CN114317730A

CN114317730A - Application of TTPAL as gastric cancer monitoring or prognosis judgment marker

Info

Publication number: CN114317730A
Application number: CN202011051380.3A
Authority: CN
Inventors: 于君; 苟红艳
Original assignee: Shenzhen Research Institute of CUHK
Current assignee: Shenzhen Research Institute of CUHK
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2022-04-12
Anticipated expiration: 2040-09-29
Also published as: CN114317730B

Abstract

The present application provides methods and corresponding kits for monitoring or prognosticating gastric cancer in an individual by measuring the expression level of TTPAL protein or fragments thereof. The application also provides a method for detecting the expression level of TTPAL protein or fragments thereof. In addition, the present application also provides a pharmaceutical composition comprising an agent for specifically inhibiting the expression of TTPAL protein and its use for inhibiting gastric cancer cell growth.

Description

Application of TTPAL as gastric cancer monitoring or prognosis judgment marker

Technical Field

The present application relates to the fields of medicine and biotechnology. Specifically, the application provides a kit for monitoring or prognosis judgment of gastric cancer and related applications.

Background

Gastric cancer is the fourth most common cancer worldwide, with approximately 1,000,000 cases diagnosed each year. Gastric cancer is a high mortality disease with significantly higher morbidity in men and in developing countries, including many asian countries. Gastric cancer is usually asymptomatic or exhibits only nonspecific symptoms in an early stage, and thus diagnosis cannot be made until the disease progresses to an advanced stage in many cases. This leads to a generally poor prognosis.

The stage of stomach cancer TNM is currently the most important clinical prediction index for judging the prognosis of stomach cancer. However, many patients have a potential for tumor recurrence even if the TNM stage is in the early stage. Therefore, there is a need to develop new prognostic markers for gastric cancer monitoring or risk assessment.

Summary of The Invention

In one aspect, the present application provides a kit for monitoring or prognosing gastric cancer in an individual comprising reagents for detecting the expression level of alpha-Tocopherol Transfer Protein Analog (TTPAL) protein or a fragment thereof, or the mRNA expression level of said TTPAL protein or a fragment thereof, in a sample from said individual.

In another aspect, the present application provides the use of an agent for detecting the expression level of TTPAL protein or a fragment thereof, or the mRNA expression level of TTPAL protein or a fragment thereof, in a sample from an individual in the manufacture of a kit or medicament for monitoring or prognosing gastric cancer in said individual.

In some embodiments, the amino acid sequence of the TTPAL protein is shown as SEQ ID NO. 1 or 2, more preferably, the amino acid sequence of the TTPAL protein is shown as SEQ ID NO. 1. In some embodiments, the fragment of the TTPAL protein comprises amino acid residues 1-179 of the sequence shown in SEQ ID NO. 1 or 2.

In some embodiments, the agent comprises a binding agent that specifically binds to said TTPAL protein or fragment thereof, preferably said binding agent is an antibody to TTPAL protein or fragment thereof. In other embodiments, the agent comprises a polynucleotide probe that specifically hybridizes to mRNA of TTPAL or a fragment thereof.

In some embodiments, the sample is selected from gastric tissue, serum, plasma, or cell culture supernatant, and in particular embodiments, the sample is a fresh tissue sample or an embedded specimen of gastric cancer.

In some embodiments, the kit further comprises one or more additional reagents for immunochemical staining of the tissue.

In some embodiments, poor prognosis of gastric cancer in the individual is indicated if the level of expression of TTPAL protein or a fragment thereof, or mRNA of TTPAL protein or a fragment thereof, in a sample from the individual is above a certain threshold.

In another aspect, the present application provides a pharmaceutical composition for inhibiting gastric cancer cell growth, comprising an agent that specifically inhibits the expression of TTPAL protein or a fragment thereof, or the mRNA expression of TTPAL protein or a fragment thereof. In a specific embodiment, the agent is an interfering RNA.

In another aspect, the present application provides the use of an agent that specifically inhibits the expression of TTPAL protein or a fragment thereof, or the mRNA expression of TTPAL protein or a fragment thereof, in the manufacture of a medicament for inhibiting gastric cancer cell growth.

In yet another aspect, the present application provides a method for detecting the expression of TTPAL protein or a fragment thereof, or the mRNA expression of TTPAL protein or a fragment thereof, in a sample, which comprises treating said sample with the reagents of the above-described kit, in a specific embodiment, said sample is a gastric cancer tissue sample.

Brief description of the drawings

FIG. 1 shows the results of immunohistochemical staining of gastric cancer tissues (panel B) and paracancerous normal control tissues (panel A), in which TTPAL expression was significantly elevated in gastric cancer tissues (panel B) and statistically different from normal control TTPAL expression scores in gastric cancer tissues (panel C). P <0.001, compared to paracancerous normal controls.

FIG. 2 shows the results of a multifactorial COX regression analysis assessing the relevance of the expression level and clinical characteristics of TTPAL, indicating that high TTPAL expression may be an independent prognostic diagnostic factor for gastric cancer.

FIG. 3 shows a Kaplan-Meier analysis curve of gastric cancer patient survival. P <0.01, compared to TTPAL low expression group.

FIG. 4 shows the effect of TTPAL on the growth of gastric cancer cells after overexpression in the gastric cancer cell lines MGC803 and BGC823, where the growth rate of the cells was compared by one-way anova analysis,. times.P <0.001, compared to the empty vector group; cells were compared for colony forming ability by t-test, P <0.01, P <0.001, compared to the empty vector group. Panel A demonstrates the overexpression of TTPAL in MGC803 and BGC 823; panel B and panel C show the growth promoting effect of TTPAL over-expressed on gastric cancer cell MGC803 and BGC823, respectively; FIG. D shows a representative colony forming image; panel E and F show the results of an assay of colony forming ability after overexpression of TTPAL by MGC803 and BGC823, respectively.

Figure 5 shows the inhibitory effect of TTPAL knockdown on gastric cancer cell growth, where the growth rates of the cells were compared by one-way anova,. P <0.001, compared to siNC group; cells were compared for colony forming ability by t-test,. P <0.001, compared to siNC group. Panel a demonstrates reduced expression of TTPAL on gastric cancer tumor cells MKN74 and AGS; panels B and C show inhibition of gastric cancer tumor cell MKN74 and AGS growth following TTPAL knockdown, respectively; FIG. D shows a representative colony forming image; panels E and F show the results of the analysis of colony forming ability to inhibit MKN74 and AGS, respectively, after TTPAL knockdown.

FIG. 6 shows the promotion of the growth of subcutaneous transplanted tumors in nude mice by over-expression of TTPAL, wherein the growth rate of tumors was compared by one-way anova (P <0.0001, compared to the empty vector group); the weight of the tumors was compared by t-test analysis (. P <0.05, compared to the empty vector group). Panel A and B show the morphology of subcutaneous tumors before and after isolation from mice, respectively; panels C and D show the volume and weight of the subcutaneous tumor, respectively; panels E and F show images and scores of immunohistochemical staining, respectively.

DESCRIPTION OF THE SEQUENCES

1, SEQ ID NO: an amino acid sequence of TTPAL subtype-1.

2, SEQ ID NO: an amino acid sequence of TTPAL subtype-2.

3, SEQ ID NO: the cDNA sequence of TTPAL.

Detailed Description

The application provides an application of TTPAL in monitoring or prognosis of disease progression of gastric cancer patients. TTPAL was found by whole genome sequencing, also known as an alpha-tocopherol transfer protein analog, and the gene was mapped to the long arm q13.12 of chromosome 20. TTPAL contains at least two subtypes: subtype-1 and subtype-2. The amino acid sequence of TTPAL subtype-1 is shown in SEQ ID NO. 1. The amino acid sequence of TTPAL subtype-2 is shown in SEQ ID NO. 2. The cDNA sequence of TTPAL is shown in SEQ ID NO. 3.

The inventors of the present application found for the first time that the expression of TTPAL protein or a fragment thereof in gastric cancer cells correlates with the prognosis of gastric cancer. High expression of TTPAL protein or a fragment thereof in gastric cancer tissue is indicative of a poor prognosis of gastric cancer. That is, high levels of expression of TTPAL or fragments thereof are positively correlated with poor prognosis in the patient. This finding provides an important means for risk assessment or prognosis of gastric cancer. TTPAL or fragments thereof can be used as a novel prognostic predictive marker of gastric cancer.

The kits disclosed herein can be used to monitor the disease state of gastric cancer patients. For example, if TTPAL protein or a fragment thereof, or mRNA for TTPAL protein or a fragment thereof, is detected to be expressed at a high level in a gastric cancer patient using the above-mentioned kit, it indicates that the prognosis of the patient is poor, e.g., there are more risk factors, and the survival rate is low. In a specific embodiment, high expression of TTPAL can be used as an independent prognostic diagnostic factor for gastric cancer.

In certain embodiments, a fragment of a TTPAL protein comprises at least 20-300 amino acids, e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, and any number therebetween. For example, in some specific embodiments, the fragment of TTPAL protein comprises amino acid residues 1-179 of the sequence shown in SEQ ID NO. 1 or 2.

In certain embodiments, the TTPAL protein fragment differs from the amino acid sequence set forth in SEQ ID No. 1 or 2 by a substitution, deletion and/or addition of about 1-30, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In some specific embodiments, the TTPAL protein fragment is truncated at the C-terminus or N-terminal region of the amino acid sequence set forth in SEQ ID No. 1 or 2 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids.

In some embodiments, the TTPAL protein fragment has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with the amino acid sequence shown in SEQ ID NO. 1 or 2. In a preferred embodiment, the polypeptide variant has more than 99% homology with the sequence shown in SEQ ID NO. 1. "homology" as used herein is defined as the percentage of residues in an amino acid or nucleotide sequence variant that are identical, if necessary to the maximum percentage, after alignment and introduction of gaps in the sequence. Methods and computer programs for alignment are well known in the art.

In some embodiments, the kits disclosed herein comprise a binding agent that specifically binds to TTPAL protein or a fragment thereof. The phrase "specifically binds" when used in describing the binding relationship of a particular molecule to a protein or peptide refers to a binding reaction that is capable of determining the presence of the protein in a heterogeneous population of proteins and other biological products. Thus, under the specified binding test conditions, a specified binding agent (e.g., an antibody) binds to a particular protein at least twice that of the background and does not significantly bind other proteins present in the sample in significant amounts. Thus, a binding agent is said to "specifically bind" to a TTPAL protein or fragment thereof if it reacts at a detectable level (e.g., in an ELISA assay) with the TTPAL or fragment thereof and does not react detectably in a statistically significant manner with an unrelated polypeptide under similar conditions.

In particular embodiments, the binding agent is selected from the group consisting of antibodies, soluble receptors, aptamers, antibody mimetics, and the like. In a preferred embodiment, the binding agent is an antibody. Optionally, the antibody is labeled with a detectable moiety. The antibody may be a monoclonal antibody or a polyclonal antibody. In some embodiments, the kit may comprise at least two different antibodies, one for specifically binding to the TTPAL protein or fragment thereof (i.e., a primary antibody) and the other for detecting the primary antibody (i.e., a secondary antibody), the secondary antibody typically being linked to a detectable moiety. In some embodiments, the detectable moiety is selected from the group consisting of enzymes (e.g., horseradish peroxidase HRP and alkaline phosphatase AP or derivatives thereof APAAP, PAP), fluorophores (e.g., FITC, Rhodamine, TexasRed, PE, Rhodamine, Dylight, etc.), biotin, gold particles, and the like, and for Western Blot and ELISA, commonly used secondary antibodies are enzyme-labeled secondary antibodies; while in cell or tissue labeling experiments (cytoimmunochemistry, tissue immunochemistry, flow cytometry) a fluorophore-labeled secondary antibody is usually used, and in immunohistochemistry, a horseradish peroxidase or alkaline phosphatase-labeled secondary antibody can also be used. If a greater degree of amplification of the detection signal is desired, a Biotin/Avidin detection system may be used. In some fluorescent detection schemes, it is desirable to select different fluorescent labels; the secondary antibody marked by gold particles is more applied to an immune electron microscope.

In some embodiments, the binding agent, e.g., an antibody, has an affinity for the TTPAL protein or fragment thereof of at least about 1 nM. In certain exemplary embodiments, the binding agent, e.g., antibody, has an affinity for the TTPAL protein or fragment thereof of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM. In certain embodiments, the binding agent binds to an epitope within a splice junction unique to the TTPAL protein of the sequence shown in SEQ ID NO. 1 or 2.

The term "antibody" as disclosed herein refers to an antigen binding protein having a substantially four polypeptide chain structure, consisting of two heavy chains and two light chains. Antibodies have the ability to specifically bind to an antigen. The term "antibody" also refers to antigen-binding and epitope-binding fragments of antibodies, such as Fab fragments, that can be used in immunoaffinity assays. There are many antibody fragments that are well characterized. For example, pepsin digests the antibody from the C-terminus of the disulfide bond in the hinge region to give F (ab')₂I.e. dimers of Fab', which is itself linked to V by a disulfide bond_H-C _H1 linked light chain. Under mild conditions, F (ab')₂Can be reduced to break disulfide bonds in the hinge region, thereby resulting in (Fab')₂DimerConverted to Fab' monomer. The Fab' monomer is essentially a Fab with a partial hinge region (see, e.g., Fundamental Immunology, Paul, ed., Raven Press, n.y. (1993) for a more detailed description of other antibody fragments). Although various antibody fragments are defined in terms of digestion of intact antibodies, it is understood by those skilled in the art that fragments can be synthesized de novo either chemically or using recombinant DNA methods. Thus, the term antibody also includes antibody fragments produced by alteration of intact antibodies or synthesized using recombinant DNA methods. As described herein and known in the art, the term antibody includes variants of antibodies, such as FABs, human antibodies, modified human antibodies, single chain antibodies, non-human antibodies, and the like.

In a preferred embodiment, the kits disclosed herein comprise an antibody that specifically recognizes and specifically binds to the TTPAL protein of the sequence shown in SEQ ID NO. 1, significantly increasing the level of detection, including, for example, specificity and sensitivity of detection, of TTPAL protein expressed in gastric tissue.

In some embodiments, the kits disclosed herein comprise a polynucleotide probe that specifically hybridizes to TTPAL mRNA. Optionally, the polynucleotide is labeled with a detectable moiety (e.g., a fluorophore). In some embodiments, the kit may comprise at least two oligonucleotide primers for amplifying at least a segment of TTPAL DNA or mRNA by PCR, in particular RT-PCR. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence. The exact length of the primer depends on many factors, including temperature, source of primer, and method used.

Polynucleotide hybridization methods, as used herein, are methods of detecting the presence and/or amount of a predetermined polynucleotide sequence based on the ability of the polynucleotide sequence to form Watson-Crick base pairing with a polynucleotide probe of known sequence under suitable hybridization conditions. Examples of such hybridization methods include Southern blotting, Northern blotting, microarray and in situ blotting. The polynucleotide probes may be conjugated to a chemical moiety, such as, but not limited to, a fluorescent moiety or fluorophore, capable of generating a detectable fluorescent signal. In some embodiments, the probe is a molecular beacon.

In some embodiments, the kits disclosed herein may further comprise a suitable standard control. In specific embodiments, the standard control is indicative of the average value of TTPAL protein or fragment thereof or TTPAL mRNA in the gastric epithelium of a healthy individual not suffering from gastric cancer. In some embodiments, the standard control may be provided in the form of a set value. In addition, the kits of the invention can provide instructions for the user to analyze the test sample and assess the presence, risk or status of gastric cancer in the test individual.

As used herein, "standard control" refers to a predetermined amount or concentration of a polynucleotide sequence or polypeptide, such as TTPAL mRNA or protein, present in a normal disease-free tissue sample (e.g., a normal gastric epithelial tissue sample). Standard control values are suitable for use in the methods of the invention as a basis for comparison of the amount of TTPAL mRNA or protein present in a test sample. Established samples used as standard controls provide the average amount of TTPAL mRNA or protein that is typical of gastric epithelial tissue samples (e.g., gastric mucosa) of average healthy persons without any gastric disorders as conventionally defined, particularly gastric cancer. The standard control value may vary depending on the nature of the sample as well as other factors such as the sex, age, race, etc. of the individual on which the control value is based.

The present application also provides methods for detecting the expression of TTPAL protein or fragments thereof in a sample. In some embodiments, immunohistochemical staining is used to detect and assess the expression of TTPAL protein or fragments thereof. In a specific embodiment, the method comprises the steps of deparaffinizing the prepared tissue section to water, antigen retrieval, nonspecific site blocking, primary antibody incubation, secondary antibody incubation, staining nuclei, dehydrating the mounting, and the like.

The inventors of the present application also found that TTPAL can promote the growth of gastric cancer tumor cells. For example, in some embodiments, gastric cancer cell growth is accelerated and the colony forming ability of the cells is enhanced after over-expression of TTPAL in a gastric cancer cell line. In some embodiments, knockdown of TTPAL is capable of inhibiting the growth and colony forming ability of gastric cancer cells. In other embodiments, the tumor growth of a subcutaneous tumor transplant in an animal is promoted following injection of a gastric cancer cell line that overexpresses TTPAL into the animal.

Based on the above, the application also provides the application of the reagent for specifically inhibiting the expression of TTPAL protein or fragments thereof or the expression of TTPAL mRNA in inhibiting the growth of gastric cancer cells. In some embodiments, the agent that specifically inhibits a TTPAL protein is a TTPAL protein-specific binding agent, e.g., an antibody. In some embodiments, the agent that specifically inhibits TTPAL mRNA expression is an interfering RNA that specifically reduces TTPAL mRNA expression.

"individual," as used herein, refers to mammals, including, but not limited to, primates, cows, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. The methods and kits of the present application are applicable to biological samples from humans. In particular embodiments, the individual has been determined to be at risk of having cancer or suspected of having cancer.

The methods and kits of the present application can also be used to monitor the effectiveness of gastric cancer therapy. In alternative embodiments, the level of expression of TTPAL protein or TTPAL mRNA may decrease over time in an individual if the treatment regimen is effective; if the treatment regimen is ineffective, the level of the biomarker will not change or may increase over time.

In the present specification and claims, the words "comprise," "comprises," and "comprising" mean "including but not limited to," and are not intended to exclude other moieties, additives, components, or steps.

It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application may be applied to any other aspect, embodiment or example described herein unless incompatible therewith.

The following examples are illustrative only and are not intended to limit the scope of the embodiments of the present application or the scope of the appended claims.

Examples

Example 1: TTPAL can be used as a prognosis judgment marker of gastric cancer

1.1 sample treatment

The sample processing steps are as follows:

1) collecting gastric cancer tumor tissues and paracancer normal tissues (serving as controls), and preparing a gastric cancer fresh tissue sample or an embedded wax block specimen;

2) carrying out gradient dehydration on fresh tissues and embedding;

3) the embedded tissue was sliced to a thickness of 5 μm and kept ready for use.

1.2 immunohistochemical staining

Immunohistochemical staining was performed as follows

1) Slice dewaxing to water: placing the tissue slices into dimethylbenzene 1,

dimethylbenzene

2, 100%

absolute ethyl alcohol

1, 100% absolute ethyl alcohol 2, 95% absolute ethyl alcohol, 90% absolute ethyl alcohol, 80% absolute ethyl alcohol and 70% absolute ethyl alcohol in sequence, and soaking each reagent for 10 minutes; -

2) Antigen retrieval: soaking the dewaxed slices in water for 15 minutes, soaking the slices in a 3% hydrogen peroxide-methanol solution for 30 minutes, pouring out the 3% hydrogen peroxide-methanol solution, washing the slices with PBS for 3 times, 5 minutes each time, adding a citric acid antigen repairing solution, and boiling the slices with microwaves for 3 times, wherein the interval between every two times is 3 minutes; cooling to room temperature to expose the antigenic sites;

3) non-specific site blocking: cooling the glass slide to room temperature, changing the glass slide into PBS buffer solution, washing for 3 times, washing for 5 minutes each time, adding goat serum, sealing, and keeping the temperature at 37 ℃ for 1 hour;

4) primary antibody (purchased from Novus, under the accession number NBP1-92544) was incubated overnight: the slide glass was taken out, the back and front sides were wiped with absorbent paper, and a primary antibody, which is an antibody against the gastric cancer prognosis marker TTPAL, was added to the negative control group. Incubating overnight in a refrigerator at 4 ℃;

5) and (3) secondary antibody incubation: taking out the glass slide, putting the glass slide into PBS buffer solution, washing the glass slide for 3 times, wiping the glass slide for 5 minutes each time, adding a secondary antibody (the secondary antibody is a peroxidase-labeled secondary antibody corresponding to a primary antibody), and incubating the glass slide in an incubator for 1 hour at 37 ℃;

6) dyeing: performing color development on the tissue slices by using a DAB color developing solution;

7) dyeing the core: marking the number of cells, staining the tissue section with hematoxylin for 1-3 minutes; then washing with tap water for 15 minutes;

8) dewatering and sealing: placing the dyed tissue slices into dimethylbenzene 1,

dimethylbenzene

2, 100% absolute ethyl alcohol 2, 70% absolute ethyl alcohol, 80% absolute ethyl alcohol, 90% absolute ethyl alcohol, 95% absolute ethyl alcohol, 100%

absolute ethyl alcohol

3, 100% absolute ethyl alcohol 4, dimethylbenzene 3 and dimethylbenzene 4 in sequence, soaking each reagent for 5 minutes, sealing the slices with a sealing agent, and drying in the air for later use.

1.3 reading score

Scoring the sealed tissue sections, wherein the staining intensity score is 0 points (negative), 1 points (1+), 2 points (2+), 3 points (3 +); the positive staining rate scores were scored as 0 (negative), 1 (i.e., 1-25% of the cells stained), 2 (26-50% of the cells stained), 3 (51-75% of the cells stained), and 4 (76-100% of the cells stained). The total score was calculated as the product of the "staining intensity score" and the "staining positive rate score". The total score is less than or equal to 4, and the total score is more than 4, and the total score is a low expression group, and the total score is more than 4, and the total score is a high expression group.

1.4 results of the experiment

1) As shown in fig. 1, the immunohistochemical staining analysis of 86 gastric cancer tissue (fig. 1B) and paracancerous normal control tissue (fig. 1A) revealed that the expression of TTPAL was significantly increased in the gastric cancer tissue. The TTPAL expression score of gastric cancer tissues was significantly higher than that of control tissues (fig. 1C, P < 0.001).

2) As described above, immunohistochemical score of 4 or less is low expression group, and total score of 4 or more is high expression group. In 86 gastric cancer tissues, 30 of the tissues with high expression level of TTPAL (i.e. score > 4) were evaluated, and further the correlation between the expression level of TTPAL and clinical features including the size of tumor, pathological grade, TNM stage, etc. of patients was evaluated, and the result of multi-factor COX regression analysis (as shown in FIG. 2) indicated that high expression of TTPAL could be used as an independent prognostic diagnostic factor for gastric cancer.

3) FIG. 3 shows the Kaplan-meier analysis curve for the prognosis of gastric cancer patients. As can be seen from the figure, the survival rate of the patients with high expression level of TTPAL in tumor tissues is lower than that of the patients with low expression level (P <0.01), which indicates that the prognosis of the gastric cancer patients can be judged according to the expression level of TTPAL.

Example 2 TTPAL promotes gastric cancer tumor cell growth in vitro

The following specific procedures of the MTT method used in this example were as follows: cells were seeded in 96-well plates at a cell density of 1000 cells/well and 5 plates were simultaneously seeded, one plate was removed every 24 hours, 10. mu.l of MTT solution (5mg/ml in PBS) was added to each well, incubation was continued for 4 hours, the culture was terminated, and the culture supernatant in the wells was carefully aspirated off. Centrifugation is required for suspension cells and culture supernatant is aspirated from the wells. Add 200. mu.l DMSO/well and shake for 10 minutes to fully melt the crystals.

The specific procedures for the cell colony formation experiments used in this example are as follows: the cells were seeded at a cell density of 1000 cells/well in 6-well plates, cultured for 7-10 days, fixed with methanol, stained with 0.1% crystal violet, observed for the number of clones and counted.

The steps for over-expressing TTPAL in this example are as follows:

1) construction of overexpression of TTPAL lentiviral vector: using PCMV6-TTPAL DNA as a template, cloning TTPAL molecules to a PLV-Puro lentiviral vector, and preparing a virus solution after completely correct sequencing.

2) Packaging virus liquid: transfecting a target gene plasmid, a virus packaging plasmid PSPAX2 and PMD2G into HEK293FT cells by Lipofectmine2000 according to a ratio of 3:2:1, changing the cells for 4-6h, supplementing the cells for 24h, centrifugally collecting cell culture supernatant for 48h, and filtering to obtain the packaged virus liquid.

3) Virus-mediated transfection and selection of stably expressing TTPAL cell lines: adding virus solution and 10% FBS culture medium into cells according to the proportion of 1:1, performing resistance screening on the transfected cells after 48 hours, taking normal untransfected cells as a control, performing expanded culture after all normal controls are killed by antibiotics, verifying by using PCR and Western blot, and using the cell model for follow-up research after successful construction.

The specific operation process of knocking down TTPAL described in this example is as follows: the siNC (control plasmid) and the alternative siTTPAL-1 (siTTPAL)^#2) And sittPAL-1 (siTT)PAL^#1) Respectively transfecting to TTPAL high-expression gastric cancer cell lines, verifying interference efficiency by using q-PCR and RT-PCR, selecting siTTPAL interference sequences with high interference efficiency (the knockdown is more than 70%) to transfect cells, extracting cell total RNA 24h after transfection, and detecting the mRNA expression of TTPAL by RT-PCR; total cellular protein was extracted 48h after transfection and TTPAL protein expression was detected by Western Blot.

2.1The influence of TTPAL overexpression on the growth of gastric cancer tumor cells is studied by MTT method and cell colony formation experiment. Specifically, we found that, after over-expressing TTPAL in two gastric cancer cell lines MGC803 and BGC823 (fig. 4A), the expression of TTPAL was increased to promote the growth of gastric cancer cells and increase the cell viability by one-way anova (fig. 4B and 4C, P)<0.001). A representative colony forming image is shown in fig. 4D. Analysis by t-test showed that the over-represented TTPAL increased the colony-forming ability of the cells (FIGS. 4E and 4F,. SP)<0.01,***P<0.001)。

2.2The influence of TTPAL knockdown on the growth of gastric cancer tumor cells is researched by an MTT method and a cell colony formation experiment. Specifically, TTPAL in gastric cancer tumor cells AGS and MKN74 was knocked down by small interfering RNA siTTPAL. Following decreased expression of TTPAL (as shown in FIG. 5A), growth of gastric cancer cells was inhibited and cell viability was decreased by one-way anova (FIGS. 5B and 5C, P)<0.001). Analysis by t-test showed that TTPAL knockdown inhibited the colony forming ability of gastric cancer cells (fig. 5E and 5F,. beta.p)<0.001)。

Example 3 TTPAL promotes gastric cancer tumor cell growth in vivo

In this example, we randomized the control cell line MGC 823/vector or MGC823/TTPAL (1X 10)⁶Individual cells) were injected dorsal side of nude mice and tumor growth was compared. Tumor volumes were measured every 2 days for 15 days, and the longest and shortest tumor diameter lengths were measured and tumor volumes were calculated as the square of 0.5X the largest diameter X the smallest diameter. On day 15 of the experiment, nude mice were sacrificed, subcutaneous tumor tissue was collected, size measured, weighed, and the embedded tissue was scored by immunohistochemical staining. FIGS. 6A and 6B show subcutaneous tumors before isolation from mice, respectivelyAnd isolated forms. Fig. 6C and 6D show the volume and weight of the subcutaneous tumor, respectively. It can be seen that the tumor growth rate was significantly increased in the MGC823/TTPAL group compared to the MGC 823/vector group (FIG. 6C, one-way ANOVA, p)<0.0001). Furthermore, the tumor weight was also significantly increased in the MGC823/TTPAL group compared to the MGC 823/vector group (FIG. 6D, t test, p)<0.05). At the same time, we also stained the tissues using the immunohistochemical staining method described previously (fig. 6E) and scored (fig. 6F), which showed that the MGC823/TTPAL group score was significantly higher than the MGC 823/vector group.

Various modifications and equivalents may be made to the embodiments disclosed herein without departing from the spirit and scope of the disclosure. Unless the context indicates otherwise, any feature, step, or embodiment of an embodiment of the present disclosure may be used in combination with any other feature or embodiment.

Sequence listing

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Application of <120> TTPAL as gastric cancer monitoring or prognosis judgment marker

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<210> 2

<211> 308

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 2

Met Ser Glu Glu Ser Asp Ser Leu Arg Thr Ser Pro Ser Val Ala Ser

1 5 10 15

Leu Ser Glu Asn Glu Leu Pro Pro Pro Pro Glu Pro Pro Gly Tyr Val

20 25 30

Cys Ser Leu Thr Glu Asp Leu Val Thr Lys Ala Arg Glu Glu Leu Gln

35 40 45

Glu Lys Pro Glu Trp Arg Leu Arg Asp Val Gln Ala Leu Arg Asp Met

50 55 60

Val Arg Lys Glu Tyr Pro Asn Leu Ser Thr Ser Leu Asp Asp Ala Phe

65 70 75 80

Leu Leu Arg Phe Leu Arg Ala Arg Lys Phe Asp Tyr Asp Arg Ala Leu

85 90 95

Gln Leu Leu Val Asn Tyr His Ser Cys Arg Arg Ser Trp Pro Glu Val

100 105 110

Phe Asn Asn Leu Lys Pro Ser Ala Leu Lys Asp Val Leu Ala Ser Gly

115 120 125

Phe Leu Thr Val Leu Pro His Thr Asp Pro Arg Gly Cys His Val Val

130 135 140

Cys Ile Arg Pro Asp Arg Trp Ile Pro Ser Asn Tyr Pro Ile Thr Glu

145 150 155 160

Asn Ile Arg Ala Ile Tyr Leu Thr Leu Glu Lys Leu Ile Gln Ser Glu

165 170 175

Glu Thr Gln Asp Gly Phe Pro Ile Arg Ile Lys Ala Val His Val Val

180 185 190

Asn Glu Pro Arg Ile Phe Lys Gly Ile Phe Ala Ile Ile Lys Pro Phe

195 200 205

Leu Lys Glu Lys Ile Ala Asn Arg Phe Phe Leu His Gly Ser Asp Leu

210 215 220

Asn Ser Leu His Thr Asn Leu Pro Arg Ser Ile Leu Pro Lys Glu Tyr

225 230 235 240

Gly Gly Thr Ala Gly Glu Leu Asp Thr Ala Thr Trp Asn Ala Val Leu

245 250 255

Leu Ala Ser Glu Asp Asp Phe Val Lys Glu Phe Cys Gln Pro Val Pro

260 265 270

Ala Cys Asp Ser Ile Leu Gly Gln Thr Leu Leu Pro Glu Gly Leu Thr

275 280 285

Ser Asp Ala Gln Cys Asp Asp Ser Leu Arg Ala Val Lys Ser Gln Leu

290 295 300

Tyr Ser Cys Tyr

305

<210> 3

<211> 6248

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 3

gtcacttccg gcgcgagagg ccgggcaggc cgggcaggga gtgcgggtcg gttctgcgtg 60

cgctgccgga cgaggctccc gccgccgatt gacccgcgct ccgcccgtag tcgggccgtt 120

ctgttccaag agataaccat tgggaccttg gtagctaatg tccgaagaaa gtgactctct 180

gagaaccagc ccttctgtgg cctcactctc tgaaaatgag ctgccaccac cacctgagcc 240

tccgggctat gtgtgctcac tgacagaaga cctggtcacc aaagcccggg aagagctgca 300

ggaaaagccg gaatggagac ttcgagatgt gcaggccctt cgtgacatgg tgcggaagga 360

gtaccccaac ctgagcacat ccctcgacga tgccttcctg ctgcgcttcc tccgagcccg 420

caagtttgat tacgaccggg ccctgcagct cctcgtcaac taccacagct gtagaagaag 480

ctggcccgaa gtcttcaata acttgaagcc atcagcctta aaagatgtcc ttgcttccgg 540

gttcctcacc gtgctgcccc acactgaccc caggggctgc catgtcgtct gcatccgccc 600

agacagatgg ataccaagca actatccaat tactgaaaac atccgagcca tatacttgac 660

cttagaaaaa ctcattcagt ctgaagaaac ccaggtgaat ggaattgtaa ttcttgcaga 720

ctacaaagga gtgagtttat caaaagcatc tcactttggc ccttttatag ccaaaaaggt 780

gattggcatc ctccaggatg gtttccccat tcggataaaa gcagtccatg tggtgaatga 840

acctcgaata tttaaaggca tttttgccat cataaaacca tttctaaagg agaaaatagc 900

aaacagattc ttcctccatg ggtctgactt gaactctctc cacacaaacc ttccaagaag 960

catcctcccc aaggagtatg ggggcacggc tggggagctg gacactgcca cctggaacgc 1020

ggtactgctg gcttcagaag acgattttgt gaaagagttc tgccaacctg ttcctgcctg 1080

tgacagcatc ctgggccaga cgctgctgcc cgagggcctg acctcagatg cacagtgtga 1140

cgactccttg cgagctgtga agtcacagct gtactcctgc tactagcccg tcccccaggg 1200

tcaccatctt taattctttt ccttcttttc tttggagagg cacaaggaga atttaagggt 1260

ccatggattc agtcttgctc cttgtaatta aactgcagga tggaggaaca gcctgagata 1320

tgagcatgag cccattttgg ggtaagcctt tggttacttt aattactcca tggaagacat 1380

ggaaaatgtc cccactgatt cttaaacatt tggaatccca gtctgcaact attaatctgg 1440

aggctatatc tattttgttt tgctttttgg ttggggggtg gtgatctggt tcttacacat 1500

cttggaagca agaacaatca ggaccaaagt cactttgatc ccacttttcc aggagaaaaa 1560

ccacctgttt ggccagtgag aactacttgt atgaaataat ttggccaaac cttcagtgtg 1620

accaaatgtg agactgggag tttgtgtttt tcacaggaac cctaagtata gacctctgct 1680

gctcatcagg aaacttactg gagatgaagg ccccagctgt tgtcaccggg tttggaaagc 1740

accttaactg aatcatgtaa gcatcaggac ataagcagca ctttgtggtc aaatgtggaa 1800

gccggagact tcaaagcacc tctgggaccc actggttgaa gtttgcaata gaaacttaag 1860

ttttcccaaa tccataaagc cttagccctg gttctcaata gaatcaggga cctagcagga 1920

aatgatttta ctcaacctaa aatgctggat cccaggcccg tgtagctata agaattctgg 1980

cctggatccc aggtgtacaa ctatggacaa gatatgggcc tctactttct cctctataaa 2040

atgaggctgg atgaaatgtc agctagggcc attttggctg ctgaggctct gggatttggt 2100

ttagttactg aatgttagat tttctgccta gaaagataac tatctagata caagtggttg 2160

gatcctgttt ttgtttgtgg tacatgtgtc tttccaagag agatgtgtca ccaattagcc 2220

ctgcctttaa agaaactatt atgtgtattc ctgggactca ctgacaccaa ttttcttttt 2280

atagtgatgg ttcaattttg aaaagatggc ttttgtgagg ccaggttaag gtgaccagga 2340

tcttgtatga tgaattcctt ccatccctga gactctggta ctatattgta aacctggcta 2400

cagtagttaa ttacttgaga ttctttaatt ttggtctctg agctgggcgt ggtggttcat 2460

gcctgtaatc ccagcacttt gggaggccaa ggtgtgcgga tcacaaggtc aggagttcga 2520

gacccgcctg gccaagatgg tgaaacccca tctctactaa aaatacaaaa attagctggg 2580

cgtggtggcg ggctcctgta gtcccagcta ctcgggaggc tgaggcagaa gaatcacgtg 2640

aacccaggag gcagaagtgg cagtgagcca agatcgcacc actgcactcc agcctgggcg 2700

acagagcaag actctgcctc aaaaaaaaaa aaaaaaaaaa aaaatttttt tttttttggt 2760

ctctggaaat gaacacaagg gcaggttatt cctgggtcac ttctgggccc ccctgccctc 2820

ccagccccac ttgagtttct ctctctggtg tgggtgaacc agtcagcctg aatgttctgc 2880

atttcagcac tttagaacct ccctgtgaag attttagcct tagcccaaac atcaaattag 2940

acggttcaca tgatggtttt tgacctattt cctttctaat gtattccaca tgatcatggt 3000

gttaaatagt gaaaagtact gtgttgtgtg tgcaccttct ccgtgcattt attagactaa 3060

ccagtcaagc agacagctca gttagggaga aaacaatact ctgaaatttg aaggccaatc 3120

tgttgttact aagctgttta tctctattgc ctttttaaat gtctggataa gttgttggtg 3180

gaaattaagt tacttaacct cattaatacc aattctagag aaagttcttt tcaccatgga 3240

tagtaaccct ggatcctcta cggtactggc tgagctggaa gtgccaaaaa gcactcctgg 3300

ctgcttctgg ttccatctga tgatgatgtg acacacactg ctgaaaaggc ccaagcaggg 3360

caagtgggat ggctgaagga gggaaggagg gggttcagaa cccactggcc tggatgggag 3420

aactgggtgg aggcttcccc aagagggaag acagataaac aaaacaaaac aaaaactggg 3480

taaagaggaa tgaatcactc agccctgatg tttcaattct acactgcatt cctggccagt 3540

cgcatttgtt ttaatgcagg catggccaca gctctcctag agaattatct caaagaccag 3600

aagggacctg gagaggccta tttcttaggt ttttccagtt ggacaaggaa ggagtggttt 3660

cactcagctt ctagaaggag ttggagccta agtttatctg cctccgggag ctgcttgctt 3720

tgttttggct ccgaagaggt atcagatagt tttgacacct caggaaactt gaaccaagct 3780

gtgaaaccaa gacctccctg cgtgaaaatc aaggtggtct ccttgtggct tcaccaggat 3840

gtttgtgtca ggctgtctca gcagggtggg gaatgaccag ccagggagca cagtgagcct 3900

tactcagcac tggggagcgc actggtgagg caaacccatg aacctcaaga actgggagta 3960

tgttccttca gggagaagtt ctggcccatt gcacaaacac ttggaaatta acttttccct 4020

aaattcaaga tagtgtggtg tcggaaggaa atgggacagt aaattaggag acgcgggctc 4080

cacctttacc ttactacgtg gcctttgatg agctggctta acatccctga gtgattccat 4140

gatagagatc tatactccaa actttattcc tggtatctag ttggcttcac tgccacagac 4200

actgtactct ctctttttag aagttttttt cctttttttt ccccctcaat tggcttattt 4260

agtaaattta ggtctgaatg aattggtacc taaaagcttc caaatctata catttcagaa 4320

tatgggaatt ttcttcctct tcttccccca tatcccaaca tggaattctg gaaaactgtg 4380

cctcttcccc tgttctgcct tcatggggga gagactggat gaaatctaca aaaacagcca 4440

aaagtgccac cctggcttca tgtcttgaat ttctaacttg ctcttggcaa aggtcgcttt 4500

attttttaat ttttttcctt gcattttctt ttttattcta ttgctgctgc aaaaattaag 4560

gcaaaagtag ctttcgatct ttcatatttc atcctggttt cacaaggagt cacttatctt 4620

aggaggtctg taagtcaggt tacaaggccg ggagcggtgg ctcacacctg taatcccaac 4680

actttgagcg gctgaggtgg gtgtatcacc tgaagtccgg agtttgagac cagcctggcc 4740

aacatggtga aaccccgtct ctactaaaaa tacaaaaaat tggctgggca tggtggtggg 4800

tgcctgtaat cccagctact tgggaagctg aagcaggaga atcacttaaa cccaggtggc 4860

ggaggttgca gtgagccgac atcgcgccat tgcactccag cctgggtgac gagtgaaact 4920

ccatctcaaa aataaaaatt gaaaaatcag gttacaaaac accatttttc ccgaaataag 4980

acaataagag gcttttctct gaattccttt atattgagcc tttcagaatt ctccctgggt 5040

gggcaatttc tttaaatagt atttgaccct cagatcaatc ctgggaattt ttttcatttg 5100

ggtagcaaaa gctagagtat tgctgtggcg attataatac ttttaaaaag ttttaccatt 5160

ttaaagttgc caacatttaa ttaaggtttt cctttgaagc ctcctttaat ttagggagta 5220

aaatgttagc taaaccaatt atatactata tactatacac tgtatctcct gtggccatga 5280

gaggtgtggc tataccgaac agaaacatgc ctactgttca ggaaagatgt cagttctggt 5340

aacacctctc tgtattggga tctgttaatt ttgtaaatct aaattcttct gctcttggcc 5400

aggtgcagta gttcatgccc tgtaatccta gaactttggg aggctgaagg gggggcggat 5460

catttgaggt caggagtttg agaccagcct ggccaacatg gtgaaacccc atctctacta 5520

aaaagtacaa aaattagctg ggtgtggtgg tgcgtgcctg taatcccaac tacttgggag 5580

gctgaggcag gagaatcgct tgaacctggg aggcagcggt tgcagtaagc caagactatg 5640

ccactgcact cccgcctggg tgacaaagca atattctgtg tcaaataaat aaattcattc 5700

ttctgctctc ctgacttaga gaaatggttt gcttaaaatg ctagtaacaa acatcacagt 5760

caacaggagc ttgcttcatg cgaaggatca atgtgatttg tggatggaga tgatagtgat 5820

gaaattcctg tttcatgggg ctgtttttct tttcatctca ctgggcagca ggtttagtga 5880

ggcagtgaga tgctgctgct gtggattctt gtagctatgc ctcggcttct tggcatatca 5940

ggtaggaacc tgttacaagt gaaatacttg aaacctctct gaccaagagc ctctgatgga 6000

gtgggaggtg agctaattct ctgaccagct tggggcactg tttcagccac tggtcacatt 6060

ccttgcttca aactgaaatt cagtttggct ttgagtatag ggatacatgg tggattcatg 6120

tacttcagtg tttgttttga ccaaagttta tttttctagt gcattttcta agtcaaagtg 6180

gtgaaaatat gtaataattt tagtatgcat gactcagtct gaaacaataa aaatctctga 6240

aaaatgtg 6248

Claims

1. A kit for monitoring or prognosing gastric cancer in an individual comprising reagents for detecting the expression level of TTPAL protein, or a fragment thereof, in a sample from said individual, preferably the amino acid sequence of said TTPAL protein is as set forth in SEQ ID No. 1 or 2, and preferably the fragment of said TTPAL protein comprises amino acid residues 1-179 of the sequence as set forth in SEQ ID No. 1 or 2.

2. The kit of claim 1, wherein said agent comprises a specific binding agent to said TTPAL protein or fragment thereof, preferably said binding agent is an antibody.

3. The kit according to claim 2, wherein the antibody is capable of specifically recognizing a protein having the sequence shown as SEQ ID NO 1 or 2 or a protein fragment comprising amino acid residues 1-179 of the sequence shown as SEQ ID NO 1 or 2, preferably the antibody is capable of specifically recognizing a protein having the sequence shown as SEQ ID NO 1.

4. The kit according to any one of claims 1 to 3, wherein the sample is selected from gastric tissue, serum, plasma or cell culture supernatant, preferably the sample is a fresh tissue sample of gastric cancer or an embedded specimen.

5. The kit of claim 4, wherein the kit further comprises one or more additional reagents for immunochemical staining of the stomach tissue sample.

6. Use of a reagent for detecting the expression level of a TTPAL protein or a fragment thereof in the manufacture of a kit or medicament for monitoring or prognosticating gastric cancer in an individual.

7. The use of claim 6, wherein a poor prognosis of gastric cancer in said subject is indicated if the expression level of TTPAL protein or a fragment thereof in a sample from said subject is greater than a threshold value.

8. A pharmaceutical composition for inhibiting gastric cancer cell growth comprising an agent that specifically inhibits the expression of TTPAL protein or a fragment thereof, preferably said agent is an antibody.

9. Use of an agent that specifically inhibits the expression of a TTPAL protein or a fragment thereof in the manufacture of a medicament for inhibiting the growth of gastric cancer cells.

10. A method for detecting the expression of TTPAL protein or a fragment thereof in a sample, comprising treating said sample with the reagents of the kit of any one of claims 1 to 5, preferably wherein said sample is a gastric cancer tissue sample.