CN114622014A

CN114622014A - Application of PCP4 as tumor differentiation marker of neuroblastoma

Info

Publication number: CN114622014A
Application number: CN202011470245.2A
Authority: CN
Inventors: 董瑞; 詹镛; 杨然; 李凯; 董岿然
Original assignee: Childrens Hospital of Fudan University
Current assignee: Childrens Hospital of Fudan University
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-06-14

Abstract

The invention belongs to the technical field of tumor molecular detection, and relates to a tumor cell marker related to neuroblastoma and application thereof. The invention discloses a tumor cell marker PCP4 related to the differentiation degree of neuroblastoma, and experiments show that the expression level of PCP4 is increased along with the increase of the differentiation degree of neuroblastoma tumor cells, so that a diagnostic kit for neuroblastoma can be prepared. The PCP4 of the present invention can accurately determine the expression level of PCP4 as a tumor marker related to neuroblastoma, and can assist in the judgment of the degree of differentiation of neuroblastoma. The NB-related tumor marker PCP4 and the potential value thereof as a marker for diagnosis, detection and prognosis evaluation have profound clinical significance and application prospect.

Description

Application of PCP4 as tumor differentiation marker of neuroblastoma

Technical Field

The invention belongs to the technical field of tumor molecular detection, and relates to a tumor cell marker related to neuroblastoma and application thereof. The invention discloses a tumor cell marker PCP4 related to the differentiation degree of neuroblastoma, the expression level of PCP4 is increased along with the increase of the differentiation degree of neuroblastoma tumor cells, and a diagnostic kit for neuroblastoma can be further prepared.

Background

The prior art discloses that the pediatric tumors are important problems threatening the health of children, and the pediatric tumors become the second leading cause of death of children after trauma, wherein Neuroblastoma (NB) is the most common abdominal malignant tumor of children, accounts for 8-10% of all tumors of children, accounts for 28% of the incidence rate of total cancers of infants, is the most common malignant solid tumor of children treated most thoroughly so far in the infant stage.

Studies have shown that neuroblastoma is highly heterogeneous in clinical setting, and patients in low-risk groups benefit from surgical resection or even observation alone; patients in the middle-risk group can also obtain good prognosis through surgical resection and medium-dose chemotherapy; however, patients in the high-level group have great clinical heterogeneity, and specific sensitivity markers are not identified at present; in addition, NB has unique cell differentiation characteristics, and the previous literature reports that NB has intratumoral heterogeneity and different degrees of differentiation, with lower differentiation and higher malignancy.

PCP4(Gene ID:5121), a calmodulin regulatory protein, capable of binding to the c-domain of calmodulin (CaM) via the IQ motif and regulating its Ca via the acidic sequence²⁺Binding properties, thereby modulating CaM signaling pathways and their broad downstream targets. PCP4 has been reported to play a positive regulatory role in neuronal differentiation and to have a function of promoting neuronal cell differentiation. In the aspect of tumor, it is reported that PCP4 is a cytokine negatively correlated with tumor growth, and its expression is reduced in various tumors, including prostate cancer, bladder cancer, thyroid tumor, etc. However, although the CaM pathway is reported to be related to the differentiation of NB tumor cells in the literature, no study on PCP4 in NB is reported.

Based on the current state of the prior art, the inventors of the present application intend to provide a tumor cell marker related to neuroblastoma and applications thereof, and particularly, the expression level of the tumor cell marker PCP4, PCP4 related to the differentiation degree of neuroblastoma is increased along with the differentiation degree of the tumor cell of neuroblastoma, so that a diagnostic kit for neuroblastoma can be further prepared.

Disclosure of Invention

The invention aims to provide a novel tumor marker related to neuroblastoma, which is PCP4 and can be used as a tumor marker for evaluating the differentiation degree of NB tumor cells based on the current state of the prior art.

It is a second object of the present invention to provide antibodies against the above tissue tumor markers.

The third purpose of the invention is to provide the application of the antibody of the tissue tumor marker in neuroblastoma.

It is a further object of the present invention to provide a kit for detecting neuroblastoma.

In previous researches of the inventor, the expression level of PCP4 is found to be increased along with the increase of the differentiation degree of neuroblastoma tumor cells, and the PCP4 is expected to be a tumor marker for evaluating the differentiation degree of NB tumor cells. Therefore, it is important to explore the application of the PCP4 detection in NB clinical diagnosis and prognosis evaluation.

The invention provides a tumor marker related to neuroblastoma, which is Pcp4 and can be further used for preparing a neuroblastoma diagnostic kit.

The invention provides application of PCP4 in preparing a medicament for diagnosing or treating neuroblastoma.

Preferably, PCP4 is a diagnostic marker for the degree of differentiation of neuroblastoma.

Alternatively, PCP4 is a marker for screening drugs for the treatment of neuroblastoma.

The medicine for treating neuroblastoma is a substance for inhibiting the increase of the expression level of PCP 4.

The invention provides a diagnostic kit for neuroblastoma, which contains a Pcp4 detection reagent.

The diagnostic kit is used for assisting in judging the differentiation degree of the neuroblastoma; the Pcp4 detection reagent is a substance for identifying or quantifying Pcp4 nucleic acid or protein.

The invention provides a method for detecting PCP4, which comprises the following steps:

obtaining a neuroblastoma tumor tissue sample to be detected;

separating and extracting nucleic acid or protein components of a neuroblastoma tumor tissue sample to be detected;

PCP4 was quantified or characterized using the diagnostic kit described above.

Preferably, immunohistochemically stained NB tissue samples are detected using PCP4 antibody.

More specifically, the invention collects tissue samples meeting the standard by a Standard Operation Procedure (SOP), systematically collects complete clinical data and the like, and adopts an immunohistochemical staining method for verification.

The experimental method mainly comprises the following parts:

selection of study samples:

(1) cases of neuroblastoma clearly diagnosed by pathology;

(2) after being confirmed by more than 2 pathologists, the cells are divided into a differentiated group and an undifferentiated/low-differentiated group;

(3) collecting a neuroblastoma tumor tissue sample of a patient;

immunohistochemically stained NB tissue samples using Pcp4 antibody:

(1) preparing a neuroblastoma tumor tissue section;

(2) dewaxing: placing the glass slide into dimethylbenzene-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol in sequence, wherein the main dewaxing action is the dimethylbenzene, and the placing time is adjusted by the pattern air temperature, for example, the placing time can be reduced by the heat of the weather, and conversely, the dewaxing time is properly prolonged by the cold weather, generally 12-15 min;

(3) antigen retrieval: washing in clear water for a period of time after dewaxing, adding 3% H2O2, soaking for 10min to remove endogenous catalase, pouring out H2O2, washing twice in clear water, adding a citric acid buffer solution, putting into a microwave oven, cooking for 3min (medium fire), boiling, cooling to room temperature, cooking again, cooling to room temperature, and cooking for exposing antigen sites;

(4) serum blocking: cooling to room temperature, pouring out the citric acid buffer solution, washing with water for 2 times, placing the glass slide in PBS for 5min, washing for 2 times, wiping off the PBS around the tissue, adding serum to block nonspecific sites, placing in a 37 ℃ incubator for half an hour, and diluting the serum 10 times (900 μ l PBS: 100 μ l serum blocking solution);

(5) plus primary antibody (Pcp4 antibody): taking out the glass slide in the incubator, wiping the serum around the tissues on the back side and the front side of the glass slide by using absorbent paper, adding a first antibody, and storing in the incubator at 4 ℃ overnight;

(6) adding a secondary antibody: taking out the glass slide from the refrigerator, putting the glass slide into PBS for washing for 3 times, 5min each time, wiping off PBS around tissues, adding secondary antibody, and then putting the glass slide in a 37 ℃ incubator for half an hour;

(7) adding SABC, taking out the slices from the incubator, washing in PBS for 3 times (5 min each time), wiping off PBS around the tissues, adding SABC, and placing in an incubator at 37 deg.C for half an hour. SABC dilution 100 fold (990. mu.l PBS: 10. mu.l SABC);

(8) adding a color developing agent: taking out the slices from the incubator, washing in PBS for 3 times, each time for 5min, wiping off PBS around the tissues, adding color-developing agent (configuration of color-developing agent: adding 1 drop of color-developing agent A in 1ml of water, shaking up, then adding 1 drop of color-developing agent B, shaking up, then adding 1 drop of color-developing agent C, shaking up) A: and (3) DAB: H2O 2C: a phosphate buffer;

(9) counterdyeing: washing the developed slices with clear water, soaking in hematoxylin for dyeing, wherein the animal tissue is half a minute, and the plant tissue is 3-5 min;

(10) and (3) dehydrating: washing the dyed piece in water, sequentially placing the slide glass in 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene, placing each reagent for 2min, soaking in xylene, and placing in a ventilation hood;

(11) sealing: dripping neutral gum on the side of tissue, covering with cover glass, flattening one side, putting down the other side to avoid generating bubbles, sealing, and air drying in a fume hood.

Statistical analysis was performed using the H-score scoring system:

the immunohistochemically stained sections were observed under a mirror and the staining of the cells was counted. The intensity expression is respectively scored according to the coloring intensity of positive cells from weak to strong, the nucleus has no yellow granular-like precipitate for 0min, the faint yellow for 1 min, the brown yellow for 2min and the dark brown yellow for 3 min; the total number of intact epithelial cells and the number of positive cells in the grid were counted under a microscope. H-score ═ intensity × total number of positive cells/epithelial cells × 100%. Statistical analysis of H-score scores was performed using Wilcoxon rank sum test.

The invention provides application of a tumor cell marker PCP4 as a tumor marker related to neuroblastoma. Experiments show that the expression level of PCP4 is increased along with the increase of the differentiation degree of neuroblastoma tumor cells, and the PCP4 can be used as a diagnostic kit for neuroblastoma. The PCP4 of the present invention can accurately determine the expression level of PCP4 as a tumor marker related to neuroblastoma, and can assist in the judgment of the degree of differentiation of neuroblastoma. The invention discloses that an NB-related tumor marker PCP4 has potential values for diagnosis, detection and prognosis evaluation for the first time, and has profound clinical significance and application prospects.

Drawings

FIG. 1 is a schematic diagram showing the results of immunohistochemical staining of neuroblastoma tumor tissue Pcp4, wherein the left panel shows a tumor tissue section of a differentiated group and the right panel shows a tumor tissue section of an undifferentiated/poorly differentiated group.

Fig. 2 is a H-score box line plot of two panels of neuroblastoma Pcp4 immunohistochemical staining, wherein UN: undifferentiated, PDN: hypodifferentiation, DN: and (4) differentiation. P-value < 0.01.

FIG. 3 shows that the expression of PCP4 is correlated with the degree of differentiation and proliferation ability of tumor cells.

Figure 4 shows that children with high expression of PCP4 have a better prognosis than children with low expression of PCP4 (P ═ 0.019).

FIG. 5, immunofluorescent staining showing Pcp4 expression vs cell proliferation potency,

it is shown that expression of PCP4 decreases with increased cell proliferation capacity (expressed MKI 67).

FIG. 6 shows, by cell proliferation assay results, that down-regulation of PCP4 expression enhances the proliferative capacity of NB tumor cell lines.

Detailed Description

Example 1: single cell sequencing data-based exploration of relation between PCP4 and neuroblastoma differentiation degree

1) Sequencing NB tumor tissues by using a single cell transcriptome sequencing technology, and drawing a tumor map;

2) dividing the tumor into two types of a group with lower differentiation degree (Root cells) and a group with higher differentiation degree (End cells) by performing pseudo-time sequence analysis on the tumor cells;

3) by exploring the mRNA expression profile, MKI67 was found to be expressed in less differentiated cell types, indicating an increase in the proliferative capacity of this population of cells; while PCP4 is expressed in cells with higher differentiation degree, which indicates that the expression of PCP4 is related to the differentiation degree and proliferation capacity of tumor cells.

Example 2: survival analysis of neuroblastoma based on mRNA data in GEO database

1) Downloading mRNA expression profiles of 498 NB patients in the data set GSE49710 via a GEO database;

2) dividing 492 patients (6 cases lack PCP4 expression data) into a high expression group and a low expression group through the expression condition of PCP4 mRNA, and carrying out survival analysis by combining survival data;

3) the results showed that children with high expression of PCP4 had better prognosis than children with low expression of PCP4 (P0.019)

Example 3 immunofluorescent staining to show the relationship between Pcp4 expression and cell proliferation potency

The expression of PCP4 and MKI67 is mutually exclusive as shown by immunofluorescence staining of tumor tissues, and the expression of PCP4 is reduced along with the increase of the cell proliferation capacity (the expression of MKI 67).

Example 4 investigation of the Effect of the PCP4 Gene on NB tumor cell lines

A PCP4 expression-reduced SK-N-BE2C cell line and a no-load plasmid-introduced control group SK-N-BE2C cell line are constructed by utilizing a lentivirus shRNA transfection technology, and cell proliferation experiments show that the PCP4 expression can BE reduced to enhance the proliferation capacity of an NB tumor cell line.

The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

Use of PCP4 in the manufacture of a medicament for the diagnosis or treatment of neuroblastoma.
2. The use of claim 1, wherein PCP4 is a diagnostic marker of the degree of neuroblastoma differentiation.
3. The use of claim 1, wherein PCP4 is a marker for screening for a medicament for the treatment of neuroblastoma.
4. The use of claim 3, wherein the agent for treating neuroblastoma is an agent that inhibits an increase in the expression level of PCP 4.
5. A diagnostic kit for neuroblastoma, characterized in that it contains Pcp4 detection reagent.
6. The diagnostic kit according to claim 5, wherein the diagnostic kit is used for assisting in the judgment of the degree of differentiation of neuroblastoma; the Pcp4 detection reagent is a substance for identifying or quantifying Pcp4 nucleic acid or protein.
7. A method of detecting PCP4, the method comprising:

obtaining a neuroblastoma tumor tissue sample to be detected;

separating and extracting nucleic acid or protein components of a neuroblastoma tumor tissue sample to be detected;

quantitative or qualitative PCP4 using the diagnostic kit of claim 5 or 6.
8. The method of claim 7, wherein the immunohistochemically stained NB tissue sample is detected using PCP4 antibody.
9. The method of claim 7, wherein the method comprises the steps of:

(1) preparing a neuroblastoma tumor tissue section;

(2) dewaxing: sequentially placing the glass slide into xylene-100% ethanol-95% ethanol-90% ethanol-80% ethanol-70% ethanol for 10min-15 min;

(3) antigen retrieval: washing in clear water for a period of time after dewaxing, adding 3% H2O2, soaking for 10min to remove endogenous catalase, pouring out H2O2, washing in clear water, adding citric acid buffer solution, putting in a microwave oven, cooking to boil, cooling to room temperature, cooking once again, and cooling to room temperature;

(4) serum blocking: after cooling to room temperature, the citric acid buffer was poured off, washed with water 2 times, and the slides were placed in PBS for 5min, washed 2 times, wiped to dry the PBS surrounding the tissue, immediately serum was added to block some non-specific sites, and then placed in a 37 ℃ incubator for half an hour. Serum is diluted by 10 times;

(5) addition of the Pcp4 antibody: taking out the glass slide in the incubator, wiping serum around tissues on the back surface and the front surface of the glass slide by using absorbent paper, adding a Pcp4 antibody, and storing in the incubator at 4 ℃ overnight;

(6) adding a secondary antibody: taking out the glass slide from the refrigerator, putting the glass slide into PBS for washing for 3 times, 5min each time, wiping off PBS around tissues, adding secondary antibody, and then putting the glass slide in a 37 ℃ incubator for half an hour;

(7) adding SABC, namely taking the slices out of the incubator, putting the slices into PBS for rinsing, wiping off PBS around tissues, adding SABC, and then putting the tissues in the incubator at 37 ℃ for half an hour;

(8) adding a color developing agent: taking out the slices from the incubator, rinsing the slices in PBS, wiping off PBS around tissues, and adding a color developing agent;

(9) counterdyeing: washing the developed slices with clear water for a period of time, and soaking in hematoxylin for staining, wherein animal tissue is half a minute, and plant tissue is 3-5 min;

(10) and (3) dehydrating: after the counterdyed slices are placed in water for washing, the glass slides are sequentially placed in 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene. Placing each reagent for 2min, soaking in xylene, and moving to a fume hood;

(11) sealing: dripping neutral gum on the side of the tissue, covering with a cover glass, flattening one side, slightly laying down the other side to avoid bubbles, sealing the sheet, and air drying in a fume hood;

statistical analysis was performed using the H-score scoring system.
10. The method of claim 7, wherein the H-score scoring system statistically analyzes as follows:

observing the immunohistochemical staining section under a mirror, and counting the staining condition of cells; the intensity expression is respectively scored according to the coloring intensity of positive cells from weak to strong, the nucleus has no yellow granular-like precipitate for 0min, the faint yellow for 1 min, the brown yellow for 2min and the dark brown yellow for 3 min;

counting the total number of complete epithelial cells and the number of positive cells in the grid under a microscope;

h-score ═ intensity × total number of positive cells/epithelial cells × 100%.