CN103743909B - The influence research method that furin inhibitor grows lung adenocarcinoma cell and shifts - Google Patents

Abstract

The present invention relates to a method for studying the influence of a furin inhibitor on the growth and metastasis of lung adenocarcinoma cells, which specifically comprises the following steps: (1) detecting cell proliferation by MTT method; (2) detecting A549 cell clone formation ability; (3) ) Hochest33342/PI double staining method to detect cell apoptosis; (4) Monolayer cell migration experiment (wound? healing); (5) Transwell invasion experiment; (6) Western? Blot detection of cell migration-related protein expression levels; (7) enzyme-linked immunosorbent assay; (8) statistical analysis. The present invention can better utilize Furin inhibitor a1-PDX to suppress the expression of Furin in lung adenocarcinoma A549 cells by studying the influence of furin inhibitors on the growth and metastasis of lung adenocarcinoma A549 cells, thereby effectively providing a therapeutic solution for cancer. in accordance with.

Description

Effects of Furin Inhibitors on the Growth and Metastasis of Lung Adenocarcinoma Cells

technical field

The invention relates to a research method for the influence of a furin inhibitor on the growth and metastasis of lung adenocarcinoma cells, belonging to the field of biotechnology.

Background technique

Lung cancer is a malignant tumor that seriously threatens human health and life in the world today, and its incidence is increasing year by year. The incidence and mortality of lung cancer rank first among malignant tumors in many countries. It is estimated that the number of patients who die of lung cancer exceeds the total number of prostate, breast and colorectal cancers in a year. About 85% of lung cancer patients are non-small cell lung cancer (non-small cell lung cancer, NSCLC), which is divided into adenocarcinoma, squamous cell carcinoma, large cell carcinoma and so on according to histopathology. Most patients have locally advanced or distant metastases at the time of diagnosis. Metastasis and recurrence are the main causes of death in lung cancer patients, and about 90% of lung cancer patients die from tumor metastasis. Therefore, it is necessary to conduct in-depth research on the molecular mechanism of invasion and metastasis of lung adenocarcinoma.

Furin is an important member of the protein precursor processing enzyme family. Many important physiological processes require the participation of Furin, such as the synthesis and secretion of polypeptide and protein hormones, the maturation of membrane receptors, the activation of plasma protein precursors, etc.; The occurrence of diseases is also closely related to Furin, such as the activation of viral coat protein and bacterial exotoxin processing and tumor metastasis. Before these proteins can be active, they need to be cleaved by protein convertases, and then they can become functional proteins. Many proteins closely related to tumorigenesis and development, including Notch, Wnt, MT1-MMP, and VEGF, must be cleaved by protein convertases such as Furin during the maturation process in vivo to exert biological activity. Some members of these proteins are closely related to the occurrence and development of tumors. Furin is highly expressed in a variety of tumors and can be used as a marker in the process of tumor progression and to some extent an indicator of tumor prognosis.

Contents of the invention

The purpose of the present invention is to provide a method for studying the influence of furin inhibitors on the growth and metastasis of lung adenocarcinoma cells, so as to better utilize the Furin inhibitor a1-PDX to inhibit the expression of Furin in lung adenocarcinoma A549 cells, thereby effectively provide a basis for cancer treatment.

In order to achieve the above object, the technical solution of the present invention is as follows.

A research method for the influence of a furin inhibitor on the growth and metastasis of lung adenocarcinoma cells, specifically comprising the following steps:

(1) MTT method to detect cell proliferation:

A549 cells in the logarithmic growth phase were seeded into 96-well plates, 5×10 ³ per well, and 200nM or 400nM different concentrations of a1-PDX were added after 24h to continue culturing for 24h-96h; 20μl of 5mg/ml MTT reagent solution was added to each well, and incubated at 37°C for 4 hours. Add 150 μL of DMSO to each well to dissolve the formazan crystals, and measure the optical density at 490 nm after shaking and dissolving.

(2) Detection of A549 cell clone formation ability:

Take monolayer cultured cells in the logarithmic growth phase, digest with 0.25% trypsin and pipette into single cells, suspend the cells in 1640 culture medium containing 10% fetal bovine serum, inoculate at a cell density of 1×10 ³ /ml In a petri dish; add different concentrations of a1-PDX (200 nM, 400 nM) and place in a saturated humidity environment of 37° C. and 5% CO 2 for static culture for 2 weeks. Discard the supernatant, carefully soak twice in PBS; fix with methanol for 15min. Remove the fixative, stain with Giemsa staining solution for 10 minutes, rinse slowly with running water and air-dry, count clones directly with naked eyes and perform statistical analysis.

(3) Hochest33342/PI double staining method to detect cell apoptosis:

A549 cells in the logarithmic growth phase were seeded on coverslips treated with poly-lysine at a concentration of 1×104 cells/ml. After treatment with different concentrations of a1-PDX for 48 hours, discard the culture medium, add pre-cooled PBS to wash the cells twice; adjust the concentration of Hoechst33342/PI staining solution to a final concentration of 5 μg/ml, and stain for 15 minutes at 37°C in the dark; discard the staining solution, Add 4% paraformaldehyde to fix at 4°C for 5min. Observation and photography under a fluorescence microscope.

(4) Monolayer cell migration experiment (woundhealing):

A549 cells were inoculated in a 6-well plate, and when the confluence reached 100%, a sterile 200 μl tip was used to make a wound, and the cell debris was washed with PBS. Cells were treated the same as before. Cell migration in the injured area was photographed with a digital camera equipped with an inverted microscope at the indicated times.

(5) Transwell invasion experiment:

A549 cells (1×10 ⁵ ) treated with different concentrations of a1-PDX were inoculated in the upper chamber of a Transwell chamber containing 200 μl of RPMI1640 medium without 10% FBS. The lower chamber of the Transwell was filled with 500 μl of complete RPMI1640 medium with 10% FBS. Cells were allowed to migrate for 48 hours, then fixed with 4% formaldehyde and incubated for 15 minutes at room temperature. After washing with deionized water, stain with 0.1% crystal violet. Migrated clones were photographed under a light microscope.

(6) Western blot detection of cell migration-related protein expression levels:

Cells were collected by adding RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% TritonX-100, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, 100 mM sodium fluoride) and protease inhibitors, ice Incubate for 30 minutes, and centrifuge at 13200rpm for 30min. The supernatant was collected and the protein concentration was determined by BCA method (Pierce, USA). The total cell protein was transferred to PVDF membrane after 12% SDS-PAGE gel electrophoresis, and blocked with 5% skimmed milk at room temperature for 1 h. Antibodies to MT1-MMP, VEGF-C, VEGF-D and GAPDH (1:1000) were incubated overnight at 4°C. After washing with PBST, HRP-labeled goat anti-mouse secondary antibody was added and incubated at room temperature for 1 h. After washing with PBST, after incubation with ultra-sensitive luminescence solution (Pierce, USA), the LAS3000 imager (Fuji, Japan) took pictures.

(7) Enzyme-linked immunosorbent assay:

The treatment of A549 cells was the same as described above, and the cell culture supernatant was collected for subsequent detection according to the instructions of the MMP-9, MMP-2, VEGF-cELISA detection kit. Each sample was repeated 5 times.

(8) Statistical analysis:

SPSS13.0 software was used for statistical processing, measurement data were used paired t-test and one-way analysis of variance, count data were used chi-square test, P<0.05 was considered statistically significant.

The beneficial effect of the invention lies in that the present invention can better utilize the Furin inhibitor a1-PDX to inhibit the expression of Furin in lung adenocarcinoma A549 cells by studying the effects of furin inhibitors on the growth and metastasis of lung adenocarcinoma A549 cells, So as to effectively provide a basis for cancer treatment.

Description of drawings

Fig. 1 is a graph showing the effect of a1-PDX on the proliferation of A549 cells in the embodiment of the present invention.

Fig. 2 is a graph showing the effect of a1-PDX on the colony formation of A549 cells in the embodiment of the present invention.

Fig. 3 is a graph showing the effect of a1-PDX on apoptosis of A549 cells in the example of the present invention (control group).

Fig. 4 is a graph showing the effect of a1-PDX on apoptosis of A549 cells in the example of the present invention (200nMa1-PDX treatment group).

Fig. 5 is a graph showing the effect of a1-PDX on apoptosis of A549 cells in the example of the present invention (400nMa1-PDX treatment group).

Fig. 6 is a graph showing the effect of a1-PDX on A549 cell apoptosis in the example of the present invention (statistical analysis of cell apoptosis rate in three independent experiments).

Fig. 7 is a graph showing the influence of a1-PDX on cell migration in the monolayer cell scratch detection in the embodiment of the present invention.

Fig. 8 is a statistical analysis chart of three independent experiments of the effect of a1-PDX on the migration of A549 cells in the embodiment of the present invention.

Fig. 9 is a graph showing the influence of a1-PDX on the infiltration ability of A549 in the embodiment of the present invention (control group).

Fig. 10 is a graph showing the influence of a1-PDX on the infiltration ability of A549 in the embodiment of the present invention (200nMa1-PDX treatment group).

Fig. 11 is a graph showing the influence of a1-PDX on the infiltration ability of A549 in the embodiment of the present invention (400nMa1-PDX treatment group).

Fig. 12 is a statistical analysis chart of the cell infiltration ability of a1-PDX to A549 in three independent experiments in the embodiment of the present invention.

Fig. 13 is a graph showing the effect of a1-PDX on the expression of migration-related proteins in A549 cells in the example of the present invention.

Fig. 14 is a graph showing the effect of a1-PDX on the expression of MMP-9 in A549 cells in the embodiment of the present invention.

Fig. 15 is a graph showing the effect of a1-PDX on the expression of MMP-2 in A549 cells in the embodiment of the present invention.

Detailed ways

Specific embodiments of the present invention will be described below in conjunction with the accompanying drawings, so as to better understand the present invention.

Example

A research method for the influence of a furin inhibitor on the growth and metastasis of lung adenocarcinoma cells, specifically including the following aspects:

(1) Cell culture process: the human lung adenocarcinoma cell line A549 was purchased from the Union Cell Bank of the Chinese Academy of Medical Sciences. Grow in RPMI1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS) and 100 units/mL penicillin, 100 μg/mL streptomycin, and cultivate at 37°C, 5% CO2, and saturated humidity.

(2) The main reagents used include: Furin inhibitor a1-PDX (Merck product number: 126850-2.5MG) was dissolved in DMSO to prepare a 5 mM stock solution. Mouse anti-human VEGF-C, VEGF-D, MT1-MMP and GAPDH antibodies were all purchased from Santa Cruz Company (Santa Cruz, USA). Anti-mouse IgG antibody-HRP and anti-rabbit IgG-HRP were purchased from Sigma (Sigma, USA); MTT, Hochest33342, and Transwell were purchased from Promega, USA; MMP2 and MMP9 ELISA detection kits were purchased from Nanjing Jiancheng Biological Reagent Company. All other reagents were of domestic analytical grade.

(3) Specific implementation process and methods Experimental method:

(3a) MTT method to detect cell proliferation:

A549 cells in the logarithmic growth phase were inoculated into 96-well plates (5×10 ³ per well), and after 24 hours, different concentrations of a1-PDX (200 nM, 400 nM) were added and cultured for 24 hours to 96 hours. 20 μl of MTT reagent (5 mg/ml) solution was added to each well and incubated at 37° C. for 4 hours. Add 150 μL of DMSO to each well to dissolve the formazan crystals, and measure the optical density at 490 nm after shaking and dissolving.

(3b) Detection of A549 cell clone formation ability:

Take monolayer cultured cells in the logarithmic growth phase, digest with 0.25% trypsin and pipette into single cells, suspend the cells in 1640 culture medium containing 10% fetal bovine serum, inoculate at a cell density of 1×10 ³ /ml in a petri dish. Different concentrations of a1-PDX (200 nM, 400 nM) were added, placed in a saturated humidity environment of 37° C. and 5% CO 2 , and cultured statically for 2 weeks. Discard the supernatant, carefully soak in PBS twice. Methanol fixation for 15min. Remove the fixative, stain with Giemsa staining solution for 10 minutes, rinse slowly with running water, air dry, and directly count clones with naked eyes and perform statistical analysis.

(3c) Hochest33342/PI double staining method to detect cell apoptosis:

A549 cells in the logarithmic growth phase were inoculated at a concentration of 1×10 ⁴ cells/ml on coverslips treated with poly-lysine in advance. After treatment with different concentrations of a1-PDX for 48 hours, discard the culture medium, add pre-cooled PBS to wash the cells twice; adjust the concentration of Hoechst33342/PI staining solution to a final concentration of 5 μg/ml, and stain for 15 minutes at 37°C in the dark; discard the staining solution, Add 4% paraformaldehyde to fix at 4°C for 5min. Observation and photography under a fluorescence microscope.

(3d) Monolayer cell migration experiment (woundhealing):

A549 cells were inoculated in a 6-well plate, and when the confluence reached 100%, a sterile 200 μl tip was used to make a wound, and the cell debris was washed with PBS. Cells were treated the same as before. Cell migration in the injured area was photographed with a digital camera equipped with an inverted microscope at the indicated times.

(3e) Transwell invasion experiment:

A549 cells (1×10 ⁵ ) treated with different concentrations of a1-PDX were inoculated in the upper chamber of a Transwell chamber containing 200 μl of RPMI1640 medium without 10% FBS. The lower chamber of the Transwell was filled with 500 μl of complete RPMI1640 medium with 10% FBS. Cells were allowed to migrate for 48 hours, then fixed with 4% formaldehyde and incubated for 15 minutes at room temperature. After washing with deionized water, stain with 0.1% crystal violet. Migrated clones were photographed under a light microscope.

(3f) Westernblot detection of cell migration-related protein expression levels:

A549 cells were cultured and treated as before. Cells were collected by adding RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% TritonX-100, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, 100 mM sodium fluoride) and protease inhibitors, ice Incubate for 30 minutes, and centrifuge at 13200rpm for 30min. The supernatant was collected and the protein concentration was determined by BCA method (Pierce, USA). The total cell protein was transferred to PVDF membrane after 12% SDS-PAGE gel electrophoresis, and blocked with 5% skimmed milk at room temperature for 1 h. Antibodies to MT1-MMP, VEGF-C, VEGF-D and GAPDH (1:1000) were incubated overnight at 4°C. After washing with PBST, HRP-labeled goat anti-mouse secondary antibody was added and incubated at room temperature for 1 h. After washing with PBST, after incubation with ultra-sensitive luminescence solution (Pierce, USA), the LAS3000 imager (Fuji, Japan) took pictures.

(3g) ELISA:

The treatment of A549 cells was the same as described above, and the cell culture supernatant was collected for subsequent detection according to the instructions of the MMP-9, MMP-2, VEGF-cELISA detection kit. Each sample was repeated 5 times.

(4) Statistical analysis:

SPSS13.0 software was used for statistical processing, measurement data were used paired t-test and one-way analysis of variance, count data were used chi-square test, P<0.05 was considered statistically significant.

(5) Implementation results:

(5a) Effects of a1-PDX on proliferation and colony formation of A549 cells:

After different concentrations of a1-PDX acted on A549 for different times, the survival of the cells was detected by the MTT method, as shown in Figure 1 . The results showed that when a1-PDX was applied to A549 cells for more than 48 hours, the cell growth was significantly inhibited. The colony formation assay was used to detect the colony formation of A549 cells. The results showed that the colony-forming ability of a1-PDX-treated A549 cells was significantly reduced compared with the control group, as shown in Figure 2.

(5b) Effect of a1-PDX on apoptosis of A549 cells:

To explore the mechanism of a1-PDX inhibiting the growth of A549 cells, Hochest33342/PI double staining was used to detect the apoptosis of A549 cells. The results showed that a1-PDX could induce apoptosis of A549 cells, as shown in Figure 3, Figure 4, Figure 5, and Figure 6.

(5c) Effects of a1-PDX on migration and invasion of A549 cells:

In order to detect whether a1-PDX can regulate the migration and invasion of A549 cells, woundHealing and Transwell experiments were used to further detect the migration of A549 cells. Both experimental results showed that a1-PDX significantly reduced the migration and invasion abilities of A549 cells, as shown in Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, and Figure 12.

(5d) Effect of a1-PDX on expression of cell migration-related proteins:

Many proteins are involved in the regulation of tumor cell migration in vivo. In order to fully understand the molecular mechanism of a1-PDX regulating the migration of A549 cells, the protein expression levels of MT1-MMP, MMP2, MMP9, VEGF-c, and VEGF-d were detected by Western Blot and ELISA. As shown in Figure 13, a1-PDX significantly reduced the expression of MT1-MMP, VEGF-c, and VEGF-d in cells. At the same time, the concentrations of MMP2 and MMP9 in the cell culture supernatant were significantly lower than those of the control group, as shown in Figure 14 and Figure 15 . These results indicated that the inhibition of A549 cell migration by a1-PDX may be related to the decreased expression of MT1-MMP, MMP2/9, and VEGF-C/D.

In an embodiment of the present invention, the Furin inhibitor a1-PDX can induce proliferation inhibition and apoptosis of A549 cells. In addition, the migration ability of A549 cells was significantly reduced after incubation with a1-PDX. The expression levels of these matrix metalloproteinases effectively reflect the invasive ability of tumor cells. a1-PDX can not only inhibit the expression of MMP2 and MMP9, but also reduce the expression of MT1-MMP. a1-PDX reduces the activity of Furin enzyme, thereby reducing the maturation and activation of MT1-MMP, and further inhibiting the maturation of MMP2 and MMP9. The expression of VEGF-C and VEGF-D proteins in a1-PDX-treated cells was significantly reduced. Down-regulation of Furin enzyme activity, thereby inhibiting the expression of MMPs and VEGFs protein in A549 cells may be the mechanism of its inhibition of A549 growth, migration and invasion.

The above description is a preferred embodiment of the present invention, and it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also considered Be the protection scope of the present invention.

Claims (1)

1. furin inhibitor to lung adenocarcinoma cell growth and transfer an influence research method, it is characterized in that: specifically comprise the following steps:

(1) mtt assay detects the propagation of cell: exponential phase A549 cell is inoculated in 96 orifice plates, every hole 5 × 10 ³, the a1-PDX starting to add 200nM or 400nM variable concentrations after 24h continues to cultivate 24h-96h; Every hole adds 20 μ l5 mg/ml MTT reagent solutions, and in 37 DEG C of incubations 4 hours; Every hole adds the DMSO Rong Xie formazan crystal of 150 μ L, and concussion detects the optical density at 490nm place after dissolving;

(2) A549 Cell clonality detects: the single-layer culturing cell in growth period of taking the logarithm, and blows and beats into individual cells, cell is suspended in the RPMI-1640 containing 10% hyclone, with 1 × 10 with 0.25% Trypsin Induced ³the cell density of/ml is inoculated in double dish; The a1-PDX adding 200nM or 400nM variable concentrations puts 37 DEG C, 5%CO ₂saturated humidity environment under, quiescent culture 2 weeks; Abandoning supernatant, PBS carefully embathes 2 times; Methyl alcohol fixes 15min; Remove immobile liquid, Giemsa stain dyeing 10min, after flowing water slowly rinses rear air oxygen detrition, naked eyes directly count clone and statistical study;

(3) the two dye method of Hochest33342/PI detects Apoptosis: exponential phase cell A549 is by 1 × 10 ⁴individual/ml concentration is inoculated on the cover plate in advance with poly-D-lysine process; After the a1-PDX process 48h of variable concentrations, incline nutrient solution, adds precooling PBS washed cell 2 times; Adjust Hoechst33342/PI dye liquor concentration to final concentration 5 μ g/ml, 37 DEG C of lucifuges dyeing 15min; Discard dye liquor, add 4% paraformaldehyde, 4 DEG C of fixing 5min; Take at fluorescence microscopy Microscopic observation;

(4) cell monolayer migration experiment: A549 cell is seeded in 6 orifice plates, in time growing to degree of converging and reach 100%, makes cut with the Tip head point of 200 aseptic μ l, and with PBS washed cell fragment; In the cell migration situation of the digital camera shooting injured area that the time of specifying is equipped with inverted microscope;

(5) Transwell Matrigel: the A549 cell of the a1-PDX process of variable concentrations is inoculated in the upper chamber of Transwell cell, and upper chamber contains the RPMI1640 nutrient culture media of 200 μ l, but not containing 10%FBS; Transwell cell lower chamber is filled with the complete RPMI1640 nutrient culture media of 500 μ l, containing 10%FBS; Make cell migration 48 hours, then with 4% formaldehyde, cell is fixed, incubated at room temperature 15 minutes; After deionized water washing, 0.1% violet staining; Take the clone of migration under an optical microscope;

(6) Westernblot detects cell shifting related protein expression: collecting cell adds RIPA damping fluid and protease inhibitors, this RIPA damping fluid be 50mM TrispH7.4,150mM sodium chloride, 1% TritonX-100,0.1%SDS, 1% NaTDC and 5mMEDTA, the sodium fluoride potpourri of 100mM, hatch 30 minutes on ice, the centrifugal 30min of 13200rpm; Collect supernatant and BCA method mensuration protein concentration; Total protein of cell transfers to pvdf membrane after 12%SDS-PAGE gel electrophoresis, and under room temperature, 1h closed by 5% skim milk; The antisera overnight of MT1-MMP, VEGF-C, VEGF-D and GAPDH is hatched at 4 DEG C; The sheep anti mouse two adding HRP mark after PBST washing resists, and after incubated at room temperature 1h, PBST washing, super quick luminescent solution is hatched rear employing LAS3000 imager and taken pictures;

(7) enzyme linked immunosorbent assay: collecting cell culture supernatant also carries out follow-up detection according to MMP-9, MMP-2, VEGF-cELISA detection kit instructions; Each sample repeats 5 times;

(8) statistical study: application SPSS13.0 software carries out statistical procedures, and measurement data adopts paired t-test and one-way analysis of variance, and enumeration data adopts Chi-square Test, and P < 0.05 has statistical significance.