Application of GCDH in preparation of products for diagnosis, prognosis prediction and treatment of lung adenocarcinoma

Abstract

The invention belongs to the technical field of biological medicines, and discloses a biomarker GCDH gene or a GCDH gene coding product which can be used for auxiliary diagnosis and prognosis prediction of lung adenocarcinoma. According to the present invention, the expression level of the GCDH protein in the lung adenocarcinoma cancer tissue is significantly lower than the expression level in the paracancerous normal tissue, and the overall survival (OS) of the lung adenocarcinoma patient with the high GCDH expression is superior to the overall survival (OS) of the lung adenocarcinoma patient with the low GCDH expression, such that the GCDH gene or the GCDH gene coding product can be used for the auxiliary diagnosis and the prognosis prediction of the lung adenocarcinoma. Besides, the invention also finds that the GCDH is related to the proliferation, migration and invasion abilities of lung adenocarcinoma cells, and the over-expressed GCDH gene has an obvious inhibition effect on the proliferation, migration and invasion of the lung adenocarcinoma cells, so that the malignant progression of the lung adenocarcinoma cells is inhibited, and a new way is provided for clinical treatment of the lung adenocarcinoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of GCDH in preparing products for diagnosis, prognosis prediction and treatment of lung adenocarcinoma. Background Art

[0002] Lung adenocarcinoma is the most common subtype of lung cancer, accounting for approximately 40%-50% of lung cancer cases worldwide. The incidence of lung adenocarcinoma in my country is also showing a clear upward trend. Due to its insidious onset, lung adenocarcinoma is often asymptomatic in the early stages, and by the time it is discovered, it has already become an advanced metastatic disease with a poor prognosis. If lung adenocarcinoma can be clearly diagnosed early, it will be of great significance for lung adenocarcinoma patients to receive personalized treatment, prolong survival, improve quality of life, and reduce the social medical burden. Therefore, finding key molecules that can be used for early and accurate diagnosis and targeted treatment of lung adenocarcinoma is currently urgently needed by lung adenocarcinoma patients.

[0003] The GCDH (Glutaryl-CoA Dehydrogenase) gene is located on human chromosome 19p13.2 and comprises 11 exons with a total length of approximately 45 kb. The GCDH gene encodes glutaryl-CoA dehydrogenase (GCDH protein), a mitochondrial matrix enzyme that is key in the catabolism of lysine, hydroxylysine, and tryptophan. It catalyzes the conversion of glutaryl-CoA to crotonyl-CoA and participates in mitochondrial fatty acid β-oxidation and the tricarboxylic acid (TCA) cycle. Numerous studies have shown that GCDH is consistently expressed at low levels in various tumors, including renal and liver cancers, and GCDH expression has been widely linked to cancer through mutations in classic oncogenes. However, the role of GCDH in the development and progression of lung adenocarcinoma has not been reported. Summary of the Invention

[0004] In view of the problems and deficiencies in the prior art, the present invention aims to provide an application of GCDH in the preparation of products for the diagnosis, prognosis prediction and treatment of lung adenocarcinoma.

[0005] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a biomarker that can be used for auxiliary diagnosis and prognosis prediction of lung adenocarcinoma. The molecular marker is the GCDH gene (NCBI gene ID: 2639) or a product encoded by the GCDH gene. The product encoded by the GCDH gene includes GCDH mRNA and GCDH protein.

[0007] The expression of GCDH protein in lung adenocarcinoma tissue and adjacent normal tissue was detected by immunohistochemical staining. It was found that the expression level of GCDH protein in lung adenocarcinoma tissue was significantly lower than that in adjacent normal tissue, indicating that GCDH protein expression was downregulated to varying degrees in lung adenocarcinoma tissue.

[0008] Western blot was used to detect the expression of GCDH in lung adenocarcinoma cell lines A549, H1299, H3122 and H1975 and normal lung epithelial cell line BEAS-2B. It was found that the expression level of GCDH in lung adenocarcinoma cell lines was significantly lower than that in normal lung epithelial cell lines, indicating that GCDH expression was downregulated to varying degrees in lung adenocarcinoma cell lines.

[0009] An analysis of the survival information of lung adenocarcinoma patients in the TCGA database found that the overall survival (OS) of lung adenocarcinoma patients with high GCDH expression was better than that of lung adenocarcinoma patients with low GCDH expression, indicating that the GCDH gene can be used to assist in predicting the prognosis of lung adenocarcinoma.

[0010] The second aspect of the present invention provides the use of a detection reagent for the GCDH gene or a product encoded by the GCDH gene in the preparation of a product for auxiliary diagnosis and / or prognosis prediction of lung adenocarcinoma.

[0011] According to the above application, preferably, the product detects the expression level of GCDH gene or GCDH gene encoding product in the sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern Blot, Western Blot, chip, high-throughput sequencing platform, immunohistochemistry or enzyme-linked immunosorbent assay.

[0012] According to the above application, preferably, the product contains an antibody that specifically binds to the GCDH protein, or contains primers that specifically amplify the GCDH gene, or contains a probe that specifically detects the GCDH gene.

[0013] According to the above application, preferably, the nucleotide sequence of the primer for specific amplification of the GCDH gene is as follows:

[0014] Upstream primer: 5′-CAGGTCGGCGATGAGTGT-3′ (SEQ ID NO. 1);

[0015] Downstream primer: 5'-GGTCTTGGTCCCATTGAGG-3' (SEQ ID NO. 2).

[0016] According to the above application, preferably, the antibody is a polyclonal antibody, a monoclonal antibody or a single domain antibody;

[0017] According to the above application, preferably, the sample includes (but is not limited to) tissue, cell, serum. More preferably, the sample is tissue or serum.

[0018] According to the above application, preferably, the product is a chip, a preparation or a kit.

[0019] According to the above application, preferably, the GCDH gene encoding product includes GCDH mRNA and GCDH protein.

[0020] In the present invention, unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a "target polynucleotide") through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. In the present invention, the term "primer" refers to a nucleic acid sequence with a free 3' hydroxyl group that can complementarily bind to a template and enable reverse transcriptase or DNA polymerase to initiate template replication. A primer is a nucleotide sequence with a sequence complementary to the nucleic acid sequence of a specific gene.

[0021] The third aspect of the present invention provides the use of the GCDH gene or the product encoded by the GCDH gene in any of the following:

[0022] (A1) Use in the preparation of a medicament for preventing, alleviating and / or treating lung adenocarcinoma;

[0023] (A2) Use in the preparation of a product for inhibiting the proliferation of lung adenocarcinoma cells;

[0024] (A3) Use in the preparation of a product for inhibiting the migration of lung adenocarcinoma cells;

[0025] (A4) Use in the preparation of a product for inhibiting the invasion of lung adenocarcinoma cells;

[0026] (A5) Application in screening candidate drugs for preventing, alleviating and / or treating lung adenocarcinoma, the screening method comprising: screening drugs or agents to be screened using GCDH as a target, and selecting drugs or agents that can increase the expression level of the GCDH gene, increase the content of the GCDH protein, or increase the activity of the GCDH protein as candidate drugs for preventing, alleviating and / or treating lung adenocarcinoma.

[0027] According to the above application, preferably, the GCDH gene encoding product includes GCDH mRNA and GCDH protein.

[0028] A fourth aspect of the present invention provides the use of a GCDH expression promoter in any of the following:

[0029] (B1) Use in the preparation of a medicament for preventing, alleviating and / or treating lung adenocarcinoma;

[0030] (B2) Use in the preparation of a product for inhibiting the proliferation of lung adenocarcinoma cells;

[0031] (B3) Use in the preparation of a product for inhibiting the migration of lung adenocarcinoma cells;

[0032] (B4) Use in the preparation of a product for inhibiting the invasion of lung adenocarcinoma cells;

[0033] The GCDH expression promoter is one or more substances that increase GCDH gene expression, increase GCDH protein content, and increase GCDH protein activity.

[0034] According to the above application, preferably, the GCDH expression promoter comprises an expression vector carrying a nucleotide sequence encoding the GCDH protein or a recombinant virus containing a nucleotide sequence encoding the GCDH protein or an agonist of the GCDH protein.

[0035] The fifth aspect of the present invention provides a drug for preventing, alleviating and / or treating pancreatitis, which contains a GCDH gene, a GCDH protein, and a GCDH expression promoter; the GCDH expression promoter is one or more substances that increase GCDH gene expression, increase GCDH protein content, and increase GCDH protein activity.

[0036] According to the above-mentioned drug, preferably, the GCDH expression promoter comprises an expression vector carrying a nucleotide sequence encoding the GCDH protein or a recombinant virus containing a nucleotide sequence encoding the GCDH protein or an agonist of the GCDH protein.

[0037] According to the above-mentioned medicine, preferably, the medicine further comprises other drugs compatible with the GCDH gene, GCDH protein or GCDH expression promoter, as well as pharmaceutically acceptable carriers and / or excipients.

[0038] According to the above-mentioned drug, preferably, the carrier / excipient includes (but is not limited to): diluent, excipient (such as lactose, sodium chloride, glucose, urea, starch, water, etc.), filler (such as starch, sucrose, etc.); binder (such as monosaccharide syrup, glucose solution, starch solution, cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone); wetting agent (such as glycerol); disintegrant (such as dry starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate); absorption accelerator (such as quaternary ammonium compound, sodium lauryl sulfate, etc.); surfactant (such as polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, cetyl alcohol, etc.); wetting agent (such as glycerol, starch, etc.); adsorption carrier (such as starch, lactose, bentonite, silica gel, kaolin and bentonite, etc.); lubricant (such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, etc.).

[0039] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0040] (1) The present invention discovered for the first time that the expression of GCDH in lung adenocarcinoma tumor tissue is significantly downregulated compared with adjacent normal tissue, and the expression level of GCDH in lung adenocarcinoma cell lines is significantly lower than that in normal lung epithelial cell lines. Therefore, GCDH can be used as a molecular marker for auxiliary diagnosis of lung adenocarcinoma; by detecting the expression level of GCDH in samples, early diagnosis of lung adenocarcinoma can be achieved, providing a reference basis for clinicians to diagnose lung adenocarcinoma.

[0041] (2) The present invention discovered for the first time that the overall survival (OS) of lung adenocarcinoma patients with high GCDH expression was significantly better than that of lung adenocarcinoma patients with low GCDH expression. Therefore, the expression level of GCDH gene or GCDH protein can be used to assist in predicting the prognosis of lung adenocarcinoma, thereby providing a new approach for the judgment of lung adenocarcinoma prognosis prediction and a reference basis for clinicians to analyze the condition of lung adenocarcinoma.

[0042] (3) The present invention also found that GCDH is related to the proliferation, migration and invasion ability of lung adenocarcinoma cells. GCDH can inhibit the proliferation, migration and invasion of lung adenocarcinoma cells. Overexpression of the GCDH gene has a significant inhibitory effect on the proliferation, migration and invasion of lung adenocarcinoma cells, thereby inhibiting the malignant progression of lung adenocarcinoma cells. Therefore, by overexpressing the expression level of GCDH in lung adenocarcinoma patients, the effect of alleviating and treating lung adenocarcinoma can be achieved, providing a new approach for the clinical treatment of lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Representative results of immunohistochemical detection of GCDH protein expression levels in adjacent normal tissues and lung adenocarcinoma tumor tissues;

[0044] Figure 2 Figure 2 is the Kaplan-Meier survival curve of the effect of GCDH on the overall survival of patients with lung adenocarcinoma; L represents the GCDH low expression group, and H represents the GCDH high expression group;

[0045] Figure 3 The figure shows the results of Western blot detection of GCDH protein expression levels in normal lung epithelial cell lines and lung adenocarcinoma cells;

[0046] Figure 4 This is the result of RT-qPCR detection of GCDH protein expression level in GCDH-OV H1975 cell line;

[0047] Figure 5The figure shows the results of Western blot detection of GCDH protein expression level in GCDH-OV H1975 cell line;

[0048] Figure 6 The results of a CCK8 experiment examining the effect of GCDH overexpression on the proliferation of lung adenocarcinoma cells. The left figure is a bar graph of cell viability (OD value at 450 nm) measured after 48 hours, and the right figure is a curve showing the OD value changes of the cells at 0, 24, 48, and 72 hours.

[0049] Figure 7 Figure 1 shows the results of a clone formation experiment to detect the effect of overexpression of the GCDH gene on the proliferation ability of lung adenocarcinoma cells. Figure A shows a representative image of the clone formation experiment, and Figure B shows the statistical analysis results of the clone number.

[0050] Figure 8 The figures show the results of the Transwell migration experiment to detect the effect of overexpression of the GCDH gene on the migration of lung adenocarcinoma cells. A is a representative image of the Transwell migration experiment, showing the cell migration of the control group (GCDH-CON) and the GCDH overexpression group (GCDH-OV); B is the statistical analysis result of the number of migrating cells; CON represents the GCDH-CON group, and OV-GCDH represents the GCDH-OV group. DETAILED DESCRIPTION

[0051] The following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0052] It should be noted that the terms used herein are intended only to describe specific embodiments and are not intended to limit the exemplary embodiments of the present invention. Experimental methods in the following examples, where specific conditions are not specified, were performed using conventional techniques in the art or in accordance with the conditions recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products. All experimental results are averages of multiple experiments.

[0053] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0054] Example 1: Study on the expression of GCDH in lung adenocarcinoma tumor tissue and adjacent normal tissue

[0055] Immunohistochemical staining analysis was performed on lung adenocarcinoma tissue chips (including 25 lung adenocarcinoma adjacent normal tissue samples and 50 lung adenocarcinoma tumor tissue samples) to analyze the expression of GCDH protein in lung adenocarcinoma and adjacent normal tissues.

[0056] 1. Sample collection:

[0057] Tumor tissue samples were collected from 50 patients with histologically confirmed primary lung adenocarcinoma and 25 adjacent normal tissue samples from patients with lung adenocarcinoma. None of the patients had received endocrine therapy, radiotherapy, or chemotherapy before surgery, and surgery was the preferred treatment option. In accordance with the protocols established by the institutional review committee, each patient provided signed informed consent before sampling.

[0058] 2. Experimental methods

[0059] Immunohistochemical staining was used to detect GCDH protein expression levels in lung adenocarcinoma tumor tissues and adjacent normal tissues. The specific steps for tissue microarray immunohistochemical staining are as follows:

[0060] 1) Dewaxing and rehydration of paraffin sections: Chips containing lung adenocarcinoma tumor tissue and paired adjacent normal tissue were sequentially immersed in xylene solution twice for 15 minutes each, then immersed in anhydrous ethanol twice for 5 minutes each, followed by immersion in 85% ethanol for 5 minutes and 75% ethanol for 5 minutes, and rinsed once with distilled water.

[0061] 2) Antigen retrieval: Soak the dewaxed sections in EDTA antigen retrieval solution (pH 9.0) and microwave on low heat for 10-15 minutes. After cooling to room temperature, wash the sections three times with PBS (5 minutes each time).

[0062] 3) After antigen retrieval, add 3% hydrogen peroxide to the slides, incubate for 5 minutes, place in water to terminate the reaction, and rinse three times with PBS (5 minutes each time).

[0063] 4) Blocking: Remove excess PBS solution with absorbent paper and block with 5% goat serum at room temperature for 30 minutes.

[0064] 5) Primary antibody incubation: Aspirate the blocking solution and incubate with GCDH primary antibody. Place the sections flat in a humidified chamber and incubate at 4°C for 12 hours.

[0065] 6) Secondary Antibody Incubation: Sections were placed in PBS and washed three times on a shaker (5 minutes each time). Excess PBS was removed and HRP-labeled goat anti-rabbit secondary antibody was added and incubated at room temperature for 2 hours.

[0066] 7) DAB color development: After the secondary antibody incubation, the sections were rinsed three times in PBS (5 minutes each time) and developed using DAB.

[0067] 8) Differentiation and blueing: After the developed tissue chip is rinsed with PBS, incubated in hematoxylin for 2 minutes, washed three times with distilled water (5 minutes each time), incubated in hydrochloric acid for 10 seconds, and finally rinsed with distilled water for 15-20 minutes.

[0068] 9) Dehydration and clearing: Place the sections in 70%, 80%, 90%, 95% and 100% alcohol gradient jars for 5 minutes each. Finally, incubate the sections in xylene for 10 minutes.

[0069] 10) Sealing: Place the slices in a ventilated place to dry, cover them with neutral gum, seal them with a coverslip, and dry them naturally at room temperature.

[0070] 11) Use a tissue slide scanner to acquire images.

[0071] 3. Data processing and analysis

[0072] Immunostaining results were independently evaluated microscopically by two pathologists. Scoring criteria required a comprehensive consideration of both the staining intensity and percentage of positive cells. The immunohistochemical staining score was calculated by summing the percentage of positive cells and the staining intensity.

[0073] The percentage of positively stained cells was scored as follows: 0% of tumor cells showed positive staining, which was 0 points; (1%-30%) of tumor cells showed positive staining, which was 1 point; (30%-60%) of tumor cells showed positive staining, which was 2 points; and (60%-100%) of tumor cells showed positive staining, which was 3 points.

[0074] The staining intensity was scored as follows: no staining was scored as 0, yellow was scored as 1, brownish yellow was scored as 2, and reddish brown was scored as 3.

[0075] The final score was recorded as the color intensity score + percentage score; 0 was negative (-), 1 was weakly positive (-+), 2-4 was positive (+), and 5-6 was strongly positive (++).

[0076] 4. Experimental results

[0077] Representative results of immunohistochemical analysis of 50 lung adenocarcinoma tissues and 25 adjacent normal tissues are shown in the figure. Figure 1 shown.

[0078] Depend on Figure 1It can be seen that compared with the normal lung epithelial tissue adjacent to the cancer, the number of positively stained cells in lung adenocarcinoma tumor tissue was significantly reduced, and the staining intensity was significantly lighter, suggesting that the expression level of GCDH in lung adenocarcinoma is relatively low; further statistics on the expression levels of GCDH protein in normal lung epithelial tissue samples and lung adenocarcinoma tumor tissue samples were performed based on the immunohistochemical staining score results, and it was found that the expression level of GCDH protein in normal lung epithelial tissue samples was significantly higher than that in lung adenocarcinoma tumor tissue samples, and the difference was statistically significant.

[0079] Example 2: Bioinformatics data analysis of GCDH expression in lung adenocarcinoma

[0080] 1. Data collection and collation

[0081] We systematically searched and screened datasets related to lung adenocarcinoma in the TCGA public database. For gene chip data, we downloaded CEL raw data, and for high-throughput transcriptome sequencing (RNA-Seq) data, we downloaded expression count matrices. The downloaded data underwent preprocessing, including quality control, background correction, normalization, and gene annotation. Finally, we analyzed data from 490 lung adenocarcinoma tumor tissue samples obtained from the TCGA database.

[0082] 2. Analysis of the relationship between GCDH and prognosis of lung adenocarcinoma:

[0083] The lung adenocarcinoma patients with complete follow-up data screened in step 1 were divided into a GCDH high expression group and a low expression group (with the median expression level of GCDH as the dividing point). The overall survival information of the lung adenocarcinoma patients in the GCDH high expression and GCDH low expression groups was then analyzed and the Kaplan-Meier survival curve was drawn. Figure 2 shown.

[0084] Depend on Figure 2 It can be seen that the overall survival (OS) of lung adenocarcinoma patients with high GCDH expression is better than that of lung adenocarcinoma patients with low GCDH expression. This shows that the GCDH gene can be used to predict the prognosis of lung adenocarcinoma.

[0085] Example 3: Study on the expression of GCDH in lung adenocarcinoma cells and normal lung epithelial cell lines

[0086] 1. Western blot detection of GCDH protein expression in lung adenocarcinoma cells and normal lung epithelial cell lines

[0087] Western blot was used to detect the expression of GCDH protein in lung adenocarcinoma cell lines and normal lung epithelial cells.

[0088] (1) Cell selection and culture:

[0089] Human normal lung epithelial cell line BEAS-2B and lung adenocarcinoma cell lines A549, H1299, H3122, and H1975 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and 1% penicillin and streptomycin. All five cell lines were cultured at 37°C, 5% CO₂, and saturated humidity. The medium was changed every two days, and cells were passaged at a 1:3 ratio.

[0090] (2) Experimental methods:

[0091] Western blot was used to detect the expression level of GCDH in lung adenocarcinoma cells and normal lung epithelial cell lines. The specific steps are as follows:

[0092] 1) PAGE gel preparation: Add equal volumes of the lower gel solution and lower gel buffer to a beaker, mix thoroughly, then add a coagulant and mix again. Pour the lower gel mixture onto a glass plate. After the lower gel solidifies, add equal volumes of the upper gel solution and upper gel buffer to a beaker, mix thoroughly, add a coagulant, and mix again. Pour the mixed upper gel mixture onto a glass plate and insert a tooth comb.

[0093] 2) Protein Sample Preparation: Collect the normal lung epithelial and lung adenocarcinoma cells in the logarithmic growth phase described above, wash twice with PBS, then add RIPA lysis buffer to extract total protein. Use the BCA protein quantification kit for protein quantification, then add 200uL to each well to be tested (including standard wells and sample wells) and incubate at 37°C for 30 minutes. Measure the absorbance of the sample wells at 562nm using a microplate reader, prepare a protein concentration curve based on the OD value, and finally calculate the concentration of the protein to be tested. Depending on the protein concentration, add 5X loading buffer, mix thoroughly, denature at 95°C for 10 minutes, and store at -20°C for a short period of time for subsequent experiments.

[0094] 3) Gel Running: Mount the gel on the electrophoresis apparatus, add electrophoresis buffer, remove the comb, and add 15 μL of protein sample to each well. Maintain a constant voltage of 70 V until the indicator enters the separating gel. Run the gel at 120 V until the end of the run.

[0095] 4) Transfer: Soak the transfer apparatus and filter paper in transfer solution. Activate the PVDF membrane in methanol for 15-30 seconds. Remove and place on the gel, ensuring no air bubbles form between the gel and the PVDF membrane. Place the transfer apparatus in a basin of ice water, add transfer solution, and place the apparatus in a constant current of 250 mA for 90 minutes.

[0096] 5) Blocking: After the transfer is completed, remove the gel and PVDF membrane, block with 5% skim milk, and shake at room temperature for 2 hours.

[0097] 6) Primary antibody incubation: Add GCDH antibody (1:1000 dilution) and internal reference GAPDH (1:10000 dilution), and incubate on a shaker at 4°C overnight.

[0098] 7) Secondary antibody incubation: Wash three times with TBST (10 minutes each time); add HRP-labeled goat anti-rabbit and anti-mouse secondary antibodies (1:5000) and incubate at room temperature for 1 hour.

[0099] 8) Exposure: Wash three times with TBST (10 minutes each time); visualize the protein using a luminescent solution. Calculate the relative expression level of the target protein using VisionWorks software, using GAPDH as the internal reference protein.

[0100] Western blot was used to detect the expression level of GCDH in lung adenocarcinoma cells and normal lung epithelial cell lines. Figure 3 shown.

[0101] Depend on Figure 3 It can be seen that the expression level of GCDH protein in the normal lung epithelial cell line BEAS-2B is significantly higher than that in the lung adenocarcinoma cell lines A549, H1299, H3122 and H1975; moreover, the expression level of GCDH protein in the lung adenocarcinoma cell line H1975 is relatively low. Therefore, the H1975 cell line was selected as a tool cell for subsequent experimental studies.

[0102] Example 4: Construction of a lung adenocarcinoma cell line overexpressing the GCDH gene

[0103] 1. Cell selection and culture:

[0104] Human lung adenocarcinoma cell line H1975 was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C, 5% CO₂, and 90% relative humidity. The medium was changed every two days, and cells were passaged using digestive enzymes.

[0105] 2. Construction of a lung adenocarcinoma cell line overexpressing the GCDH gene (denoted as GCDH-OV H1975 cell line)

[0106] (1) Transformation and plasmid amplification

[0107] 1) Thaw 100 μL of competent cells on ice;

[0108] 2) Add 2.5 μL of human GCDH overexpression plasmid (purchased from Beijing Sino Biological Science and Technology Co., Ltd., Cat. No.: HG14093-ACG) to the competent cells, gently pipette to mix, and incubate on ice for 30 minutes;

[0109] 3) Heat shock in a 42°C metal bath for 45 seconds, then immediately place in an ice bath for 3 minutes;

[0110] 4) Add 900 μL of LB medium and incubate on a shaker at 37°C for 1 hour at 200 rpm.

[0111] 5) After recovery, centrifuge the bacterial suspension at 4000 rpm for 3 minutes, discard the supernatant, and use 200 μL of the remaining pellet to evenly distribute the bacterial pellet. Inoculate the bacterial suspension evenly into solid LB medium containing kanamycin (if the plasmid corresponds to kanamycin resistance, confirm the actual resistance) and invert the medium to culture overnight.

[0112] 6) The next morning, select a colony and inoculate it into 100 mL of liquid LB medium. Incubate on a shaker at 37°C (200 rpm) for 12-16 hours.

[0113] (2) Plasmid extraction:

[0114] 1) Remove the bacterial solution from the shaker and centrifuge;

[0115] 2) Discard the supernatant and proceed according to the selected kit instructions (if no corresponding kit is available here, follow the conventional similar procedures: add an appropriate amount of buffer containing RNase A, mix thoroughly, and then aliquot into 1.5 mL EP tubes);

[0116] 3) Add lysis buffer and gently invert several times to completely lyse the bacteria;

[0117] 4) Add the precipitant (pre-cooled), gently invert, and observe the white flocculent precipitate;

[0118] 5) After standing, centrifuge at an appropriate speed (e.g., 12,000 rpm) for a certain time (e.g., 15 minutes);

[0119] 6) Take an appropriate number of plasmid DNA extraction columns, pour the supernatant into the column, let it stand, centrifuge, and discard the filtrate;

[0120] 7) Add washing solution to the column, centrifuge, and discard the filtrate;

[0121] 8) Repeat the washing steps;

[0122] 9) After centrifugation again, transfer the column to a new 1.5 mL EP tube;

[0123] 10) Add preheated elution buffer, let stand and then centrifuge to obtain the plasmid.

[0124] (3) Transfection: This plasmid is not a lentiviral plasmid and is directly transfected into cells. The specific steps are as follows:

[0125] 1) H1975 cells were plated in a six-well plate. A control group and an overexpression group were set up, with two wells in each group. Transfection was performed when the cell density reached 50%.

[0126] 2) Add 2 μg of control plasmid (the control plasmid is an invalid nucleotide sequence that does not affect GCDH expression, and the target sequence is: 5'-AAUUCUCCGAACGUGUCACGU-3') or the human GCDH overexpression plasmid extracted in step (2) into 200 μL transfection buffer, mix well, and centrifuge;

[0127] 3) Add 4 μL of transfection reagent to the above buffer, pipette several times to mix, and let it stand for 10 minutes;

[0128] 4) After replacing the H1975 cell culture medium, add the buffer containing the plasmid and transfection reagent to the corresponding wells and gently shake to mix.

[0129] 5) Replace the culture medium 4 hours after transfection;

[0130] 6) 48 h / 72 h after transfection, observe the cells for green fluorescence under a microscope and add kanamycin for selection to obtain the GCDH-OV H1975 lung adenocarcinoma cell line and the corresponding control line (the control line is designated as GCDH-CON).

[0131] 3. RT-qPCR detection of GCDH gene expression level in GCDH-OV H1975 cell line

[0132] RT-qPCR was used to detect the expression level of the GCDH gene in the GCDH-OV H1975 cell line and the GCDH-CON control line. The specific steps are as follows:

[0133] 1) Cellular RNA Extraction: Collect cells (GCDH-OVH1975 cell line or GCDH-CON control line) in the logarithmic phase from a 6-cm dish. Discard the culture medium and add 1 mL of Trizol. After thorough lysis, transfer the cells to an Eppendorf tube. Add 200 μL of chloroform and mix. Let stand at room temperature for 10 minutes. Transfer the Eppendorf tube to a centrifuge and centrifuge at 12,000 rpm for 10 minutes. Aspirate 400 μL of the supernatant and transfer it to a new Eppendorf tube. Add an equal volume of isopropanol, let stand at room temperature for 15 minutes, and centrifuge at low temperature. After centrifugation, a white precipitate is obtained, which is discarded. Resuspend the precipitate in 1 mL of 75% ethanol and centrifuge at low temperature. Discard the supernatant again, air-dry the precipitate until it becomes transparent, and dissolve it in an appropriate amount of nuclease-free water. Assay the concentration and purity of total RNA using a NanoDrop 2000.

[0134] 2) Reverse transcription to synthesize cDNA:

[0135] A. Remove 4×gDNA wiperMix and 5×HiScript III qRT SuperMix from HiScript III RT SuperMix for qPCR (+gDNA wiper), centrifuge briefly, and place on ice.

[0136] Prepare the genomic DNA removal reaction solution according to the components in Table 1. Perform the reaction on ice and gently pipette to mix thoroughly. Incubate at 42°C for 2 minutes.

[0137] Table 1 Reaction solution system for removing genomic DNA

[0138] Reagents Usage 4xgDNAwiperMix 4uL Total RNA 100ng-1000ng <![CDATA[RNaseFreeddH20]]> to 16 μL

[0139] B. After the reaction is completed, prepare the reverse transcription reaction solution according to the components in Table 2 and proceed on ice:

[0140] Table 2 Reverse transcription reaction system

[0141] Reagents Usage Step 1) reaction solution 16uL 5xHiScriptIIIqRTSuperMix 4uL Total 20 μL

[0142] c. Gently mix the prepared mixture. Turn on the PCR instrument and program the following cycle. Place the samples one by one into the instrument and begin the reverse transcription reaction at: 37°C for 15 minutes, 85°C for 5 seconds. After the reaction is complete, remove the reverse transcription reaction solution (i.e., cDNA) and dilute it 1:10 for use in the qPCR reaction. (When adding the reverse transcription reaction solution to the RT-qPCR reaction system in the next step, do not add more than 1 / 10 of the RT-qPCR reaction volume (v / v)).

[0143] 3) Fluorescence quantitative detection:

[0144] Using GAPDH as an internal reference, the relative expression levels of the GCDH gene in different cell lines were detected by RT-qPCR reaction, and the expression differences between them were compared.

[0145] The nucleotide sequences of the specific amplification primers for GCDH are as follows:

[0146] Upstream primer: 5′-CAGGTCGGCGATGAGTGT-3′ (SEQ ID NO. 1);

[0147] Downstream primer: 5'-GGTCTTGGTCCCATTGAGG-3' (SEQ ID NO. 2).

[0148] The nucleotide sequences of the GAPDH-specific amplification primers are as follows:

[0149] Upstream primer: 5′-CCTTCCCAGCCTCGCGTCCCGAGTTTGTCA-3′;

[0150] Downstream primer: 5'-GAAGCAGCCCAGGAGCTCCCCGTC-3'.

[0151] Prepare the RT-qPCR reaction system (20 μL system) according to the ratio in Table 3.

[0152] Table 3 RT-qPCR reaction system

[0153]

[0154] The two-step RT-qPCR method was used to generate melting curves. The program settings are shown in Table 4.

[0155] Table 4 Two-step RT-qPCR reaction system program settings

[0156]

[0157] According to the original RT-qPCR detection results, the relative expression level of the GCDH gene was calculated according to the 2^-ΔΔCT method, that is, the difference in the transcription level of the target gene GCDH in the GCDH-OV H1975 cell line and the GCDH-CON control line.

[0158] All data of RT-qPCR detection are expressed as mean ± standard deviation (mean ± SD). The comparison between two groups was performed by two-sided Student's t test, and three groups or more were compared by one-way analysis of variance. All results were plotted using GraphPad Prism 6 software, with P < 0.05 as the test level, and when P < 0.05, it was considered statistically significant. Figure 4 shown.

[0159] Depend on Figure 4 It can be seen that compared with the GCDH-CON control strain, the expression level of the GCDH gene in the GCDH-OVH1975 cell line overexpressing the GCDH gene was significantly upregulated.

[0160] 4. Western blot detection of GCDH protein expression level in GCDH-OV H1975 cell line

[0161] Western blot was used to detect the expression level of GCDH protein in GCDH-OV H1975 cell line. The specific operation method of Western blot was the same as that in Example 3.

[0162] Western blot test results Figure 5As shown. Figure 5 It can be seen that compared with the GCDH-CON control strain, the expression level of GCDH protein in the GCDH-OV H1975 cell line overexpressing the GCDH gene was significantly upregulated.

[0163] Example 5: Effects of GCDH on cell proliferation, migration and invasion of lung adenocarcinoma cells

[0164] 1. Study on the effect of GCDH on the proliferation of lung adenocarcinoma cells

[0165] The GCDH-OV H1975 cell line overexpressing the GCDH gene constructed in Example 4 and the GCDH-CON control line were subjected to a CCK8 assay to detect the effect of GCDH on the proliferation of lung adenocarcinoma cells.

[0166] The specific operation of the CCK8 experiment is as follows:

[0167] 1) The experiment was divided into two groups: negative control group (GCDH-CON group) and GCDH-OV group. Each group had five replicate wells. GCDH-CON control strain and GCDH-OV H1975 cell line suspension (concentration of 5×10 3 / mL), add 100 μL to each well.

[0168] 2) After 8 hours of incubation, discard the old culture medium from the five wells in each group. Add 20 μL of fresh culture medium containing CCK8 reagent to each well of the 96-well plate in the dark. Gently shake the plate and continue incubation in the incubator. Remove the plate after 2 hours and measure the OD value at 450 nm using a microplate reader. Repeat the same method to measure the absorbance of the cells at 24, 48, and 72 hours.

[0169] The results of CCK8 experiments are as follows Figure 6 As shown. Figure 6 It can be seen that compared with the GCDH-CON group, the cell proliferation in the GCDH-OV group was significantly inhibited (P < 0.05), indicating that overexpression of GCDH has a significant inhibitory effect on the proliferation of H1975 cells.

[0170] 2. Study on the effect of GCDH on the clone-forming ability of lung adenocarcinoma cells

[0171] The GCDH-OV H1975 cell line overexpressing the GCDH gene constructed in Example 4 and the GCDH-CON control line were subjected to a clone formation experiment to detect the effect of GCDH on the clone formation ability of lung adenocarcinoma cells.

[0172] The specific operation of the clone formation experiment is as follows:

[0173] 1) The experiment was divided into two groups: a negative control group (GCDH-CON group) and a GCDH-OV group, with three replicates per group. 2 mL of complete medium was added to the corresponding wells of the GCDH-CON and GCDH-OV groups in a 6-well plate. Then, 1500 cells of the GCDH-CON control cell suspension and GCDH-OV H1975 cell suspension were inoculated into the corresponding wells of the GCDH-CON and GCDH-OV groups, respectively. After mixing, the cells were cultured in a 37°C, 5% CO2 incubator. The medium was changed every 5 days. Visible cell colonies formed after 2 weeks.

[0174] 2) Remove the culture medium, wash once with PBS, and add 1 mL of pre-cooled 4% paraformaldehyde to each well for 15 minutes. After fixation, remove the fixative and add 0.5% crystal violet for staining for 20 minutes. After staining, remove the crystal violet and gently rinse the remaining crystal violet with clean water. After drying, place under bright light and photograph with a camera.

[0175] The results of the clone formation experiment are as follows Figure 7 As shown. Figure 7 It can be seen that compared with the GCDH-CON group, the cell proliferation ability of the GCDH-OV group was significantly reduced (P < 0.05), indicating that overexpression of GCDH has a significant inhibitory effect on the proliferation ability of H1975 cells.

[0176] 3. Study on the effect of GCDH on the migration of lung adenocarcinoma cells

[0177] The GCDH-OV H1975 cell line overexpressing the GCDH gene constructed in Example 4 and the GCDH-CON control line were subjected to a Transwell chamber migration experiment. The effect of the GCDH gene on cell migration ability was verified by detecting the migration of the GCDH-OV H1975 cell line into the serum-containing medium in the Transwell chamber.

[0178] The specific method of Transwell chamber migration assay is as follows:

[0179] 1) The experiment was divided into two groups: negative control group (GCDH-CON group) and GCDH-OV group. Each group had three replicate wells. The number of cells added to the upper chamber was 5×10 4 Prepare a serum-free cell suspension (migration experiment) for each cell, add 700uL of culture medium containing 10% fetal bovine serum to the lower chamber, and culture in a constant temperature incubator at 37°C for 48 hours.

[0180] 2) Remove the Transwell chamber, wash three times with PBS, fix with 4% paraformaldehyde for 30 minutes, and then wash three times with PBS. Stain with crystal violet for 30 minutes, then discard the crystal violet solution. Observe the staining intensity under a microscope, rinse with purified water, and observe under a fluorescence microscope, photograph, and count.

[0181] Data processing and analysis: All data are expressed as mean ± standard deviation. Comparisons between two groups were performed using a two-sided Student's t-test, and comparisons between three or more groups were performed using one-way analysis of variance. All results were plotted using GraphPad Prism 6 software. P < 0.05 was considered statistically significant.

[0182] The results of the Transwell chamber migration experiment were as follows Figure 8 As shown. Figure 8 It can be seen that compared with the GCDH-CON group, the cell migration ability of the GCDH-OV group was significantly reduced (P < 0.05), indicating that overexpression of GCDH has a significant inhibitory effect on the migration ability of lung adenocarcinoma cell H1975.

[0183] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above technical content as a guide to make changes or modifications. These are equivalent embodiments of equivalent modifications. However, any simple modifications, equivalent changes, and modifications to the above embodiments that do not depart from the technical concept of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the claims of the present invention.

Claims

1. Use of a detection reagent for the GCDH gene or its encoded product in the preparation of a product for auxiliary diagnosis and / or prognosis prediction of lung adenocarcinoma.

2. The use according to claim 1, characterized in that The product detects the expression level of the GCDH gene or the GCDH gene encoding product in a sample through RT-PCR, real-time quantitative PCR, in situ hybridization, Northern Blot, Western Blot, chip, high-throughput sequencing platform, immunohistochemistry or enzyme-linked immunosorbent assay.

3. The use according to claim 2, characterized in that The product contains an antibody that specifically binds to the GCDH protein, or contains a primer that specifically amplifies the GCDH gene, or contains a probe that specifically detects the GCDH gene.

4. The use according to claim 3, characterized in that The nucleotide sequences of the primers for specific amplification of the GCDH gene are as follows: Upstream primer F: 5'-CAGGTCGGCGATGAGTGT-3', Downstream primer R: 5′-GGTCTTGGTCCCATTGAGG-3′; The antibody is a polyclonal antibody, a monoclonal antibody or a single domain antibody.

5. The use according to claim 2, characterized in that The samples include tissues, cells, and serum; the products are chips, preparations, or kits; and the GCDH gene-encoded products include GCDH mRNA and GCDH protein.

6. Use of the GCDH gene or its encoded product in any of the following: (A1) Use in the preparation of a drug for preventing, alleviating and / or treating lung adenocarcinoma; (A2) Use in the preparation of a product for inhibiting the proliferation of lung adenocarcinoma cells; (A3) Use in the preparation of a product for inhibiting the migration of lung adenocarcinoma cells; (A4) Use in the preparation of a product for inhibiting the invasion of lung adenocarcinoma cells; (A5) Application in screening candidate drugs for preventing, alleviating and / or treating lung adenocarcinoma, the screening method comprising: screening drugs or agents to be screened using GCDH as a target, and selecting drugs or agents that can increase the expression level of the GCDH gene, increase the content of the GCDH protein or increase the activity of the GCDH protein as candidate drugs for preventing, alleviating and / or treating lung adenocarcinoma.

7. Use of the GCDH expression promoter in any of the following: (B1) Use in the preparation of drugs for preventing, alleviating and / or treating lung adenocarcinoma; (B2) Use in the preparation of a product for inhibiting the proliferation of lung adenocarcinoma cells; (B3) Use in the preparation of a product for inhibiting the migration of lung adenocarcinoma cells; (B4) Use in the preparation of a product for inhibiting the invasion of lung adenocarcinoma cells; The GCDH expression promoter is one or more substances that increase GCDH gene expression, increase GCDH protein content, and increase GCDH protein activity.

8. The use according to claim 7, characterized in that: The GCDH expression promoter comprises an expression vector carrying a nucleotide sequence encoding the GCDH protein, a recombinant virus containing a nucleotide sequence encoding the GCDH protein, or an agonist of the GCDH protein.

9. A drug for preventing, alleviating and / or treating pancreatitis, characterized in that: The medicine contains GCDH gene, GCDH protein and GCDH expression promoter; the GCDH expression promoter is one or more substances that increase GCDH gene expression, increase GCDH protein content and increase GCDH protein activity.

10. The drug according to claim 9, characterized in that The GCDH expression promoter comprises an expression vector carrying a nucleotide sequence encoding the GCDH protein, a recombinant virus containing a nucleotide sequence encoding the GCDH protein, or an agonist of the GCDH protein.

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