CN111665358A

CN111665358A - Application of NALCN protein in prognosis prediction of esophageal squamous cell carcinoma

Info

Publication number: CN111665358A
Application number: CN202010555474.8A
Authority: CN
Inventors: 王立东; 靳艳; 范宗民; 宋昕; 杨苗苗; 王苒; 周福有; 李恩民; 高社干; 秦艳茹; 杨道科; 常志伟; 陈志国; 李秀敏
Original assignee: Anyang Tumor Hospital; First Affiliated Hospital of Zhengzhou University; First Affiliated Hospital of Henan University of Science and Technology; Xinxiang Medical University; Shantou University Medical College
Current assignee: Anyang Tumor Hospital; First Affiliated Hospital of Zhengzhou University; First Affiliated Hospital of Henan University of Science and Technology; Xinxiang Medical University; Shantou University Medical College
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2020-09-15
Anticipated expiration: 2040-06-17
Also published as: CN111665358B

Abstract

The invention belongs to the technical field of medical biology, and particularly discloses a marker for predicting esophageal squamous cell carcinoma prognosis, namely NALCN protein and application of a detection reagent of the NALCN protein in preparation of a marker for predicting esophageal squamous cell carcinoma prognosis. The NALCN protein is negatively expressed in normal epithelial tissues beside the esophageal squamous carcinoma, has positive expression of different degrees in esophageal squamous carcinoma tissues, and the survival period of patients with the esophageal squamous carcinoma positively expressed by the NALCN protein is short. By detecting the expression of the NALCN protein in the tissue sample, the survival period of the esophageal squamous cell carcinoma patient can be effectively distinguished, so that a new way is provided for judging the prognosis prediction of the esophageal squamous cell carcinoma, and a reference basis is provided for a clinician to analyze the condition of the esophageal squamous cell carcinoma.

Description

Application of NALCN protein in prognosis prediction of esophageal squamous cell carcinoma

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to application of NALCN protein in prognosis prediction of esophageal squamous cell carcinoma.

Background

Esophageal cancer is one of the common digestive tract malignancies in humans. The main histopathological types of the esophageal cancer comprise esophageal squamous carcinoma and esophageal adenocarcinoma, wherein the esophageal squamous carcinoma is taken as a main type in China, the esophageal adenocarcinoma is taken as a main type in western countries, and the Chinese is one of the countries with the highest morbidity and mortality of the esophageal squamous carcinoma. Esophageal squamous carcinoma is latent in onset and has unobvious early symptoms, and patients who see a doctor are mostly in middle and late stages, so the prognosis of esophageal squamous carcinoma patients is extremely poor, and the 5-year survival rate after operation is less than 25%. The prognosis of esophageal squamous cell carcinoma is closely related to the course of disease when the diagnosis is confirmed, and early detection, early diagnosis and early treatment are important for improving the life cycle of patients with esophageal squamous cell carcinoma. At present, a molecular target for prognosis prediction of esophageal squamous cell carcinoma is lacked in clinical diagnosis and treatment, and a tumor marker related to esophageal squamous cell carcinoma with high sensitivity and strong specificity is urgently needed to be found and used as a basis for clinical diagnosis, prognosis judgment and individualized treatment.

Therefore, a new tumor marker related to esophageal squamous cell carcinoma prognosis is found, prognosis of patients with esophageal squamous cell carcinoma is effectively distinguished, clinical treatment can be effectively guided, individualized and accurate treatment is realized, and the method is of great importance for improving survival rate of patients with esophageal squamous cell carcinoma and improving survival quality of patients. The currently discovered tumor markers related to esophageal squamous cell carcinoma are few and are less clinically accepted and applied, and the auxiliary diagnosis of the tumor markers on esophageal squamous cell carcinoma patients is still in an exploration stage. The research team of the inventor finds that the NALCN gene and the expression product thereof are closely related to the occurrence, development and prognosis of esophageal squamous cell carcinoma, but the research report of the NALCN gene in the related field of digestive tract tumors is blank, in particular to the research field of esophageal squamous cell carcinoma.

Disclosure of Invention

The invention aims to provide a marker for prognosis prediction of esophageal squamous cell carcinoma and application thereof.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention firstly provides a molecular marker capable of predicting prognosis of an esophageal squamous carcinoma patient, wherein the molecular marker is NALCN protein encoded by NALCN gene (called as VGCNL 1). The NALCN protein (full name Sodium leak channel non-selective protein) has GenBank accession number NP-001337677.1.

Detection of NALCN protein expression by immunohistochemistry has the following characteristics:

(1) the NALCN protein is negatively expressed in normal epithelial tissues beside the esophageal squamous carcinoma and positively expressed in different degrees in esophageal squamous carcinoma tissues;

(2) through correlation analysis with clinical pathological characteristics, the positive expression of the NALCN protein in the esophageal squamous carcinoma tissue and the tumor infiltration degree are found to be related;

(3) the survival time of the esophageal squamous carcinoma patient with positive NALCN protein expression is short.

The invention also provides application of the detection reagent of the NALCN protein in preparing a product for predicting esophageal squamous cell carcinoma prognosis.

According to the above-mentioned application, preferably, the product detects the expression level of NALCN protein in the sample by Western Blot (Western Blot), immunohistochemistry or enzyme-linked immunosorbent.

According to the above use, preferably the product comprises an antibody that specifically binds to the NALCN protein.

Preferably, the antibody is a polyclonal antibody or a monoclonal antibody according to the above-mentioned use.

According to the above-mentioned use, preferably, the antibody is a monoclonal antibody, which is an antibody prepared by a method known in the art or a purchased antibody. More preferably, the NALCN antibody is purchased from Novus Biologicals under the accession number NBP 1-90028.

According to the above-mentioned use, preferably, the sample is a tissue sample, serum or cells.

According to the above application, preferably, the sample is an esophageal squamous carcinoma surgical resection tissue sample or an endoscopic biopsy tissue sample.

According to the above application, preferably, the detection of the tissue sample is carried out by detecting the NALCN protein in the tissue sample by an immunohistochemical method, and determining the expression level of the NALCN protein in the tissue sample.

The invention also provides a product for predicting the prognosis of esophageal squamous carcinoma, which is characterized by comprising a reagent for detecting the expression level of NALCN protein.

Preferably, the agent is an antibody that specifically binds to the NALCN protein according to the above-mentioned product.

Preferably, the antibody is a polyclonal antibody or a monoclonal antibody according to the above-mentioned product.

According to the above product, preferably, the antibody is a monoclonal antibody, which is an antibody prepared by a method known in the art or a purchased antibody. More preferably, the NALCN antibody is purchased from Novus Biologicals under the accession number NBP 1-90028.

Preferably, the product is a chip, a preparation or a kit according to the above.

The method for judging the prognosis prediction of the esophageal squamous cell carcinoma comprises the following steps: if NALCN protein is positively expressed, the survival time of the esophageal squamous carcinoma patient is short (the short survival time of the esophageal squamous carcinoma patient means that the survival time is shorter than that of a patient with negative NALCN protein expression); the NALCN protein expression is negative, so that the survival period of the esophageal squamous carcinoma patient is long; the judgment standard of NALCN protein expression positivity is known in the field, and when the expression positivity is judged, a positive control of NALCN protein expression positivity and a negative control of NALCN protein non-expression can be set. The survival time is calculated in a manner known in the art, from the time when the patient is diagnosed with the pathological diagnosis to the time when the patient dies for any reason or is healthy from the time when the patient is diagnosed with the pathological diagnosis to the time of the last follow-up.

Compared with the prior art, the invention has the following positive beneficial effects:

(1) the invention provides a molecular marker NALCN protein capable of effectively distinguishing the survival period of an esophageal squamous cell carcinoma patient, wherein the survival time of a patient group with NALCN protein positive expression is shorter than that of a patient group with NALCN negative expression (P is less than 0.05), which indicates that the positive expression of NALCN protein is related to the bad prognosis of the esophageal squamous cell carcinoma, so that the prognosis of the esophageal squamous cell carcinoma patient can be predicted by detecting the expression level of the NALCN protein in a tissue sample, thereby providing a new way for judging the prognosis prediction of the esophageal squamous cell carcinoma and providing a reference basis for a clinician to analyze the condition of the esophageal squamous cell carcinoma.

(2) The kit for predicting the prognosis of the esophageal squamous cell carcinoma, provided by the invention, can be used for quickly detecting the expression level of NALCN protein in an esophageal squamous cell carcinoma surgical resection tissue sample or an endoscopic biopsy tissue sample, and can be used for predicting the prognosis of the esophageal squamous cell carcinoma. The kit is simple to operate and convenient to use, so that prognosis prediction of esophageal squamous cell carcinoma is more convenient and easier.

Drawings

Figure 1 is a photograph of immunohistochemical staining of NALCN protein in esophageal squamous carcinoma tissues at different staining scores.

FIG. 2 is a photograph of immunohistochemical staining micrographs of NALCN protein in esophageal squamous carcinoma tissue and paired paracancerous normal squamous epithelial tissue.

FIG. 3 is a Kaplan-Meier analysis survival graph of NALCN protein expression in esophageal squamous carcinoma tissues.

FIG. 4 is a Kaplan-Meier analysis survival curve graph of NALCN protein expression in esophageal squamous cell carcinoma endoscopic biopsy tissue.

Detailed Description

The invention will be further illustrated by the following examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention.

The experimental methods in the following examples, which do not indicate specific conditions, all employ conventional techniques in the art, or follow the conditions suggested by the manufacturers; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The SPSS 25.0 software was used for data statistics and analysis. P <0.05 represents statistically significant results. The relationship between NALCN protein expression levels and clinical pathology characteristics was analyzed using the chi-square test. The Kaplan-Meier curve and the Log-rank test are used for analyzing the correlation between the NALCN protein expression level and the survival time of the esophageal squamous cell carcinoma patient, and analyzing the value of the NALCN protein expression level for the prognosis evaluation of the esophageal squamous cell carcinoma. And carrying out single-factor and multi-factor regression analysis by adopting a Cox risk ratio regression model.

The pathological grading of esophageal squamous carcinoma in the invention is graded according to the pathological standard of UICC (the Union for International cancer control) stage sixth edition, specifically, the primary tumor (T) stage, Tx: primary tumors cannot be assessed; t0: no evidence of primary tumors; and Tis: carcinoma in situ: intraepithelial or intramucosal cancer does not penetrate the muscularis mucosae to reach the submucosa; t1: tumor invasion and submucosa; t2: tumor invasion and muscle layer; t3: the tumor penetrates the muscular layer and reaches the serosa; t4: tumors invade adjacent tissues, distant organs metastasize. Lymph node metastasis (N) stage, Nx: regional lymph nodes cannot be evaluated; n0: no regional lymph node metastasis; n1: regional lymph node metastasis. Distant metastasis (M) staging, Mx: distant metastasis cannot be assessed; m0: no distant metastasis;

m1: there is distant metastasis. The final clinical stage is stage I: T1N0M 0; stage IIA: T2-3N 0M 0; in stage IIB: T1-2N 1M 0; stage III: T3N1M0, T4 any NM 0; stage IV: any T any NM 1.

The first embodiment is as follows: detection method of esophageal squamous carcinoma prognosis prediction marker NALCN protein expression level

The expression level of NALCN protein is detected by an immunohistochemical method, and the specific steps are as follows:

1. preparing a paraffin tissue specimen:

fixing normal tissues beside esophageal squamous carcinoma and paired carcinoma with 10% neutral formalin fixing liquid for 48h, washing with tap water, dehydrating with gradient ethanol (70% ethanol reagent for 2 h, 80% ethanol reagent for 2 h, 90% ethanol reagent for 3 h, 95% ethanol reagent for 3 h, anhydrous ethanol reagent for 2 h, and anhydrous ethanol reagent for 1 h), transparent xylene (xylene reagent for 2 h and xylene reagent for 2 h), wax-dipping at 54-58 deg.C (wax-dipping I (54-56 deg.C) for 30 min, wax-dipping II (56-58 deg.C) for 1h, and wax-dipping III (56-58 deg.C) for 1 h), embedding into wax block, slicing into 5um thick slices with a slicer, sticking on glass slide, and placing in a 65 deg.C drying cabinet for overnight.

2. HE (hematoxylin-eosin) staining:

tissue chip sections were baked at 65 ℃ for 30 minutes, xylene I reagent was dewaxed for 15 minutes, xylene II reagent was dewaxed for 15 minutes, and in order to wash off dissolved paraffin and xylene, reduced-gradient ethanol hydration was used (anhydrous ethanol I reagent for 15 minutes, anhydrous ethanol reagent II for 15 minutes, 95% ethanol I reagent for 5 minutes, 95% ethanol II reagent for 5 minutes, 85% ethanol reagent for 5 minutes, 75% ethanol reagent for 5 minutes, tap water). Placing the tissue chip into hematoxylin staining solution for 3-5 minutes, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue liquid to blue for 30 seconds, washing with running water, staining in eosin staining solution for 5 minutes, placing the tissue chip into ascending gradient ethanol for dehydration (85% ethanol reagent for 5 minutes, 95% ethanol reagent for 5 minutes, 100% ethanol reagent for 5 minutes), making xylene reagent for 5 minutes transparent, taking out from xylene, air drying, sealing with neutral gum, observing under microscope, determining tumor tissue pathological type, selecting esophageal squamous carcinoma, and making into tissue chip at later stage.

3. Preparing an esophageal squamous carcinoma tissue chip:

the pathological type of esophageal squamous carcinoma tissues is determined on the HE section by a pathologist, and the position of the required cancer nest tissues is marked. Marking the sampling part at the corresponding position of the paraffin specimen, taking out the tissue cores one by one from the marked part by using a perforating needle with the diameter of 1.5mm, putting the tissue cores into a pre-designed array module, and arranging the tissue cores into a tissue chip module. Slicing into 4 μm thick slices with a microtome, sticking on the anti-slip glass, placing in a 60 deg.C drying oven overnight, performing HE staining again, and rechecking to determine whether the spot position of the chip is accurate, wherein the qualified chip can be used for later immunohistochemical staining.

4. Immunohistochemistry:

tissue chips were baked at 60 ℃ for 30 minutes, xylene I reagent was dewaxed for 15 minutes, xylene II reagent was dewaxed for 15 minutes, and in order to wash off dissolved paraffin and xylene, reduced gradient ethanol hydration was used (anhydrous ethanol I reagent for 15 minutes, anhydrous ethanol II reagent for 15 minutes, 95% ethanol I reagent for 5 minutes, 95% ethanol II reagent for 5 minutes, 85% ethanolReagent 5 min, 75% ethanol reagent 5 min, distilled water wash). Performing high-pressure repair on sodium citrate (PH6.0) antigen repair liquid (heating and boiling by an electromagnetic oven, putting a specimen, putting the specimen in a pressure cooker, continuously heating, timing for 2 minutes after an air jet valve jets air, stopping heating, naturally cooling to room temperature), putting the slices into a PBS buffer solution (PH7.4) decoloring shaking table, and shaking and washing for 3 times, 5 minutes each time. 3% H₂O₂After incubation for 25 minutes at room temperature in the dark, the sections were washed 3 times in PBS buffer (pH7.4) with shaking for 5 minutes each, and the sections were spun off slightly, NALCN antibody (antibody purchased from Novus Biologicals, Inc., Cat. NBP1-90028, dilution ratio 1:100) was added and placed in a wet box overnight at 4 ℃. The next day the wet box was removed, returned to room temperature, and the sections were washed 3 times in PBS buffer (pH7.4) with shaking in a shaking table for 5 minutes each. After the section is slightly dried, adding a secondary antibody (HRP mark) of the corresponding species of the primary antibody, incubating for 50 minutes at room temperature, and putting the section into a PBS buffer solution (PH7.4) to decolor and wash for 3 times, 5 minutes each time by shaking in a shaking table; and (3) slightly spin-drying the slices, adding a Diaminobenzidine (DAB) solution under a mirror to develop color, flushing the slices with tap water to stop developing the color, re-dyeing with a hematoxylin dyeing solution for 3 minutes, returning the hematoxylin blue to the blue, and flushing with running water. The slices are sequentially put into gradient ethanol for dehydration (5 minutes of 75% ethanol reagent, 5 minutes of 85% ethanol reagent, 5 minutes of 100% ethanol I reagent and 5 minutes of 100% ethanol II reagent), the xylene I reagent is transparent for 5 minutes, the xylene II reagent is transparent for 5 minutes, the slices are taken out of xylene and slightly dried, and the slices are sealed by neutral gum and observed under a microscope.

5. Determination of NALCN protein expression levels

The tissue chips were evaluated for staining according to current standards. Grading standard: the color development intensity and percentage of the positive cells are comprehensively considered.

Immunohistochemical color intensity: the cell-free cytoplasm color development is 0 point, the weaker cell cytoplasm color development (light yellow) is 1 point, the medium cell cytoplasm color development (brown) is 2 points, and the stronger cell cytoplasm color development (brown) is 3 points.

Percentage of positive tumor cells: 0-3% of tumor cells have cytoplasmic staining of 0, 4-25% of tumor cells have cytoplasmic staining of 1, 26-50% of tumor cells have cytoplasmic staining of 2, and 51-75% of tumor cells have cytoplasmic staining of 3; > 75% of the tumor cells showed cytoplasmic staining of 4 points.

Marking the color development intensity score multiplied by the percentage score as a final score result; wherein 0 is negative (-), 1-4 are weak positive (+), 5-8 are medium positive (+), and 9-12 are strong positive (+ +++).

Fig. 1 is a NALCN protein immunohistochemical staining micrograph of different staining scores in esophageal squamous carcinoma tumor tissues, wherein fig. 1A and 1B are negative expression of the NALCN protein, fig. 1C and 1D are weak positive expression of the NALCN protein, fig. 1E and 1F are positive expression in the NALCN protein, and fig. 1G and 1H are strong positive expression of the NALCN protein. As can be seen from FIG. 1, NALCN protein was expressed positively to varying degrees in esophageal squamous carcinoma tissues.

And (3) evaluating the expression level of the NALCN protein in the esophageal squamous carcinoma according to the final scoring result, wherein 0 is negative expression, and more than or equal to 1 is positive expression.

Example two: application of NALCN protein in prediction of esophageal squamous cell carcinoma prognosis

1. A retrospective study: research for detecting expression condition of NALCN protein in esophageal squamous carcinoma tissue and paired paracarcinoma normal squamous epithelial tissue by immunohistochemical method

The expression level of NALCN protein in 150 cases of esophageal squamous carcinoma tissues and matched paracancer normal epithelial tissues is detected by an immunohistochemical method, and the NALCN protein is scored and analyzed.

(1) Collecting samples:

esophageal squamous carcinoma cancer tissues and paracarcinoma paired normal squamous epithelial tissues of 150 esophageal squamous carcinoma patients (150 patients are confirmed to be diagnosed after postoperative case diagnosis determined by case experts) from national key laboratories of provincial co-construction of Zhengzhou university are collected, the tissue samples are tissue samples surgically excised from esophageal squamous carcinoma patients, and the esophageal squamous carcinoma patients are not subjected to radiotherapy and chemotherapy before surgery. Each patient signed an informed consent prior to sampling, according to the rules prescribed by the ethical review board.

2. Experimental methods

The expression level of UGT3A2 protein in 150 cases of cardiac adenocarcinoma tissues and matched paracancer normal epithelial tissues is detected by an immunohistochemical method, and the expression level is scored and analyzed.

The detailed procedures of immunohistochemical method and scoring criteria are the same as those in the first embodiment, and are not repeated herein.

3. Results of the experiment

Immunohistochemical examination of the cancer tissue and paracarcinoma squamous cell epithelium of 150 patients with esophageal squamous carcinoma is shown in Table 1.

TABLE 1150 histological chip information and NALCN protein immunohistochemical detection results of cancer tissue and paracarcinoma squamous epithelial tissue of patients with esophageal squamous carcinoma

As can be seen from Table 1, NALCN protein is negatively expressed in normal squamous epithelial tissue beside esophageal squamous carcinoma; the positive expression rate in the esophageal squamous carcinoma tissue is 68.7% (103/150). Statistical analysis of the data in table 1 was further performed using Chi-square test (Chi-square test) and found that there was a significant difference in the expression levels of NALCN protein between esophageal squamous carcinoma tissue and paracarcinoma normal squamous epithelial tissue (P < 0.001). The experimental result shows that the NALCN protein has obvious difference between the esophageal squamous carcinoma tissue and the normal epithelial tissue, and the UGT3A2 protein has obvious correlation with the esophageal squamous carcinoma.

FIG. 2 is a microscopic image of representative immunohistochemical staining of esophageal squamous carcinoma tissues and paired paracarcinoma normal squamous epithelial tissues, FIGS. 2A and 2B show negative expression of NALCN protein in paracarcinoma normal squamous epithelia, and FIGS. 2C and 2D show positive expression of NALCN protein in esophageal squamous carcinoma tissues.

2. A retrospective study: analysis of relation between NALCN protein expression and clinical pathological characteristics and prognosis survival of esophageal squamous carcinoma patients

In order to determine the clinical significance of the NALCN protein expression in the esophageal squamous cell carcinoma, clinical data statistics, NALCN protein expression level statistics in a tissue sample of surgical excision and postoperative follow-up statistics are further carried out on 902 cases of esophageal squamous cell carcinoma patients, and the relationship between the NALCN protein expression level in the 902 cases of esophageal squamous cell carcinoma patient samples and the clinical pathological characteristics and survival time of the esophageal squamous cell carcinoma patients is analyzed according to statistical results.

(1) And (4) follow-up statistics:

follow-up visits are carried out by means of outpatient reexamination, telephone follow-up visits, home visits, letters, public security information and the like, and the follow-up visits are carried out by 12 months and 28 days in 2019. Death was used as the endpoint event, loss of visit and survival as tail-biting data. The clinical data and postoperative statistical results of 902 patients with esophageal squamous carcinoma are shown in table 2 (only 200 test results of 902 patients with esophageal squamous carcinoma are listed in table 2).

Wherein, the NALCN protein expression level in the tissue samples of 902 patients with esophageal squamous carcinoma after surgical resection is detected by an immunohistochemical method; the detailed procedures of immunohistochemical method and scoring criteria are the same as those in the first embodiment, and are not repeated herein.

TABLE 2902 clinical data and postoperative follow-up statistics for 200 of patients with esophageal squamous carcinoma

Note: "NA" in Table 2 is missing this information and replaced by NA.

(2) Relationship between NALCN protein expression and clinical phenotype in cancer tissues of esophageal squamous carcinoma patients:

the correlation between NALCN protein expression and clinical pathological characteristics in cancer tissues of 902 patients with esophageal squamous carcinoma was analyzed by chi-square test, and the results are shown in Table 3.

TABLE 2 relationship between NALCN protein expression of patients with esophageal squamous cell carcinoma and clinical pathological features of patients with esophageal squamous cell carcinoma

As can be seen from Table 3, the NALCN protein expression level and the infiltration depth are significantly related (P <0.001), and the detection of the NALCN protein expression can be used for judging the infiltration depth of the esophageal squamous carcinoma, and the positive expression rate of the NALCN protein is gradually increased (54.2%, 70.4%, 72.9%, 77.3%) along with the increase of the infiltration depth.

(3) Relationship between NALCN protein expression and survival time in cancer tissues of esophageal squamous carcinoma patients:

the Kaplan-Meier method and the Log-rank test are adopted to analyze the relationship between the NALCN protein expression level and the survival time of 902 patients with esophageal squamous cell carcinoma, the value of the NALCN protein expression level for predicting the prognosis of the esophageal squamous cell carcinoma is evaluated, and the result is shown in figure 3.

As can be seen from fig. 3, the survival time of the patient group with positive expression of NALCN protein was shorter (P ═ 0.009) than that of the patient group with negative expression of NALCN protein, and the survival prognosis was poor.

(4) Possibility evaluation of NALCN protein expression level as an independent prognosis prediction index of esophageal squamous carcinoma:

and (3) evaluating the possibility that the NALCN protein expression level is used as an independent prognosis prediction index of the esophageal squamous cell carcinoma by adopting a Cox proportional risk model.

Table 4 is the result of a one-way Cox regression analysis performed on the data in table 2. As can be seen from Table 4, NALCN positive expression, age of 60 or more, low differentiation, deep infiltration, lymph node positive metastasis and distant organ metastasis all can become risk factors for poor prognosis of esophageal squamous cell carcinoma.

TABLE 4 relationship of different prediction index and prognosis survival in patients with esophageal squamous cell carcinoma by one-way Cox regression analysis

The esophageal squamous carcinoma-related prognosis influencing factors in the table 4 are subjected to collinearity diagnosis, collinearity factors are eliminated, the remaining factors are included in multi-factor Cox regression analysis, and the multi-factor Cox regression analysis result is shown in the table 5.

TABLE 5 Multi-factor Cox regression analysis of the relationship between different prognostic indicators and prognosis survival in patients with esophageal squamous cell carcinoma

As can be seen from table 5, the expression, age, differentiation degree, infiltration degree, lymph node metastasis and distant organ metastasis of NALCN protein can be used as independent prognostic factors for esophageal squamous cell carcinoma, and can be used for prognosis evaluation, survival time prediction, and therapeutic effect evaluation of patients.

3. A retrospective study: immunohistochemical method for detecting expression of NALCN protein in endoscopic biopsy cancer tissue of esophageal squamous carcinoma patient

Biopsy cancer tissues of 315 patients with endoscopic biopsy and pathological diagnosis of esophageal squamous carcinoma are prepared into tissue chips, and the NALCN protein expression level is detected by an immunohistochemical method. 315 patients with esophageal squamous carcinoma may or may not have undergone esophageal squamous carcinoma resection (e.g., radical or palliative surgery) and may undergo only chemotherapy or radiation therapy. The detailed procedures of immunohistochemical method and scoring criteria are the same as those in the first embodiment, and are not repeated herein. The immunohistochemical results are shown in table 6.

TABLE 6315 immunohistochemical detection results of NALCN protein in tumor tissue samples of endoscopic biopsy esophageal squamous cell carcinoma patients

Note: "NA" in Table 6 is missing from the patient and replaced by NA.

As can be seen from Table 6, in the biopsy tissue samples of 315 patients with esophageal squamous cell carcinoma, the NALCN protein was positively expressed in the patients with esophageal squamous cell carcinoma, and the positive rate was 68.6% (226/315).

The statistical data in Table 6 were subjected to survival analysis using the Kaplan-Meier method and the log-rank test, and the results are shown in FIG. 4. As can be seen from fig. 4, the survival time of the patient group with positive expression of NALCN protein was shorter than that of the patient group with negative expression of NALCN protein in the esophageal squamous carcinoma endoscopic biopsy tissue sample (P ═ 0.037).

Therefore, the prognosis can be predicted by detecting the NALCN protein expression of patients who have not undergone esophageal squamous carcinoma surgery (such as radical or palliative surgery) and have undergone chemotherapy, radiotherapy and chemoradiotherapy.

In conclusion, the NALCN protein expression level can effectively distinguish the life cycle of the esophageal squamous cell carcinoma patient, thereby providing a new way for prognosis prediction of the esophageal squamous cell carcinoma and providing a reference basis for a clinician to analyze the condition of the esophageal squamous cell carcinoma. The NALCN protein is used as a detection marker and has the following characteristics: when the tissue sample immunohistochemical staining score of the esophageal squamous carcinoma sample is more than or equal to 1 minute, judging the NALCN protein positive expression, and prompting that the life cycle of an esophageal squamous carcinoma patient is short; and on the contrary, when the tissue sample immunohistochemical staining score of the esophageal squamous carcinoma sample is 0, judging that the NALCN protein is negatively expressed, and prompting the long life of the esophageal squamous carcinoma patient.

Example three: preparation of kit for predicting esophageal squamous carcinoma prognosis

A kit for predicting prognosis of esophageal squamous carcinoma, the kit comprising an antibody capable of specifically binding to NALCN protein, said NALCN antibody being purchased from Novus Biologicals under the code NBP 1-90028. The kit adopts an immunohistochemistry method to detect the NALCN protein in an esophageal squamous cell carcinoma surgical resection tissue sample or an endoscopic biopsy tissue sample, determines the expression level of the NALCN protein in the esophageal squamous cell carcinoma surgical resection tissue sample or the endoscopic biopsy tissue sample, and distinguishes the life cycle of an esophageal squamous cell carcinoma patient according to the expression level of the NALCN protein. Further, the kit or the reagent also contains a secondary antibody for detection, an antigen retrieval solution, a buffer solution and a color development reagent.

The kit judges the expression level of NALCN protein according to the detected tissue sample immunohistochemical staining score (the scoring judgment method is shown in the first embodiment). When the tissue sample immunohistochemical staining score is larger than or equal to 1 minute, judging the NALCN protein positive expression, and prompting that the life cycle of the esophageal squamous cell carcinoma patient is short; and on the contrary, when the tissue sample immunohistochemical staining score of the esophageal squamous carcinoma sample is 0, judging that the NALCN protein is negatively expressed, and prompting the long life of the esophageal squamous carcinoma patient.

The kit can quickly detect the expression level of NALCN protein in an esophageal squamous cell carcinoma surgical resection tissue sample or an endoscopic biopsy tissue sample, and can be used for prognosis prediction of esophageal squamous cell carcinoma. The kit is simple to operate and convenient to use, so that prognosis prediction of esophageal squamous cell carcinoma is more convenient and easier.

Although specific embodiments of the invention have been described in detail, those of ordinary skill in the art will understand that. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. The application of the detection reagent of the NALCN protein in preparing products for predicting esophageal squamous cell carcinoma prognosis.

2. The use according to claim 1, wherein the product detects the expression level of NALCN protein in a sample by Western Blot, immunohistochemistry or enzyme-linked immunosorbent assay.

3. Use according to claim 2, wherein the product comprises an antibody that specifically binds to a NALCN protein.

4. The use of claim 3, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

5. The use of any one of claims 1-4, wherein the sample is a tissue sample, serum, or cells.

6. The use of claim 5, wherein the sample is an esophageal squamous carcinoma surgically excised tissue sample or an endoscopic biopsy tissue sample.

7. A product for predicting prognosis of esophageal squamous carcinoma, comprising a reagent for detecting the expression level of NALCN protein.

8. The product of claim 7, wherein the agent is an antibody that specifically binds to a NALCN protein.

9. The product of claim 8, wherein the product is a chip, a formulation or a kit.