CN111879950A - Application of DDRGK1 as molecular marker for predicting prognosis of gastric cancer patient by using platinum chemotherapeutic drugs - Google Patents
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Abstract
The invention researches the relation between the expression condition of DDRGK1 in gastric cancer tissues and the sensitivity of platinum chemotherapeutic drugs for the first time, finds that the protein expression quantity of DDRGK1 is closely related to the drug resistance and the prognostic index of the platinum chemotherapeutic drugs for patients with gastric cancer in clinical treatment, and the patients with DDRGK1 high expression have good treatment effect and low drug resistance when using the platinum chemotherapeutic drugs, indicate good prognosis and are suitable for chemotherapy by using the platinum chemotherapeutic drugs; the prognosis and treatment effect of the low-expression gastric cancer patient using the platinum chemotherapeutic drugs are poor, the possibility of drug resistance is high, the prognosis is poor, and the low-expression gastric cancer patient is not suitable for chemotherapy using the platinum chemotherapeutic drugs; meanwhile, personalized treatment schemes can be formulated for different patients, so that the treatment effect is improved, and the life quality of the patients is improved; the invention has great value for clinical application and drug development.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of DDRGK1 as a molecular marker for predicting the prognosis of a gastric cancer patient by using a platinum chemotherapeutic drug; the invention also relates to application of the reagent for detecting DDRGK1 in preparing a kit and a composition for predicting the prognosis of a gastric cancer patient by using a platinum chemotherapeutic drug.
Background
Gastric cancer is one of common malignant tumors in the world, the mortality rate of the gastric cancer is the third highest in tumor median ranks, and the gastric cancer is the third highest in China. Currently, surgical resection is the only possible method for curing gastric cancer, but is only effective on early gastric cancer, and most patients are diagnosed at middle and late stages, so that the chance of surgical cure is lost. The platinum medicine is one of first-line chemotherapy medicines for middle and late gastric cancer patients and is widely applied to clinic. However, the 5-year survival of such patients is still less than 30%, the main reasons for this being tumor resistance and adverse drug reactions. Therefore, new molecules for regulating and controlling the sensitivity and the drug resistance of the gastric cancer cells to the platinum chemotherapeutic drugs are further discovered, the molecular mechanism of the action of the molecules is clarified, the selection of the individualized treatment scheme of the gastric cancer patients is facilitated, the treatment effect is improved, and the life quality of the patients is improved.
DDRGK1 is mainly localized in endoplasmic reticulum, participates in endoplasmic reticulum stress through multiple signal pathways, is an important protein for maintaining tissue and cell homeostasis, and other functions of DDRGK1 have been revealed in recent years, and it has been found that it has a great relationship with diseases such as myocardial infarction, skeletal dysplasia, blood system diseases, schizophrenia, parkinson's disease, intestinal inflammation, and ultraviolet radiation injury. However, there is no literature disclosing studies on the prognosis of DDRGK1 with tumor resistance and with chemotherapeutic drugs in tumor patients.
Therefore, there is a need to provide a new technical solution to overcome the above-mentioned drawbacks.
Disclosure of Invention
The invention aims to research the difference of protein expression quantity of DDRGK1 in gastric cancer cells and predict whether gastric cancer patients in middle and late stages are suitable for treatment by using platinum chemotherapeutic drugs, so that the gastric cancer patients are treated in a layered manner, an individual treatment scheme is selected according to the difference between the gastric cancer patients, the treatment effect is improved, and the tumor-free survival time of the gastric cancer patients after chemotherapy by using the platinum chemotherapeutic drugs is prolonged; meanwhile, the application of the reagent for detecting the DDRGK1 in the preparation of a composition and a kit for predicting the prognosis of a gastric cancer patient after using a platinum chemotherapeutic drug and the preparation of a gastric cancer treatment drug is developed.
In order to achieve the technical purpose, the invention adopts the following technical route and scheme:
the invention firstly proves that DDRGK1 can obviously increase the drug sensitivity of gastric cancer cells to platinum chemotherapeutic drugs by using a gene overexpression and gene knockdown method in the gastric cancer cells MGC803, BGC823, SGC7901 and AGS cells; then, the protein expression quantity of DDRGK1 in the gastric cancer tissues of 124 patients using platinum chemotherapeutic drugs after operation is detected by using an immunohistochemical method, and the survival period analysis result shows that the high expression of DDRGK1 can obviously prolong the tumor-free survival period of the patients; therefore, based on the analysis of the results, the invention provides the application value of DDRGK1 as a molecular marker for predicting the prognosis of gastric cancer patients using platinum chemotherapeutic drugs.
In clinical treatment, by detecting the protein expression amount of DDRGK1 in gastric cancer tissues, the prognosis of gastric cancer patients with platinum chemotherapeutic drugs can be evaluated in advance, and the method comprises the following steps: pre-evaluating the prognosis of a gastric cancer patient after chemotherapy by using a platinum chemotherapeutic drug; provides theoretical basis for better personalized chemotherapy scheme for gastric cancer patients with high DDRGK1 expression; better layered treatment is carried out on the gastric cancer patient, and finally the prognosis of the gastric cancer patient is improved.
Therefore, the invention provides the application of DDRGK1 as a molecular marker for predicting the prognosis of gastric cancer patients by using platinum chemotherapeutic drugs; also provides application of the reagent for detecting DDRGK1 in preparing a kit and a composition for predicting the prognosis of a gastric cancer patient by using a platinum chemotherapeutic drug.
The reagent for detecting DDRGK1 is an antibody against DDRGK 1.
Preferably, the anti-DDRGK 1 antibody is a DDRGK1 rabbit anti-human polyclonal antibody.
Preferably, the kit or the composition detects the protein expression level of the molecular marker DDRGK1 by an immunohistochemical method.
The judgment of the chemotherapy prognosis comprises the following steps: the chemotherapy sensitivity and chemotherapy prognosis of patients with gastric cancer using platinum chemotherapy drugs are evaluated.
As clinical detection application of the DDRGK1 molecular marker, the invention provides a kit for predicting the prognosis of gastric cancer patients after using platinum chemotherapeutic drugs, which is used for detecting the protein expression level of DDRGK1 in gastric cancer tissues and predicting the prognosis of gastric cancer patients after using platinum chemotherapeutic drugs.
The kit comprises: a rabbit anti-human polyclonal antibody specific to DDRGK1 and a secondary antibody and a color development reagent required by an immunohistochemical technology.
Immunohistochemical agents include: xylene, absolute ethyl alcohol, normal rabbit serum, EDTA antigen repair liquid, citric acid antigen repair liquid, 3% hydrogen peroxide, DDRGK1 rabbit anti-human polyclonal antibody, HRP-labeled goat anti-rabbit secondary antibody, hematoxylin staining liquid, hematoxylin differentiation liquid, hematoxylin rewet liquid, neutral gum, DAB color developing agent and PBS buffer solution.
Based on the kit, the invention provides an immunohistochemical detection method and a scoring standard, which comprise the following steps:
the method comprises the following steps: carrying out immunohistochemical staining on the gastric cancer tissue section by using an immunohistochemical reagent;
the method specifically comprises the following steps:
paraffin section dewaxing to water: sequentially placing the gastric cancer tissue slices into dimethylbenzene I15min → dimethylbenzene II15min → dimethylbenzene III15min → absolute ethyl alcohol I5 min → absolute ethyl alcohol II 5min → 85% alcohol 5min → 75% alcohol 5min → distilled water washing.
Antigen retrieval: placing the gastric cancer tissue slices in a repairing box filled with citric acid antigen repairing buffer solution (PH6.0) for antigen repairing in a microwave oven, wherein the medium fire is 8min till the gastric cancer tissue slices are boiled, stopping the fire for 8min, keeping the temperature, and then turning to the medium fire for 7min, wherein the buffer solution is prevented from being excessively evaporated in the process. After natural cooling, the slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.
Blocking endogenous peroxidase: the gastric cancer tissue slices are put into 3% hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides are placed in PBS (PH7.4) and washed for 5min each time by shaking on a decoloration shaking table.
And (5) serum blocking, namely dripping 3% BSA (bovine serum albumin) into a histochemical ring to uniformly cover the tissues, and blocking for 30min at room temperature. (Primary antibody was goat-derived blocked with rabbit serum, other sources with BSA)
Adding a primary antibody: the blocking solution was gently spun off, DDRGK1 primary antibody (1:200) prepared in a certain ratio with PBS was added dropwise to the slices, and the slices were incubated overnight at 4 ℃ in a wet box. (Small amount of water added in wet box to prevent evaporation of antibody)
Adding a secondary antibody: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section was slightly spun dry, a secondary antibody (HRP-labeled) to the corresponding species was added dropwise to the ring to cover the tissue, and the mixture was incubated at room temperature for 50 min.
DAB color development: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive color is brown yellow, and the section is washed by tap water to stop color development.
Counterstaining cell nuclei: counter-staining with hematoxylin for about 3min, washing with tap water, differentiating with hematoxylin differentiation solution for several seconds, washing with tap water, returning the hematoxylin to blue, and washing with running water.
Dewatering and sealing: and (3) putting the gastric cancer tissue slices into 75% alcohol for 5min → 85% alcohol for 5min → absolute ethanol I for 5min → absolute ethanol II for 5min → xylene I for 5min in sequence, dehydrating and transparentizing, taking the gastric cancer tissue slices out of the xylene, slightly drying, and sealing with neutral gum.
Step two: microscopic examination is carried out by using a microscope, and images are collected;
step three: analysis using biological image processing software gave immunohistochemical scores.
Protein expression of DDRGK1 was semi-quantitatively evaluated according to the following scoring criteria based on the percentage of positive cells and staining intensity in gastric cancer tissues (3 random fields at 400-fold).
Cells in the gastric cancer tissue section are light yellow to dark brown and are taken as positive cell markers. The staining intensity was scored as the staining property exhibited by most cells (the staining intensity was compared with the background staining): no coloration is defined as 0 points, yellowish is defined as 1 points, tan is defined as 2 points, and tan is defined as 3 points. Percentage of positive cells i.e. average number of positive cells in 5 fields (400 x) of a certain type of cells: less than 10% is defined as score 1, 10% to 25% is defined as score 2, 25% to 50% is defined as score 3, 50% to 75% is defined as score 4, > 75% is defined as score 5. And multiplying the two scores to obtain a semi-quantitative analysis result, wherein the low expression is obtained when the score is less than or equal to 9, and the high expression is obtained when the score is more than 9.
According to the experimental results, the high-expression DDRGK1 gastric cancer patient has good treatment effect by using platinum chemotherapeutic drugs, and the prognosis has long tumor-free survival period; and the low-expression gastric cancer patient has poor treatment effect by using platinum chemotherapeutic drugs, and the prognosis tumor-free survival time is short.
Compared with the prior art, the invention has the following beneficial effects:
the invention researches the relation between DDRGK1 expression in stomach cancer tissues and the sensitivity of stomach cancer patients using platinum chemotherapeutic drugs for the first time, finds that DDRGK1 has different protein expression amounts in stomach cancer tissues of different stomach cancer patients, and the protein expression amount of DDRGK1 is closely related to the drug resistance and the prognosis index of the stomach cancer patients using the platinum chemotherapeutic drugs for clinical treatment, and the patients with high DDRGK1 expression have good treatment effect and low drug resistance when using the platinum chemotherapeutic drugs, indicate good prognosis and are suitable for using the platinum chemotherapeutic drugs for chemotherapy; and the low-expression gastric cancer patient has poor prognosis and treatment effect by using the platinum chemotherapeutic drugs, has high possibility of drug resistance, indicates poor prognosis and is not suitable for chemotherapy by using the platinum chemotherapeutic drugs. Therefore, DDRGK1 is used as a molecular marker for predicting the prognosis of the gastric cancer patient using the platinum chemotherapeutic drug, the kit for predicting the prognosis of the gastric cancer patient using the platinum chemotherapeutic drug is provided, whether the gastric cancer patient is suitable for chemotherapy using the platinum chemotherapeutic drug can be judged in advance before chemotherapy, and therefore layered treatment can be performed on the patient, personalized treatment schemes can be formulated for different patients, the treatment effect is improved, and the life quality of the patient is improved; meanwhile, the method has great value for clinical application and drug development.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below.
FIG. 1A is a graph showing that stable overexpression of DDRGK1 in the present invention increases the drug sensitivity of gastric cancer cell MGC803 to cisplatin. CCK8 cell proliferation experiments demonstrated changes in MGC803 cell viability following different doses of cisplatin treatment for 24h or 48h, stably over-expressing DDRGK1(DDRGK1) and its control (pLVX);
FIG. 1B is a graph showing that DDRGK1 shown in FIG. 1A is successfully and stably overexpressed in gastric cancer cell MGC803, and pLVX is a negative control in the present invention;
FIG. 1C shows that over-expression of DDRGK1 increases the drug sensitivity of gastric cancer cell BGC823 to cis-platin in accordance with the present invention. CCK8 cell proliferation experiment verifies that after different doses of cisplatin treatment for 24h or 48h, the BGC823 cell viability of over-expressing DDRGK1(Myc-DDRGK1, Myc is a label) and its control (pcDNA3.1) is changed;
FIG. 1D is a verification in the present invention that DDRGK1 in FIG. 1C was successfully over-expressed in gastric cancer cell BGC823 (the expression of DDRGK1 was detected by Myc tag), pcDNA3.1 is a negative control;
FIG. 2A is a graph of the drug sensitivity of DDRGK1 knockdown in the present invention to reduce gastric cancer cell SGC7901 to cisplatin. The CCK8 cell proliferation experiment verifies that after different doses of cisplatin treatment are carried out for 24h, BGC823 cell viability changes of DDRGK1(siDDRGK1) and a control (siNC) thereof are knocked down;
FIG. 2B is a graph showing that DDRGK1 shown in FIG. 2A was successfully knocked down in gastric cancer cell SGC7901, and sinC is a negative control in the present invention;
FIG. 2C shows that DDRGK1 knocks down the drug sensitivity of gastric cancer cell AGS to cisplatin in the present invention. The CCK8 cell proliferation experiment demonstrated changes in AGS cell viability with different doses of cisplatin 24h after knockdown of DDRGK1(siDDRGK1) and its control (siNC);
FIG. 2D is a graph demonstrating the successful knockdown of DDRGK1 in gastric cancer cell AGS in FIG. 2C, with sinC as a negative control, in accordance with the present invention;
FIG. 3A is a graph showing that stable overexpression of DDRGK1 reduces the formation of MGC803 clones in the presence of cisplatin in accordance with the present invention. Clonogenic experiments demonstrated the change in clonogenic of MGC803 that stably overexpressed DDRGK1(DDRGK1) and its control (pLXV) 24h after cisplatin treatment;
FIG. 3B is a statistical result of the 3 biological replicates of FIG. 3A in the present invention. Results statistical tests were performed on student's t-test with mean ± standard deviation, pLVX as control: p < 0.05;
FIG. 4A is a graph showing that stable knock-down of DDRGK1 in the present invention increases the clonal formation of SGC791 in the presence of cisplatin. Clonogenic experiments demonstrated that after cisplatin treatment for 24h, the clonogenic changes of SGC7901 for DDRGK1(shDDRGK1) and its control (shNC) were stably knocked down;
FIG. 4B is a statistical result obtained from 3 biological replicates of FIG. 4A in the present invention. Results were statistically tested by student t-test with mean ± sd, shNC as control, x: p < 0.05;
figure 5A is a graph of DDRGK1 overexpression in the present invention increasing the drug sensitivity of gastric cancer cells MGC803 to oxaliplatin. CCK8 cell proliferation experiment detects UFBP1 stable overexpression (UFBP1) and change of control (pLVX) cell viability after oxaliplatin with different concentrations is treated for 24 hours, and IC is calculated50；
FIG. 5B is the IC of the present invention obtained from 4 biological replicates of FIG. 5A50The statistical result of (2). Results statistical tests were performed on student's t-test with mean ± standard deviation, pLVX as control: p<0.05；
Figure 6A is a graph of the knockdown of DDRGK1 in the present invention reduces the drug sensitivity of gastric cancer cell MGC803 to oxaliplatin. CCK8 cell proliferation experiment detects UFBP1 stable knockdown (shUFBP1) and the change of control (shNC) cell activity after oxaliplatin with different concentrations is treated for 24 hours;
FIG. 6B is the IC of the present invention obtained from 3 biological replicates of FIG. 6A50The statistical result of (2). Results were statistically tested by student t-test with mean ± sd, shNC as control, x: p<0.05；
Figure 7A is a graph of UFBP1 in accordance with the present invention increasing the tumor-free survival of advanced gastric cancer patients following surgery with platinum-based chemotherapeutic agents. Dividing protein expression of UFBP1 into high expression group (n-57) and low expression group (n-67), and plotting relationship between protein expression of UFBP1 and tumor-free survival time of gastric cancer patients by GraphPadprism;
fig. 7B is a typical photograph of immunohistochemistry with high and low expression of UFBP1 in gastric cancer tissue as described in fig. 7A, scale: 100 μm.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments.
The application of DDRGK1 as a molecular marker for predicting the prognosis of gastric cancer patients with platinum chemotherapeutic drugs will be described in detail below with reference to the accompanying drawings. Unless otherwise specified, the specific experimental procedures mentioned in the examples are conventional procedures, and the experimental reagents used are purchased from conventional companies. One of them was anti-DDRGK 1 rabbit anti-human polyclonal antibody.
Example 1
Correlation analysis between the difference in protein expression level of DDRGK1 in gastric cancer cells and the drug sensitivity of gastric cancer cells to platinum-based chemotherapeutic drugs
Detecting an object: gastric cancer cells MGC803, BGC823, SGC7901 and AGS
The experimental method comprises the following steps:
1. CCK-8 cell proliferation assay
Inoculating gastric cancer cells into 96-well plate, wherein the cell amount of each well is 1 × 103Individual cells (cell proliferation assay) or 8X 103Setting 4-5 multiple wells for each cell (drug sensitivity experiment), collecting cells at different time points after the cells adhere to the wall (12h), and detecting the cell proliferation condition after adding cisplatin with different concentrations and control treatment (DMSO) cells (drug sensitivity experiment). During detection, the CCK-8 premix is prepared firstly, then the 96-well plate is taken out, the original culture medium in the hole is removed, 100mL of the CCK-8 premix is added into each hole, the 96-well plate is continuously placed at 37 ℃ and 5% CO2After the cells are cultured in the constant-temperature incubator for 1h, the light absorption value is measured by an enzyme-labeling instrument at the wavelength of 450nm, and the cell proliferation condition is calculated.
2. Clone formation experiments
Complete media containing a final concentration of cisplatin of 0.625. mu.g/mL (MGC803) or 0.313. mu.g/mL (SGC7901) and its corresponding concentration of DMSO (negative control without cisplatin) were prepared. Will be steadily rotatedGastric cancer cells were seeded into small dishes (3.5cm) with a cell count of 1X 10 per dish3Setting 4 repeated dishes for each group of cells, after the cells are attached to the wall (12h), discarding the original culture medium, changing the culture medium into a complete culture medium containing DMSO or cisplatin (hereinafter cis-platinum is used for replacing), continuing to culture the cells for 24h, discarding the culture medium containing the drug, changing the culture medium into a new complete culture medium without the drug, continuing to culture for 10 days (MGC803) or 7 days (SGC7901 cells), changing the medium for 2-3 times, then taking out the cells, removing the original culture medium by suction, carefully washing the cells twice by using 1 XPBS, then adding 800mL of 4% paraformaldehyde, fixing for 20min at normal temperature, and (3) sucking away the fixing solution, carefully washing the cells once with 1 XPBS, adding 800mL of 0.1% crystal violet, dyeing at normal temperature for 20-30 min, sucking away the dyeing solution, carefully washing the cells twice with 1 XPBS, opening the culture dish into a fume hood, naturally airing, photographing and calculating the number of formed clones.
3、IC50Measurement experiment
Using the experimental procedure of the CCK8 cell proliferation experiment in example 1, the proliferation of cells after 24 hours of addition of different concentrations of oxaliplatin and its control treatment (DMSO) was obtained, and then IC was calculated by GraphPad Prism50The value is obtained.
We tested the changes in viability of 24h and 48h cells treated with different doses of cisplatin in MGC803 cells stably overexpressing DDRGK1 and BGC823 cells transiently overexpressing DDRGK1 using CCK-8 cell proliferation assays; further, we also used siRNA to transiently knock down DDRGK1(siDDRGK1) in gastric cancer cells SGC7901 and AGS, and also examined the change in viability of 24h cells treated with different doses of cisplatin using the CCK-8 cell proliferation assay.
The experimental results show that:
as shown in fig. 1A to 1D, stable overexpression of DDRGK1 significantly increased the cytotoxicity of cisplatin towards MGC803 and BGC823 cells; as shown in fig. 2A to 2D, the knockdown of DDRGK1 significantly reduced cisplatin cytotoxicity on SGC7901 and AGS cells, and this increase or decrease in cytotoxicity was time and dose dependent.
We next examined the effect of cisplatin treatment on clonogenic behavior of MGC803 stably overexpressing DDRGK1 and SGC7901 mixed clones stably knockdown DDRGK1 and their corresponding control cells using a clonogenic assay.
The experimental results show that:
as shown in fig. 3A and 3B, over-expression of DDRGK1 in the presence of cisplatin significantly reduced clone formation; as shown in fig. 4A and 4B, the knockdown of DDRGK1 significantly increased the formation of clones in the presence of cisplatin.
Oxaliplatin (Oxaliplatin) is a3 rd generation platinum anticancer drug and is a platinum compound of diaminocyclohexane, namely 1, 2-diaminocyclohexane group is used for replacing amino of cisplatin, the action of the platinum compound is the same as that of cisplatin, DNA is used as an action target, and platinum atoms and the DNA form cross connection to antagonize the replication and transcription of the platinum compound. At present, the platinum in the first-line chemotherapy drugs for middle and late gastric cancer patients is mainly oxaliplatin, and we have proved that DDRGK1 can increase the drug sensitivity of gastric cancer cells to cisplatin, and in order to more directly determine the relationship between DDRGK1 and the sensitivity of gastric cancer to platinum chemotherapy drugs, we further investigate whether DDRGK1 affects the drug sensitivity of gastric cancer cells to oxaliplatin through CCK-8 cell proliferation experiments.
The experimental results show that:
as shown in FIGS. 5A and 5B, we found that over-expression of DDRGK1 in MGC803 significantly reduced the IC of cells treated with oxaliplatin for 24h50(pLVX: 22.91. mu.M, DDRGK1: 12.65. mu.M); as shown in FIGS. 6A and 6B, knocking down DDRGK1 in SGC7901 significantly increased IC of cells treated with oxaliplatin for 24h50(shNC: 64.63. mu.M, shDDRGK1: 95.84. mu.M). Therefore, like cisplatin, DDRGK1 also significantly increased the drug sensitivity of gastric cancer cells to oxaliplatin.
In summary, these experiments used different methods of gene processing including stable or transient transformants, over-expressed or knockdown; various biochemical experiments, including CCK-8 cell proliferation and clonogenic experiments, demonstrated that DDRGK1 significantly increased the drug sensitivity of gastric cancer cells to platinum-based chemotherapeutic drugs from the cellular level.
Wherein the IC50The method comprises the following steps: half inhibitory concentration (concentration of drug that inhibits half of cell growth); pLVX is: negative control stably overexpressing DDRGK1(ii) a pcDNA3.1 is a negative control for transient overexpression of DDRGK 1; shNC is: negative control with stable knockdown of DDRGK 1; siNC is a negative control for transient knockdown of DDRGK 1.
Example 2
Detection of protein expression level of DDRGK1 in 124 collected gastric cancer tissue specimens by immunohistochemical method
Collecting patient data: screening the cases from the electronic case system of the second hospital affiliated with suzhou university, the inclusion criteria of the cases included: pathological diagnosis is stomach cancer through electron microscope; surgical resection of gastric cancer was performed between 2011 and 2015 at 12 months; post-operative platinum-based chemotherapy regimens alone; the platinum based chemotherapy regimen was used for 3 cycles or more. Patients with gastric cancer that met the inclusion criteria were subjected to TNM staging according to AJCC seventh edition, and at the same time, patients were subjected to electronic case review to obtain Disease Free Survival (DFS) for the patients.
Detection of protein expression of DDRGK1 in gastric cancer tissue by SP method (streptavidin-peroxidase): formalin-fixed, paraffin-embedded gastric cancer tissue specimens were obtained from patients meeting the selection criteria, and protein expression of DDRGK1 in the specimens was detected by immunohistochemical analysis.
The method comprises the following specific steps:
the method comprises the following steps: carrying out immunohistochemical staining on the gastric cancer tissue section by using an immunohistochemical reagent;
the method specifically comprises the following steps:
paraffin section dewaxing to water: sequentially placing the gastric cancer tissue slices into dimethylbenzene I15min → dimethylbenzene II15min → dimethylbenzene III15min → absolute ethyl alcohol I5 min → absolute ethyl alcohol II 5min → 85% alcohol 5min → 75% alcohol 5min → distilled water washing.
Antigen retrieval: placing the gastric cancer tissue slices in a repairing box filled with citric acid antigen repairing buffer solution (PH6.0) for antigen repairing in a microwave oven, wherein the medium fire is 8min until the tissue slices are boiled, stopping the fire for 8min, keeping the temperature, and turning to the medium and low fire for 7min, wherein the buffer solution is prevented from being excessively evaporated in the process, and the dry slices are not cut. After natural cooling, the slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.
Blocking endogenous peroxidase: the gastric cancer tissue slices are put into 3% hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides are placed in PBS (PH7.4) and washed for 5min each time by shaking on a decoloration shaking table.
And (5) serum blocking, namely dripping 3% BSA (bovine serum albumin) into a histochemical ring to uniformly cover the tissues, and blocking for 30min at room temperature. (Primary antibody was goat-derived blocked with rabbit serum, other sources with BSA)
Adding a primary antibody: the confining liquid is gently thrown off, DDRGK1 primary antibody prepared according to a certain proportion is dripped on the slices, and the slices are flatly placed in a wet box for incubation at 4 ℃ overnight. (Small amount of water added in wet box to prevent evaporation of antibody)
Adding a secondary antibody: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section was slightly spun dry, a secondary antibody (HRP-labeled) to the corresponding species was added dropwise to the ring to cover the tissue, and the mixture was incubated at room temperature for 50 min.
DAB color development: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive color is brown yellow, and the section is washed by tap water to stop color development.
Counterstaining cell nuclei: counter-staining with hematoxylin for about 3min, washing with tap water, differentiating with hematoxylin differentiation solution for several seconds, washing with tap water, returning the hematoxylin to blue, and washing with running water.
Dewatering and sealing: and (3) putting the gastric cancer tissue slices into 75% alcohol for 5min → 85% alcohol for 5min → absolute ethanol I for 5min → absolute ethanol II for 5min → xylene I for 5min in sequence, dehydrating and transparentizing, taking the gastric cancer tissue slices out of the xylene, slightly drying, and sealing with neutral gum. The experiment was performed with IgG antibody from the same source as a negative control in place of the primary antibody.
Step two: and (5) microscopic examination is carried out by using a microscope, and the image is collected.
Step three: analyzing by using biological image processing software to give an immunohistochemical score;
thirdly, a grading method: protein expression of DDRGK1 was semi-quantitatively evaluated according to the following scoring criteria based on the percentage of positive cells and staining intensity in gastric cancer tissues (3 random fields at 400-fold).
Cells in the gastric cancer tissue section are light yellow to dark brown and are taken as positive cell markers. The staining intensity was scored as the staining property exhibited by most cells (the staining intensity was compared with the background staining): no coloration is defined as 0 points, yellowish is defined as 1 points, tan is defined as 2 points, and tan is defined as 3 points. Percentage of positive cells i.e. average number of positive cells in 5 fields (400 x) of a certain type of cells: less than 10% is defined as score 1, 10% to 25% is defined as score 2, 25% to 50% is defined as score 3, 50% to 75% is defined as score 4, > 75% is defined as score 5. And multiplying the two scores to obtain a semi-quantitative analysis result, wherein the low expression is obtained when the score is less than or equal to 9, and the high expression is obtained when the score is more than 9.
Based on the experimental results, we found that the protein expression level of DDRGK1 was different in the gastric cancer group of different patients, 57 cases were high expression, and 67 cases were low expression. The data were statistically significant (p <0.01) after statistical chi-square testing.
Fourthly, analyzing results: according to the median of protein expression of DDRGK1, dividing patients into a DDRGK1 high expression group and a DDRGK1 low expression group, combining DFS of corresponding patients, performing KKaplan-MeierPluter survival time analysis by using Graphpad software (V6.02), performing Log-Rank test and drawing a survival curve, and analyzing the relation between DDRGK1 expression and DFS of gastric cancer patients.
Experimental results as shown in fig. 7A-7B, we found that high expression of DDRGK1 significantly prolonged tumor-free survival in patients (P0.0173) with statistically significant differences (P <0.05) using Kaplan-Meier Plotter survival analysis. The experimental results further reveal that DDRGK1 can significantly influence the prognosis of gastric cancer patients with platinum-based chemotherapy, and can be used as a molecular marker for predicting the prognosis of gastric cancer patients with platinum-based chemotherapy.
Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention.
Claims (9)
- Use of DDRGK1 as a molecular marker for predicting the prognosis of gastric cancer patients with platinum-based chemotherapeutic drugs, characterized in that: the application is to predict the prognosis of gastric cancer patients with platinum chemotherapeutic drugs by detecting the protein expression level of DDRGK1 in gastric cancer cells.
- 2. Use according to claim 1, characterized in that: comparing the detected protein expression quantity of the DDRGK1 with a preset protein expression quantity threshold value of DDRGK1, wherein the condition that the protein expression quantity is higher than the threshold value is evaluated as that the gastric cancer patient is sensitive to the platinum chemotherapeutic drug and/or the prognosis of the platinum chemotherapeutic treatment is good, and the condition that the protein expression quantity is lower than the threshold value is evaluated as that the gastric cancer patient is not sensitive to the platinum chemotherapeutic drug and/or the prognosis of the platinum chemotherapeutic treatment is not good.
- 3. The application of a reagent for detecting DDRGK1 in preparing a kit for predicting the prognosis of a gastric cancer patient by using a platinum chemotherapeutic drug.
- 4. Use of an agent that detects DDRGK1 in the manufacture of a composition for predicting prognosis of a gastric cancer patient with a platinum-based chemotherapeutic agent.
- 5. Use according to claim 3 or 4, characterized in that: the reagent for detecting DDRGK1 is an antibody against DDRGK 1.
- 6. The kit for detecting the expression level of DDRGK1 in gastric cancer tissue according to claim 5, wherein: the anti-DDRGK 1 antibody is a DDRGK1 rabbit anti-human polyclonal antibody.
- 7. The kit for detecting the expression amount of DDRGK1 in gastric cancer tissue according to claim 3 or 4, wherein: the kit or the composition detects the protein expression amount of the molecular marker DDRGK1 in gastric cancer tissues by an immunohistochemical method.
- 8. The use of claim 7, wherein the immunohistochemical method comprises the following steps:the method comprises the following steps: carrying out immunohistochemical staining on the gastric cancer tissue section by using an immunohistochemical reagent;step two: microscopic examination is carried out by using a microscope, and images are collected;step three: analysis using biological image processing software gave immunohistochemical scores.
- 9. The use according to claim 8, wherein the percentage of positive cells and the staining intensity in gastric cancer tissues are determined based on immunohistochemical results, and the protein expression of DDRGK1 is evaluated semi-quantitatively according to the following scoring criteria;the cells in the gastric cancer tissue section are light yellow to brown and are taken as positive cell markers;staining intensity was scored as the staining property exhibited by most cells: no coloration is defined as 0 point, light yellow is defined as 1 point, tan is defined as 2 points, and tan is defined as 3 points;percent positive cells is the average number of positive cells in a plurality of randomly selected cell fields: less than 10% is defined as score 1, 10% -25% is defined as score 2, 25% -50% is defined as score 3, 50% -75% is defined as score 4, > 75% is defined as score 5;and multiplying the two scores to obtain a semi-quantitative analysis result, wherein the low expression is obtained when the score is less than or equal to 9 points, and the high expression is obtained when the score is more than 9 points.
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