variants in castration-resistant prostate cancer (CRPC). Recent studies indicate that AR-V7 plays an important role in the development of CRPC and in the drug resistance development process, and can be used as a molecular marker for guiding drug selection in CRPC patients. However, the application of these biomarkers in prostate cancer diagnosis and prognosis still needs to be studied more intensively, and other novel biomarkers for prostate cancer diagnosis and prognosis still need to be found.
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 prostate 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 prostate cancer, presents challenges, and not all cancer-related markers can be detected in circulating tumor cells.
Therefore, a highly sensitive and specific less invasive method for detecting prostate cancer is a social development need, and a more effective method for rescuing prostate cancer patients and improving their quality of life is needed.
Disclosure of Invention
To solve the above problems, the present inventors have found that the expression levels of DHFRL a and TMEM87A in prostate cancer patients are significantly higher than in healthy people. More particularly, the present inventors have found that high levels of DHFRL a and/or TMEM87A are expressed with good sensitivity and specificity when used for diagnosing prostate cancer and are correlated with prognosis of prostate cancer patients and thus can be used as biomarkers for prostate cancer diagnosis and prognosis.
As used herein DHFRL is an abbreviation for dihydrofolate reductase 2 (dihydrofolate reductase, DHFRL 1), which has NCBI Gene ID 200895.
As used herein TMEM87A is an abbreviation for transmembrane protein 87A (transmembrane protein a, TMEM 87A), with NCBI Gene ID 25963.
In particular, the present invention provides a biomarker for prostate cancer diagnosis, wherein the biomarker is DHFRL a and/or TMEM87A.
In other aspects, the invention also provides a biomarker for prognosis of prostate cancer, wherein the biomarker is DHFRL a and/or TMEM87A.
In other aspects, the invention also provides a kit for diagnosis of prostate cancer comprising reagents for detecting DHFRL a and/or TMEM87A expression.
In other aspects, the invention also provides a kit for prognosis of prostate cancer, the kit comprising reagents for detecting DHFRL a and/or TMEM87A expression.
In other aspects, the invention also provides the use of an agent that detects DHFRL a and/or TMEM87A expression in the manufacture of a tool for diagnosis of prostate cancer.
In other aspects, the invention also provides the use of an agent that detects DHFRL a and/or TMEM87A expression in the manufacture of a tool for prognosis of prostate cancer.
Further, the diagnosis of prostate cancer includes the steps of:
(1) Collecting a sample of a test subject, and collecting a control sample;
(2) Detecting and comparing the expression level of DHFRL a and/or TMEM87A in the test subject sample and the control sample;
Diagnosing a test subject as having or at risk of having prostate cancer if the expression level of DHFRL1 in the sample of the test subject is increased compared to the expression level of DHFRL1 in the control sample and/or the expression level of TMEM87A in the sample of the test subject is increased compared to the expression level of TMEM87A in the control sample.
Further, the control sample is derived from healthy tissue of a healthy population or test subject.
Further, the prognosis of the prostate cancer includes the steps of:
(1) Collecting samples of a patient with the prognosis of the prostate cancer as a group to be tested, and taking the samples of the patient with the pre-prostate cancer as a control group;
(2) Detecting and comparing the expression level of DHFRL a and/or TMEM87A in the samples of the test group and the control group;
If the expression level of DHFRL1 in the test group sample is reduced compared to the expression level of DHFRL1 in the control group sample and/or the expression level of TMEM87A in the test group sample is reduced compared to the expression level of TMEM87A in the control group sample, then the prognosis of the test group is judged to be 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 prostate tissue of the test subject, such as a prostate biopsy sample, and the control sample is derived from prostate tissue of a healthy subject, such as a prostate 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-prostate 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-prostate cancer patient comprises prostate tissue of the subject to be tested, such as a prostate biopsy sample. In a preferred embodiment, the sample of the prognostic and pre-prostate cancer patient is a circulating tumor cell.
As used herein, the reagent for detecting expression of DHFRL a and/or TMEM87A in a sample of a test subject is not particularly limited and is a reagent for detecting expression of DHFRL a and/or TMEM87A 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 DHFRL a and/or TMEM87A 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 DHFRL a and TMEM87A in prostate cancer patients are significantly higher than in healthy humans. More particularly, the present inventors have found that high levels of expression of DHFRL a and/or TMEM87A, especially both, when used in combination, have good sensitivity and specificity for diagnosing prostate cancer and are correlated with prognosis of prostate cancer patients and thus can be used as biomarkers for prostate cancer diagnosis and prognosis. In addition, the present invention also finds that prostate cancer can be diagnosed and prognostic as by harvesting circulating tumor cells from a subject and detecting expression levels of DHFRL and TMEM87A therein.
Drawings
FIG. 1 shows the expression levels of DHFRL and TMEM87A in prostate cancer tissue samples and paracancerous normal tissue samples.
Fig. 2 shows the detection DHFRL of expression of TMEM87A and DHFRL1 in prostate cancer tissue samples and paracancerous normal tissue samples by Immunohistochemical (IHC) experiments.
FIG. 3 shows the expression levels of DHFRL and TMEM87A in circulating tumor cells of prostate cancer patients.
Fig. 4 shows the expression levels of DHFRL and TMEM87A in human prostate cancer cells LNCap and human normal prostate epithelial cells RWPE 1.
Fig. 5 shows the change in the migratory and invasive capacity of human prostate cancer cells LNCap after interfering DHFRL with expression of TMEM87A and/or DHFRL.
Fig. 6 shows ROC curve analysis of DHFRL a and TMEM87A, alone and in combination, in prostate cancer patients and healthy humans.
Figure 7 shows analysis of Kaplan-Meier survival curves for DHFRL a and TMEM87A, alone and in combination, in prostate cancer patients and healthy humans.
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 prostate cancer and paired normal tissues
Tumor genome map (TCGA) project, which was planned to be co-initiated in 2006 by U.S. National Cancer Institute (NCI) and National Human Genome Research Institute (NHGRI), used large-scale experiments with 36 cancers using large-scale sequencing-based genomic analysis techniques, 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 78 whole gene expression profile data and clinical information of prostate cancer tissues and normal tissues, statistical analysis adopts R language (version 3.1.1) software, program packages (heatmap, venndiagram, hist, etc.) to be installed and loaded, and then uses DESeq and edge program packages to analyze, find out differentially expressed genes. Judgment standard: (1) expression level of cancer/paracancerous region < -2, (2) P <0.05, and (3) have not been reported in prostate cancer. Two genes that were significantly highly expressed in prostate cancer, DHFRL and TMEM87A, were finally selected.
Example 2: DHFRL 1A and TMEM87A are highly expressed in prostate cancer
Clinical 55 cases of prostate cancer tissue samples and 58 cases of paracancerous normal tissue samples were collected, RNA of the prostate cancer tissue samples and the paracancerous normal tissue samples were extracted by TRIzol method, and mRNA levels of DHFRL A and TMEM87A were detected by RT-qPCR method, respectively. The results are depicted in fig. 1, which demonstrates that DHFRL and TMEM87A are highly expressed in prostate cancer.
Expression of DHFRL and TMEM87A in prostate cancer tissue samples and paracancerous normal tissue samples was further confirmed by immunohistochemical experiments.
The immunohistochemical experiments were performed as follows:
(1) Conventional paraffin sections, deparaffinized to water, incubated for 10min at 3%H 2O2 room temperature, endogenous peroxidase activity eliminated, PBS washed 3 times, 5 min/time, then autoclaved with antigen retrieval solution (EDTA/sodium citrate) for 5min, naturally cooled to room temperature, PBS washed 3 times, 5 min/time.
(2) Adding blocking solution, blocking at room temperature for 30min, and washing with PBS for 3 times and 5 min/time.
(3) A working solution of DHFRL/TMEM 87A antibody (ex Abcam) diluted in the appropriate ratio was added dropwise, incubated for 1h at 37℃and washed 3 times with PBS for 5 min/time.
(4) Adding goat anti-rabbit secondary antibody working solution, and incubating for 30min at 37 ℃.
(5) Washing with PBS for 3 times, 5 min/time, developing with DAB, and washing with PBS after developing.
(6) Hematoxylin dye counterstains the cell nuclei, and the cells were washed thoroughly with water and soaked in PBS for 2min.
(7) Alcohol dehydration treatment with different concentration gradients.
(8) Permeabilization: xylene I for 10min and xylene II for 10min.
(9) Sealing the sheet with neutral resin, drying and photographing with a positive microscope.
Expression of DHFRL a and TMEM87A in paracancerous tissue, prostate cancer tissue was examined by Immunohistochemical (IHC) experiments described above and scored and quantified as a result, see fig. 2. It can be seen that DHFRL a and TMEM87A have higher expression in prostate cancer tissue.
Example 3: detection of DHFRL and TMEM87A expression levels in circulating tumor cells of prostate cancer patients
1) Extracting 10mL of venous blood of a prostate cancer patient into an ACD anticoagulation tube, and conventionally centrifugally separating plasma for later use;
2) Enrichment and separation of CTC cells in plasma comprises the following specific steps: extracting a single cell layer in the blood plasma by adding a sample density separating liquid (CYTELLIGEN) into the blood plasma, then adding immune cell removing magnetic beads to remove CD45 + immune cells in the extracted single cell layer, and concentrating and enriching CTC in the single cell layer by 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 was measured and expression of DHFRL and TMEM87A in CTC cells was measured by RT-qPCR and compared to expression of DHFRL and TMEM87A in cells harvested from normal prostate tissue, as shown in fig. 3, which demonstrates that DHFRL1 and TMEM87A are highly expressed in CTC cells from prostate cancer patients.
Example 4: DHFRL 1A and TMEM87A affect invasion and migration of prostate cancer cells
Human prostate cancer cells LNCap and human normal prostate epithelial cells RWPE1 were cultured in an RPMI-1640 medium containing 10% fetal bovine serum (containing 100 U.mL -1 penicillin and 0.1 mg.mL -1 streptomycin) at 37℃in a constant temperature incubator with 5% CO 2.
After digestion and collection of the cultured cells, RNA was extracted and expression of DHFRL and TMEM87A 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 DHFRL and TMEM87A are significantly more expressed in human prostate cancer cells LNCap than in human normal prostate epithelial cells RWPE 1.
Expression of DHFRL and TMEM87A in prostate cancer cells was interfered with by siRNA (siRNA sequence ：siNC：5'-UUCUCCGAACGUGUCACGUUCAUACTT-3'(SEQ ID No.1);siDHFRL1：5'-CUCAAGGAACCUCCACAAGGAGCUCTT-3'(SEQ ID No.2); siTMEM87A：5'-CCGGCAACCGUAGCUGCUGCCGACCTT-3'(SEQ ID No.3))( interference results are shown in fig. 4), followed by Transwell cell migration and invasion assay verification. The results are shown in fig. 5, which shows that the migration and invasion capacity of human prostate cancer cells LNCap decreased significantly after the expression of DHFRL and TMEM87A genes were interfered with individually and in combination, wherein the decrease after the combined interference was more significant.
Example 5: DHFRL1 diagnostic value of prostate cancer of 1 and TMEM87A
MRNA levels of DHFRL1 and TMEM87A in prostate 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 DHFRL1 and TMEM 87A. The results are shown in fig. 6, which shows that DHFRL (sensitivity 63.64%, specificity 68.97%) and TMEM87A (sensitivity 69.09%, specificity 75.86%) mRNA expression have independent diagnostic effects on prostate cancer, but the combined diagnostic effect is better, the area AUC (area under the ROC curve) = 0.9251 under ROC curve, the sensitivity can reach 80%, and the specificity can reach 91.38%. From this result, DHFRL a and TMEM87A alone have a certain diagnostic effect, but the diagnostic specificity and sensitivity are insufficient, and higher sensitivity and specificity can be achieved when the two are used in combination. Thus DHFRL and TMEM87A can be used for the diagnosis of prostate cancer alone and in combination.
Example 6: DHFRL1 and TMEM87A relationship with clinical prognosis of prostate cancer
The relationship of DHFRL and TMEM87A to overall survival of prostate cancer patients was statistically analyzed using the mRNA levels of DHFRL1 and TMEM87A in prostate cancer tissue samples and paracancerous normal tissue samples measured in example 2. As a result, as shown in fig. 7, it can be seen that the five-year overall survival rate of the group of prostate cancer patients in which DHFRL1 was Low-expressed (DHFRL low+tme87A High) or TMEM87A was Low-expressed (DHFRL high+tme87A Low) was significantly higher than that of the group of DHFRL and TMEM87A High-expressed (DHFRL 1 high+tmem87 AHigh), and that the five-year overall survival rate of the group of prostate cancer patients in which DHFRL1 was Low-expressed and simultaneously TMEM87A was Low-expressed (DHFRL 1low+tmem87 ALow) was highest. This illustrates: DHFRL1 high expression of 1 and/or TMEM87A can lead to poor prognosis for prostate cancer patients.
It should be noted that while the present invention has been illustrated in the drawings and described in connection with the preferred embodiments thereof, it is to be understood that the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but are to be construed as providing a full breadth of the disclosure. 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 that detects DHFRL a and TMEM87A expression in the manufacture of a tool for diagnosis of prostate cancer.
2. The use according to claim 1, characterized in that the diagnosis of prostate cancer comprises the following steps:
(1) Collecting a sample of a test subject, and collecting a control sample;
(2) Detecting and comparing the expression levels of DHFRL a and TMEM87A in the test subject sample and the control sample;
If the expression level of DHFRL1 in the sample of the test subject is increased compared to the expression level of DHFRL1 in the control sample and the expression level of TMEM87A in the sample of the test subject is increased compared to the expression level of TMEM87A in the control sample, diagnosing that the test subject has or is at risk of having prostate cancer.
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, pus, organs, biopsy samples, circulating tumor cells, circulating tumor DNA or urine abscission cells.
4. Use of an agent that detects DHFRL a and TMEM87A expression in the manufacture of a tool for prognosis of prostate cancer.
5. The use according to claim 4, wherein the prognosis of prostate cancer comprises the steps of:
(1) Collecting samples of a patient with the prognosis of the prostate cancer as a group to be tested, and taking the samples of the patient with the pre-prostate cancer as a control group;
(2) Detecting and comparing the expression levels of DHFRL a and TMEM87A in the samples of the test group and the control group;
If the expression level of DHFRL1 in the test group sample is reduced compared to the expression level of DHFRL1 in the control group sample and the expression level of TMEM87A in the test group sample is reduced compared to the expression level of TMEM87A in the control group sample, then the prognosis of the test group is judged to be good.
6. The use according to claim 5, wherein the sample of the patient with prognosis and preprostatic cancer is one or more of serum, plasma, whole blood, pus, organ, biopsy sample, circulating tumor cells, circulating tumor DNA or urine shed cells.
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