CN112159850B - PCR kit for diagnosing gastric cancer - Google Patents

Abstract

The invention discloses a PCR kit for diagnosing gastric cancer, which comprises: andSCTAGgene matched PCR primer, the PCR is RT-PCR, qRT-PCR or real-timePCR, the primer is: 5'-AGAGCCGAGCAGATATTACC-3', respectively; 5'-TCTACATCGCGTCTGTACCT-3', the kit for diagnosing gastric cancer further comprises an internal reference primer: 5'-CCACCCAGAAGACTGTGGAT-3', respectively; 5'-TTCTAGACGGCAGGTCAGGT-3' are provided. The positive detection rate of the kit for detecting tumor tissues reaches up to 93.8 percent, and the positive detection rate of tissues beside cancer reaches up to 60.4 percent.

Description

PCR kit for diagnosing gastric cancer

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a PCR kit for diagnosing gastric cancer.

Background

Gastric cancer is one of the most prominent cancers worldwide, with over 1,000,000 new cases and about 783,000 deaths due to gastric cancer each year worldwide. Early onset symptoms of the gastric cancer are extremely hidden, and a reliable detection means which is effective for early gastric cancer diagnosis is lacked, so that patients are mostly developed to the advanced stage when being diagnosed, thereby losing the best treatment opportunity and seriously reducing the overall survival time of the patients. The detection of serum tumor markers is the only way for the early detection of asymptomatic micro-tumors at present. Therefore, the search for tumor markers with high sensitivity and strong specificity is an important method for improving early gastric cancer diagnosis.

Cancer/Testis Antigen (CTA) is one of tumor-associated antigens, and is physiologically present only in Testis tissue of a normal male body, but can be specifically expressed in tumor tissue. The special expression mode makes CTA possess the features of tumor expression specificity and high immunogenicity. Recent studies show that CTA also plays a positive role in promoting tumor proliferation, invasion and metastasis. In conclusion, the discovery of CTA molecules provides unprecedented opportunities for further research and clinical development in the fields of tumor diagnosis and immunotherapy. CTA is expressed in most types of tumors, but different CTAs are expressed in large differences between different tumor types. The CT-X antigen is more likely to be expressed in bladder, lung, ovarian and melanoma tissues. However, gastric and colorectal cancers are considered to be cancer types where CTA expresses "poverty". Tumor markers specific for gastric cancer include: serum Pepsinogen (PG), gastrin (G), a gastric tumor tissue marker, an Epidermal Growth Factor Receptor (EGFR), a TP53 gene and p53 antibody, an interstitial epithelial cell transformation factor (EMT), microsatellite instability (MSI) and the like; novel gastric tumor markers include: microRNA/miR/miRNA, long-chain non-coding RNA (lncRNA), Matrix Metalloproteinase (MMP) and the like; the relatively low frequency of expression of CTA in gastric cancer has also led to the hampered clinical use of CTA in gastric cancer, and a great deal of research work is still required to explore CTA highly expressed in gastric cancer patients.

SCTAG (super-reactive Cancer/Testis antibenery for Gutdiagnosis) is a novel CTA, is the most abundant nucleoprotein in male sperms in a physiological state, and plays a role in regulating and controlling sperm chromatin condensation, transcription termination and the like.

Disclosure of Invention

The invention aims to solve the problems that the detection positive rate of the existing tumor marker is low and the diagnosis is easy to miss when the existing tumor marker is used as a gastric cancer screening means, and provides a PCR kit for diagnosing gastric cancer.

A PCR kit for diagnosing gastric cancer, comprising: andSCTAGPCR primers for gene matching;

the PCR is RT-PCR, qRT-PCR or real-timePCR;

the primer is as follows:

5’-AGAGCCGAGCAGATATTACC-3’

5’-TCTACATCGCGTCTGTACCT-3’；

the PCR kit for diagnosing gastric cancer further comprises an internal reference primer:

5’-CCACCCAGAAGACTGTGGAT-3’

5’-TTCTAGACGGCAGGTCAGGT-3’。

the present invention provides a PCR kit for diagnosing gastric cancer, which comprises: andSCTAGthe PCR primer matched with the gene is RT-PCR, qRT-PCR or real-timePCR, and the primer is as follows: 5'-AGAGCCGAGCAGATATTACC-3', respectively; 5'-TCTACATCGCGTCTGTACCT-3', the kit for diagnosing gastric cancer further comprises an internal reference primer: 5'-CCACCCAGAAGACTGTGGAT-3', respectively; 5'-TTCTAGACGGCAGGTCAGGT-3' are provided. The positive detection rate of the kit for detecting tumor tissues reaches up to 93.8 percent, and the positive detection rate of tissues beside cancer reaches up to 60.4 percent.

Drawings

FIG. 1 shows the PCR reaction melting curve of SCTAG gene and reference gene GAPDH in normal tissue of stomach cancer and paracarcinoma;

FIG. 2 DNA agarose gel electrophoresis analysis results; g: internal reference GAPDH, 1: patient No. 1; 2: patient No. 2; 3: patient No. 3; a: expression of gastric cancer tissue SCTAG-mRNA; b: expression of SCTAG-mRNA in stomach cancer adjacent tissue; c: expression of SCTAG-mRNA in stomach normal tissue;

FIG. 3 distribution of positive rates of SCTAG mRNA expression; (T) cancerous tissue from a patient with gastric adenocarcinoma, (P) paracancerous tissue, (N) normal tissue;

FIG. 4 comparison of SCTAG mRNA expression levels in cancer, paracancerous and normal tissues of patients with gastric adenocarcinoma;

FIG. 5 SCTAG mRNA expression in healthy human testicular tissue; m = DNA molecular marker, a = SCTAG mRNA, b = internal reference. 1= testis; 2= placenta; 3= heart; 4= brain; 5= thyroid; 6= trachea; 7= salivary glands; 8= fetal brain; 9= uterus; 10= colon; 11= stomach; 12= spleen; 13= small intestine; 14= bone marrow;

FIG. 6 is a schematic diagram of a dilution process for standard multiple ratios;

FIG. 7 is the relationship between SCTAG protein concentration and clinical pathological characteristics in the serum of gastric adenocarcinoma patients; (A) the concentration of SCTAG protein in gastric adenocarcinoma patients is obviously higher than that of healthy control groups; relationship between SCTAG protein concentration in serum of gastric adenocarcinoma patients and various clinical variables: (B) age; (C) the degree of cell differentiation; (D) depth of attack (T); (E) lymph node metastasis (N); (F) clinical staging (TNM); (G) tumor size; (H) vascular invasion; (I) the spirit is subject to invasion;

FIG. 8 relationship between SCTAG protein concentration in serum of gastric adenocarcinoma patients and tissue gastric tumor markers: (A) an EGFR; (B) p53; (C) HER-2; (D) ki 67;

fig. 9 evaluation of the diagnostic ability of serum SCTAG protein on gastric adenocarcinoma. (A) Diagnostic ability of serum SCTAG protein in patients with gastric adenocarcinoma in general; diagnostic capacity (AUC: area under curve) of serum SCTAG protein in patients with gastric adenocarcinoma at clinical stage I (B), clinical stage II (C) and clinical stages III-IV (D), respectively;

figure 10 comparison of SCTAG protein levels in male and female serum and serum SCTAG protein for the diagnostic ability of male and female patients;

FIG. 11 analysis of the diagnostic ability of clinical serum tumor markers for gastric adenocarcinoma; (A) CEA; (B) CA 724; (C) CA 199; (D) CA 242; (E) CA 50; (F) AFP; (G) gastric cancer three CEA, CA724 and CA 199; (H) gastric cancer four CEA, CA724, CA199, and CA 242; (I) six digestive tract tumors, CEA, CA724, CA199, CA242, CA50 and AFP;

fig. 12 post-operative serum SCTAG protein concentration changes;

fig. 13 immunohistochemical detection of expression of SCTAG protein in gastric cancer tissues (a) anti-SCTAG negative control in gastric adenocarcinoma; (B) normal stomach tissue SCTAG is not expressed; (C) SCTAG weakly positive in gastric dysplastic tissue; (D-F) high, medium and low differentiated adenocarcinoma of gastric adenocarcinoma is SCTAG resistant and strong positive.

Detailed Description

Example 1 gastric adenocarcinoma tissue Total mRNA extraction and transcription

First, collection of tissue samples

Gastric adenocarcinoma tissue and its corresponding paracancerous and normal tissues were selected from the tissue specimen bank of the Mitsui Leidai Hospital, Jilin university for extraction of mRNA from the tissues. (the paracarcinoma tissues were taken from the part other than the gastric cancer margin by 1.5cm, and the normal tissues were taken from the part other than the gastric cancer margin by 5 cm). Simultaneously selecting pathological wax blocks of gastric adenocarcinoma tissues for a section immunohistochemical staining experiment; a total of 48 tissues (including cancer, paracarcinoma and normal) were collected from patients with gastric adenocarcinoma.

Second, extraction of total mRNA of gastric adenocarcinoma tissue

The TRIzol method is used for extracting total mRNA in tissues, and comprises the following specific steps:

(1) 50mg of the tissue was cut into small pieces, put into 1mL of TRIzol, and the tissue was sufficiently pulverized with a dispersion homogenizer and mixed well. Standing at room temperature for 5 min;

(2) adding 200uL chloroform into the mixed solution, shaking vigorously for 15s to mix them uniformly, and standing for 3 min at room temperature;

(3) centrifuging 12000g of the mixed solution after standing for 15 min at 4 ℃, taking 450uL of upper-layer water phase, adding 450uL of precooled isopropanol with the same volume, fully mixing, and standing for 10min at room temperature;

(4) centrifuging 12000g of the mixed solution after standing for 10min at 4 ℃, removing supernatant and keeping precipitate;

(5) adding 1mL of 75% ethanol into the precipitate, centrifuging at 4 ℃ for 5min at 7500g, and removing the supernatant;

(6) repeating the step 5 once;

(7) finally, adding 30uL of RNAse-free (RNase) DEPC water into the precipitate, fully dissolving in 55 ℃ metal bath for 10min, taking out, placing on ice for subsequent experiments, or storing in a refrigerator at-80 ℃ for freezing and storing;

thirdly, reverse transcription of mRNA of gastric adenocarcinoma tissue into cDNA

Reverse transcription is carried out on the total mRNA extracted from the tissue sample to obtain corresponding total cDNA. The specific method comprises the following steps:

(1) firstly, the concentration detection and quality control are carried out on the total mRNA of a tissue sample (the ratio of RNA: 260/280 is selected to be in the range of 1.9-2.0, and the ratio of 260/230 is 1.8-2.2);

(2) according to the total mRNA concentration, 1ug of mRNA samples are added to ensure the consistency of the mRNA loading concentration of each sample, and the mixture is prepared according to the following system:

(3) heating the reaction system at a constant temperature of 70 ℃ for 5min, and then immediately placing on ice for 5 min;

(4) preparing a mixture according to the following system, adding the mixture into the reaction system, and fully and uniformly mixing;

(5) and (3) heating the uniformly mixed reaction system at a constant temperature of 42 ℃ for 60 min, and then heating at a constant temperature of 70 ℃ for 15 min. Taking out cDNA, and placing on ice for subsequent experiment, or storing in refrigerator at-20 deg.C for freezing;

the above steps can be completed by a common PCR instrument, and 25. mu.L of cDNA sample is finally obtained.

Immunohistochemical staining of gastric adenocarcinoma tissue

The reagents used were: 4% formalin; paraffin wax; glass slide; ethanol water solutions with different concentrations; 3% hydrogen peroxide; TE buffer solution; 5% goat serum; SCTAG primary antibody resistance; DAB color development liquid; dyeing with hematoxylin; and (4) encapsulating tablets. SCTAG proteins and monoclonal antibodies Hup1N, Hup1M, Hup2N were purchased from Briar patch biosciences.

1. Dyeing step

(1) Slicing: embedding the tissue soaked by formalin with paraffin, and continuously slicing wax blocks of single cancer tissue into slices with the thickness of 3 mu m.

(2) Dewaxing: the paraffin sections on the slides were baked at 65 degrees for 2 hours. Dewaxing with xylene rapidly for 3 times, each time for 2 min,

(3) serial ethanol rehydration: after completion of deparaffinization, tissue sections were rehydrated with a series of dilutions of ethanol: absolute ethanol 2 min × 3 times → 95% ethanol 2 min × 3 times → 80% ethanol 2 min × 1 times → 70% ethanol 2 min × 1 times → PBS wash 2 min × 3 times.

(4) Blocking endogenous peroxidase: tissue sections were soaked in 3% hydrogen peroxide for 10min to quench endogenous peroxide. Washed again with PBS 2 min X3 times.

(5) Antigen retrieval: the sections were placed in an autoclave containing TE buffer (1 mM EDTA and 10 mM Tris, pH 9.2), heated at 98 ℃ for 30min, and washed with PBS 2 min X3 times.

(6) Blocking of non-specific proteins: a closed circle was drawn along 2mm around the tissue using an immunohistochemical section pen, the liquid around the sample was gently wiped off with filter paper, 5% goat serum (same source of secondary antibody) was added dropwise and placed in a wet box at room temperature for 30 min. Do not wash.

(7) Primary antibody incubation: the goat serum on the section is thrown off, the residual serum around the tissue is wiped dry by filter paper, and the anti-SCTAG primary antibody is directly added. Mouse IgG was added to the control group. Then placed in a wet box on a shaker and shaken at 4 ℃ overnight. Taking out from the environment at 4 ℃ and rewarming for 30min at room temperature. The primary antibody was removed and rinsed 5min x 3 times with PBS shaker.

(8) And (3) secondary antibody incubation: wiping the residual liquid around the tissue with filter paper, adding horseradish peroxidase-labeled secondary antibody, placing in a wet box, and shaking for 30min at room temperature. The secondary antibody liquid was removed and rinsed 5min x 3 times with PBS shaker.

(9) DAB color development: the residual liquid around the tissue was wiped dry with filter paper and DAB was added (quickly drop-in, observe the staining change, pour off the staining solution). The color change condition is observed under a microscope to control the color reaction time, and the color reaction time is basically controlled at room temperature for 2-5 min. The PBS was rinsed 5min X3 times after the tap water rinsing.

(10) Counterdyeing: a large drop of hematoxylin staining solution was added dropwise to the sections, and the sections were rinsed with tap water after staining until complete removal of hematoxylin.

(11) Dewatering step by step, and sealing: dewatering in reverse order to dewaxing and rehydrating, immersing the slices in xylene for transparency after dewatering, and sealing the slices with neutral resin. Labeling, and marking the sample information, the antibody information and the time.

(12) Microscopic examination: after the neutral resin was completely dried, the sections were placed under an optical microscope for observation.

2. Result judgment

1) Immunohistochemical positive determination

The results of Immunohistochemical (IHC) staining were examined by 2 pathologists without knowledge of clinical data, with at least two independent reads of tissue sections from each individual. After comparing the results of the two, they reread the sections whose results differ until consensus is reached. Briefly, IHC staining was semi-quantitatively labeled as "-" (negative: no or less than 5% positive cells), "+" (6-25% positive cells), "+ +" (26-50% positive cells)), "+ + +" (more than 50% positive cells). The three groups of the last "+", "+ + + +", were classified as positive for IHC staining; "-" was assigned to IHC staining negative group.

2) Calculation of immunohistochemical Positive Strength

The intensity of positive staining in tissues was analyzed by Integrated Optical Density (IOD) using Image-Pro Plus software (version 6.0). Under the same light microscope and the same exposure intensity, 10 digital images were taken from each sample. After Image-Pro Plus staining, total IOD value of positive staining and total Area of positive staining, Area (um)²). The IOD/Area ratio of each image is taken to represent the staining intensity of the image. Finally, the average IOD/Area ratio of 10 digital images was taken as the value of staining intensity of the section.

Example 2 detection of SCTAG Gene in gastric adenocarcinoma tissue

First, design and synthesis of primer

Forward and reverse oligonucleotide primers were designed for SCTAG and internal reference GAPDH gene sequences using Primer Express version 3.0 software. And synthesized by Shanghai Biotech company; and finally diluting the primer concentration to a working concentration (10 mu M) according to the content of different primers. The specific primer sequence information is as follows (table 1):

establishment of two, qRT-PCR reaction system

The following reaction system was added to the PCR octal tube:

and (3) reacting and amplifying the uniformly mixed reaction system according to the following qPCR amplification program. Totally completing 40 cycles;

three wells were made for each sample, and the average Ct (cycle threshold) value was taken as the Ct value for that sample.

Third, Ct value calculation in qRT-PCR experiment

The expression level of the target cDNA was determined by taking the Δ Ct value as a quantitative value, and Δ Ct = Ct (target gene: SCTAG) -Ct (reference gene: GAPDH). The Δ Ct value of gastric cancer tissue was used as a control group, and Δ Δ Ct = Δ Ct (paracancer or normal tissue) - Δ Ct (cancer tissue). The relative expression amount is calculated by a-delta-Ct method, when the-delta-Ct > 0, the expression amount of the SCTAG mRNA in the paracancer/normal tissue is higher than that in the cancer tissue.

Expression of SCTAG mRNA in gastric cancer tissue

Primers were designed for the SCTAG and GAPDH gene sequences using Primer Express version 3.0 software, and their dissolution peaks were shown to be unimodal by the dissolution curve (fig. 1); the primers are reasonable in design and good in unicity; electrophoresis results also showed that the length of the expression band of SCTAG corresponded to the fragment length expected by primer design (fig. 2); and sending the qPCR product to Shanghai bio-corporation for product sequencing, wherein the obtained sequence result is consistent with the sequence of the target gene.

A total of 45 gastric cancer tissues were detected for the presence of SCTAGmRNA (93.75%, 45/48); CTA gene SCTAGmRNA is also expressed in a certain proportion in non-tumor tissue regions: para-cancerous (60.42%, 29/48) and normal (25.00%, 12/48) tissues; chi-square test results showed that the more proximal regions to the central part of the tumor, the higher the positive frequency of SCTAGmRNA expression (p < 0.001) (table 2). Meanwhile, the experiment also finds that normal tissues expressing the SCTAGGmRNA, and paired cancer tissues and tissues beside the cancer certainly express the SCTAG mRNA. The cancer tissues adjacent to the cancer tissues expressing the SCTAG mRNA also express the SCTAG mRNA certainly in the paired cancer tissues, but normal tissues do not necessarily express the SCTAG mRNA; similarly, cancer tissues expressing SCTAG mRNA did not predict whether their paired paracancerous and normal tissues could positively express SCTAG mRNA (fig. 3).

^※ PLess than 0.001, and has statistical significance.

The expression levels of SCTAG mRNA in different tissues are different, and in 29 cases of gastric cancer patients with cancer tissues and paracancerous tissues expressing SCTAG mRNA at the same time, the expression level of the SCTAG mRNA in the paracancerous tissues is generally higher than that of the cancer tissues (FIG. 4A); in 12 patients with SCTAG mRNA expressed in cancer, both paracancer and normal tissues, SCTAG mRNA expression levels were expressed as paracancer > normal > tumor tissue (fig. 4B). In conclusion, the expression level of SCTAG mRNA in the cancer side tissue is higher than that of the normal tissue and the tumor tissue, which indicates that SCTAG plays a role in the processes of cell differentiation and tumorigenesis. FIG. 5 shows the expression of SCTAG mRNA in healthy human testicular tissue; the results showed that SCTAG mRNA was expressed only in testis tissue and not in other tissues and organs in healthy humans.

Example 3 detection of SCTAG protein in human serum

The specific operation flow is as follows:

(1) transferring the ELISA plate, sample diluent and other reagents to room temperature (18-25 ℃), and balancing for at least 30 min; centrifuging the standard substance for 30-60s, adding 1mL of sample diluent, fully diluting into 2000pg/mL of standard substance stock solution, and balancing for at least 10min after dilution;

(2) dilution of standard products in multiple proportions: as shown in fig. 6, 250uL of sample diluent was removed into each tube (nos. 0-6); and sucking 250uL of standard stock solution, adding the standard stock solution into a number 6, fully blowing, beating and uniformly mixing, sucking 250mL of mixed solution from a number 6 pipe, adding the mixed solution into a number 5 pipe after thorough mixing, and sequentially transmitting the mixed solution to a number 1 pipe. The undiluted standard solution was used as the highest concentration standard (2000 pg/mL). The sample dilution was used as a zero concentration standard (0 pg/ml);

(3) sample dilution: serum samples collected were diluted with sample diluent at a rate of 1: 5, dilution in proportion;

(4) sample adding: respectively provided with a standard sample hole and a sample hole to be detected. Adding 100uL of standard solution or a sample to be detected into each hole of a lining gun, wherein the standard hole is used as a double-hole, and the sample hole to be detected needs to be used as a triple-hole; gently shaking and mixing, covering the upper plate with the paste, and incubating for 2h at 37 ℃;

(5) discarding the liquid, inverting the plate for spin-drying, and sucking dry with clean paper towels without washing;

(6) 100uL of biotin-labeled antibody working solution (1X) was added to each well. And covering with a new board sticker. Incubating at 37 ℃ for 1 h;

(7) discarding the liquid in the wells, spin-drying, adding washing buffer (200 uL) to each well for washing, soaking for 2 min each time, and spin-drying at 200 uL/well. Washing the plate for 3 times;

(8) 100uL of horse radish peroxidase-labeled avidin (HRP-avidin) working solution (1X) was added to each well. Covered with a new panel sticker. Incubating at 37 ℃ for 1 h;

(9) repeating the step 7-8, and washing the plate for 5 times;

(10) to each well, 90ul of TMB substrate solution was added sequentially. Incubating at 37 deg.C for 15-30min, and developing in dark. (reaction time control: reaction termination was carried out at the time when the blue color change appeared in the well No.1 (31.25 pg/mL) and not in the well No. 0 (0 pg/mL);

(11) add 50uL of stop solution to each well in sequence, gently tap the plate to ensure adequate mixing;

(12) within 5min after the reaction is ended, measuring the absorbance value (OD value) of each hole by using an enzyme-labeling instrument at a wavelength sequence of 450 nm;

(13) results of ELISA data calculation analysis: respectively calculating the average absorbance OD values of the standard wells and the sample wells, wherein the detection value of a single well is not more than 20% of the average value; the OD value of well number 0 (at a concentration of 0 pg/mL) was subtracted from the average for each standard and sample; establishing a standard curve by taking the absorbance OD value of the standard as a horizontal coordinate (X) and the SCTAG protein concentration of the corresponding standard as a vertical coordinate (Y); the SCTAG protein content of the sample to be detected can be converted into corresponding concentration from the standard curve according to the OD value; the final actual concentration of the sample should be the concentration obtained on the standard curve multiplied by the dilution factor.

Correlation between serum SCTAG protein and clinical pathological characteristics of gastric adenocarcinoma patient

Compared with the SCTAG protein (94.30 +/-88.70 pg/mL) in the serum of a normal human, the SCTAG protein (1040.31 +/-826.52 pg/mL) in the serum of a gastric adenocarcinoma patient is obviously increased (P is less than 0.001; figure 7A); meanwhile, the independent t test and Kruskal-Wallis test result show that: the levels of SCTAG protein in the serum of gastric cancer patients have no significant correlation with other clinical pathological features, such as: age (P = 0.271), degree of cell differentiation (P = 0.531), depth of invasion (T) (P = 0.919), lymph node metastasis (N) (P = 0.150), clinical stage (P = 0.662), tumor size (P = 0.100), no statistical difference in the relationship between vascular invasion (P = 0.742) and neural invasion (P = 0.609) (fig. 7B-I); also, there were no statistical differences in the relationship between serum SCTAG protein concentrations and the tissue tumor markers EGFR (P = 0.384), P53 (P = 0.925), HER-2 (P = 0.603), ki67 (P = 0.1353) (fig. 8).

Example 4 evaluation of the diagnostic ability of serum SCTAG protein for patients with gastric adenocarcinoma

First, diagnostic ability for early stage patients

When the sensitivity and specificity of the serum SCTAG protein of all gastric adenocarcinoma patients are drawn on a ROC curve, the area under the ROC curve (AUC) is 0.9937 (figure 9A), which shows that the serum SCTAG protein has higher sensitivity and specificity for the diagnosis of gastric adenocarcinoma patients and extremely strong diagnostic capability; meanwhile, in order to evaluate the diagnostic ability of serum SCTAG protein on early gastric cancer, three groups of clinical variables of clinical stage I, clinical stage II and clinical stages III-IV are further subjected to subgroup analysis, and the diagnostic ability of the serum SCTAG protein is found to be the highest in patients with gastric adenocarcinoma of clinical stages III-IV (AUC =0.9963, FIG. 9D); in addition, the serum SCTAG protein has high diagnostic capability in early gastric cancer (clinical stage I) (AUC =0.9941, figure 9B); if the Mean value of serum SCTAG protein concentration of a healthy control group plus 3-fold standard deviation (Mean +3 SD) is taken as the critical value of the positive diagnosis of the gastric cancer, the positive rate of the serum SCTAG protein in the diagnosis of gastric adenocarcinoma patients can reach 93.5 percent (159/170), and the positive rate of the early gastric cancer patients (clinical stage I) in the diagnosis is 96.3 percent (26/27).

Secondly, the diagnostic ability is influenced by the sex factors of patients

In normal organisms, SCTAG protein is only present in males (testicular sites), for which we compared differences in serum SCTAG protein concentration between male and female healthy persons, and between male and female patients, to confirm whether SCTAG could be suitable as a serum tumor biomarker for diagnosis in both male and female populations. The results showed no difference in serum SCTAG protein concentration levels between the healthy controls of males (97.08 ± 89.60 pg/mL) and females (91.64 ± 88.71 pg/mL) (P = 0.771) (fig. 10A). Also there was no difference in serum SCTAG protein concentration levels (P = 0.660) between male (1021.99 ± 771.74 pg/mL) and female (1083.05 ± 949.09 pg/mL) patients with gastric adenocarcinoma (fig. 10B). Furthermore, we plot ROC curves to further analyze the diagnostic ability of serum SCTAG proteins for patients of different sexes. The results showed that AUC values for men were 0.9960 (fig. 10C) and AUC values for women were 0.9885 (fig. 10D), both of which had substantially similar diagnostic abilities of serum SCTAG protein. All the data show that the diagnosis capability of the serum SCTAG protein concentration on the gastric adenocarcinoma is not influenced by the sex factor of the patient, so that the method is applicable to wide population screening and is very hopeful to become a serum marker capable of diagnosing the gastric adenocarcinoma.

Third, the diagnostic ability of serum SCTAG protein to multi-index combined screening

Furthermore, we also analyzed the clinical diagnostic ability of clinical tumor markers for gastric cancer patients with detected serum SCTAG antigens and found that CA724 (AUC = 0.6917) and CA50 (AUC = 0.7293) both had moderate diagnostic ability (fig. 11B, E). But all other tumor markers were relatively poor in diagnostic ability, such as CEA (AUC = 0.5721); CA199 (AUC = 0.5152); CA242 (AUC = 0.5856); AFP (AUC = 0.5731) (fig. 11A, C, D, F). For the gastric cancer with difficult screening, the detection rate of the gastric cancer can be effectively improved by multi-index combined screening. The diagnostic abilities of three gastric cancers (CEA, CA199 and CA 724) and four gastric cancers (CEA, CA724, CA199 and CA 242) were improved to AUC =0.6689 and AUC =0.6680, respectively (fig. 11G, H). The diagnosis ability of six digestive tract tumors can reach 0.9076 (FIG. 11I). In comparison, the serum SCTAG protein has stronger diagnosis capability on gastric adenocarcinoma and has good sensitivity and specificity.

Fourth, change of secretion level of SCTAG protein in serum of gastric adenocarcinoma patients before and after operation

In 20 cases of patients with gastric adenocarcinoma, 18 patients with radical gastric cancer resection were treated, and 2 patients with extensive metastasis of the tumor in the abdominal cavity were treated. The serum SCTAG protein concentration (415.05 + -207.33 pg/mL) of the 18 patients with routine gastric carcinoma radical operation at 7 days after operation is obviously reduced (P < 0.001) compared with the serum SCTAG protein concentration (788.11 + -214.34 pg/mL) before operation; however, the SCTAG protein concentration in post-operative serum (1757.47 ± 160.29 pg/mL) was significantly higher in 2 gastric cancer patients undergoing switching than in pre-operative serum SCTAG protein concentration (801.78 ± 47.39 pg/mL) (fig. 12). The research result also verifies that the serum SCTAG protein has the capability of diagnosing the gastric adenocarcinoma again, and can be used for monitoring whether gastric tumor cells remain after operation and detecting the existence of the gastric adenocarcinoma in real time.

Fifth, SCTAG expression in stomach adenocarcinoma tumor tissue

We found that SCTAG protein is widely expressed in gastric cancer tissues, 98 samples out of 120 samples have SCTAG protein expression, and the positive rate is as high as 81.7% (table 2); and immunohistochemical staining showed that SCTAG protein positive staining was located in the cytoplasm of gastric adenocarcinoma tissue (fig. 12). No expression of SCTAG protein was found in normal tissues (0/96, 0%) (Table 2; FIG. 13B). Interestingly, when we observed that gastric cancer tissue sections expressed SCTAG protein, we found that there was also a small amount of SCTAG protein expressed in the dysplastic tissues of the stomach with a positive rate of 61.6% (table 2; fig. 13C); the expression frequency of SCTAG protein in gastric adenocarcinoma, dysplasia and normal tissue is in a downward trend (P < 0.001).

Chi fang analysis results show that: the positive expression rate of SCTAG protein in tissues is extremely closely related to the degree of pathological differentiation of cells (table 3, P = 0.004). With the increase of the malignancy degree of cell differentiation, the expression positive rate of the SCTAG protein is also in a descending trend: high differentiation (100%), medium differentiation (93.2%), low differentiation (71.2%) (table 3). Meanwhile, the positive expression rate of SCTAG protein was significantly correlated with the clinical staging of gastric cancer (table 3, p = 0.023). The more clinically staged gastric adenocarcinoma patients express SCTAG protein more frequently. SCTAG protein is also more positively expressed in tissues affected by tumors in vessels (P = 0.044) and nerves (P = 0.033). While the positive frequency of SCTAG protein expression in gastric adenocarcinoma tissue was not statistically different from the patient's age (P = 0.94), gender (P = 0.119), depth of invasion (T stage) (P = 0.064), lymph node metastasis (P = 0.073), gross typing (P = 0.744) and tumor size (P = 0.082) (table 3). Meanwhile, the positive frequency of expression of SCTAG protein in gastric cancer tissues was not significantly correlated with the expression of tumor markers EGFR (P = 0.329), P53 (P = 0.544), and HER-2 (P = 0.474) in gastric cancer tissues (table 3).

When observing the gastric cancer tissue section, the following results are found: positive staining for SCTAG protein was found in the glandular cavity of gastric adenocarcinoma, which was thought to be caused by secretion of SCTAG protein into the glandular cavity by gastric adenocarcinoma cells (fig. 13, arrows).

Sequence listing

<110> Jilin university

<120> PCR kit for diagnosing gastric cancer

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 510

<212> DNA/RNA

<213> human (Homo sapiens)

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agagctggcc cctgactcac agcccacaga gttccacctg ctcacaggtt ggctggctca 60

gccaaggtgg tgccctgctc tgagcattca ggccaagccc atcctgcacc atggccaggt 120

acagatgctg tcgcagccag agccggagca gatattaccg ccagagacaa agaagtcgca 180

gacgaaggag gcggagctgc cagacacgga ggagagccat gagtaagtgg gcccagctga 240

gggtgggctg gggctgaggc tgggagctct cagggcccag ccttcctctc accacttttc 300

ttggtctcac cagggtgctg ccgccccagg tacagaccgc gatgtagaag acactaattg 360

cacaaaatag cacatccacc aaactcctgc ctgagaatgt taccagactt caagatcctc 420

ttgccacatc ttgaaaatgc caccatccaa taaaaatcag gagcctgcta aggaacaatg 480

ccgcctgtca ataaatgttg aaaagtcatc 510

<210> 2

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<213> human (Homo sapiens)

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Met Ala Arg Tyr Arg Cys Cys Arg Ser Gln Ser Arg Ser Arg Tyr Tyr

1 5 10 15

Arg Gln Arg Gln Arg Ser Arg Arg Arg Arg Arg Arg Ser Cys Gln Thr

20 25 30

Arg Arg Arg Ala Met Arg Cys Cys Arg Pro Arg Tyr Arg Pro Arg Cys

35 40 45

Arg Arg His

50

Claims (3)

1. DetectionSCTAGThe application of the gene expression amount reagent in preparing a PCR kit for diagnosing gastric cancer is characterized in that: saidSCTAGThe gene is shown in a sequence table SEQ ID NO. 1; the reagent is a primer:

5’-AGAGCCGAGCAGATATTACC-3’

5’-TCTACATCGCGTCTGTACCT-3’。

2. use according to claim 1, characterized in that: the PCR is RT-PCR, qRT-PCR or real-timePCR.

3. Use according to claim 1, characterized in that: also comprises an internal reference primer:

5’-CCACCCAGAAGACTGTGGAT-3’

5’-TTCTAGACGGCAGGTCAGGT-3’。

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