CN103992987A - Method for regulation of EGFR expression of pancreatic cancer cell through Notch pathway - Google Patents

Abstract

A method for regulation of EGFR expression of pancreatic cancer cells through the Notch pathway is by interference of pancreatic cancer cells by Notchl siRNA to obviously decrease EGFR. And through ChIPseq, it is found that Notch and CSL combine together on an EGFR promoter. This shows that the Notch pathway regulates the EGFR expression in pancreatic cancer cells through CSL, thereby inhibiting the growth of tumor. The method clarifies for the first time the relationship between the expression of Notch pathway and EGFR expression in pancreatic cancer cells and provides important targets for tumor targeting treatment.

Description

A kind of method that regulates pancreatic cancer cell EGFR to express by Notch path

Technical field

The present invention relates to the method that regulates pancreatic cancer cell EGFR to express by Notch path, belong to oncomolecularbiology field.

Background technology

The related gene of EGF-R ELISA (epithelial growth factor receptor, EGFR) in can active cell core, thus cell fission propagation promoted.Have been found that at present in cancer of the stomach, mammary cancer, bladder cancer and incidence squama cancer, EGFR expresses and increases, and illustrates that EGF-R ELISA is to play a crucial role in the developing of many tumours.

Notch signal path is one highly evolves and conservative path, the interaction between mediated cell and cell.In the time of adjacent cell ligand and acceptor interaction, can cause Notch albumen continuous cracking, discharge born of the same parents' inner segment (NICD) of acceptor.NICD proceeds in nucleus, and in nucleus with DBP CSL (CBFi/RBP-J κ, Suppressor of Hairless, the acronym of Lag-1) combination, start the transcribing of target gene.Our expression that studies have shown that EGFR is subject to the regulation and control of Notch-1, can reduce the expression of EGFR by regulation and control Notch-1.

Summary of the invention

The technical problem to be solved in the present invention is propagation and the growth for controlling tumour cell, how to block the signal path that EGFR mediates.

Technical solution of the present invention, for disturbing after pancreatic cancer cell with Notch1siRNA, can make EGFR express obviously and decline.

Present method has following advantage and effect:

1. present method can make EGFR express obviously decline.

2. present method, has good stability, acts on obvious feature.

Brief description of the drawings

PCR electrophoresis result (the M:100bp DNA Marker of Fig. 1 ChIP sample checking; 1: the positive controls of making of RNA pol II antibody; The sample sets of 2:Flag antibody precipitation Notch1; The sample sets of 3:Flag antibody precipitate C SL; 4: the negative control group that non-specific IgG antibody does);

Fig. 2 CSL binding site schematic diagram in fragment (compare after CSL binding site among fragment that we check order, we have found to have two places consistent with CSL binding site sequence) that checks order.

Embodiment

1. synthetic interference fragment

Synthetic specificity is for siRNA and the negative control siRNA of Notch1 gene mRNA.

2. transfection

In 1640 substratum that contain 10% foetal calf serum, cultivate BxPC3 cell, in cell culture incubator, under 37 DEG C, 50mL/L C02 saturated humidity, cultivate, the recombinant expression vector plasmid that obtains in the test group transfection the first step, control group transfection pcDNA3 empty plasmid.Cell goes down to posterity in advance for 24 hours, in six orifice plates behind degrees of fusion to 80% left and right, according to Lipofectamine ^tM2000 specification sheetss carry out the transfection of negative control siRNA sequence and Notch1 siRNA.

3.WESTERN BLOT detects

In above-mentioned pancreatic cancer cell, add after RIPA protein lysate lysing cell, we require to extract total protein according to test kit, make the concentrated glue of polyacrylamide and separation gel, the equal protein sample of getting different groups carries out electrophoresis, then carries out successively transferring film, sealing, primary antibodie, two and anti-ly hatches and wash that (wherein primary antibodie weaker concn is: 1: 500; Two anti-weaker concns are: 1: 3000).Finally, colour developing exposure is also developed a film by the exposure of x line, compares through the gray-scale value of the fixed each histone band of autophoresis gel analysis system scan well surveying, and EGFR expresses decline.

4. chromatin immunoprecipitation experiment

(1) (every dish approximately 1 × 10 when the degree of converging of cultivating BxPC3 cell is 80% ⁷individual, use RPMI1640 substratum), start preparing experiment.

(2) with the transfection reagent of Roche company by 5 kinds of plasmids respectively transfection enter in BxPC3 cell, every kind of plasmid at least transfection 2 coils cell, 37 degree thermostat containers are cultivated harvested cell after 48h.

(3) in the day before yesterday on-test by the sealing treatment of Protein G agarose ball: get the swelling good Protein G agarose ball of 300 μ L, of short durationly abandon supernatant after centrifugal, add 1mL distilled water, on vortex mixer, 4 DEG C of rotation 3min wash away residual salt and ethanol, repeat to wash 3 times.Add distilled water 900 μ L, 10mg/mL BSA100 μ L, on vortex mixer, 4 DEG C of rotation overnight incubation, for subsequent use.

(4) in substratum, add 4% formaldehyde solution, making its final concentration is 1%, after mixing, at room temperature leaves standstill crosslinked 10 minutes.Add 1.25M glycine, making its final concentration is 0.125M again, at room temperature leaves standstill 5 minutes and stop crosslinking reaction after mixing.Outwell reaction solution, by culture dish as on ice.

(5) with appropriate 4 DEG C of 1 × PBS, every dish cell is washed 2 times, blotted washing lotion.Every dish adds 1.2mL4 DEG C of 1 × PBS afterwards, scrapes cell with cell, moves in 1.5mL EP pipe.4 DEG C of centrifugal 5min of 5000rpm, abandon supernatant, obtain cell precipitation.

(6) in every tube cell precipitation, add 300 μ L4 DEG C Lysis buffer, final concentration be 1 × 25 × cocktail and the final concentration PMSF that is 1mM, blow and beat resuspended mixing, vortex vibration 3-5 time, at standing cracking 10min on ice.4 DEG C of centrifugal 5min of 8000rpm, abandon supernatant afterwards, obtain nucleus precipitation.

(7) in every tube cell core precipitation, add 300 μ L4 DEG C nucleus lysate, final concentration be 1 × 25 × cocktaiL and the final concentration PMSF that is 1mM, blow and beat resuspended mixing, vortex vibration 3-5 time, at standing cracking 10min on ice.

(8) on ice ultrasonic 3 times, ultrasound intensity 50, each 5s, every minor tick 1min.4 DEG C of centrifugal 10min of 12000rpm, get supernatant afterwards.

(9) every pipe equal-volume supernatant that takes a morsel, is incorporated in 1 pipe as Input sample cumulative volume 50 μ L.Add 50 μ L distilled waters, the NaCL that final concentration is 0.2M and 2 μ L RNase A, mix latter 37 DEG C and hatch 30min again.Add afterwards 2 μ L Proteinase Ks, separate crosslinked spending the night for 65 DEG C.The extracting of phenol/chloroform, 1% agarose gel electrophoresis is identified ultrasonic clip size, is advisable with 200-500bp.

(10) the homogeneous Protein G agarose ball suspension (total consumption 500 μ L) that adds equal-volume to seal in the ultrasonic supernatant liquor of every pipe, final concentration is 1 × 25 × cocktail, on vortex mixer, 4 DEG C of rotations are hatched and within 2 hours, are carried out pre-washing, with the nonspecific immunity sphaeroprotein in the system of removing.4 DEG C of centrifugal 2min of 5000rpm afterwards, collect supernatant.

(11) every pipe add final concentration be 1 × 25 × cocktail, again in wherein adding 2 μ g Normal Rabbit IgG non-specific antibodies as negative control in a pipe, in other every pipes, add 2 μ g Notchl antibody, 4 DEG C of rotation overnight incubation on vortex mixer.

(12) second half Protein G agarose ball suspension of remainder is mixed to rear equivalent and adds in each pipe, then add final concentration be 1 × 25 × cocktail, on vortex mixer, 4 DEG C of rotations are hatched and within 2 hours, are carried out immunoprecipitation.4 DEG C of centrifugal 2min of 5000rpm, abandon supernatant afterwards, obtain being combined with the Protein G agarose ball precipitation of DNA-transcription factor-transcription factor antibody complex.

(13) under 4 DEG C of environment, on vortex mixer, Protein G agarose ball precipitation is respectively washed 1 time with 1mL low salt buffer, high-salt buffer and LiCl damping fluid respectively, finally use TE (pH=8.0) damping fluid to wash twice, each 5min, 4 DEG C of centrifugal 2min of 5000rpm, abandon supernatant.

(14) prepare elutriant.In every tubulin G agarose ball precipitation, add 300 μ L elutriants resuspended, on heating vortex mixer, 65 DEG C, 1200rpm vibration 30min carry out wash-out.The centrifugal 2min of normal temperature 5000rpm afterwards, collects supernatant.In every pipe supernatant, adding final concentration is NaCL and the 2 μ L Proteinase Ks of 0.2M, separates crosslinked spending the night for 65 DEG C, and phenol/chloroform extracting obtains the target DNA of purifying.

6. on ILLUMINA HIseq2000, carry out high-flux sequence.

7., after the data obtained is analyzed by self-programmed software, we find that there is Partial Fragment and drop on the promoter region that has EGFR on No. 7 karyomit(e)s of people.Entered and the comparison of CSL binding site (TGGGAAA, TTTCCCA), binding site is all among fragment that we check order (seeing Fig. 2) again.The mixture binding specificity of this explanation Notch and CSL in the front promoter region of egfr gene (chr7: 55054219-55192138), the expression of regulation and control EGFR.

Claims (3)

1. a method that regulates pancreatic cancer cell EGFR to express by Notch path, is characterized in that using after Notchl siRNA interference effect pancreatic cancer cell, and EGFR expresses obviously and declines.

2. detection method according to claim 1, what it is characterized in that use is that Notchl siRNA disturbs pancreatic cancer cell.

3. detection method according to claim 2.