CN115951056A - Pancreatic cancer prognosis molecular marker NLRC4, detection kit and application of detection kit - Google Patents

Abstract

The invention provides a pancreatic cancer prognostic molecular marker NLRC4, a detection kit and application thereof. According to the invention, the gene screening and immunohistochemical experiments are adopted to determine that the NLRC4 has bad prognosis when being highly expressed in a pancreatic cancer tumor tissue chip, so that the NLRC4 can be used as a molecular marker for predicting the tumor curative effect or prognosis. The pancreatic cancer prognosis molecular marker and the detection kit containing the molecular marker antibody provided by the invention can be applied to pancreatic cancer curative effect prediction or prognosis, and have the advantages of strong specificity, strong sensitivity, high accuracy and the like.

Description

Pancreatic cancer prognosis molecular marker NLRC4, detection kit and application thereof

Technical Field

The invention relates to the field of tumor molecular biology, in particular to a pancreatic cancer prognosis molecular marker NLRC4, a detection kit and application thereof.

Background

Pancreatic cancer is one of the most malignant tumors, has the defects of hidden onset, rapid progress, low surgical resection rate, poor chemoradiotherapy effect and extremely poor prognosis, has the overall survival rate of less than 5 percent in 5 years, accounts for the 13 th site in the global cancer incidence rate, and ranks as the fourth site of all malignant tumor lethal causes. Most pancreatic cancer patients often have had lymphoid, hepatic or blood-borne metastases diagnosed, thereby losing the opportunity for radical surgical treatment. The main cause of death of patients with pancreatic cancer is the local invasion or distant metastasis of tumors at an early stage, and the main factor for restricting the prognosis of patients. At present, the invasion and metastasis mechanism of pancreatic cancer is not clear, the malignant biological behavior generation mechanism of pancreatic cancer is understood, and appropriate intervention measures are given, so that the method has very important practical significance for improving the clinical diagnosis and treatment level of pancreatic cancer and improving the prognosis of patients.

At present, the diagnosis and prognosis judgment of pancreatic cancer is mainly carried out clinically by detecting the change of tumor markers (CA 199, CEA and CA 125) in serum and combining clinical symptoms, imaging diagnosis and pathology detection, but the level change of the pancreatic cancer cannot directly represent the change of tumor tissue molecular biology, and the accuracy and the specificity of early diagnosis and treatment prognosis evaluation of pancreatic ductal adenocarcinoma are low, so that a large amount of manpower and financial support is needed. Wherein: the operation specimen of a pancreatic cancer patient can be used for detecting various tumor markers, so that other markers which can simply, conveniently, accurately and specifically judge the prognosis of the pancreatic cancer patient are urgently needed to evaluate the treatment condition and the prognosis condition of the tumor patient and select the optimal treatment mode so as to improve the life quality and the survival rate of the patient.

Disclosure of Invention

Aiming at the fact that no ideal molecular indexes are clinically available for assessing pancreatic cancer prognosis and risk of metastasis, the technical problem to be solved by the invention is to provide a novel biomarker capable of accurately and specifically judging pancreatic cancer prognosis, a detection kit and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows: a molecular marker for predicting pancreatic cancer treatment effect or prognosis, wherein the molecular marker is NLRC4. Wherein the pancreatic cancer is preferably pancreatic ductal adenocarcinoma.

NLRC4 is a member of the NOD-like receptor family, and mainly senses components of flagellin and III-type secretion system of bacteria, thereby triggering immune response to invasion of related pathogenic bacteria. NLRC4 is in a resting state under normal conditions through self-inhibition; when the pathogenic bacteria component enters into the cell, the pathogenic bacteria component is recognized and activated by another NOD-like receptor family protein, namely NAIP subfamily protein, so that NLRC4 is further activated; activated NLRC4 self-multimerizes and recruits Caspase-1 to form inflammasome, which generates a series of immune responses. The invention screens one of the genes promoting pancreatic cancer invasion and metastasis by a plurality of gene screening methods: NLRC4; on the basis, the expression of NLRC4 in pancreatic cancer tumor cells is determined through an immunohistochemical test, and the expression quantity is highly related to pancreatic cancer invasion and metastasis; wherein: patients with high NLRC4 expression in pancreatic cancer tumor cells are associated with postoperative recurrent metastasis, and have short recurrence-free Survival (RFS) and Overall Survival (OS); therefore, the molecular marker can be used as a molecular marker for judging the prognosis of pancreatic cancer, and can be used as an important index for judging the prognosis of a pancreatic cancer patient alone or in cooperation with other molecular markers to guide postoperative administration of the pancreatic cancer patient, so that the survival quality of the pancreatic cancer patient is improved.

The pancreatic cancer prognostic molecular marker NLRC4 can be applied to preparation of a pancreatic cancer prognostic detection kit; the detection kit comprises NLRC4, a fragment of NLRC4 or NLRC4 antibody.

The invention provides an immunohistochemical kit capable of rapidly detecting the NLRC4 expression quantity, which comprises an enzyme-labeled primary antibody, an enzyme-labeled secondary antibody, an antibody diluent, a developing solution and a phosphate buffer solution; wherein: the enzyme-labeled primary antibody is an enzyme-labeled NLRC4 antibody; the enzyme-labeled secondary antibody is goat anti-mouse IgG or rabbit IgG; the color developing solution is an HRP color developing solution.

Compared with the prior art, the invention has the following advantages and effects:

1. according to the invention, a gene NLRC4 for promoting pancreatic cancer invasion and metastasis is obtained by screening through a plurality of different gene screening methods; and 104 cases of primary operation human pancreatic cancer tissue chips are selected, NLRC4 immunohistochemistry is carried out, the histochemical result is scored and divided into an NLRC4 high expression group and an NLRC4 low expression group, OS and RFS survival curves are drawn according to follow-up statistics, experiments verify that the expression quantity of NLRC4 in pancreatic cancer mesenchyme is highly related to pancreatic cancer invasion and metastasis and is negatively related to OS and RFS, and therefore when the NLRC4 is highly expressed in pancreatic cancer tumor cells, pancreatic cancer patients have poor prognosis, the NLRC4 can be used as a molecular marker for predicting the pancreatic cancer curative effect or prognosis and is used for judging or cooperating with other molecular markers to jointly evaluate the treatment condition and the prognosis condition of pancreatic cancer so as to improve the survival quality of the patients.

2. The molecular marker NLRC4 for pancreatic cancer curative effect prediction or prognosis and the immunohistochemical kit containing the molecular marker antibody provided by the invention can be applied to pancreatic cancer curative effect prediction or prognosis, and have the advantages of high sensitivity, strong specificity, high accuracy and the like.

3. The immunohistochemical kit provided by the invention can quickly detect the expression quantity of NLRC4, quickly evaluate the expression quantity of NLRC4 by a scoring method, directly judge the diagnosis and treatment effect, disease progression and prognosis evaluation of pancreatic cancer patients, further guide the postoperative administration of the pancreatic cancer patients, improve the survival rate of the patients and reduce the recurrence risk.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a process diagram of screening gene NLRC4 promoting pancreatic cancer invasion and metastasis according to the present invention;

FIG. 2 is a data statistics chart of the gene NLRC4 for promoting pancreatic cancer invasion and metastasis screened by the present invention; wherein: a) Screening a liver transfer related gene data statistical chart by using a CRISPR library; b) Screening a volcano chart of the liver metastasis related genes by a single cell sequencing method; c) Enrichment analysis of a transcriptome sequencing KEGG differential gene scattergram; d) Gene intersection result graphs obtained by the three screening methods;

FIG. 3 is a photograph of pancreatic cancer tissue chip NLRC4 immunohistochemical staining; wherein: FIG. (a) is a photograph of immunohistochemical staining of NLRC4-high group; panel (b) is a photograph of an NLRC4-low group immunohistochemical staining.

FIG. 4 is a plot of RFS and OS curves for NLRC4-high NLRC4-low NLRC group.

FIG. 5 is a graphical depiction of curves for different NLRC4 expression sets RFS, OS in the TCGA database.

Fig. 6 is a statistical analysis of the appearance of liver metastasis during follow-up for different NLRC4 expression groups.

Detailed Description

The present invention will be described in further detail with reference to examples, which are illustrative of the present invention and are not to be construed as being limited thereto.

Test example: 1. screening out a gene NLRC4 for promoting pancreatic cancer invasion and metastasis, wherein the specific method comprises the following steps:

as shown in fig. 1 and 2, the present application screens liver transfer related genes by using CRISPR library (fig. 2A); screening liver transfer related genes by a single cell sequencing method (figure 2B); enrichment analysis of differential genes using transcriptome sequencing KEGG (figure 2C); the gene intersection of the above three screening methods was used to obtain 39 genes, as shown in FIG. 2D, and the statistical results of part of the data are shown in the following table: (all of the above three gene screening methods are prior art and will not be described in detail in the present invention.)

TABLE 1

Cene_ID	sgRNA_num	Pos|p-value	Pos|goodsgrna	Pos|lfc	Pos|rank
						NLRC4	6	0.00004271	5	6.7344	1
BAI2	3	0.00005618	3	5.8727	2
						PIK3R5	3	0.00008178	3	6.0038	3
FAM83B	3	0.00014658	3	6.2699	5
						ADAM17	3	0.00028506	3	6.2371	6
TM4SF1	3	0.00074021	8	4.1183	7

The genes with the top rank are obtained by taking the gene intersection of the three screening methods, and the NLRC4 has the first rank, so the NLRC4 is taken as the gene for promoting the pancreatic cancer invasion and metastasis by final screening.

2. NLRC4 is used as a molecular marker for judging the curative effect or prognosis of pancreatic cancer treatment, and the diagnosis and treatment effect, disease progression and prognosis evaluation of a pancreatic cancer patient are judged according to the expression quantity of NLRC4.

(1) The expression level of NLRC4 is rapidly detected by immunohistochemical staining:

through the immunohistochemical method, the HRP coupling NLRC4 primary antibody is used for rapid detection (the result can be obtained within 2 hours), the detection precision is improved, the detection speed is increased, the detection means is simplified, the detection cost is reduced, and the specific test scheme is as follows:

collecting samples:

adopting a pancreatic cancer tissue chip after 104 patients;

the operation method comprises the following steps:

experimental reagent: primary, ready-to-use Max vision secondary antibody: goat anti-mouse IgG and rabbit IgG, (or plain biotinylated secondary and tertiary antibodies); distilled water and 0.01M Phosphate Buffered Saline (PBS) at pH7.4 to dilute the antibody and wash the slide; xylene, absolute ethyl alcohol (and gradient alcohol prepared from absolute ethyl alcohol), hydrochloric acid, ammonia water, and hematoxylin dye solution; antigen retrieval solution: citric acid buffer or citric acid buffer (pH 6.0); H2O2, inactivating endogenous peroxidase; antibody diluent; and (4) HRP color development liquid. Wherein: the primary antibody is enzyme-labeled NLRC4 antibody.

The method comprises the following specific steps:

1. slicing the paraffin-embedded tissue block, wherein the thickness of the slice is 4 mu m, and baking the slice for 2 hours on a baking sheet machine;

2. dewaxing the paraffin sections to water (15 min for xylene 1-2, 5-10min for absolute ethyl alcohol 1-2, 95%, 85%, 75%, 60% and 5min for distilled water respectively) by a conventional method;

3. quickly immersing the hydrated flakes into a stainless steel cup containing an antigen repairing solution (citric acid or citric acid buffer solution), tightly covering the pot cover, heating until air is sprayed, and timing for 2min;

4. naturally cooling to room temperature, placing the slices into a hydrogen peroxide immersion frame box, incubating for 10min at room temperature in the dark to block the activity of endogenous peroxidase;

5, soaking and washing the slices with PBS for 3 times, and 5-10min each time (reducing the PH environment);

6. diluting primary antibody (NLRC 4 primary antibody) with antibody diluent, placing the slices in a wet box in parallel, dripping the primary antibody (the antibody covers 1mm of the whole tissue edge) into the tissue area, and standing overnight at 4 ℃;

7. before the operation of the same antibody, adding dropwise a Max vision secondary antibody working solution marked by ready-to-use HPR, and putting a wet box into a 37 ℃ incubator for incubation for 30min;

8. preparing DAB dyeing, dripping DAB dyeing liquid into a tissue area (the DAB dyeing liquid is required to cover the edge of all tissues by 1 mm), and observing under a microscope until the color of yellow brown appears for about 3-10min;

9. quickly washing with distilled water after color development, counterstaining with hematoxylin for 10 mm, washing with tap water, washing with hydrochloric acid and ethanol (color becomes red), washing with distilled water, and washing with ammonia water to turn blue (3 min);

10. gradient alcohol dehydration (gradient alcohol 60%, 75%, 85%, 95%, absolute ethanol 1-2 for 20min, and xylene 1-2 for 10 min). Neutral gum seals (take care not to have air bubbles when tabletting).

(2) Method for rapidly determining expression quantity of NLRC4 by scoring method

The expression quantity of the NLRC4 is rapidly evaluated through grading, the prognosis of the patient is judged, the detection result is judged in an immunohistochemical grading mode and is compared with a standard value in the existing database, the expression quantity level of the NLRC4 of the patient is obtained, the prognosis of the patient is judged, and a corresponding treatment scheme can be formulated according to the prognosis condition of the patient.

The specific scheme is as follows: the section has no impurity staining, takes the positive cells of the brown yellow or brown particles appearing on the normal staining part of the antibody, and randomly selects 4 visual fields under a 20X10 times light microscope to observe and score the interstitial positive staining part. Scoring according to the proportion and distribution condition of the positive cells for 1-5% =1, wherein 5% -10% is 2, 10% -20% =3, and >20% is 4; target protein staining intensity: weak positive is 1 point, moderate positive is 2 points, high positive is 3 points, strong positive is 4 points. The percentage of positive cells in each field is multiplied by the staining intensity score for the protein of interest and summed.

After the surgical specimen chip of pancreatic ductal adenocarcinoma patients was subjected to section IHC staining, the specimens were classified into NLRC4-high group and NLRC4-low group according to the expression level of NLRC4, as shown in FIG. 3; performing follow-up statistics on patients corresponding to the NLRC4-high group and the NLRC4-low group, and drawing OS and RFS curves as shown in FIG. 4; as can be seen from fig. 4, the OS and RFS of the NLRC4-high group are significantly shortened compared to the NLRC4-low group, and the following conclusions are drawn: NLRC4 expression level is negatively correlated with prognosis (OS, RFS) of pancreatic cancer patients; NLRC4 can be used as an important index for judging the prognosis of a pancreatic cancer patient.

Further, as shown in FIG. 5, the statistical analysis of pancreatic cancer data in TCGA data (tumor gene family map) library of the present application can also reach the above conclusion. Wherein: NLRC4-high is NLRC4 high expression group, NLRC4-low is NLRC4 low expression group; censored is censored data, i.e., individuals who died immediately before a relapse occurred, individuals who were interviewed, and individuals who did not experience a relapse.

Further, as shown in fig. 6, the present application performs statistical analysis on 90 patients with liver metastasis during long-term follow-up, and performs chi-square test on the relationship between the occurrence of liver metastasis and NLRC4 expression, finds that the liver metastasis ratio of the NLRC4 high-expression group is significantly higher than that of the NLRC4 low-expression group, and further verifies that the expression of NLRC4 is negatively correlated with the survival prognosis of pancreatic cancer patients, and NLRC4 can be used as a molecular marker for predicting the efficacy or prognosis of pancreatic cancer.

In addition, it should be noted that the specific embodiments described in the present specification may be different in terms of the parts, the shapes of the components, the names of the components, and the like. All equivalent or simple changes of the structure, the characteristics and the principle of the invention which are described in the patent conception of the invention are included in the protection scope of the patent of the invention. Various modifications, additions and substitutions for the specific embodiments described may be made by those skilled in the art without departing from the scope of the invention as defined in the accompanying claims.

Claims (6)

1. A molecular marker for assessing the prognosis of pancreatic cancer, wherein the molecular marker is NLRC4.

2. Application of pancreatic cancer prognosis molecular marker NLRC4 in preparation of pancreatic cancer prognosis detection kit.

3. A pancreatic cancer prognostic assay kit comprising NLRC4, a fragment of NLRC4 or an NLRC4 antibody.

4. An immunohistochemical kit for detecting NLRC4 expression quantity is characterized by comprising an enzyme-labeled primary antibody, an enzyme-labeled secondary antibody, an antibody diluent, a developing solution and a phosphate buffer solution; wherein: the enzyme-labeled primary antibody is an enzyme-labeled NLRC4 antibody.

5. The immunohistochemistry kit of claim 4, wherein the enzyme-labeled secondary antibody is goat anti-mouse IgG or rabbit IgG.

6. The immunohistochemistry kit of claim 5, wherein the chromogenic solution is an HRP chromogenic solution.

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