CN115851947A - Application of DAGLA in diagnosis and treatment of liver cancer - Google Patents
Application of DAGLA in diagnosis and treatment of liver cancer Download PDFInfo
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Abstract
The invention belongs to the technical field of disease diagnosis and treatment and molecular biology, and particularly relates to application of DAGLA in diagnosis and treatment of liver cancer. According to research, the expression level of DAGLA in human liver cancer tissues is obviously higher than that of paracancer normal tissues; meanwhile, the total survival time and the recurrence-free survival time of liver cancer patients with high-expression DAGLA are obviously shortened, the number of tumors is more, the differentiation degree is worse, and the vascular invasion is more likely to occur. Meanwhile, over-expression of DAGLA can promote proliferation, migration and invasion capacity of the liver cancer cells, and conversely, knocking down DAGLA can obviously inhibit proliferation, invasion and metastasis of the liver cancer cells. The DAGLA discovered by the invention is helpful for deeply understanding the pathogenesis of the liver cancer, and is also helpful for providing potential biomarkers and treatment targets for clinical diagnosis and prognosis, and preventing and treating the liver cancer, so that the DAGLA has good value of practical application.
Description
Technical Field
The invention belongs to the technical field of disease diagnosis and treatment and molecular biology, and particularly relates to application of DAGLA in diagnosis and treatment of liver cancer.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Hepatocellular carcinoma (hereinafter, liver cancer) is one of the most common malignant tumors in the world and is also the lethal cause of the third largest malignant tumor. In recent years, clinical treatment means of liver cancer are abundant, early liver cancer is expected to be cured radically through operation and radio frequency ablation, but ideal treatment measures are not provided for patients with late liver cancer, the overall prognosis of the patients with liver cancer is still not satisfactory, and recurrence and metastasis are still the most serious problems affecting the prognosis of the patients with liver cancer. With the intensive research, the molecular targeted therapy and immunotherapy of liver cancer have made breakthrough progress, and targeted drugs represented by ranvatinib and sorafenib and immune checkpoint inhibitors represented by acilizumab and convilumab have been approved as first-line treatment schemes for patients with advanced liver cancer in combination with bevacizumab. However, the objective response rate of liver cancer to the drugs is low, the survival time of patients is prolonged by single drug therapy for only a few months, and the economic burden of the concomitant adverse drug reactions also limits the clinical application. Therefore, the screening and targeted intervention of key regulatory molecules influencing the liver cancer progression have important significance for reducing tumor drug resistance and improving the prognosis of patients.
Diacylglycerol lipase α (DAGLA) is an important endocannabinoid synthase that specifically hydrolyzes Diacylglycerol to the endocannabinoid 2-arachidonic acid glycerol (2-AG) and free fatty acids. There are few reports of the role of DAGLA in tumorigenesis and cancer progression, and researchers have found that DAGLA promotes malignant progression of Oral Squamous Cell Carcinoma (OSCC), exacerbating its malignant phenotype by modulating cell cycle progression. At present, the role of DAGLA in hepatocellular carcinoma progression is not reported in the literature.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor provides the application of DAGLA in diagnosis and treatment of liver cancer through long-term technical and practical exploration. According to the invention, the DAGLA can be used as a diagnosis and prognosis marker of liver cancer and an action target point for preparing an anti-liver cancer medicament. The present invention has been completed based on the above results.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided use of a substance for detecting DAGLA-encoding genes and expression products thereof in the preparation of a product for diagnosing, detecting, monitoring or predicting the prognosis of liver cancer.
Both the DAGLA-encoding gene and the expression product thereof may be of human origin, and the expression product of the DAGLA-encoding gene may be a DAGLA protein, i.e., diacylglycerol lipase α.
In a second aspect of the invention, there is provided a product for diagnosing, detecting, monitoring or predicting the prognosis of liver cancer, comprising detecting the transcription of a DAGLA-encoding gene in a sample based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; and/or a substance for detecting the expression of diacylglycerol lipase alpha in a sample based on an immunoassay.
In a third aspect of the present invention, there is provided a system for diagnosing, detecting, monitoring or predicting the prognosis of liver cancer, the system comprising at least:
i) An analysis module, the analysis module comprising: a detection substance for determining the expression level of a gene selected from the above-mentioned DAGLA-encoding genes and expression products thereof in a test sample of a subject, and;
ii) an evaluation module comprising: determining the subject's condition based on the expression levels of the DAGLA-encoding genes and their expression products determined in i).
In a fourth aspect of the present invention, the DAGLA coding gene and the use of the expression product thereof as a target for preparing or screening liver cancer drugs are provided.
In a fifth aspect of the invention, there is provided the use of a substance which inhibits the DAGLA-encoding gene and its expression product in any one or more of:
(a1) Inhibiting the proliferation of liver cancer cells or preparing products for inhibiting the proliferation of liver cancer cells;
(a2) Inhibiting the formation of liver cancer cell colonies or preparing products for inhibiting the formation of liver cancer cell colonies;
(a3) Inhibiting the invasion and metastasis of liver cancer cells or preparing products for inhibiting the invasion and metastasis of the liver cancer cells;
(a4) Inhibiting the growth of liver cancer or preparing products for inhibiting the growth of liver cancer;
(a5) Treating liver cancer or preparing products for treating liver cancer.
In a sixth aspect of the present invention, there is provided a product comprising as an active ingredient at least a substance inhibiting DAGLA-encoding gene and expression products thereof.
The function of the product is any one or more of the following:
(a1) Inhibiting proliferation of hepatocarcinoma cell;
(a2) Inhibiting the formation of hepatoma carcinoma cell colonies;
(a3) Inhibiting invasion and metastasis of liver cancer cells;
(a4) Inhibiting the growth of liver cancer;
(a5) Treating liver cancer.
Compared with the prior art, one or more technical schemes have the following beneficial effects:
according to the technical scheme, various molecular biology technologies are utilized, and the expression level of DAGLA in human liver cancer tissues is found to be remarkably higher than that of paracancer normal tissues; meanwhile, the total survival time and the recurrence-free survival time of liver cancer patients with high-expression DAGLA are obviously shortened, the number of tumors is more, the differentiation degree is worse, and the vascular invasion is more likely to occur. Meanwhile, over-expression of DAGLA can promote proliferation, migration and invasion capacity of the liver cancer cells, and conversely, knocking down DAGLA can obviously inhibit proliferation, invasion and metastasis of the liver cancer cells.
In conclusion, the DAGLA discovered by the technical scheme is helpful for deeply understanding the pathogenesis of the liver cancer, and is also helpful for providing potential biomarkers and therapeutic targets for clinical diagnosis and prognosis, and preventing and treating the liver cancer, so that the DAGLA has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph showing the expression levels of DAGLA in liver cancer tissue (T) and paracancerous normal liver tissue (N) obtained by fluorescence real-time quantitative Polymerase Chain Reaction (PCR) in the example of the present invention.
FIG. 2 shows the expression level of DAGLA in liver cancer tissue (Tumor) and paracancerous Normal liver tissue (Normal) by immunohistochemical staining in the present example.
FIG. 3 shows that the patients were divided into high-expression group and low-expression group by using the median of DAGLA expression in the liver cancer tissue as a boundary, and the difference between the total survival time (Overall survival) and the Recurrence-free survival time (Recurrence-free survival) was analyzed.
FIG. 4 is a graph showing the sex ratio (A), tumor invasion vessels (B), tumor body number (C) and differentiation degree (D) of DAGLA-highly expressed liver cancer patient population and DAGLA-less expressed liver cancer patient population according to the present invention.
FIG. 5 shows that the influence of DAGLA on the proliferation capacity of tumor cells is detected in two human hepatoma cell lines, PLC/PRF/5 and Hep3B in the embodiment of the invention.
FIG. 6 shows the effect of DAGLA on the colony forming ability of tumor cells detected in two human hepatoma cell lines, PLC/PRF/5 and Hep3B in the present invention.
FIG. 7 shows that the influence of DAGLA on the tumor cell migration ability is detected in two human hepatoma cell strains, namely PLC/PRF/5 and Hep3B in the embodiment of the invention.
FIG. 8 shows that DAGLA is detected in two human hepatoma cell lines of PLC/PRF/5 and Hep3B in the embodiment of the invention to have the influence on the migration and invasion capacity of tumor cells.
FIG. 9 shows that a model of subcutaneous hepatoma was constructed in BALB/c nude mice in the present invention, PLC/PRF/5-con and Hep3B-con were negative control cells transfected with corresponding control lentiviral vectors, PLC/PRF/5-OE was overexpressed cells transfected with over-expressed DAGLA lentiviral vectors, and Hep3B-shDAGLA was knockdown cells transfected with knockdown DAGLA lentiviral vectors. Wherein A is the gross tumor, B is the tumor volume measurement analysis chart, and C is the tumor mass measurement analysis chart.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In a typical embodiment of the present invention, there is provided a use of a substance for detecting DAGLA-encoding genes and expression products thereof for preparing a product for diagnosing, detecting, monitoring or predicting prognosis of liver cancer.
Both the DAGLA-encoding gene and the expression product thereof can be of human origin, and the expression product of the DAGLA-encoding gene can be a DAGLA protein, namely diacylglycerol lipase alpha.
Wherein the liver cancer is hepatocellular carcinoma.
The prediction of liver cancer prognosis at least comprises the evaluation of liver cancer clinical pathological characteristics and the survival period of a subject.
The clinical pathological characteristics of the liver cancer at least comprise a liver cancer tumor state, and the liver cancer tumor state comprises tumor invasion blood vessel condition, tumor body number and tumor differentiation degree.
The subject survival includes at least subject Overall Survival (OS) and relapse-free survival (RFS). Research shows that the tumor of the patient with high expression DAGLA is more likely to invade blood vessels, the tumor number is more, and the differentiation degree is worse compared with the patient with low expression DAGLA. Meanwhile, kaplan-meier survival analysis results show that the total survival time (Overall survival) and Recurrence-free survival (Recurrence-free survival) of patients in a DAGLA high expression group are remarkably shortened.
In yet another embodiment of the present invention, there is provided a product for diagnosing, detecting, monitoring or predicting prognosis of liver cancer, comprising at least detecting transcription of DAGLA gene in a sample based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or a substance for detecting the expression of diacylglycerol lipase alpha in a sample based on an immunoassay.
In yet another embodiment of the present invention, the substance is a substance for detecting the transcription of DAGLA gene in a sample using methods including, but not limited to, northern hybridization method, liquid phase hybridization, mRNA expression profiling chip, RAKE method, in situ hybridization; or, the substance is a substance for detecting the expression of the diacylglycerol lipase alpha in the sample by using a reagent strip including but not limited to Immunohistochemistry (IHC), ELISA, colloidal gold and a protein chip.
The sample is a liver (cancer) cell or liver (cancer) tissue of a subject; the subject may be a liver cancer patient or a population at potential risk for liver cancer.
Such products include, but are not limited to, primers, probes, reagents, kits, devices (e.g., oligonucleotide probes or an assembly thereof, high-throughput detection chips on chip substrates or detection substrates, microfluidic detection chips, and the like), and devices.
In another embodiment of the present invention, a system for diagnosing, detecting, monitoring, and predicting prognosis of liver cancer is provided, the system at least comprising:
i) An analysis module, the analysis module comprising: a detection substance for determining the expression level of a gene selected from the group consisting of the above-mentioned DAGLA-encoding genes and expression products thereof in a test sample from a subject, and;
ii) an evaluation module comprising: determining the subject's condition based on the expression levels of the DAGLA-encoding genes and their expression products determined in i).
Wherein the subject can be a liver cancer patient or a population at potential risk of liver cancer; the sample may be liver (cancer) cells or liver (cancer) tissue of the subject.
As mentioned above, the liver cancer is specifically hepatocellular carcinoma.
The clinical pathological characteristics of the liver cancer at least comprise a liver cancer tumor state, and the liver cancer tumor state comprises tumor invasion blood vessel condition, tumor body number and tumor differentiation degree.
The subject survival includes at least subject Overall Survival (OS) and relapse-free survival (RFS).
In another embodiment of the present invention, the application of the DAGLA as a target in preparing or screening a liver cancer prevention and treatment drug is provided.
In another embodiment of the present invention, the method for screening a drug for preventing and treating liver cancer comprises:
1) Treating the expression and/or DAGLA containing system with a candidate substance; setting a parallel control without candidate substance treatment;
2) After the step 1) is completed, detecting the expression level of DAGLA in the system; compared with a parallel control, if the expression level of the DAGLA in the system treated by the candidate substance is obviously reduced, the candidate substance can be used as a candidate liver cancer prevention and treatment drug.
In another embodiment of the present invention, there is provided a use of a substance inhibiting DAGLA-encoding genes and expression products thereof for any one or more of:
(a1) Inhibiting the proliferation of liver cancer cells or preparing products for inhibiting the proliferation of liver cancer cells;
(a2) Inhibiting the formation of liver cancer cell colonies or preparing products for inhibiting the formation of liver cancer cell colonies;
(a3) Inhibiting the invasion and metastasis of liver cancer cells or preparing products for inhibiting the invasion and metastasis of the liver cancer cells;
(a4) Inhibiting the growth of liver cancer or preparing products for inhibiting the growth of liver cancer;
(a5) Treating liver cancer or preparing products for treating liver cancer.
Substances that inhibit DAGLA-encoding genes and their expression products include, but are not limited to, RNA interference molecules or antisense oligonucleotides against DAGLA, small molecule inhibitors, sirnas, shrnas, substances that perform lentiviral interference or gene knock-outs, and specific antibodies against DAGLA itself or molecules upstream and downstream thereof, such as anti-DAGLA antibodies.
In still another embodiment of the present invention, the above product may be a drug or a test reagent, which is used for basic research.
In still another embodiment of the present invention, there is provided a product comprising as an active ingredient at least a substance inhibiting DAGLA-encoding genes and expression products thereof.
The function of the product is any one or more of the following:
(a1) Inhibiting proliferation of hepatocarcinoma cell;
(a2) Inhibiting the formation of hepatoma carcinoma cell colonies;
(a3) Inhibiting invasion and metastasis of liver cancer cells;
(a4) Inhibiting the growth of liver cancer;
(a5) Can be used for treating hepatocarcinoma.
The substance for inhibiting the DAGLA-encoding gene and the expression product thereof includes, but is not limited to, RNA interference molecules or antisense oligonucleotides against DAGLA, small molecule inhibitors, siRNA, shRNA, substances for performing lentiviral interference or gene knock-out, and specific antibodies against DAGLA itself or molecules upstream and downstream thereof, such as anti-DAGLA antibodies.
In another embodiment of the present invention, the product can be a drug or a test reagent, which can be used for basic research.
When the product is a medicament, the medicament may further comprise one or more pharmaceutically or dietetically acceptable auxiliary materials. The adjuvants can be solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, pills and suppositories. Powders and tablets may contain from about 0.1% to about 99.9% of the active ingredient. Suitable solid excipients may be magnesium carbonate, magnesium stearate, talc or lactose. Tablets, powders, pills and capsules are solid dosage forms suitable for oral administration. Liquid form preparations include solutions, suspensions and emulsions, examples of which are aqueous parenteral solutions or water-propylene glycol solutions, or oral solutions with the addition of sweeteners and contrast agents. In addition, it can be made into small water injection for injection, lyophilized powder for injection, infusion solution or infusion solution.
The subject to which the medicament is administered may be a human or non-human mammal, such as rat, mouse, guinea pig, rabbit, dog, monkey, chimpanzee, etc.
The present invention is further illustrated by the following specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; the present invention is not particularly limited, and may be commercially available.
Examples
Experimental methods
RNA extraction
1.1 extracting cell RNA using Feijie FAST 200RNA rapid extraction kit, the specific operation steps are as follows: (1) For about 1X 10 6 A number of cells in logarithmic growth phase were collected by trypsinization into 1.5ml Eppendorf tubes, centrifuged at 800g for 5min, the supernatant was aspirated off, and the cell pellet was washed 2 times with precooled PBS. (2) Adding 500 μ l RA2 cell lysate into the cell precipitate, thoroughly blowing, mixing, standing at room temperature for 3min, and transferring the lysate into the inner sleeve of a new column-type filter sleeve. (3) centrifugation at 14,000g for 1min at 4 ℃. Taking out the inner sleeve, sucking away the liquid in the outer sleeve, and inserting the inner sleeve back into the outer sleeve. (4) Mu.l of washing buffer was added to the inner cannula and centrifuged at 14,000g for 1min at 4 ℃. Taking out the inner sleeve, sucking and discarding the liquid in the outer sleeve, and inserting the inner sleeve back into the outer sleeve. And (5) repeating the step (4) once. (6) The inner cannula was centrifuged at 14,000g for 1min at 4 ℃ without any added reagent. (7) The inner cannula was removed and inserted into a new 1.5ml Eppendorf tube, depending on the number of cells, at the bottom of the inner cannula25-50 mul of elution buffer solution is dripped into the center of the filter membrane and is kept stand for 3min at room temperature. Centrifuge at 14,000g for 1min at 4 ℃. (8) Taking out the inner sleeve, namely taking the extracted RNA sample in an Eppendorf tube, and carrying out subsequent experiments or freezing and storing at-80 ℃.
1.2 extracting tissue RNA by using Trizol method, the concrete operation steps are as follows: (1) Weighing 50-100 mg of fresh hepatocyte liver cancer specimen or paracancer normal specimen, putting into a sterile enzyme-free 1.5ml Eppendorf tube, adding 1ml of Trizol reagent and 3 grains of zirconia ceramic grinding beads into each tube, grinding for 30 seconds at 70Hz in a low-temperature tissue grinder, standing for 30 seconds, and circulating for 30 times. (2) After the grinding, 200. Mu.l of chloroform was added to each tube, the mixture was inverted and mixed, and shaken on ice for 20min. (3) centrifugation at 14000g for 15min at 4 ℃. (4) Carefully aspirate 500. Mu.l of the upper clear liquid and transfer it into a fresh sterile, enzyme-free Eppendorf tube. Adding equal volume of isopropanol into each tube, mixing by inversion, and shaking on ice for 20min. (5) centrifugation at 14000g for 20min at 4 ℃. (6) The supernatant was removed by aspiration, 1000. Mu.l of absolute ethanol was added to each tube, shaken well and shaken well on ice for 20min. (7) centrifuge at 14000g for 15min at 4 ℃. (8) repeating the step (6) once. (9) And (4) after centrifugation, a white crystal precipitate is visible at the bottom of the Eppendorf tube, and the Eppendorf tube is reversely buckled on absorbent paper and is kept stand for 3min. (10) Adding 50-100 μ l DEPC water into each tube, shaking to dissolve to obtain extracted RNA sample, and performing subsequent experiment or freezing at-80 deg.C.
2. Fluorescent real-time quantitative Polymerase Chain Reaction (PCR)
2.1 reverse transcription PCR Using TAKARA reverse transcription kit, the specific procedure is as follows:
(1) Reaction for removing genomic DNA
A reaction mixture was prepared on ice according to the following composition.
The genomic DNA removal reaction was performed in a conventional PCR instrument, and the procedure was as follows:
maintaining at 42 deg.C for 2min (or room temperature for 5 min) → 4 deg.C
(2) Reverse transcription PCR reaction
A reaction mixed solution is prepared on ice according to the following components, and the reaction mixed solution is put into a common PCR instrument immediately for reverse transcription after being gently and uniformly mixed.
The reverse transcription reaction procedure of the common PCR instrument is as follows:
15min at 37 ℃ → 5sec at 85 ℃ → maintenance at 4 ℃
2.2 fluorescent real-time quantitative PCR reaction Using Genecopoeia kit, the specific procedures were as follows:
preparing a reaction mixed solution on ice according to the following components, and carrying out subsequent on-machine detection.
The reaction program of the fluorescent real-time quantitative PCR instrument is as follows:
the related primer sequences are as follows:
3. immunohistochemical staining
(1) The temperature of the baking machine is set to be 65 ℃, and the slices are baked for 60min until the paraffin is fully melted. (2) Placing the slices in environment-friendly tissue clearing agent I (10 min), environment-friendly tissue clearing agent II (10 min), anhydrous ethanol (5 min), 95% ethanol (5 min), 80% ethanol (5 min), 70% ethanol (5 min), and PBS sequentially, and soaking for three times, each time for 5min. (3) The prepared 1 XEDTA antigen retrieval solution is poured into the antigen retrieval box and sliced. Putting the mixture into a microwave oven for antigen retrieval, wherein the medium-high fire lasts for 3-5 min, and the low fire lasts for 15min. Taking out, standing at room temperature and cooling. (4) three washes with PBS for 5min each. (5) The sections were transferred to a wet box, endogenous catalase blocking agent was added dropwise, and incubation at room temperature for 15min. PBS was washed three times for 5min each. (6) 2M HCl is added dropwise and incubated at room temperature for 20min. PBS was washed three times for 5min each. (7) Tris-HCl (pH = 8.5) was added dropwise and incubated at room temperature for 20min. PBS was washed three times for 5min each. (8) adding goat serum blocking solution dropwise, and incubating at room temperature for 20min. And (4) throwing off the liquid on the surface of the slide without washing. (9) preparing primary antibody according to the corresponding proportion, and incubating overnight at 4 ℃. (10) three washes with PBS for 5min each. (11) Adding biotin-labeled goat anti-rabbit and mouse IgG polymer dropwise, and incubating at room temperature for 20min. PBS was washed three times for 5min each. (12) And (4) dripping horseradish enzyme labeled streptavidin working solution, and incubating for 20min at room temperature. PBS was washed three times for 5min each. (13) Preparing DAB dye solution, throwing off liquid on the surface of the slide, dripping the DAB dye solution, observing the dyeing degree under a microscope, recording the time, and putting PBS into the slide for stopping color development after the color development is finished. And (14) dropwise adding hematoxylin staining solution, and standing for 2min. The tap water washes back to blue. (15) Dripping 1% hydrochloric acid alcohol solution, observing differentiation effect under microscope, repeating for several times until the effect is satisfied. (16) Placing the tissue slices in 70% ethanol (5 min), 80% ethanol (5 min), 95% ethanol (5 min), anhydrous ethanol (5 min), environment-friendly tissue clearing agent I (10 min), and environment-friendly tissue clearing agent II (10 min) in sequence, and dehydrating. (17) the mounting is dried and analyzed by photography.
4. Lentiviral transfection
(1) The cells are transfected in a six-well culture plate, and the cell density is preferably 25 to 40 percent. (2) Old medium was aspirated off, washed twice with autoclaved PBS and 1000. Mu.l of complete medium was added. (3) Add 40. Mu.l of lentivirus infection enhancing fluid per well. And (4) calculating the dosage of the lentivirus. Lentivirus dose = MOI value × number of transfected cells/lentivirus titer, unit: μ l. The MOI value (Multiplicity of Infection) is an index for measuring the proportion of virus infected cells, and the proper MOI is determined by drawing up different MOI values to detect the transfection efficiency through a preliminary experiment in advance. (5) The calculated volume of virus was added to the corresponding well and shaken well by gentle shaking. Putting the mixture into a cell culture box for normal culture. (6) After 12-16 h of transfection, the six-well plate is taken out, and the cell state is observed under a microscope. If the cell state is normal, replacing a normal complete culture medium for continuous culture after 24 hours of transfection; the cell state is poor, and the normal culture medium is replaced in time. (7) After transfection for 36-48 h, observing transfection efficiency under a fluorescence microscope, and transferring to a culture flask for continuous culture. If the transfection efficiency is not satisfactory, corresponding screening drugs with proper concentration are added into the culture medium according to the resistance label of the lentiviral vector for screening the stable transgenic cell strain.
CCK-8 experiment
(1) Collecting cells in logarithmic growth phase, adjusting the concentration to about 1X 10 4 ～2×10 4 One per ml. (2) 1000-2000 cells are added into each hole of a 96-hole cell culture plate, each hole volume is 100-200 mu l, each cell line in each group is provided with 6 multiple holes, and the cell numbers in the experimental group and the control group are kept consistent. The total time of setting was 5 days, and 5 groups were used. Putting the mixture into a cell culture box for continuous culture. And (3) taking out the culture plate after the cells are attached to the wall (about 6-12 h). Preparing CCK-8 working solution, and diluting CCK-8 stock solution to 10% by using a blank culture medium. (4) Old medium in the first day (group) wells was aspirated, 100. Mu.l of CCK-8 working solution was added to each well, and incubation was carried out at 37 ℃ for 60min. The absorbance value of the sample well at 450nm was measured with a microplate reader. (5) Taking the first measurement time as first data, and repeating the steps (3) and (4) and recording data every 24h.
6. Clone formation assay
(1) Collecting cells in logarithmic phase, blowing and beating the cells uniformly by using proper complete culture medium, and counting the cells. (2) The cells are planted in a six-hole plate with about 500-1000 cells per hole, the culture medium is filled to 2ml, and the cells are put into an incubator for culture after being fully shaken up. (3) Periodically observing under a microscope, and timely replacing the culture medium. (4) After about 10d to 14d, when the cells grew in distinct colonies and the colony size was visible to the naked eye, the cells were removed from the incubator. (5) washing with PBS for three times, and adding 1ml of methanol into each hole for fixing for 20min. PBS was washed three times. 1ml of 1% crystal violet dye solution is added into each hole, and dyeing is carried out for 12h. (6) And (4) absorbing the crystal violet dye solution, soaking and washing the six-hole plate by using double distilled water, washing off the redundant dye solution, and airing and taking a picture.
7. Scratch healing test
(1) Cells were cultured in six-well plates and, when the cell density was about 80%, the blank medium was replaced and starved for 24h. (2) And three horizontal straight lines of Mark strokes are used for assisting in marking on the back of the six-hole plate. The surfaces of six-well plates were scribed with 3 scratches using 200 μ l autoclaved tips perpendicular to the mark lines, washed twice with autoclaved PBS and replaced with blank medium. (3) Taking a picture under a microscope at 0h after the scratch, selecting a proper visual field and recording the position, then taking pictures at the same position respectively at 12h,24h,36h and 48h, and measuring and recording the change of the scratch width by using a ruler.
Transwell experiment
(1) The Transwell experiment was classified into two types of phenotype verification experiments, invasion and migration, according to whether the Matrigel matrix gel was pre-laid. The matrigel was slowly melted in advance at 4 ℃ and the transwell chamber was pre-cooled. (2) For the invasion experiment, the melted matrigel and the blank culture medium are uniformly mixed according to the proportion of 1, 50 mu l of the matrigel is dripped on the upper surface of each cell filter membrane, bubbles are avoided as much as possible, a sterile forceps is used for clamping the cells and rotating the cells to enable the matrigel to completely cover the upper surface of the filter membrane, and the cells are placed in a cell culture box for 60min to enable the matrigel to be crosslinked and solidified. For migration experiments, 100. Mu.l of blank medium was added to the transwell chamber and placed in a cell incubator for 60min of humidification. (3) Collecting cells in logarithmic growth phase, resuspending with blank medium, and controlling cell density at 2 × 10 5 ～5×10 5 Each/ml. (4) The transwell chamber was removed, the liquid in the chamber was carefully aspirated, 600. Mu.l of a medium containing 20% serum was added to the lower layer of the chamber, and the chamber was placed in the well with sterile forceps, preferably with the lower layer of the chamber in contact with the medium. Each chamber is provided with 2 x 10 4 ～5×10 4 Individual cells, no more than 200. Mu.l in volume. The chamber was placed in a cell incubator and incubation continued. (5) The corresponding group of cells was removed at 24h,36h and 48h, respectively, and placed in a new 24-well plate and rinsed 3 times for 5min each with PBS. The cell was placed in a well containing 1ml of methanol and fixed for 20min. The cells were washed 3 times with PBS for 5min each. (6) The liquid in the chamber was spun dry and dyed in 1% crystal violet dye for 24h at room temperature. After taking out, the upper surface of the chamber was lightly wiped with a cotton swab, and the photograph was taken under a microscope.
9. Subcutaneous tumor formation experiment of nude mice
(1) The 5-6 week-old athymic BALB/c nude mice were collected, and each group had 6 mice, for a total of four groups. (2) The experimental cell lines were cultured normally in petri dishes, collected by trypsinization, resuspended in blank medium and counted. According to the counting result, according to 1 × 10 7 At a ratio of one cell to 100. Mu.l/cell, cell suspensions were prepared again from the blank medium and placed on ice until use. (3) The skin of the right armpit of the nude mouse was disinfected with iodophor, and 100. Mu.l of the cell suspension was aspirated with a 1ml syringe and injected subcutaneously into the right armpit. (4) The growth state of the nude mice was observed every 3-5 days, and the presence or absence of tumor formation in the axilla was noted. The mice were sacrificed 28-35 days later, subcutaneous tumors were isolated, weighed and tumor volume calculated, and recorded by photography.
10. Statistical analysis
Statistical analysis was performed using GraphPad 8.0 and SPSS 20.0. The significance of the difference between the two sets of data was analyzed using the t-test. The DAGLA expression data and clinicopathologic feature associations were evaluated using the chi-square test. Survival curves were plotted using the Kaplan-meier method. Each set of data was replicated three times and the results were expressed as mean ± standard deviation, where P <0.05 was considered statistically significant.
Results of the experiment
In this example, the fluorescent real-time quantitative Polymerase Chain Reaction (PCR) confirmed that the expression of DAGLA in liver cancer tissue (T) is significantly higher than that in paracancerous normal liver tissue (N), and the results are shown in fig. 1. Meanwhile, as shown in fig. 2, it was confirmed by immunohistochemical staining that DAGLA was significantly higher expressed in liver cancer tissue (Tumor) than paracancerous Normal liver tissue (Normal). Indicating that the DAGLA can be used as a liver cancer diagnosis marker.
The median of DAGLA expression in the liver cancer tissue is taken as a boundary, the patients are divided into a high expression group and a low expression group, and the Kaplan-meier survival analysis result shows that the total survival time (Overall survival) and the Recurrence-free survival (Recurrence-free survival) of the patients in the DAGLA high expression group are obviously shortened (see figure 3). And as shown in fig. 4, compared with the DAGLA low expression group, the DAGLA high expression liver cancer patient population has a higher male proportion (a), tumors are more likely to invade blood vessels (B), the tumor body number is more (C), and the differentiation degree is worse (D). This suggests that DAGLA may be used for prognostic evaluation of liver cancer.
Furthermore, in order to explore the role of DAGLA in the development of liver cancer, the application of DAGLA in the development of liver cancer is firstly explored at a cellular level. The influence of DAGLA on the proliferation capacity of tumor cells is detected in two human hepatoma cell lines of PLC/PRF/5 and Hep 3B. PLC/PRF/5-con and Hep3B-con are negative control cells transfected with corresponding control lentiviral vectors, PLC/PRF/5-OE are over-expression cells transfected with DAGGA lentivirus vectors, and Hep 3B-shDAGGA is knockdown cells transfected with DAGGA lentivirus vectors. CCK-8 proliferation experiment results show that DAGLA is over-expressed to promote liver cancer cell proliferation (FIG. 5 left), whereas DAGLA is knocked down to obviously inhibit liver cancer cell proliferation (FIG. 5 right). Meanwhile, the influence of DAGLA on the colony forming capability of tumor cells is detected in two human liver cancer cell lines, namely PLC/PRF/5 and Hep 3B. The results of the clonogenic experiments are shown in fig. 6, and DAGLA can promote the hepatoma cell colony-forming ability, and the knockdown DAGLA can inhibit hepatoma cell colony-forming. And simultaneously, detecting the influence of DAGLA on the migration capacity of the tumor cells in two human hepatoma cell strains of PLC/PRF/5 and Hep 3B. Cell scratch healing experiments show that the migration ability of the liver cancer cells is promoted by DAGLA, and the liver cancer cell migration is remarkably inhibited by knocking down DAGLA (see figure 7). The influence of DAGLA on the migration and invasion capacity of tumor cells is detected in two human liver cancer cell lines, namely PLC/PRF/5 and Hep 3B. Transwell migration (above fig. 8) and invasion (below fig. 8) experiments show that DAGLA promotes migration and invasion of liver cancer cells, and knockdown DAGLA significantly inhibits liver cancer invasion and metastasis.
In order to further verify the effect of DAGLA in liver cancer, a liver cancer subcutaneous tumor model is constructed in a BALB/c nude mouse. By carrying out dissection and measurement statistical analysis on tumor bodies, we find that compared with a control group, tumor bodies formed by the liver cancer cells over-expressing DAGLA are remarkably increased, and the tumor forming capability of the liver cancer cells is remarkably inhibited by knocking down DAGLA (A is a gross tumor body, B is a tumor body volume measurement analysis diagram, and C is a tumor body mass measurement analysis diagram).
In conclusion, the invention discloses an application of an endocannabinoid synthetase DAGLA in predicting prognosis of a liver cancer patient and serving as a potential target of a novel anti-tumor drug, wherein the DAGLA chromosome position is chr11:61,680,391-61,747,001 (human genome map: GRCh38.P14, database source, https:// www.ncbi.nlm.nih.gov/gene /). According to the invention, by utilizing various molecular biological technologies, the expression level of DAGLA in human liver cancer tissues is found to be remarkably higher than that of paracancer normal tissues; the total survival time and the recurrence-free survival time of liver cancer patients with high-expression DAGLA are obviously shortened, the number of tumors is more, the differentiation degree is worse, and the vascular invasion is more likely to occur.
Then, the invention adopts the human hepatoma cell strain with stable over-expression and DAGLA knock-down as a research object, and detects the influence of DAGLA on the behavioral phenotype of the hepatoma cell. In vitro research results show that the over-expression of DAGLA can promote the proliferation, migration and invasion capacities of the liver cancer cells, and conversely, the knocking-down of DAGLA can obviously inhibit the proliferation, invasion and metastasis of the liver cancer cells.
The invention constructs a subcutaneous tumor formation model in a BALB/c immunodeficient nude mouse, and proves that the liver cancer proliferation is promoted after the DAGLA is over-expressed in the nude mouse, and the liver cancer progression can be obviously inhibited by knocking down the DAGLA. The results show that DAGLA can be used as a molecular marker for evaluating the prognosis of a liver cancer patient and is expected to become a potential action target of a novel anti-tumor drug.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. Application of substances for detecting DAGLA coding genes and expression products thereof in preparing products for diagnosing, detecting, monitoring or predicting liver cancer prognosis.
2. The use of claim 1, wherein the DAGLA-encoding gene and its expression product are of human origin;
the liver cancer is specifically hepatocellular carcinoma.
3. The use of claim 1, wherein said prognosis of liver cancer comprises at least an assessment of the clinical pathology of liver cancer and the survival of the subject;
specifically, the clinical pathological characteristics of the liver cancer at least comprise a liver cancer tumor state, wherein the liver cancer tumor state comprises tumor invasion blood vessel condition, tumor body number and tumor differentiation degree;
the subject survival includes at least overall survival and relapse-free survival of the subject.
4. A product for diagnosing, detecting, monitoring or predicting the prognosis of liver cancer, comprising at least the step of detecting the transcription of DAGLA gene in a sample based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or a substance for detecting the expression of diacylglycerol lipase alpha in a sample based on an immunoassay.
5. The product of claim 4, wherein the substance is a substance that detects the transcription of the DAGLA gene in the sample using methods including Northern hybridization, solution phase hybridization, mRNA expression profiling, RAKE, in situ hybridization; or, the substance is a substance for detecting the expression condition of the diacylglycerol lipase alpha in the sample by adopting the methods including but not limited to immunohistochemistry, ELISA, colloidal gold test paper and protein chips;
the sample is a liver (cancer) cell or liver (cancer) tissue of a subject; the subject is a liver cancer patient or a population with potential risk of liver cancer.
6. A system for diagnosing, detecting, monitoring, predicting the prognosis of a liver cancer, the system comprising:
i) An analysis module, the analysis module comprising: a detection substance for determining the expression level of a gene selected from the above-mentioned DAGLA-encoding genes and expression products thereof in a test sample of a subject, and;
ii) an evaluation module comprising: determining the subject's condition based on the expression levels of the DAGLA-encoding genes and their expression products determined in i).
7. The system of claim 6, wherein the liver cancer is particularly hepatocellular carcinoma;
the clinical pathological characteristics of the liver cancer at least comprise a liver cancer tumor state, wherein the liver cancer tumor state comprises tumor invasion blood vessel condition, tumor body number and tumor differentiation degree;
the subject survival includes at least overall survival and relapse-free survival of the subject.
8, the application of DAGLA as a target spot in preparing or screening liver cancer prevention and treatment medicines.
9. Use of a substance that inhibits a DAGLA-encoding gene and its expression product in any one or more of:
(a1) Inhibiting the proliferation of the liver cancer cells or preparing products for inhibiting the proliferation of the liver cancer cells;
(a2) Inhibiting the formation of liver cancer cell colonies or preparing products for inhibiting the formation of liver cancer cell colonies;
(a3) Inhibiting the invasion and metastasis of liver cancer cells or preparing products for inhibiting the invasion and metastasis of the liver cancer cells;
(a4) Inhibiting the growth of liver cancer or preparing products for inhibiting the growth of liver cancer;
(a5) Treating liver cancer or preparing products for treating liver cancer.
10. The use according to claim 9, wherein the substance inhibiting DAGLA-encoding genes and their expression products comprises RNA interfering molecules or antisense oligonucleotides against DAGLA, small molecule inhibitors, siRNA, shRNA, substances performing lentiviral interference or gene knock-out, and specific antibodies against DAGLA itself or molecules upstream and downstream thereof, including anti-DAGLA antibodies.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116637198A (en) * | 2023-07-27 | 2023-08-25 | 济南市中心医院 | Application of TFAM K76 locus acetylation modification in liver cancer diagnosis and treatment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116637198A (en) * | 2023-07-27 | 2023-08-25 | 济南市中心医院 | Application of TFAM K76 locus acetylation modification in liver cancer diagnosis and treatment |
| CN116637198B (en) * | 2023-07-27 | 2023-10-13 | 济南市中心医院 | Application of TFAM K76 locus acetylation modification in liver cancer diagnosis and treatment |
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