CN108642015B - Real-time apoptosis observation model of breast cancer cells induced by aromatase inhibitor and construction method - Google Patents

Abstract

The invention relates to an aromatase inhibitor induced breast cancer cell real-time apoptosis observation model and a construction method thereof, wherein the model comprises two cell lines, namely an MCF7/VC3AI-CYP19 cell line and a ZR7530/VC3AI-CYP19 cell line. The model of the invention can continuously observe the process of apoptosis occurrence and development in real time and can be used for screening the optimal administration mode and the optimal administration time. Based on cell level exploration, the invention further constructs a living body imaging animal model by constructing the luciferase over-expression tumor cells in the future, detects the growth and metastasis conditions of the tumor in real time at the animal level, and further researches and explores the optimal administration mode and the optimal administration time of the anti-tumor drug. On the basis of reducing or not changing the given dosage, the optimal administration strategy of early attenuation and synergism is sought.

Description

Real-time apoptosis observation model of breast cancer cells induced by aromatase inhibitor and construction method

Technical Field

The invention relates to the field of gene apoptosis models, in particular to an aromatase inhibitor induced breast cancer cell real-time apoptosis observation model and a construction method thereof.

Background

Breast cancer is one of the most common malignancies in women. About 70% of breast cancer patients are hormone receptor positive breast cancer, and tamoxifen endocrine therapy is the most commonly used endocrine treatment drug for premenopausal hormone receptor positive breast cancer patients. For postmenopausal hormone receptor positive breast cancer patients, Aromatase Inhibitor (AI) drugs such as letrozole are superior in curative effect to tamoxifen and serve as first-line endocrine treatment drugs. Postmenopausal patients have reduced levels of estrogen in their circulating blood, but aromatase can further promote breast tumor growth by converting androstenedione and testosterone (testostone) to estrogen. It has been found that the stimulation of tumor tissue by in situ estrogen is more pronounced than that of estrogen in circulating blood. Aromatase inhibitors that block estrogen synthesis are therefore an important endocrine treatment modality for postmenopausal hormone receptor positive breast cancer.

Breast cancer is a tumor with high heterogeneity at the molecular level, and breast cancer patients with simultaneous overexpression of Estrogen Receptor (ER) and human epidermal growth factor receptor (HER2) will consider a dual-target treatment model of endocrine therapy in combination with targeted therapy. The research on the action mechanism of letrozole on cells is the basis of combined research. Research shows that aromatase inhibitor can induce apoptosis, so that the establishment of a real-time observation model of letrozole for inducing apoptosis of breast cancer cells is the basis of all research.

Aromatase is a key metabolic enzyme that converts androgens to estrogens and is the major cause of elevated estrogen levels in postmenopausal breast cancer patients. The research shows that the expression level of aromatase in the tumor tissues of breast cancer patients is increased, the enzyme activity is higher, and the estrogen level in peripheral blood is increased. Aromatase inhibitors may promote apoptosis. The therapeutic position of the aromatase inhibitor letrozole in postmenopausal hormone receptor positive breast cancer is well established. Clinical studies indicate that endocrine therapy resistance is an important factor in hormone receptor positive breast cancer recurrence and metastasis. Many clinical basic studies will combine letrozole with other drugs in the future to find new therapeutic approaches to overcome drug resistance. At present, cancer cells separated and purified from tumor tissues of breast cancer patients are generally used in laboratories for in vitro research.

In order to make in vitro experiments better simulate in vivo environment, a good tool model cell is of great importance. As with the results of the other groups, the expression of aromatase was low in MCF7 cells and ZR7530 cells. This greatly limits the study of the mechanism of action of aromatase inhibitors in the cell line and molecular level of the present invention. In order to better simulate the action and mechanism exploration of the aromatase inhibitor in an in vitro test. It is necessary to construct aromatase-overexpressing cell lines. A large number of researches show that the drug effect and the adverse reaction are influenced by the medication mode of the chemotherapeutic drugs, the medication sequence of the combined drugs and the like. Many researchers have endeavored to find the best way to use drugs to minimize side effects with the expectation of achieving maximum efficacy.

Disclosure of Invention

The invention aims to provide an aromatase inhibitor induced breast cancer cell real-time apoptosis observation model and a construction method thereof, and the method provides a model basis for real-time observation and measurement of letrozole drug effect, screening of combined drugs and search of an optimal drug administration mode.

The technical scheme for realizing the purpose of the invention is as follows:

compared with the prior art, the invention has the following advantages and positive effects:

the model of the invention can continuously observe the process of apoptosis occurrence and development in real time and can be used for screening the optimal administration mode and the optimal administration time. Based on cell level exploration, the invention further constructs a living body imaging animal model by constructing the luciferase over-expression tumor cells in the future, detects the growth and metastasis conditions of the tumor in real time at the animal level, and further researches and explores the optimal administration mode and the optimal administration time of the anti-tumor drug. On the basis of reducing or not changing the given dosage, the optimal administration strategy of early attenuation and synergism is sought.

The real-time fluorescent quantitative RCR detection result shows that the expression quantity of the mRNA of the aromatase in the two cell models is obviously higher than that of the tool cell. The results of the western blot method show that the expression level of aromatase protein is obviously increased in two cell models. MTT method detection results show that the cell model over expressing aromatase is stimulated to proliferate fast by Testosterone (Testosterone) and estrogen. Letrozole inhibits the proliferation of testosterone-induced ZR7530/VC3AI-CYP19 cells in a concentration-dependent manner, a real-time observation model of AI drug letrozole-induced breast cancer cell apoptosis is successfully established, and an important experimental basis is laid for researching the action mechanism of letrozole.

Drawings

FIG. 1 is a diagram showing the identification of aromatase-overexpressing cell lines; FIG. 1a shows the Western blot method for detecting the expression level of aromatase protein in cells; FIGS. 1b and 1c show qPCR for detecting the transcription level of aromatase in cells;

FIG. 2 is a graph showing the effect of estrogen androgen on the proliferative capacity of aromatase-overexpressing cells; FIG. 2a is the effect of testosterone (T) on the proliferative capacity of aromatase-overexpressing cells FIG. 2b is the effect of estrogen (E2) on the proliferative capacity of aromatase-overexpressing cells;

FIG. 3 letrozole inhibits aromatase-overexpressing cell proliferation;

FIG. 4 is a graph showing the green fluorescence of cells treated with different concentrations of letrozole in ZR7530/VC3AI-CYP19 for 48 h.

Detailed Description

To further understand the contents, features and functions of the present invention, the following embodiments are provided and described in detail with reference to the accompanying drawings, it should be noted that the embodiments are illustrative and not restrictive, and the protection scope of the present invention should not be limited thereby.

The invention aims to construct an aromatase over-expression breast cancer cell model and an aromatase inhibitor AI-induced breast cancer cell real-time apoptosis observation model.

The invention adopts a lentivirus envelope mediated gene transfection method to construct tool cell lines MCF7/VC3AI and ZR7530/VC3AI for stable expression of apoptosis fluorescent indicator protein VC3 AI. Meanwhile, the cell line is used for constructing AI induced breast cancer cell real-time apoptosis observation models MCF7/VC3AI-CYP19 and ZR7530/VC3AI-CYP 19. Real-time fluorescent quantitative PCR and Western blotting are adopted to detect and verify the expression of aromatase (aromase/CYP19) in the cells. Respectively observing the in vitro proliferation capacity of ZR7530/VC3AI-CYP19 cells under the action of testosterone and estradiol and the cell proliferation capacity under the action of Aromatase Inhibitor (AI) letrozole.

In order to more clearly illustrate the above solution, the present invention provides the following specific operation modes.

Materials (I) and (II)

Main drugs, reagents: estradiol (E2), testosterone (testosterone) were purchased from Sigma company, usa, aromatase inhibitor letrozole was purchased from Selleck company, aromatase CYP19 antibody was purchased from ABcam company, reverse transcription kit and fluorescent quantitative PCR kit were purchased from total gold biotechnology limited. Puromycin was purchased from Invitrogen, usa.

2. Cell line: human embryonic kidney cells 293T and human breast cancer cells MCF-7 and ZR7530 were cryopreserved by the laboratory. The over-expression plasmid pCDH-CMV-VC3AI [19] carrying the gene of apoptosis-indicating fluorescent protein was obtained from Lenzi professor laboratories. The full-length open reading frame cloning plasmid PGEM-CYP19A1, human CYP19A1, was purchased from Beijing Yinqiao Shenzhou technologies, Inc. The integration plasmid pCMV-dR8.91 and the envelope plasmid pCMV-VSV-G, as well as the overexpression VECTOR pCDH-puro-VECTOR, were maintained in the laboratory.

qPCR primers: synthesized by Jinweizhi corporation. Wherein the sequence of the upstream primer of the aromatase is 5'-GACCAATGAATCGGGCTATGT-3', the sequence of the downstream primer is 5'-GATGTCTGGTTTGATGAGGAGAG-3' GAPDH, the sequence of the upstream primer is 5'-GATTCCACCCATGGCAAATTC-3', and the sequence of the downstream primer is 5'-GTGAAGACGCCAGTGGAC-3'.

Second, Experimental methods

1. Cell culture:

293T and MCF-7 cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS), and ZR7530 cells were cultured in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS). The cells were cultured in a cell culture chamber with a CO2 volume fraction of 5% at 37 ℃.

Construction of the plasmid PCDH-puro-CYP19A1

PGEM-CYP19A1 is used as a template, a CYP19A1 gene product containing a specific restriction endonuclease recognition sequence is amplified through PCR, and the product is cut and recovered by specific endonuclease. Then, the PCDH-puro-vector cut by the corresponding restriction enzyme is connected with the recovered product by using T4 ligase. After transformation, bacterium selection, enzyme digestion identification and sequencing verification, the construction of the PCDH-puro-CYP19A1 is successful.

3. Cell line construction:

and (3) spreading the 293T cells in a six-well plate, and transfecting by using a calcium phosphate coprecipitation method when the cells are uniformly attached and the cell fusion degree is 80%. According to the vector plasmid: integrating plasmids: envelope plasmid 2: 1: 1 ratio for transfection. After 5h of transfection, fresh DMEM medium was replaced. After 48h, the culture broth was collected, centrifuged at 8000 rpm for 30 minutes, and virus was collected. The collected virus solution was added dropwise to Polybrene to a final concentration of 10. mu.g/mL to infect the cells. After 12h, the culture medium was replaced with fresh one. After 2d of culture, cells stably overexpressing VC3AI were selected by Blasticidin. In the same way, aromatase was overexpressed in cells expressing VC3 AI. Puromycin drug screen is used for stably expressing the breast cancer cells of the aromatase.

4. Real-time fluorescent quantitative PCR detection of mRNA levels:

collecting the cells after drug screening, and extracting the total RNA of the cells by a Trizol method. RNA quantification was performed using a Nanodrop spectrophotometer, followed by cDNA synthesis using a reverse transcription kit. GAPDH was chosen as the internal reference quantitation.

5. Western blot detection of protein expression

Collecting MCF7/VC3AI-CYP19 and ZR7530/VC3AI-CYP19 cells, centrifuging, collecting cell precipitate, adding cell lysate containing protease inhibitor for lysis, and standing on ice for 5 min, 95 deg.C, and 10 min. The protein was quantified photometrically and then subjected to polyacrylamide gel electrophoresis at 50 ug/well. The electrophoretically separated bands were transferred to PVDF membrane. Sealing with 5% skimmed milk for 1 h. Rabbit anti-human CYP19 antibody and mouse anti-human β -actin antibody were raised against TBST according to 1: 1000 dilution, 4 degrees overnight, TBST membrane washing 3 times. And then incubating with a horseradish peroxidase-labeled secondary antibody, washing the membrane, and detecting the expression of the aromatizing enzyme CYP19 protein in the cells by a chemiluminescence system. Beta-actin is used as an internal reference.

6. Cell proliferation assay

Taking ZR7530/VC3AI-CYP19 cells in logarithmic growth phase, suspending in RPMI 1640 culture solution containing deuteromycin, and culturing at 2 × 10³Cells were seeded per well in 96-well plates, with 5 parallel wells. On day 2, testosterone, estradiol or alpha-testosterone was addedAnd (4) carrying out trozole treatment, and taking an equal amount of solvent as a control. And after culturing for 48 hours in a cell culture box, adding MTT (methyl thiazolyl tetrazolium) into the culture solution, continuously culturing for 4 hours, measuring the absorbance (A) value at the 490nm wavelength of a microplate reader, and calculating the proliferation capacity of each group of cells treated by the medicine.

7. Cultured cell photographing

After the cells were treated accordingly, the cells were photographed using an EVOS FL fluorescence microscope (Advanced Microcopy Group).

Third, statistical method

Statistical analysis was performed using SPSS 19.0 statistical software, and the measurements were expressed as x + -SD. Statistical analysis of the differences between the two groups was performed using the t-test. When P is less than 0.05, the difference is statistically significant.

Fourth, experimental results

1. Successfully constructs the aromatase over-expression cell line

The real-time fluorescent quantitative PCR detection result shows that the expression quantity of the mRNA of the aromatase in MCF7/VC3AI-CYP19 and ZR7530/VC3AI-CYP19 cells is obviously higher than that of cells which are not transfected with the over-expression plasmid of the aromatase. The expression level of aromatase protein in MCF7/VC3AI-CYP19 and ZR7530/VC3AI-CYP19 cells detected by protein imprinting method is obviously higher than that of cells without transfection of aromatase over-expression plasmid, and is consistent with the result of real-time fluorescence quantitative PCR detection of the expression of aromatase mRNA (figure 1).

2. Effect of estrogens and androgens on the proliferative capacity of aromatase-overexpressing cells

MTT detection results show that ZR7530/VC3AI-CYP19 cells can promote cell proliferation by utilizing estrogen and androgen. Wherein 100nmol/L of testosterone has the most obvious effect of promoting the cell proliferation capacity. The cell proliferation rate was about 1.5 times that of the solvent control group. The enhancement of cell proliferation potency by the continued elevated concentration of testosterone was reduced and even inhibited cell proliferation was observed (fig. 2 a). Estrogen (E2) promoted cell proliferation at an optimal concentration of 10nmol/L at a rate of about 1.25 times that of the solvent control (FIG. 2 b).

3. Inhibition of aromatase-overexpressing cell proliferative capacity by letrozole

MTT detection shows that letrozole can inhibit the promotion effect of testosterone on the proliferation capacity of ZR7530/VC3AI-CYP19 cells under the condition that 100nmol/L of testosterone exists. Letrozole showed concentration-dependent inhibition of ZR7530/VC3AI-CYP19 cell proliferation (FIG. 3).

4. Fluorescence microscopy detection of apoptosis

The invention selects letrozole with different concentrations to act on ZR7530/VC3AI-CYP19 cells. After 48h, green fluorescence was detected. The results are shown in the figure (FIG. 4). Most dead cells were detected as emitting green fluorescence, and some cells died but no green fluorescence. The indication shows that the cytotoxic effect of letrozole on breast cancer cells not only promotes apoptosis, but also participates in other non-apoptotic pathways.

Because the target cell can stably express the exogenous gene for a long time through a lentivirus transfection system, in the invention, the target gene is connected into a pCDH-CMV vector to construct an over-expression vector through plasmid recombination. Then establishing MCF/VC3AI-CYP19 cells and ZR7530/VC3AI-CYP19 which stably over-express apoptosis indicating fluorescent protein and aromatizing enzyme through a lentivirus system.

The cell proliferation result detected by MTT shows that both estrogen and aromatase substrate testosterone can obviously promote the proliferation of ZR7530/VC3AI-CYP19 cells. This represents the biological function of aromatase to convert testosterone into estrogen. The aromatase inhibitor letrozole can inhibit the promoting effect of testosterone on the proliferation of ZR7530/VC3AI-CYP19 cells in a concentration-dependent manner. Apoptosis of cells is a caspase family protease cascade process in which caspase-3 activation is a key step in the apoptotic protease cascade. VC3AI can be cleaved by caspase-3 to emit green fluorescence. Studies report that letrozole can promote apoptosis of breast cancer cells. By constructing a cell line with VC3AI and CYP19 over-expressed, the process of inducing apoptosis and development of letrozole can be continuously monitored in real time on the basis of keeping cell activity. The apoptosis rate is quantified visually by the fluorescence intensity and number. Compared with other technical processes for monitoring apoptosis, the method is simpler and more convenient. The cell model can be used for screening drugs which have combined synergistic effect with letrozole so as to be used as a basis for further research of reversing drug resistance.

Claims (1)

1. An aromatase inhibitor induced breast cancer cell real-time apoptosis observation model is characterized in that: the model comprises two cell lines, namely an MCF7/VC3AI-CYP19 cell line and a ZR7530/VC3AI-CYP19 cell line; the construction method is as follows

The method comprises the following steps of:

293T and MCF-7 cells are cultured in DMEM culture solution containing 10% fetal calf serum, and ZR7530 cells are cultured in RPMI 1640 culture medium containing 10% fetal calf serum; culturing the cells in a cell culture box with the volume fraction of CO2 being 5 percent at 37 ℃;

construction of PCDH-puro-CYP19A1 plasmid

PGEM-CYP19A1 is used as a template, a CYP19A1 gene product containing a recognition sequence of a restriction endonuclease is amplified through PCR, and the product is cut and recovered by a specific endonuclease; connecting the PCDH-puro-vector after the enzyme digestion of the corresponding restriction enzyme with the recovered product by using T4 ligase; after transformation, bacterium selection, enzyme digestion identification and sequencing verification, the construction of PCDH-puro-CYP19A1 is successful;

construction of a cell line:

and (3) paving 293T cells in a six-well plate, and when the cells are uniformly attached and the cell fusion degree is 80%, transfecting by using a calcium phosphate coprecipitation method according to a carrier plasmid PCDH-puro-CYP19A 1: integration plasmid pCMV-dR8.91: envelope plasmid pCMV-VSV-G = 2: 1: 1, transfecting a vector plasmid PCDH-puro-CYP19A1, an integration plasmid pCMV-dR8.91 and an envelope plasmid pCMV-VSV-G, replacing a fresh DMEM culture solution after 5h of transfection, collecting the culture solution after 48h, centrifuging at 8000 rpm/10 min, collecting viruses, dripping Polybrene with the final concentration of 10 mu G/mL into the collected virus solution to infect MCF-7 or ZR7530 cells, replacing the fresh culture solution after 12h, culturing for 2d, and screening the cells stably over-expressing VC3AI by Blasticidin; CYP19A1 is overexpressed in cells expressing VC3 AI.

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