CN114317730B

CN114317730B - Application of TTPAL as gastric cancer monitoring or prognosis judgment marker

Info

Publication number: CN114317730B
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: 2024-01-09
Anticipated expiration: 2040-09-29
Also published as: CN114317730A

Abstract

The present application provides methods for monitoring or prognosis of gastric cancer in an individual by measuring the expression level of TTPAL protein or a fragment thereof, and corresponding kits. The present application also provides methods for detecting the expression level of a TTPAL protein or fragment thereof. In addition, the application also provides a pharmaceutical composition containing an agent for specifically inhibiting TTPAL protein expression and application of the pharmaceutical composition 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 medical and biotechnology arts. In particular, the application provides kits for monitoring or prognosis of gastric cancer and related applications.

Background

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

Gastric cancer TNM staging is currently the most important clinical predictor for judging gastric cancer prognosis. However, many patients have a potential for tumor recurrence even though TNM staging is early. Thus, 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 prognostic determination of gastric cancer in an individual, comprising reagents for detecting the expression level of an alpha-Tocopherol Transfer Protein Analog (TTPAL) protein or fragment thereof, or the mRNA expression level of the TTPAL protein or fragment thereof, in a sample from the individual.

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

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

In some embodiments, the above-described agents include a binding agent that specifically binds to the TTPAL protein or fragment thereof, preferably the binding agent is an antibody to the TTPAL protein or fragment thereof. In other embodiments, the agent comprises a polynucleotide probe capable of specifically hybridizing to mRNA of TTPAL or a fragment thereof.

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

In some embodiments, the above-described kits further comprise one or more additional reagents for immunochemical staining of tissues.

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

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

In yet another aspect, the present application provides a method of detecting expression of a TTPAL protein or fragment thereof, or expression of mRNA of a TTPAL protein or fragment thereof, in a sample, comprising treating the sample with a reagent in a kit as described above, in particular embodiments, the sample is a gastric cancer tissue sample.

Brief description of the drawings

Fig. 1 shows the results of immunohistochemical staining of gastric cancer tissue (panel B) and paracancerous normal control tissue (panel a), wherein the expression of TTPAL in gastric cancer tissue is significantly increased (panel B), and the gastric cancer tissue has a significant statistical difference in TTPAL expression scores from the normal control (panel C). P <0.001, compared to paracancerous normal controls.

FIG. 2 shows the results of a multi-factor COX regression analysis assessing the correlation of TTPAL expression levels and clinical features, indicating that TTPAL high expression can be used as 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 promotion of gastric cancer cell growth by TTPAL after overexpression of gastric cancer cell lines MGC803 and BGC823, wherein the growth rate of cells was compared by single factor analysis of variance, P <0.001, compared to empty vector group; comparing the colony forming ability of the cells by t-test, P <0.01, P <0.001, and comparing with empty vector group. Panel a demonstrates the overexpression of TTPAL in MGC803 and BGC 823; the diagram B and the diagram C respectively show the promotion effect on the growth of gastric cancer cells MGC803 and BGC823 after TTPAL is over-expressed; panel D shows a representative colony forming image; panels E and F show the results of analysis of colony forming ability after MGC803 and BGC823 overexpressing TTPAL, respectively.

Figure 5 shows the inhibition of gastric cancer cell growth by TTPAL knockdown, wherein the growth rate of the cells is compared by one-way anova, P <0.001, compared to the siNC group; comparing the colony forming ability of the cells by t-test, P <0.001, compared to siNC group. Panel a demonstrates reduced expression of TTPAL in gastric cancer tumor cells MKN74 and AGS; panel B and C show the inhibition of growth of gastric cancer tumor cells MKN74 and AGS, respectively, after TTPAL knockdown; panel D shows a representative colony forming image; panels E and F show the results of assays for the ability to inhibit colony formation of MKN74 and AGS, respectively, after TTPAL knockdown.

FIG. 6 shows the promotion of tumor growth by subcutaneous transplantation of nude mice by overexpressing TTPAL, wherein tumor growth rates were compared by one-factor anova (P <0.0001, compared to empty vector group); tumor weights were compared by t-test analysis (< P0.05 compared to empty vector group). Panels a and B show the morphology of subcutaneous tumors before and after separation from mice, respectively; panels C and D show the volume and weight of subcutaneous tumors, respectively; panels E and F show images and scores of immunohistochemical staining, respectively.

DESCRIPTION OF THE SEQUENCES

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

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

SEQ ID NO. 3: cDNA sequence of TTPAL.

Detailed Description

The application provides application of TTPAL in monitoring or prognosis judgment of disease progression of gastric cancer patients. TTPAL was found by whole genome sequencing, also known as alpha-tocopherol transferase analogues, which gene was located in 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 expression of TTPAL protein or a fragment thereof in gastric cancer cells correlates with prognosis of gastric cancer. High expression of TTPAL protein or fragments thereof in gastric cancer tissue indicates a poor prognosis of gastric cancer. That is, high levels of expression of TTPAL or fragments thereof are positively correlated with poor prognosis in patients. This finding provides an important means for risk assessment or prognosis of gastric cancer. TTPAL or fragments thereof can be used as a novel prognosis prediction marker of gastric cancer.

The kits disclosed herein can be used to monitor the disease state of a gastric cancer patient. For example, if the TTPAL protein or fragment thereof, or mRNA of the TTPAL protein or fragment thereof is detected to be expressed at high levels in a gastric cancer patient using the above-described kit, this indicates that the patient has a poor prognosis, e.g., has more risk factors, and has a lower survival rate. In particular embodiments, TTPAL high expression may be used as an independent prognostic diagnostic of gastric cancer.

In certain embodiments, fragments of the TTPAL protein comprise 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 in between. 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 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 acid substitutions, deletions, and/or additions. In some specific embodiments, the TTPAL protein fragment is truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids at the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO. 1 or 2.

In some embodiments, the TTPAL protein fragment has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth 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 identical residues in an amino acid or nucleotide sequence variant after sequence alignment and introduction of gaps, if desired, to achieve a maximum percentage of homology. 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 a TTPAL protein or 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 can determine the presence of a protein in a heterogeneous population of proteins and other biological products. Thus, under the specified binding test conditions, the specified binding agent (e.g., antibody) binds to a particular protein at least twice 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 the binding agent reacts with the TTPAL protein or fragment thereof at a detectable level (e.g., in an ELISA assay) and does not react in a statistically significant manner with an unrelated polypeptide under similar conditions.

In specific embodiments, the binding agent is selected from antibodies, soluble receptors, aptamers, antibody mimics, 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., primary antibody) and the other for detecting the primary antibody (i.e., secondary antibody), the secondary antibody typically having a detectable moiety attached thereto. In some embodiments, the detectable moiety is selected from the group consisting of enzymes (e.g., horseradish peroxidase HRP and alkaline phosphatase AP or its derivatives APAAP, PAP), fluorophores (e.g., FITC, rhodamine, texasRed, PE, rhodamine, dylight, etc.), biotin, gold particles, etc., and for Western Blot and ELISA, the commonly used secondary antibodies are enzyme-labeled secondary antibodies; while secondary antibodies labeled with fluorophores are commonly used in cell or tissue labeling experiments (cytoimmunochemistry, histoimmunochemistry, flow cytometry), secondary antibodies labeled with horseradish peroxidase or alkaline phosphatase may also be used in immunohistochemistry. If a greater degree of amplification of the detection signal is desired, a Biotin/Avidin detection system may be used. In some fluorescence detection schemes, it is desirable to select a different fluorescent label; the gold particle-labeled secondary antibodies are more applied to the 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., an antibody, has an affinity for a 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 located within the unique splice junction of the TTPAL protein of the sequence set forth in SEQ ID NO. 1 or 2.

The term "antibody" as disclosed herein refers to an antigen binding protein having a structure of substantially four polypeptide chains, consisting of two heavy chains and two light chains. Antibodies have the ability to specifically bind to antigens. 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 a number of well-characterized antibody fragments. For example, pepsin digests antibodies from the C-terminus of the disulfide bond in the hinge region, yielding F (ab') ₂ I.e., dimers of Fab', which is itself disulfide-bonded to V _H -C _H 1. Under mild conditions, F (ab') ₂ Can be reduced to break disulfide bonds in the hinge region, thereby bringing about (Fab') ₂ The dimer is converted to Fab' monomer. Fab' monomers are essentially Fab with partial hinge regions (see, e.g., fundamental Immunology, paul, ed., raven Press, n.y. (1993)) for a more thorough description of other antibody fragments. Although various antibody fragments are defined in terms of digestion of intact antibodies, it will be understood by those skilled in the art that fragments may be synthesized de novo either chemically or using recombinant DNA methods. Thus, the term antibody also includes antibody fragments produced by altering intact antibodies or synthesized using recombinant DNA methods. As described herein and in the artThe term antibody is known to include variants of antibodies, such as FAB, human antibodies, modified human antibodies, single chain antibodies, non-human antibodies, and the like.

In a preferred embodiment, the kit disclosed herein comprises antibodies that specifically recognize and specifically bind to TTPAL proteins of the sequence shown in SEQ ID NO. 1, significantly increasing the detection levels, including for example detection specificity and sensitivity, etc., for TTPAL proteins expressed in gastric tissue.

In some embodiments, the kits disclosed herein comprise a polynucleotide probe capable of specifically hybridizing 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 one 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.

The polynucleotide hybridization method used herein is a method 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 hybridization. The polynucleotide probe may be conjugated with a chemical moiety, such as, but not limited to, a fluorescent moiety or a fluorophore, capable of producing 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 a specific embodiment, the standard control is indicative of an average 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 point. In addition, the kit of the present invention may provide instructions for a 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. The established sample used as a standard control provides an average amount of TTPAL mRNA or protein that is typical of average healthy human gastric epithelial tissue samples (e.g. gastric mucosa) that do not suffer from any gastric disease, in particular gastric cancer, as conventionally defined. Standard control values may vary depending on the nature of the sample and 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 expression of a TTPAL protein or fragment thereof in a sample. In some embodiments, immunohistochemical staining is used to detect and assess expression of TTPAL protein or fragments thereof. In specific embodiments, the methods comprise dewaxing prepared tissue sections to water, antigen retrieval, non-specific site blocking, primary antibody incubation, secondary antibody incubation, staining, nuclear staining, dehydration sealing, and the like.

The inventors have also found that TTPAL is capable of promoting the growth of gastric cancer tumour cells. For example, in some embodiments, gastric cancer cells grow faster and the colony forming ability of the cells is enhanced after TTPAL is overexpressed in the gastric cancer cell line. In some embodiments, the TTPAL knockdown is capable of inhibiting the growth and colony forming ability of gastric cancer cells. In other embodiments, injection of a gastric cancer cell line that overexpresses TTPAL subcutaneously in an animal promotes tumor growth in a subcutaneous animal transplant.

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

"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 certain 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 therapies. In alternative embodiments, the level of TTPAL protein or TTPAL mRNA expression 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", "comprising" and "include" 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 particular aspects, embodiments or examples of the present application may be applied to any other aspects, embodiments or examples described herein unless contradicted by context.

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 prognosis marker of gastric cancer

1.1 sample processing

The sample processing steps are as follows:

1) Collecting gastric cancer tumor tissue and paracancerous normal tissue (as control), and preparing a fresh gastric cancer tissue sample or an embedded wax block sample;

2) Gradient dehydrating and embedding fresh tissues;

3) The embedded tissue slice is prepared for standby, wherein the slice thickness is 5 microns.

1.2 immunohistochemical staining

The immunohistochemical staining procedure was as follows

1) Slice dewaxing to water: sequentially placing the tissue slices into xylene 1, xylene 2, 100% absolute ethanol 1, 100% absolute ethanol 2, 95% absolute ethanol, 90% absolute ethanol, 80% absolute ethanol and 70% absolute ethanol, and soaking each reagent for 10 minutes; -

2) Antigen retrieval: soaking the dewaxed slices in water for 15 minutes, soaking the slices in 3% hydrogen peroxide-methanol solution for 30 minutes, pouring out the 3% hydrogen peroxide-methanol solution, washing the slices with PBS for 3 times, each time for 5 minutes, adding citric acid antigen repair liquid, and boiling the slices with microwaves for 3 times at intervals of 3 minutes; cooling to room temperature to expose the antigenic site;

3) Nonspecific site blocking: after the glass slide is cooled to room temperature, the glass slide is changed into PBS buffer solution, and the glass slide is washed for 3 times, wherein each time lasts for 5 minutes, and goat serum is added for sealing at 37 ℃ for 1 hour;

4) One antibody (purchased from Novus corporation under the trade designation NBP 1-92544) was incubated overnight: and taking out the glass slide, wiping the back side and the front side by using water absorption paper, and adding a primary antibody which is an antibody of a gastric cancer prognosis judgment marker TTPAL, and adding PBS into a negative control group. Incubating overnight in a refrigerator at 4 ℃;

5) Secondary antibody incubation: the slide glass is taken out and put into PBS buffer solution for cleaning for 3 times, each time lasts for 5 minutes, the slide glass is wiped dry, and secondary antibodies (the secondary antibodies are peroxidase-marked and correspond to the primary antibodies) are added, and the slide glass is incubated in an incubator for 1 hour at 37 ℃;

6) Dyeing: developing the tissue slice with DAB developing solution;

7) Nuclear dyeing: labeling cell number, staining the tissue slice with hematoxylin for 1-3 min; then washing with tap water for 15 minutes;

8) And (3) removing the water sealing piece: sequentially 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, soaking each reagent for 5 minutes, sealing the tablet, and airing for later use.

1.3 read scoring

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

1.4 experimental results

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

2) As described above, the total score of immunohistochemical score ∈4 was lower expression group and total score > 4 was higher expression group. In 86 gastric cancer tissues, TTPAL high expression (namely, score > 4) has 30 cases, and correlation between TTPAL expression level and clinical characteristics including tumor size, pathological grading, TNM grading and the like of patients is further evaluated, and the multi-factor COX regression analysis result (shown in figure 2) shows that TTPAL high expression can be used as an independent prognosis diagnostic factor of gastric cancer.

3) FIG. 3 shows a Kaplan-meier analysis curve for prognosis of gastric cancer patients. As can be seen from the figure, patients with high expression levels of TTPAL in tumor tissue have a lower survival rate (P < 0.01) than patients with low expression levels, indicating that prognosis of gastric cancer patients can be judged based on the expression levels of TTPAL.

Example 2 TTPAL promotes gastric cancer tumor cell growth in vitro

The specific operation procedure of the MTT method used in this example is as follows: cells were seeded at a cell density of 1000 cells/well in 96 well plates while seeding 5 plates, one plate was removed every 24 hours, 10 μl of MTT solution (5 mg/ml in PBS) was added to each well, incubation was continued for 4 hours, culturing was stopped, and the culture supernatant in the wells was carefully aspirated. The suspension cells need to be centrifuged and the culture supernatant in the well is removed. 200 μl DMSO was added to each well and the mixture was shaken for 10 minutes to allow the crystals to fully thaw.

The following procedure for the cell colony formation assay used in this example was as follows: 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, and the number of clones observed and counted.

The steps for overexpressing TTPAL in this example are as follows:

1) Construction of an overexpressed TTPAL lentiviral vector: the DNA of PCMV6-TTPAL is used as template, TTPAL molecule is cloned to PLV-Puro slow virus vector, and after sequencing is completely correct, virus liquid is prepared.

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

3) Virus-mediated transfection and selection of cell lines stably expressing TTPAL: the virus liquid and 10% FBS culture medium are added into cells in a ratio of 1:1, after 48 hours, the transfected cells are subjected to resistance screening, normal untransfected cells are used as a control, after the normal control is killed by antibiotics, the cells can be subjected to expansion culture, verification is performed by using PCR and Western blot, and after the cell model is successfully constructed, the cells can be used for subsequent research.

The specific operation procedure of the TTPAL knockdown in this embodiment is as follows: the siNC (control plasmid), the alternative siTTPAL-1 (siTTPAL) ^#2 ) And siTTPAL-1 (siTTPAL) ^#1 ) Respectively transfecting to a gastric cancer cell line with high TTPAL expression, verifying interference efficiency by q-PCR and RT-PCR, selecting a siTTPAL interference sequence transfected cell with high interference efficiency (knockdown is more than 70%), extracting total RNA of the transfected cell for 24 hours, and detecting mRNA expression of the TTPAL by RT-PCR; the total cell protein was extracted 48h after transfection, and TTPAL protein expression was detected by Western Blot.

2.1The influence of TTPAL over-expression on the growth of gastric cancer tumor cells is studied through an MTT method and a cell colony formation experiment. Concrete embodimentsAfter we overexpress TTPAL in two gastric cancer cell lines MGC803 and BGC823 (fig. 4A), we found that increased expression of TTPAL promotes growth of gastric cancer cells by single factor analysis of variance, and cell viability was increased (fig. 4B and 4C, ×p<0.001). Representative colony formation images are shown in FIG. 4D. Analysis by t-test showed that overstable TTPAL increased the colony forming ability of cells (FIGS. 4E and 4F,: P)<0.01,***P<0.001)。

2.2The influence on the growth of gastric cancer tumor cells after TTPAL knockdown is studied through 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 sitpal. After TTPAL expression was decreased (as shown in FIG. 5A), growth of gastric cancer cells was inhibited and cell viability was decreased as shown by single factor analysis of variance (FIGS. 5B and 5C, & ltP+ & gt<0.001). Analysis by t-test showed that TTPAL knockdown inhibited the colony forming ability of gastric cancer cells (fig. 5E and 5F, × P<0.001)。

Example 3 TTPAL promotes gastric cancer tumor cell growth in vivo

In this example, we randomly selected the control cell line MGC 823/vector or MGC823/TTPAL (1X 10) ⁶ Individual cells) were injected into the dorsal side of nude mice and the growth of tumors was compared. Tumor volume was measured every 2 days for 15 days, tumor diameter lengths of longest and shortest tumor were measured, and tumor volume was calculated as volume = 0.5X square of maximum diameter X minimum diameter. On day 15 of the experiment, nude mice were sacrificed, subcutaneous tumor tissues were collected, the size was measured, weighed, and the embedded part of tissues was scored for immunohistochemical staining. Fig. 6A and 6B show the morphology of the subcutaneous tumor before and after isolation from mice, respectively. Fig. 6C and 6D show the volume and weight of the subcutaneous tumor, respectively. As can be seen, the tumor growth rate of the MGC823/TTPAL group was significantly increased compared to that of the MGC 823/vector group (FIG. 6C, one-way anova, p)<0.0001). Furthermore, the tumor weight of the MGC823/TTPAL group was also significantly increased over the MGC 823/vector group (FIG. 6D, t-test, p)<0.05). Meanwhile, we also applied the immunohistochemical staining method described previously to stain and score the tissues (fig. 6E) and showed that MGC 823/ttal group scored significantly higher than MGC 823/vector group.

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

Sequence listing

<110> Shenzhen research institute at university of hong Kong Chinese

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

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 342

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 1

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 Val Asn Gly Ile Val Ile Leu Ala Asp Tyr Lys Gly Val

180 185 190

Ser Leu Ser Lys Ala Ser His Phe Gly Pro Phe Ile Ala Lys Lys Val

195 200 205

Ile Gly Ile Leu Gln Asp Gly Phe Pro Ile Arg Ile Lys Ala Val His

210 215 220

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

225 230 235 240

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

245 250 255

Asp Leu Asn Ser Leu His Thr Asn Leu Pro Arg Ser Ile Leu Pro Lys

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Gln Leu Tyr Ser Cys Tyr

340

<210> 2

<211> 308

<212> PRT

<213> Homo sapiens (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> Homo sapiens (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. Use of a reagent for detecting the expression level of a TTPAL protein, or a fragment thereof, wherein the amino acid sequence of the TTPAL protein is shown as SEQ ID No. 1 or 2, and the fragment of the TTPAL protein comprises amino acid residues 1-179 of the sequence shown as SEQ ID No. 1 or 2, in the preparation of a kit or medicament for monitoring or prognosis of gastric cancer in an individual.

2. The use of claim 1, wherein if the expression level of TTPAL protein or fragment thereof in a sample from the individual is greater than a threshold, a poor prognosis of gastric cancer in the individual is indicated.

3. The use according to claim 1 or 2, wherein the agent is a specific binding agent for the TTPAL protein or fragment thereof.

4. The use of claim 3, wherein the binding agent is an antibody.

5. The use according to claim 4, 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.

6. The use of claim 2, wherein the sample is selected from stomach tissue, serum, plasma or cell culture supernatant.

7. The use of claim 6, wherein the sample is a gastric cancer fresh tissue sample or an embedded specimen.

8. The use of claim 6, wherein the kit further comprises one or more additional reagents for immunochemical staining of gastric tissue samples.

9. Use of a reagent for specifically inhibiting expression of a TTPAL protein or a fragment thereof in the preparation of a medicament for inhibiting growth of gastric cancer cells, wherein the amino acid sequence of the TTPAL protein is shown as SEQ ID No. 1 or 2, and the fragment of the TTPAL protein comprises amino acid residues 1-179 of the sequence shown as SEQ ID No. 1 or 2.