CN113384564A - Application of p53 target-based trimethoxy dihydroxy phenanthrene compound in cervical cancer resistance and detection method - Google Patents
Application of p53 target-based trimethoxy dihydroxy phenanthrene compound in cervical cancer resistance and detection method Download PDFInfo
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- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2333/4748—Details p53
Abstract
The invention discloses an application and a detection method of a p53 target-based trimethoxy dihydroxy phenanthrene compound in the aspect of cervical cancer resistance, and discovers the effect of the compound 1,5, 6-trimethoxy-2, 7-dihydroxy phenanthrene on cervical cancer Hela cells through detection, and the compound is used for preparing, treating or preventing human cervical cancer, and cell scratch experiments prove that the compound can inhibit the migration of the Hela cells; the compound can induce apoptosis through Hoechst33258 fluorescent staining, annexin V-FITC/PI double staining and PI single staining apoptosis detection; the compound can induce G0/G1 phase block of Hela cells as proved by a PI single staining cell cycle method. Confirmed by the above various methods: the invention provides scientific theoretical basis for clinical application of trimethoxy dihydroxy phenanthrene compounds based on p53 target spots or pathways to resist cervical cancer.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of p53 target-based 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene in cervical cancer resistance and a detection method.
Background
Malignant tumors are known as cancers and are a general term for more than 100 related diseases. When a cell in the body mutates, it constantly divides and is not controlled by the body, and finally forms cancer. The morbidity and mortality of cancer is second only to cardiovascular disease. Cancer can be classified into two broad categories, one of which is that of epithelial origin, such as lung cancer, breast cancer, esophageal cancer, stomach cancer, cervical cancer, colon cancer, and the like. Among them, cervical cancer is a malignant tumor occurring in the cervical part of the uterus, which is also called cervical invasive carcinoma. It is the most serious cervical lesion and one of the three major gynecological malignancies. Not only the female reproductive organ tumor is the first disease, but also the most common diseases in various malignant tumors of the female. Cervical cancer in women is the only gynecological disease which can be found in early stage at present. Compared with endometrial cancer and ovarian cancer, the cervical cancer has obvious pathological symptoms, and can be divided into 3 types, namely mild, moderate and severe, according to the disease; the degree of disease is different, the possibility of developing into cervical cancer is different, generally, 15% of mild, 30% of moderate and 45% of severe precancerous lesions of cervical cancer can develop into cervical cancer, the more serious the degree of precancerous lesions, the higher the probability of developing into cervical cancer, and mild Zi emperor cervical precancerous lesions can be cured completely after being treated in time.
Cancer may be treated by surgical resection, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy, or other methods. The choice of treatment modality depends on the location of the tumor, the degree of malignancy, the degree of progression, and the physical state of the patient. The treatment of cancer, whether chemotherapy, surgery or radiotherapy, is a significant burden on the body and after malignant metastasis, it is difficult to completely cure in any way. Oncogene, oncogene suppressor gene discovery, and the theory of apoptosis have been developed, and their understanding has also been carried out from the cellular level to the molecular level. Cancer is related to changes of a plurality of oncogenes and cancer suppressor genes, information transmission and protease activity related to apoptosis are researched, and a new means for treating cancer is hopefully provided.
In recent years, some progress is made in the research of the cancer suppressor gene p53, and the target is widely concerned as a target drug for researching and treating cancer. The MDM2 protein is the most important negative regulator of p53, and is involved in regulating the stability and activity of p53 protein, inhibiting cell growth, inducing apoptosis and regulating cell cycle function. However, the application of the p53 target and the action pathway of MDM2-p53 protein and protein in the preparation of antitumor drugs for treating and/or preventing human cervical cancer is rarely reported.
Disclosure of Invention
The invention aims to provide a method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on a p53 target spot, and also provides application of the method in the aspect of resisting cervical cancer.
In order to realize the purpose, the following technical scheme is adopted:
apoptosis is one of the programmed forms of cell death and is an important form of cell homeostasis, whether endogenous or exogenous, induced by the target gene p 53. Combining with a flow cytometer, Hoechst33258, the method proves that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can induce Hela cells to generate apoptosis in a concentration-dependent manner, and the cell cycle is blocked at the G0/G1 stage. This is consistent with the expression that activation of the p53 pathway inhibits cell cycle, promotes damaged cellular DNA repair, and promotes apoptosis when normal cells are damaged. In order to further study the action mechanism of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for inducing Hela cells to generate apoptosis, Western Blot is used to detect the influence of the Western Blot on the expression of proteins such as p53 pathway, Bcl-2 family (Bax, Bcl-2 and Mcl-1) and the like. The results show that in Hela cells, the expression level of p53 protein is increased by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, the Bax/Bcl-2 ratio is increased, the expression level of Mcl-1 is reduced, and the expression level of a downstream target gene MDM2 of p53 is also increased. As is known, p53 and MDM2 have negative feedback regulation, and the over-expression of MDM2 protein does not inhibit the expression of p53 protein in the invention, so that p53 can play normal biological functions. Therefore, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene was shown to act on the p53-MDM2 pathway, blocking the inverse regulation of p53 by MDM 2. In order to further prove whether the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene plays a role in anti-tumor activity or not depending on p53, a cell p53 siRNA knockdown experiment is carried out on Hela cells, the result shows that the survival rate of the cells in the p53 siRNA + Drug group is obviously higher than that of the cells in the Control siRNA + Drug group, and the p53 participates in the anti-proliferation activity of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the Hela cells. Based on the research, the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene has potential to develop a therapeutic drug for resisting cervical cancer.
In conclusion, the invention proves that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene inhibits Hela cell proliferation in the G0/G1 phase through cell cycle arrest, exerts an anti-tumor effect through inducing apoptosis, and realizes an inhibition effect through activating P53, and the specific mechanism is based on a P53 target and an MDM2-P53 protein and protein action path, as shown in figure 1.
The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on a p53 target spot specifically comprises the following steps:
the following tests are not in order.
1. And detecting the influence of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the proliferation of Hela cells by adopting an IncuCyte living cell imaging system.
The method comprises the following specific steps:
according to 0.5X 104Hela cells are taken at the density of each ml, and are plated in a 96-well plate to be divided into a DMSO control group (the control group for short) and a drug concentration group (the group given 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group for short), and 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (3.125 mu M-100 mu M) with different concentrations is added into the drug concentration group. And (3) placing the 96-well plate in an IncuCyte living cell imaging system, collecting cell image dynamics at regular time, and calculating the cell fusion rate under different treatment conditions.
2. Cell scratch experiments were performed to quantify the potential for collective spreading of cells.
The method comprises the following specific steps:
according to 2X 105Taking Hela cells at the density of each ml, plating the Hela cells in a 96-well plate, culturing the cells, performing scratching and buffer washing when the cell fusion rate reaches 90-100%, treating the Hela cells by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, photographing the Hela cells at different time points (0h-24h) in an IncuCyte living cell imaging system, and calculatingThe difference between the scratch widths of 0h and 24h was calculated and the mobility of the cells was expressed as the ratio between the difference and the initial scratch width. The buffer solution is PBS, and washing is carried out for 2-3 times.
3. Hoechst33258 fluorescent staining experiments were performed to analyze morphological changes caused by apoptosis.
The method comprises the following specific steps:
(1) cell culture: according to 7.5X 104Hela cells were harvested at cell density per ml, plated in six-well plates, and cultured.
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (3.125. mu.M, 6.25. mu.M) treatment is given.
(3) Fixing: after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene intervenes in Hela cells for 48 hours, 4% of paraformaldehyde is added, and the mixture is fixed at normal temperature in a dark place.
(4) Dyeing: sucking out the fixing solution (paraformaldehyde), washing with the buffer solution, adding Hoechst33258 staining solution for staining, and incubating at 37 ℃ in a dark place.
(5) And (3) observation by a fluorescence microscope: and (5) washing with buffer solution, observing the cell morphology under a fluorescence microscope, and taking pictures.
In the step (3), the fixed time is 15 min. In the steps (4) and (5), the buffer solution is PBS, washing is carried out for 3 times, 3min is carried out each time, and incubation is carried out for 15 min.
4. An annexin V-FITC/PI (propidium iodide) double staining apoptosis detection experiment is carried out to distinguish early and late apoptosis, necrosis and normal cells.
The method comprises the following specific steps:
(1) cell culture: according to 7.5X 104Hela cells were harvested at cell density per ml, plated in six-well plates, and cultured.
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (3.125. mu.M, 6.25. mu.M, 12.5. mu.M) treatment was given when the cells grew to 60% -70%.
(3) Collecting cells: and (3) treating the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48 hours, and collecting cells.
(4) Adding 1 × Annexin V Bingding Solution: adding 1 × Annexin V binding Solution into a control group and a 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group respectively.
(5) Grouping experiments: the control component is a negative control group, an Annexin V-FITC staining group, a PI staining group, an Annexin V-FITC and PI double staining group; the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene component is Annexin V-FITC and PI double staining group.
(6) Dyeing: adding Annexin V-FITC conjugate into each group in the step (5), adding PI Solution, and culturing at room temperature in a dark place.
(7) Flow detection: adding 1 × Annexin V Binding Solution, and performing machine detection within 1 h.
5. The experiment for detecting apoptosis by PI (propidium iodide) single staining is carried out.
The method comprises the following specific steps:
(1) cell culture: according to 7.5X 104Hela cells were harvested at cell density per ml, plated in six-well plates, and cultured.
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group (3.125. mu.M) is administered after the cells grow to 60% -70%.
(3) Collecting cells: adding 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene to intervene in Hela cells for 48h, collecting the cells, reserving the supernatant, and collecting the cells according to the method of the step 4 (3).
(4) Ethanol fixation: adding 75% ethanol, and fixing at 4 deg.C.
(5) Propidium Iodide (PI) staining: after ethanol fixation, propidium iodide staining solution was added to each tube of cell sample, and the tube was incubated at 37 ℃ in the dark.
(6) Flow detection: cell cycle assays were performed using a flow cytometer over 1 h.
6. PI single staining cell cycle detection experiments were performed.
The cell cycle after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on Hela cells is detected by using a flow cytometer through single staining with PI.
(1) Cell culture: the method is the same as the step 5.
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: the method is the same as the step 5.
(3) Collecting cells: collecting cells after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene intervention is carried out for 36h, discarding the supernatant, and collecting the cells according to the method of the step 4 (3).
(4) Ethanol fixation: the method is the same as the step 5.
(5) Propidium iodide staining: the method is the same as the step 5.
(6) Flow detection: the method is the same as the step 5.
7. The target protein was detected by performing Western blotting (Western Blot) based on the principle of antigen-antibody binding by gel electrophoresis.
The method comprises the following specific steps:
(1) extraction of total cellular protein:
cell culture: according to 7.5X 104HeLa cells were taken at a cell density of one cell/ml, plated in a six-well plate, and subjected to cell culture.
1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment (3.125. mu.M, 6.25. mu.M, 12.5. mu.M) was given when the cells grew to 60% -70%.
Collecting cells: and (3) carrying out intervention on the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h, and then collecting cells.
Cell lysis: adding lysis mixture (mixture of RIPA: PMSF: phosphatase inhibitor at volume ratio of 100:1: 1), lysing for 30min, performing ultrasonic treatment, and centrifuging.
(2) BCA assay concentration
0.5mg/ml protein standard was prepared.
Diluting a standard product: 0.5mg/ml protein standard was diluted.
Dilution of the protein to be tested: and (4) diluting by 40 times.
Preparing a BCA working solution: reagent a and reagent B were 50: 1.
Loading a 96-well plate: and adding a protein standard diluent and a protein diluent to be detected into the 96-well plate, and then adding a BCA working solution.
And (3) incubation: incubate at 37 ℃ in the dark.
Measuring absorbance by an enzyme-linked immunosorbent assay (ELIAS): the absorbance of each well under A562nm was measured, and a standard curve was drawn with the concentration as the abscissa and the absorbance as the ordinate.
(3) Denaturation of proteins
5 XLoading buffer was added to the protein samples and denatured at 100 ℃.
(4) Glue preparation
And (3) leak detection: and adding deionized water, and detecting whether glue leaks or not.
Preparing glue: mixing the gel A and B, adding 10% APS solution, mixing, pouring into a mold, concentrating gel A and B, mixing, adding 10% APS solution, mixing, and pouring into the upper layer of the gel mixture.
(5) Electrophoretic preparation and sampling
The installation device comprises: adding 1 Xelectrophoresis liquid into the electrophoresis tank, pulling out the comb to remove air bubbles, and designing a loading pore channel.
And (3) carrying out Marker hole loading: the same volume of Marker as protein was added.
Loading protein sample wells: the loading volume is the loading mass/stock concentration, and the blank channel is filled with the same volume of 1 × loading.
Electrophoresis: and (3) keeping the voltage at 80V, adjusting the voltage to 120V after the bromophenol blue runs through the separation gel, and stopping electrophoresis when the bromophenol blue runs to the bottom of the concentrated gel and does not run out.
(6) Rotary film
Removing concentrated gel without protein by using a wane, spreading a layer of sponge on each of black and white transfer membrane clips, adding 4 layers of filter paper, soaking, spreading, placing the gel on the black transfer membrane clip, activating the PVDF membrane in methanol, transferring to the gel, closing the transfer membrane clip, and performing constant-current transfer in an ice bath.
(7) Milk closure
PVDF membrane was transferred to a 5mg/ml milk blocking solution and blocked on a shaker at room temperature.
(8) Washing membrane
And putting the PVDF membrane into the membrane washing solution, and washing the membrane on a shaking table.
(9) Primary antibody incubation
Primary antibody was added according to the size of the membrane and incubated on a shaker at 4 ℃.
(10) Washing membrane
And (3) putting the PVDF membrane into the membrane washing liquid, and washing the membrane.
(11) Incubation with secondary antibody
Adding secondary antibody according to the size of the membrane, and incubating in a shaking table at room temperature.
(12) Washing membrane
And (3) putting the PVDF membrane into the membrane washing liquid, and washing the membrane.
(13) Development
Mixing ECl hypersensitive luminescent solution A and solution B in equal volume, dripping onto membrane, loading onto machine, and developing.
(14) Analysis of results
Images were collected and analyzed using an Image lab gel analysis system.
8. Small interfering RNA (siRNA) cell transfection experiments are carried out, and the small interfering RNA specifically degrades target mRNA by combining with the target gene or the mRNA of the target gene, thereby silencing the target gene.
The specific method comprises the following steps:
according to 7.5X 104Hela cells are taken at the cell density of/ml, and are paved in a six-hole plate for cell culture, and after the cell fusion rate reaches 50% -70%, a transfection reagent is added for transfection. After 16h of transfection, the transfected cells are collected, counted again and inoculated into a 96-well plate, after 10h of culture, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment (3.125 mu M) is given, MTT detection is carried out after 48h, and the 96-well plate is placed into an IncuCyte zooming live cell imaging system to periodically collect images for analyzing the survival condition of the cells. The Transfection reagent is siRNA control, p53 siRNA # 1, p53 siRNA # 2, p53 siRNA # 3, jetPRIME Buffer, jetPRIME Transfection reagent.
The invention has the beneficial effects that:
(1) the invention discovers that the action mechanism of the compound 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (Mulanphenanthrene) for treating or preventing human cervical cancer is a novel action mechanism based on an MDM2-p53 protein-protein interaction signal channel and a p53 target spot, and the invention provides a scientific theoretical basis for clinical application of the compound.
(2) Cell scratch experiments prove that the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can inhibit the migration of Hela cells; the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can induce apoptosis through Hoechst33258 fluorescent staining, annexin V-FITC/PI double staining and PI single staining apoptosis detection; 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can induce G0/G1 phase block of Hela cells as proved by a PI single staining cell cycle method.
(3)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene plays an anti-tumor role by activating p53 protein, and the pathway of the anti-tumor role is the p53-MDM2 pathway and the p53 action target point.
Drawings
FIG. 1 is a schematic diagram of the p53 target and MDM2-p53 protein and protein action pathways.
FIG. 21 is a graph showing the decrease of the fusion rate of HeLa cells caused by the action of 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the concentration dependence of HeLa cells.
FIG. 31, 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene inhibits migration of HeLa cells; a: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HeLa cells for 24h, and the HeLa cells are observed under a mirror; b: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acted on HeLa cells for 24h, and the average mobility of the HeLa cells. Statistical results are expressed as mean ± standard deviation, n is 3, P <0.01 compared to control.
FIG. 4 under-the-lens observation of HeLa cell nuclei by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene under a fluorescent microscope.
FIG. 51, 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene induces apoptosis in HeLa cells; a: detecting apoptosis by Annexin V-FITC/PI double staining; b: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HeLa cells for 48 hours, and the average apoptosis rate of the HeLa cells is high. Statistical results are expressed as mean ± standard deviation, n is 3, P <0.01 compared to control.
FIG. 61, 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene induces apoptosis in HeLa cells; A. b: detecting the cell cycle by PI single staining and flow cytometry; c: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HeLa cells for 48 hours, and the proportion of each cell cycle is accounted. Statistical results are expressed as mean ± standard deviation, n is 3, P <0.01 compared to control.
FIG. 71, 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene block the HeLa cell cycle at G0/G1; A. b: detecting the cell cycle by PI single staining and flow cytometry; c: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HeLa cells for 48 hours, and the proportion of each cell cycle is accounted. Statistical results are expressed as mean ± standard deviation, n is 3, P <0.01 compared to control.
FIG. 8 protein expression; statistical results are expressed as mean ± standard deviation, n is 3, P <0.5, P <0.01, P <0.001, P <0.0001 compared to control.
FIG. 9 expression of protein.
FIG. 10 p53 mediates the inhibition of the proliferation of cervical cancer cells by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene; a: transfection p53 siRNA, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acted on HeLa cells for 48h, the fusion rate curve of HeLa cells; b: transfection of P53 siRNA, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HeLa cells for 48h, the fusion rate of HeLa cells is shown as mean. + -. standard deviation, n is 3, compared to the control group, P is < 0.01.
FIG. 11 survival rate of HeLa cells treated with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h after transfection with p53 siRNA.
FIG. 12 Standard Curve of protein concentration for BCA assay.
Detailed Description
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
The sources of drugs and reagents used in the specific examples of the invention are shown in Table 1.
TABLE 1 drugs and reagents
Examples
The detection method for the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the Hela cells of the cervical cancer based on the p53 target spot comprises the following steps:
1. and detecting the influence of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the proliferation of Hela cells by adopting an IncuCyte living cell imaging system. IncuCyte ZOOM is a novel integrated system that can be used for real-time visualization and automated analysis of cell migration in a 96-well format in culture incubators.
The method comprises the following specific steps:
taking Hela cells with good logarithmic phase growth state according to 0.5 × 104The cell density of each/ml is 100 mul per well is plated in a 96-well plate, after the cells are attached to the wall, the DMSO control group (abbreviated as control group) with 0.2% (v: v) and the drug different concentration group (1,5, 6-trimethoxy-2, 7-dihydroxy phenanthrene group) (3.125 muM, 6.25 muM, 12.5 muM, 25 muM, 50 muM, 100 muM) are designed, each group is provided with 5 duplicate wells, the old culture medium is discarded, 100 mul of 1,5, 6-trimethoxy-2, 7-dihydroxy phenanthrene with different concentrations diluted by MEM complete culture medium is added into each group, and 100 mul of DMSO with 0.2% (v: v) diluted by culture medium is added into the control group. The method comprises the steps of placing a 96-well plate to be observed in an IncuCyte zooming live cell imaging system, dynamically observing cell growth change by regularly acquiring cell images, setting the time interval of image acquisition to be 4h, acquiring images by a 10X lens, automatically identifying a cell area (setting parameters to screen out dead cell fragments) in each image by analysis software built in the system, acquiring 4 pictures with different visual fields in each hole, calculating cell fusion rate under different processing conditions, drawing a curve graph, and indirectly reflecting the cell survival condition.
An Incucyte living cell dynamic imaging system is used for acquiring images of Hela cells processed by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (0-100 mu M) with different concentrations in real time, and cell fusion rate is analyzed after 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is processed for 48 hours. The results show that: the cell fusion rate curve gradually moves downwards along with the increase of the concentration of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, which indirectly reflects that the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can generate concentration-dependent proliferation inhibition on Hela cells, and is shown in figure 2.
2. Cell scratch experiments were performed to quantify the potential for collective spreading of cells.
In vitro scratch testing is a simple and economical method of studying cell migration in vitro. This method creates a new artificial gap, the so-called "scratch", and the monolayer of cells that newly created the edge of the gap will move towards the opening to close the "scratch", observing the rate at which the remaining cell population diffuses into the empty areas. Scratch tests are commonly used to quantify the potential for cells to spread collectively.
The method comprises the following specific steps:
selecting Hela cells with good logarithmic phase growth state according to 2 × 105Cell density of one/ml, 100. mu.l/well were plated in 96-well plates, and the 96-well plates were placed at 37 ℃ in 5% CO2Culturing overnight in incubator until cell fusion rate reaches 90% -100%, scratching with scratching device, washing with PBS for 2-3 times, treating with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene after no excessive dead cells are observed under microscope, and treating with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene at concentration of 0.84 μ M (2 IC)50) Control group was DMSO at the corresponding volume. Each group was set with 5 multiple wells, observed and photographed at different time points (0h, 4h, 8h, 12h, 16h, 20h, 24h) in the IncuCyte live cell imaging system, the 0h and 24h scratch widths were calculated using the IncuCyte ZOOM software, the difference between the two was calculated, and the ratio of the difference to the initial scratch width was used to represent the cell mobility.
Metastasis is one of the basic features of malignant tumors. And (3) detecting the influence of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on Hela cell migration by adopting a cell scratch experiment. The result shows that after 24h of scratching, the scratching of the control group is narrowed, Hela cells migrate to the middle of the scratching area towards the inside of the scratching, while the migration distance of the cells of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treated group is shortened, and the scratching gap is obviously widened. 0.84. mu.M (2 IC) in comparison with control DMSO50) The mobility of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group cells is obviously reduced, as shown in figure 3,**P<0.01, the difference is statistically significant.
The results show that after 24h scratching, DMSO scratching of the control group is narrowed, the cell migration distance of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene-treated group is shortened, and the scratching gap is obviously widened. Control DMSO, cell migration 34.70. + -. 0.35%, 0.84. mu.M (2 IC)50) The cell mobility of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treated group is 25.92 +/-0.62 percent, and the cell mobility is obviously reduced (**P<0.01), indicating that the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can inhibit Hela cell migration.
3. Hoechst33258 fluorescent staining experiments were performed to analyze morphological changes caused by apoptosis.
Hoechst33258 is a fluorescent dye of the Bisbenzimidazole (Bisbenzimidazole) type, and belongs to a non-intercalating nucleic acid fluorescent dye. The dye is combined with A-T base pairs of DNA, dead cells can be immediately dyed, cell nucleuses of apoptotic cells can be seen to be densely dyed with compact granular block fluorescence under a fluorescence microscope, and living cells are evenly dispersed and fluorescent. Hoechst33258 fluorescent staining was used to analyze morphological changes caused by apoptosis.
The method comprises the following specific steps:
(1) cell culture: taking Hela cells in good growth state, according to 7.5 × 104Cell density of individual/ml, 2ml per well plated in six well plates, placed in incubator overnight.
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: when the cells grow to 60% -70%, the floating dead cells are washed away by PBS, and 2ml of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene diluted by the culture medium is added into each hole. The concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is set to be 3.125 mu M and 6.25 mu M, and the control group is DMSO with the same volume.
(3) Fixing: after 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on Hela cells for 48 hours, 1ml of 4% paraformaldehyde is added into each hole, and the mixture is fixed for 15min at normal temperature in a dark place.
(4) Dyeing: the fixative was aspirated and washed 3 times with cold PBS, 3min each. Add 500. mu.l Hoechst33258 staining solution and incubate 15min at 37 ℃ in the dark.
(5) And (3) observation by a fluorescence microscope: the cells were washed 3 times with cold PBS for 3min each. The cell morphology was observed under a fluorescence microscope and photographed.
In order to study whether 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene induces Hela cell apoptosis, the nuclei were first morphologically observed using Hoechst33258 in combination with a fluorescence microscope. The results show that Hela cells which are not treated by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene present weak and uniform blue fluorescence, after the treatment of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, the chromatin of Hela cells is condensed, and condensed and compact granular block-shaped strong fluorescence can be seen in the cells, and the apoptosis phenomenon is obvious along with the increase of the concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, as shown in figure 4.
4. An annexin V-FITC/PI (propidium iodide) double staining apoptosis detection experiment is carried out to distinguish early and late apoptosis, necrosis and normal cells.
In the early stage of apoptosis, Phosphatidylserine (PS) is transferred from the inner side of a plasma membrane to the outer layer, the PS is exposed on the surface of a cell, Annexin V-FITC is specifically combined with the PS which turns outwards in the early stage of apoptosis, FITC is used as a fluorescent probe, and the apoptosis is detected by a flow cytometer. Propidium Iodide (PI) is a nucleic acid dye, and in cells with advanced apoptosis and necrosis, PI crosses the cell membrane, inserts nucleic acid, and shows red fluorescence. Therefore, Annexin V and PI staining can be used to differentiate between early and late apoptotic and necrotic cells.
The method comprises the following specific steps:
(1) cell culture: collecting Hela cells in good log phase at 7.5 × 104Cell density of individual/ml, 2ml per well plated in six well plates, placed in incubator overnight.
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: when the cells grow to 60% -70%, the floating dead cells are washed away by PBS, and 2ml of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene diluted by the culture medium is added into each hole. The concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is set to be 3.125. mu.M, 6.25. mu.M and 12.5. mu.M, and the control group is DMSO with the same volume.
(3) Collecting cells: treating 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h, collecting cells, sucking the supernatant in a six-well plate, transferring the supernatant into a 15ml centrifuge tube, washing the supernatant for 1 time by precooled PBS (phosphate buffer solution), digesting the supernatant by pancreatin, adding a culture medium to stop digestion, centrifuging the supernatant for 5min at the temperature of 4 ℃ at 1000rmp, discarding the supernatant, adding PBS, and repeatedly centrifuging the supernatant for 1 time.
(4) Adding 1 × Annexin V Bingding Solution: 400. mu.l of 1 × Annexin VBingdi Solution was added to the DMSO group, and 100. mu.l of 1 × Annexin V binding Solution was added to the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group.
(5) Grouping experiments: mu.l each of the cell suspension obtained in step (3) was taken and added to a new flow tube. The DMSO group is respectively marked as a negative control group, an Annexin V-FITC staining group, a PI staining group, an Annexin V-FITC and PI double staining group; the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group was designated as Annexin V-FITC and PI double staining group.
(6) Dyeing: adding 5 mul Annexin V-FITC conjugate into the cell suspension according to the grouping, mixing uniformly, adding 5 mul PI Solution, mixing uniformly, and culturing at room temperature in a dark place for 15 min.
(7) Flow detection: adding 400 μ l of 1 × Annexin V Binding Solution, blowing, beating, mixing, and detecting on a machine within 1 h.
To confirm that the death mode of Hela cells caused by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is apoptosis, the death of Hela cells caused by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene was detected by double staining with Annexin V/PI using a flow cytometer. The results show that the proportion of early apoptotic cells of Hela cells is increased from 2.20% of the control group to 37.61% (1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, 12.5 mu M), the proportion of late apoptotic cells is increased from 4.95% of the control group to 55.85% (1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, 12.5 mu M), namely, after Hela cells are treated by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h, the proportion of apoptotic cells of Hela cells is increased from 7.15% (0 mu M) to 93.46% (1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, 12.5 mu M) of the control group, and the number of early and late apoptotic cells of Hela cells is increased in a dose-dependent manner. Therefore, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can induce Hela cell apoptosis, thereby remarkably inhibiting cell proliferation, as shown in FIG. 5.
5. And carrying out PI single staining to detect apoptosis experiment.
The main component of the cell cycle and apoptosis kit, PI (propidium iodide), is a fluorescent dye that can bind to DNA: the PI can be specifically combined with DNA in the cell, the greater the amount of the PI combined with the DNA, the stronger the fluorescence intensity, the content of the DNA can be judged according to the fluorescence intensity, the stained cell is detected by using a flow cytometer, and the distribution of the detected cell in different periods in the cycle is judged according to the intensity of the fluorescence. When the cell is subjected to apoptosis, DNA is degraded and broken, and a sub-small diploid peak subG1, namely an apoptosis peak, appears before the G0/G1 stage.
The method comprises the following specific steps:
(1) cell culture: collecting Hela cells in good log phase stateAccording to 7.5X 104Cell density of individual/ml, 2ml per well plated in six well plates, placed in incubator overnight.
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: when the cells grow to 60% -70%, the floating dead cells are washed away by PBS, and 2ml of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene diluted by the culture medium is added into each hole. The concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is set to be 3.125. mu.M, and the control group is DMSO with the same volume.
(3) Collecting cells: adding 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for intervention for 48h, collecting cells, sucking supernatant in a six-well plate, transferring the supernatant into a 15ml centrifuge tube, washing the supernatant for 1 time by precooled PBS, digesting the supernatant by 0.25% of pancreatin without EDTA, adding the previously collected supernatant to stop digestion, centrifuging the supernatant for 5min at 1000rmp and 4 ℃, discarding the supernatant, adding PBS, repeatedly centrifuging the supernatant for 1 time, and discarding the PBS.
(4) Ethanol fixation: 1ml of 75% ethanol was added and fixed in a refrigerator at 4 ℃ for 2h or overnight.
(5) Propidium iodide staining: an propidium iodide staining solution was prepared according to the number of samples to be tested, as shown in Table 2.
TABLE 2 preparation of propidium iodide staining solution
Centrifuging the fixed sample at 1000rmp and 4 ℃ for 5min, removing supernatant, and adding 500 cells into each tube of cell sampleμl propidium iodide staining solution, slowly and fully resuspending the cell pellet, and bathing in the dark at 37 ℃ for 30 minutes.
(6) Flow detection: cell cycle assays were performed using a flow cytometer over 1 h.
The results show that after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is treated for 48 hours, the proportion of cells in the G0/G1 phase is not obviously changed, the proportion of cells in the G2/M phase is reduced, the proportion of cells in the s phase is not obviously changed, and the proportion of cells in the sub G1 phase is increased (the proportion of cells in the sub G1 phase is increased: (the formula of the formula I)**P<0.01), which shows that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on Hela cells to cause apoptosis, and the result is consistent with the result of flow-type apoptosis, as shown in FIG. 6.
6. PI single staining cell cycle detection experiments were performed.
(1) Cell culture: the method is the same as the step 5.
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: the method is the same as the step 5.
(3) Collecting cells: adding medicine for intervening for 36h, collecting cells, discarding supernatant in a six-well plate, washing with precooled PBS for 1 time, digesting with 0.25% pancreatin without EDTA, adding culture medium to stop digestion, centrifuging at 1000rmp and 4 ℃ for 5min, discarding supernatant, adding PBS, repeatedly centrifuging for 1 time, and discarding PBS.
(4) Ethanol fixation: the method is the same as the step 5.
(5) Propidium iodide staining: the method is the same as the step 5.
(6) Flow detection: the method is the same as the step 5.
Cell cycle arrest is one of the mechanisms that inhibit tumor cell proliferation. The cell cycle after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on Hela cells is detected by using a flow cytometer through single staining with PI. The results show that the ratio of G0/G1 is increased after 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is treated for 36h (**P < 0.01), the S phase ratio decreased, and the G2/M ratio decreased, indicating that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene blocks the cell cycle at G0/G1, as shown in FIG. 7.
7. Protein imprinting is carried out, and the target protein is detected by the principle of antigen-antibody binding through gel electrophoresis.
Protein imprinting involves transferring the corresponding protein from a treated cell or biological tissue sample to a solid support by gel electrophoresis, recognizing and developing it with a specific antibody, and detecting the expression of the specific protein by analyzing the location and intensity of the coloration on the membrane.
The method comprises the following specific steps:
(1) extraction of total cellular protein:
cell culture: HeLa cells in good log phase were collected at 7.5X 104Cell density of individual/ml, 2ml per well plated in six-well plates, placed in incubator for further culture.
1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: when the cells grow to 60% -70%, the floating dead cells are washed away by PBS, and 2ml of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene diluted by the culture medium is added into each hole. The concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is set to be 3.125. mu.M, 6.25. mu.M and 12.5. mu.M, and the control group is DMSO with the same volume.
Collecting cells: after the intervention of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h, adding pancreatin for digestion, adding a culture medium to stop digestion, transferring to a 15ml centrifuge tube, centrifuging at 4 ℃ and 1000rpm for 5min, removing supernatant, adding cold PBS, blowing, beating, uniformly mixing, centrifuging once, adding 1ml PBS, blowing uniformly, transferring to a 1.5ml EP tube, and centrifuging at 4 ℃ for 15min at 7.0 XG.
Cell lysis: and centrifuging, collecting supernatant, removing cell debris, adding lysis mixed liquor, and lysing the cell debris mixed liquor on ice for 30min, wherein the volume ratio of the lysis mixed liquor to the cell debris mixed liquor is RIPA to PMSF to phosphatase inhibitor is 100:1: 1. And (3) after the cracking is finished, performing ultrasonic treatment by using 50% energy of an ultrasonic crusher to completely crack the protein, performing centrifugation after the ultrasonic treatment, and performing 12 XG centrifugation at 4 ℃ for 30 min.
(2) BCA assay concentration
0.5mg/ml protein standard preparation: the 5mg/ml protein standard BSA was diluted to 0.5 mg/ml.
Diluting a standard product: the prepared 0.5mg/ml protein standard was diluted in the following concentration gradient.
TABLE 3 dilution of protein standards
Dilution of the test protein (40-fold dilution): mu.l of deionized water was added to 2. mu.l of the protein sample.
Preparing a BCA working solution: the reagent A and the reagent B are mixed fully and uniformly according to the ratio of 50:1, and are prepared for use.
Loading a 96-well plate: and respectively adding 20 mul of protein standard diluent and protein diluent to be detected into each hole, setting three multiple holes, adding 200 mul of BCA working solution, and slowly and fully mixing uniformly to avoid bubbles.
And (3) incubation: incubate at 37 ℃ for 30min in the dark.
Measuring absorbance by an enzyme-linked immunosorbent assay (ELIAS): and (3) measuring the absorbance of each hole under A562nm by using a multifunctional microplate reader, drawing a standard curve by taking the concentration as an abscissa and the absorbance (OD) as an ordinate, and calculating the protein concentration of the sample according to the standard curve, wherein the protein concentration curve is shown in FIG. 12.
(3) Denaturation of proteins
Adding 5 Xloading buffer solution into protein sample, adding 1/4 of protein sample, mixing, and denaturing at 100 deg.C for 5 min.
(4) Glue preparation
And (3) leak detection: adding deionized water, detecting whether glue leaks or not, pouring out the water, and wiping the filter paper to dry.
Preparing glue: and (3) uniformly mixing the solution A and the solution B in a ratio of 1:1, adding 10% of APS solution into the solution A, uniformly mixing, pouring into a mold, uniformly mixing the solution A and the solution B in a ratio of 1:1, adding 10% of APS solution into the concentrated glue, uniformly mixing, and directly pouring into the upper layer of the solution A. Immediately, slowly inserting a nine-hole comb into the gel, standing for 17min, and waiting for gel polymerization.
(5) Electrophoretic preparation and sampling
The installation device comprises: adding 1 × 500ml of electrophoresis solution into an electrophoresis tank, pulling a comb to remove bubbles, and designing a loading pore channel.
And (3) carrying out Marker hole loading: the same volume of Marker as protein was added.
Loading protein sample wells: determining the sample loading quality according to the protein concentration, further determining the sample loading volume, wherein the sample loading volume calculation method comprises the following steps: the loading mass (μ g)/stock concentration, blank wells were filled with the same volume of 1 × loading.
Electrophoresis: and (3) keeping the voltage at 80V, adjusting the voltage to 120V after the bromophenol blue runs through the separation gel, and stopping electrophoresis when the bromophenol blue runs to the bottom of the concentrated gel and does not run out.
(6) Rotary film
After electrophoresis, taking out the gel plate, putting the gel plate into a white empty tray filled with 1 multiplied by membrane transferring liquid, removing concentrated gel without protein by using a wane, placing a membrane transferring clamp with black at the bottom and white at the top, respectively laying a layer of sponge on each of the black and white clamps, soaking, laying flat, respectively adding 4 layers of filter paper, soaking and laying flat, carefully placing the cut gel on the black membrane transferring clamp, cutting a PVDF membrane with corresponding size, placing the PVDF membrane into methanol for activating for 20s, transferring the gel to the gel, laying flat, closing the membrane transferring clamp, avoiding bubbles, and constantly transferring the membrane for 90min in an ice bath.
(7) Milk closure
After the membrane transfer, the PVDF membrane was transferred to a 5mg/ml milk blocking solution with plastic tweezers and blocked on a shaker at room temperature for 90 min.
(8) Washing membrane
Recovering the milk sealing liquid, and placing into a refrigerator at 4 deg.C. Putting the PVDF membrane into the membrane washing solution, and washing the membrane on a shaking table for 3 times, 10min each time.
(9) Primary antibody incubation
The antibody was diluted according to the antibody instructions, primary antibody was added in the corresponding volume according to the size of the membrane, sealed with plastic wrap, and incubated overnight in a shaker at 4 ℃.
(10) Washing membrane
And taking the membrane out of the primary antibody, recovering the primary antibody, putting the PVDF membrane into the membrane washing solution, and washing the membrane for 10min each time for 3 times by a shaking table at room temperature.
(11) Incubation with secondary antibody
After membrane washing, diluting the antibody according to the instruction, adding a secondary antibody with a corresponding volume according to the size of the membrane, sealing the opening with a plastic membrane, and incubating for 2h in a shaking table at room temperature.
(12) Washing membrane
And taking the membrane out of the secondary antibody, recovering the secondary antibody, putting the PVDF membrane into the membrane washing solution, and washing the membrane for 10min each time for 3 times by a shaking table at room temperature.
(13) Development
Mixing ECl hypersensitivity luminescent solution A and solution B in equal volume. And covering the filter paper on the PVDF membrane, and completely sucking the membrane washing liquid on the PVDF membrane. Slowly and uniformly dripping the hypersensitive luminous liquid on the film to make the luminous liquid cover the surface to be developed, and then loading the film on a machine for development.
(14) Analysis of results
Images were collected and analyzed using an Image lab gel analysis system.
The negative feedback regulation formed between p53-MDM2 is the main reason for the inactivation of wild-type p 53. Bcl-2 family proteins are key mediators of apoptosis. In order to further discuss the action mechanism of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for inducing Hela cell apoptosis, Western Blot is used for detecting p53 pathway protein and Bcl-2 family protein. The results show that p53 protein is up-regulated, MDM2 protein is up-regulated, Bax/Bcl-2 ratio is increased, and Mcl-1 protein is down-regulated. 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene may act on the p53-MDM2 pathway, and Mcl-1 participates in the apoptosis of Hela cells caused by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, as shown in FIG. 8.
The results show that p53 protein is up-regulated, MDM2 protein is up-regulated, Bcl-2 protein is down-regulated, Mcl-1 protein is down-regulated, Bax protein is not obviously changed, and the Bax/Bcl-2 ratio is increased. The up-regulation of MDM2 protein did not inhibit the expression of p53, indicating that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on the p53-MDM2 pathway. The Mcl-1 protein is involved in the regulation of apoptosis.
8. Small interfering RNA (siRNA) cell transfection experiments were performed.
Small interfering RNA (siRNA) is a technology that achieves efficient silencing of target genes via the mechanism of RNA interference (RNAi). The small interfering RNA specifically degrades target mRNA by binding with the target gene or mRNA of the target gene, thereby destroying the target mRNA and stopping the synthesis of the encoded protein, thereby silencing the target gene.
The specific method comprises the following steps:
taking Hela cells in logarithmic growth phase, according to 7.5 × 104Cell density of/ml, 2ml per well were inoculated in six-well plates and cultured overnight, and after cell fusion rate reached 50-70%, transfection reagents were added as siRNA control, p53 siRNA # 1, p53 siRNA # 2, p53 siRNA #3 (PBS wash 3 times before transfection). Cell fluid was changed 8h after transfection and fresh non-resistant medium was added. Transfection was performed with jetPRIME transfection reagent according to the manufacturer's instructions. Taking one well of a six-well plate as an example, 200. mu.l of jetPRIME Buffer was added to a 1.5ml EP tube, then 5. mu.l of siRNA was added, vortexed for 10s, 5. mu.l of jetPRIME Transfection reagent was added, vortexed for 10s, and then vortexed for 10s, incubated at room temperature for 10min, and Transfection reagent and non-antibody medium were added to the six-well plate for Transfection. After 16h of transfection, the transfected cells were collected, re-counted and inoculated into a 96-well plate, and after 10h of culture, 1,5, 6-trimethoxy-2, 7-bis was administeredAnd (3) treating with hydroxyphenanthrene (3.125 mu M), performing MTT detection after 48h, and putting a 96-well plate into an IncuCyte zooming live cell imaging system in an incubator to periodically acquire images for analyzing the survival condition of the cells.
And (4) conclusion:
1. transfection of siRNA in Hela cells silenced p53 gene:
p53 is a key regulatory factor of apoptosis, and the expression of p53 is increased most obviously after Hela cells are treated by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene. To investigate whether 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene inhibited Hela cell proliferation dependent on the expression of p53, we transfected siRNA in Hela cells to silence the p53 gene. After three siRNA (p53 siRNA # 1, p53 siRNA # 2 and p53 siRNA #3) fragments were transfected, protein expression level of p53 in Hela cells was detected using WB. The results show that: in Hela cells, the p53 siRNA # 3 fragment can obviously reduce the expression level of the p53 gene in the Hela cells, while the silencing effect of the p53 siRNA # 1 and the p53 siRNA # 2 fragment is not obvious as shown in FIG. 9. Therefore, p53 siRNA # 3 fragment was selected for subsequent experimental studies.
2. The silencing p53 gene can reverse the proliferation inhibition of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer cells:
(1) the expression of the p53 protein is reduced, so that the fusion rate of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene to Hela cells can be improved:
in FIG. 10A, the change of cell fusion rate after adding 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h was dynamically observed using IncuCyte. According to different moments, a line graph is made, and the result shows that: in Hela cells, the number of Control siRNA and p53 siRNA group cells is obviously increased, and the cell fusion rate curve of the p53 siRNA group is higher than that of the Control siRNA; after adding 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, the cell fusion rate curves of the Control siRNA + Drug group and the p53 siRNA + Drug group are moved downwards, and the cell fusion rate curve of the p53 siRNA + Drug group is higher than that of the Control siRNA + Drug group. In order to exclude the influence of the change of the p53 expression level on the fusion rate of Hela cells, the fusion rate curve of the Control siRNA and p53 siRNA groups is set as 100%, and after standardization, the results show that: the fusion rate of Hela cells in the Control siRNA + Drug group is 33.63%, and the fusion rate of cells in the p53 siRNA + Drug group isThe total rate is 48.25%, as shown in FIG. 10B, the fusion rate is significantly increased, and the difference has statistical significance: (**P<0.01)。
(2) The survival rate of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on Hela cells can be improved by down regulating the expression of the p53 protein:
after transfection of p53 siRNA in Hela cells, the cells were treated according to the Control group, p53 siRNA group, Control siRNA + Drug group and p53 siRNA + Drug group, and the cell survival rate was examined after 48h by MTT. The results show that: the survival rate of Hela cells after Control siRNA + Drug group treatment was 12.96%, while the survival rate of Hela cells after p53 siRNA + Drug treatment after p53 siRNA transfection was 35.58% (p53 siRNA #3), see FIG. 11, the difference has statistical significance: (see FIG. 11)***P<0.001)。
Based on the results, under the condition of not administering 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment, IncuCyte shows that the cell numbers of the Control group and the p53 siRNA group are obviously increased, and the cell fusion rate curve of the p53 siRNA group is higher than that of the Control group Control siRNA; after 48 hours of treatment of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, cell fusion rate curves of the Control siRNA + Drug group and the p53 siRNA + Drug group are moved downwards, and the cell fusion rate curve of the p53 siRNA + Drug group is higher than that of the Control siRNA + Drug group. The fusion rate of Hela cells in the Control siRNA + Drug group is 33.63%, the fusion rate of cells in the p53 siRNA + Drug group is 48.25%, the fusion rate is obviously increased, and the difference has statistical significance (the**P<0.01). MTT detection results show that: compared with the Control siRNA + Drug treated group, the survival rate of Hela cells after p53 siRNA + Drug treatment is improved by 22.62% (p53 siRNA # 3). 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene plays an anti-tumor role by activating p53 protein, and the pathway of the anti-tumor role is the p53-MDM2 pathway and the p53 action target point.
Claims (10)
1. The application of the p53 target-based trimethoxy dihydroxyphenanthrene compound in the aspect of resisting cervical cancer is characterized in that the trimethoxy dihydroxyphenanthrene compound is 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene.
2. A method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on a p53 target spot is characterized by comprising the following steps:
the following tests are not in sequence;
1. detecting the influence of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on Hela cell proliferation by adopting an IncuCyte living cell imaging system;
2. performing a cell scratch experiment to quantify the potential of cell mass diffusion;
3. performing a Hoechst33258 fluorescent staining experiment, and analyzing morphological changes caused by apoptosis;
4. carrying out annexin V-FITC/PI double staining detection cell apoptosis experiment to distinguish early and late apoptosis, necrosis and normal cells;
5. performing a PI single staining apoptosis detection experiment;
6. performing a PI single staining cell cycle detection experiment;
7. performing a western blot experiment, and detecting the target protein by using the principle of combining gel electrophoresis antigen and antibody;
8. and carrying out siRNA cell transfection experiment, wherein the small interfering RNA specifically degrades target mRNA by combining with the target gene or the mRNA of the target gene, thereby silencing the target gene.
3. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the Hela cells of the cervical cancer based on the p53 target spot according to claim 2, characterized in that the step 1 adopts an IncuCyte living cell imaging system to detect the effect of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the Hela cell proliferation; the method specifically comprises the following steps:
according to 0.5X 104Taking Hela cells at the density of each ml, paving the Hela cells in a 96-well plate, dividing the Hela cells into a control group and a drug concentration group, adding 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene with different concentrations into the drug concentration group, placing the 96-well plate in an IncuCyte living cell imaging system, collecting the cell image dynamics at regular time, and calculating the cell fusion rate under different treatment conditions; the concentration of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is 3.125-100 mu M.
4. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on the p53 target spot according to claim 2, wherein the step 2 is performed with a cell scratch test, and specifically comprises the following steps:
according to 2X 105Hela cells are taken at the density of each ml, the Hela cells are laid in a 96-well plate, cell culture is carried out, when the cell fusion rate reaches 90% -100%, scratching and buffer solution washing are carried out, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given, pictures are taken at different time points of 0h-24h in an IncuCyte living cell imaging system, the scratch widths of 0h and 24h are calculated, the difference value of the two is calculated, and the migration rate of the cells is represented by the ratio of the difference value to the initial scratch width.
5. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on the p53 target spot according to claim 2, wherein the step 3 is performed by a Hoechst33258 fluorescence staining test, which specifically comprises:
(1) cell culture: according to 7.5X 104Taking Hela cells at the cell density of each ml, and plating the Hela cells in a six-hole plate for culture;
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is given for treatment, and the concentrations of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene are 3.125 mu M and 6.25 mu M respectively;
(3) fixing: after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene intervenes in Hela cells for 48 hours, adding 4% paraformaldehyde, and fixing at normal temperature in a dark place;
(4) dyeing: sucking out the fixing solution, washing with a buffer solution, adding Hoechst33258 staining solution for staining, and incubating at 37 ℃ in a dark place;
(5) and (3) observation by a fluorescence microscope: and (5) washing with buffer solution, observing the cell morphology under a fluorescence microscope, and taking pictures.
6. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on the p53 target spot according to claim 2, wherein the step 4 of performing an annexin V-FITC/PI double staining detection apoptosis experiment specifically comprises the following steps:
(1) cell culture: according to 7.5X 104Taking Hela cells at the cell density of each ml, and plating the Hela cells in a six-hole plate for culture;
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given when the cells grow to 60% -70%; the concentrations of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene are 3.125 mu M, 6.25 mu M and 12.5 mu M;
(3) collecting cells: treating 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h, and collecting cells;
(4) adding 1 × Annexin V Bingding Solution: respectively adding 1 × Annexin V Bingding Solution into a control group and a 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group (drug group);
(5) grouping experiments: the control component is a negative control group, an Annexin V-FITC staining group, a PI staining group, an Annexin V-FITC and PI double staining group; the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene component is Annexin V-FITC and PI double staining group;
(6) dyeing: adding Annexin V-FITC conjugate into each group in the step (5), adding PI Solution, and culturing at room temperature in a dark place;
(7) flow detection: adding 1 × Annexin V Binding Solution, and performing machine detection within 1 h.
7. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on the p53 target spot according to claim 2, wherein the step 5 is carried out with PI single stain for detecting apoptosis, and specifically comprises the following steps:
(1) cell culture: collecting Hela cells in good log phase at 7.5 × 104Plating Hela cells with the cell density of each ml in a six-hole plate;
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is given when the cells grow to 60% -70%; the concentration of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is 3.125 mu M;
(3) collecting cells: adding 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene to intervene Hela cells for 48h, collecting the cells, reserving the supernatant, and collecting the cells according to the method of the step 4 (3);
(4) ethanol fixation: adding 75% ethanol, and fixing at 4 deg.C;
(5) PI staining: after the ethanol is fixed, adding propidium iodide staining solution into each tube of cell sample, and carrying out light-shielding warm bath at 37 ℃;
(6) flow detection: cell cycle assays were performed using a flow cytometer over 1 h.
8. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on the p53 target spot according to claim 2, wherein the step 6 is performed with PI single stain cell cycle detection experiment, and specifically comprises the following steps:
(1) cell culture: the method is the same as the step 5;
(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: the method is the same as the step 5;
(3) collecting cells: collecting cells after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene intervenes for 36h, discarding the supernatant, and collecting the cells according to the method of the step 4 (3);
(4) ethanol fixation: the method is the same as the step 5;
(5) propidium iodide staining: the method is the same as the step 5;
(6) flow detection: the method is the same as the step 5.
9. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on the p53 target spot according to claim 2, wherein the step 7 is performed by a Western blot experiment, and specifically comprises the following steps:
(1) extraction of total cellular protein:
cell culture: according to 7.5X 104Taking HeLa cells at the cell density of each ml, and plating the HeLa cells in a six-hole plate for cell culture;
1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given; the concentration of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is 3.125 mu M, 6.25 mu M and 12.5 mu M;
collecting cells: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene intervenes for 48h and then cells are collected;
cell lysis: adding cracking mixed liquor for cracking, then carrying out ultrasonic treatment and centrifuging; the cracking mixed liquor is prepared by mixing the following components in volume ratio of RIPA to PMSF: a mixture of phosphatase inhibitors 100:1: 1;
(2) BCA assay concentration
Preparing 0.5mg/ml protein standard;
diluting a standard product: diluting 0.5mg/ml protein standard;
dilution of the protein to be tested: diluting by 40 times;
preparing a BCA working solution: reagent A, reagent B is 50: 1;
loading a 96-well plate: adding a protein standard diluent and a protein diluent to be detected into a 96-well plate, and then adding a BCA working solution;
and (3) incubation: incubating at 37 ℃ in the dark;
measuring absorbance by an enzyme-linked immunosorbent assay (ELIAS): measuring the absorbance of each hole under A562nm, and drawing a standard curve by taking the concentration as an abscissa and the absorbance as an ordinate;
(3) denaturation of proteins
Adding 5 Xloading buffer solution into protein sample, and performing constant temperature denaturation at 100 ℃;
(4) glue preparation
And (3) leak detection: adding deionized water, and detecting whether glue leaks or not;
preparing glue: mixing the gel A and B, adding 10% APS solution, mixing, pouring into a mold, concentrating gel A and B, mixing, adding 10% APS solution, mixing, and pouring into the upper layer of the gel mixture;
(5) electrophoretic preparation and sampling
The installation device comprises: adding 1X electrophoresis liquid into an electrophoresis tank, pulling a comb to remove bubbles, and designing a loading pore channel;
and (3) carrying out Marker hole loading: adding a Marker with the same volume as the protein;
loading protein sample wells: loading volume is loading quality/stock solution concentration, and blank pore channels are filled by 1 × loading with the same volume;
electrophoresis: keeping the voltage at 80V, adjusting the voltage to 120V after the bromophenol blue runs through the separation gel, and stopping electrophoresis when the bromophenol blue runs to the bottom of the concentration gel and does not run out;
(6) rotary film
Removing concentrated gel without protein by using a wane, respectively paving a layer of sponge on each of the black and white color of a transfer membrane clamp, respectively adding 4 layers of filter paper, soaking, paving, placing the gel on the black transfer membrane clamp, activating the PVDF membrane in methanol, transferring the activated PVDF membrane onto the gel, closing the transfer membrane clamp, and performing constant-current transfer membrane in ice bath;
(7) milk closure
Transferring the PVDF membrane into 5mg/ml milk sealing liquid, and sealing at room temperature;
(8) washing membrane
Putting the PVDF membrane into the membrane washing liquid, and washing the membrane;
(9) primary antibody incubation
Adding primary antibody according to the size of the membrane, and incubating in an environment at 4 ℃;
(10) washing membrane
Putting the PVDF membrane into membrane washing liquid, and washing the membrane;
(11) incubation with secondary antibody
Adding a secondary antibody according to the size of the membrane, and incubating at room temperature;
(12) washing membrane
Putting the PVDF membrane into membrane washing liquid, and washing the membrane;
(13) development
Mixing ECl hypersensitive luminescent solution A and solution B in equal volume, dripping onto a membrane, loading on a machine, and developing;
(14) analysis of results
Images were collected and analyzed using an Image lab gel analysis system.
10. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cervical cancer Hela cells based on the p53 target spot according to claim 2, wherein the step 8 is carried out with siRNA cell transfection experiment, and specifically comprises the following steps:
according to 7.5X 104Paving Hela cells with the cell density of/ml in a six-hole plate, carrying out cell culture, adding a transfection reagent after the cell fusion rate reaches 50-70%, and carrying out transfection; after 16h of transfection, the transfected cells were harvested and re-transfectedCounting and inoculating the cells to a 96-well plate, culturing for 10h, then treating with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene with the concentration of 3.125 mu M, performing MTT detection after 48h, and putting the 96-well plate into an IncuCyte zooming live cell imaging system in an incubator to acquire images at regular time for analyzing the survival condition of the cells; the Transfection reagent is siRNA control, p53 siRNA #1, p53 siRNA #2, p53 siRNA #3, jet PRIME Buffer, jet PRIME Transfection reagent.
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| US20200055950A1 (en) * | 2017-09-07 | 2020-02-20 | Shanghai Yifanke Biotechnology Co., Ltd. | Use of anti-pl2l60 protein antibody in preparation of anti-tumor medicine and method for treating tumor |
| CN112870185A (en) * | 2021-02-25 | 2021-06-01 | 沈阳药科大学 | Medicine for treating or preventing cervical cancer of human, preparation method and application |
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| US20200055950A1 (en) * | 2017-09-07 | 2020-02-20 | Shanghai Yifanke Biotechnology Co., Ltd. | Use of anti-pl2l60 protein antibody in preparation of anti-tumor medicine and method for treating tumor |
| CN112870185A (en) * | 2021-02-25 | 2021-06-01 | 沈阳药科大学 | Medicine for treating or preventing cervical cancer of human, preparation method and application |
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